摘要:

Hot oV the press O O O O H OH 1 O AcO AcO R AcO H AcO O 2 R = a-OH 3 R = b-OH Robert A. Hilla and Andrew R. Pittb aDepartment of Chemistry Glasgow University Glasgow UK G12 8QQ. E-mail bobh@chem.gla.ac.uk bDepartment of Pure and Applied Chemistry Strathclyde University Thomas Graham Building 295 Cathedral Street Glasgow UK G1 1XL. E-mail a.r.pitt@strath.ac.uk An unusual linear diterpene linulatusin 1 has been found in Aster lingulatus (G. A. Cordell and co-workers Phytochemistry 1998 49 609). Two diterpenoids 2 and 3 with a new skeleton called euphoperfoliane have been isolated from Euphorbia semiperfoliata (G. Appendino et al. J. Nat. Prod. 1998 61 749). The euphoperfoliane skeleton is presumably formed by C9–C17 cyclisation of a jatrophane skeleton.H H H HO O O OH O OH O HO O O 4 Sinuflexlin 4 from the soft coral Sinularia flexibilis is a biscembranoid diterpene that may be formed via a hetero- Diels–Alder coupling of sinularin (C.-Y. Duh et al. Tetrahedron Lett. 1998 39 7121). Cyclohopenol 5 from rhizomes of Pyrrosia lingua is a representative of a new triterpenoid skeleton (K. Shiojima and co-workers Chem. Pharm. Bull. 1998 46 730). A tetracyclic diterpenoid 6 with a new carbon skeleton has been isolated from Euphorbia segatalis (M.-J. U. Ferreira et al. Phytochemistry 1998 49 179). The parent pentol has been named segetalol. Three sesquiterpenoids including clitocybulol A 7 with a new skeleton have been isolated from the Basidiomycete Clitocybula oculus (W.A. Ayer et al. Phytochemistry 1998 49 589). Four ring-Bcontracted stigmastane derivatives including taiwaniasterol A 8 have been isolated from the leaves of Taiwania cryptomerioides (J.-M. Fang and co-workers Phytochemistry 1998 48 1391). Several highly oxygenated naphthalenes and acetophenones have been isolated from the liverwort Adelanthus decipiens from Ecuador including 2,3,4,5,6-pentamethoxyacetophenone Hill and Pitt Hot oV the press HO OAc H AcO HO O H H H BzO OAc 6 5 OH OH O H HO HO HO 7 O 8 9 (D. S. Rycroft et al. Phytochemistry 1998 48 1351). The aromatic hydrocarbon 10 from the foliar epicuticular wax of Pilocarpus jaborandi is of biogenetic interest and may serve as a biomarker for this species (A.Salatino and co-workers Phytochemistry 1998 49 127). The phytoalexin o-hibiscanone 11 formed by Hibiscus cannabinus has been shown to be highly toxic towards the fungal pathogen Verticillium dahliae (A. A. Bell et al. Phytochemistry 1998 49 431). The structure of the dimeric butenolide glochidiolide 12 from Glochidion acuminatum was confirmed by X-ray analysis (H. Otsuka et al. Chem. Pharm. Bull. 1998 46 1180). O OMe MeO OMe MeO 10 OMe 9 O O O O OH O O 11 OH 12 Kenji Mori has written a review on the synthesis stereochemistry and bioactivity of semiochemicals (Eur. J. Org. Chem. 1998 1479). The chemistry of the potent carcinogen iii ptaquiloside 13 found in bracken has been reviewed ranging from the determination of its structure the formation of the actual carcinogen via loss of the glucose moiety at about pH 8 through to its carcinogenic eVect by reaction with adenine causing DNA strand scission (Scheme 1) (K.Yamada Angew. OH O H loss of D-glucose HO O O OH HO OH N 13 OH HO pH 8 HO N Scheme 1 Scheme 2 D Chem. Int. Ed. 1998 37 1818). An examination of methionine biosynthesis in plants has been published by the group of Roland Douce (S. Ravanel et al. Proc. Natl. Acad. Sci. USA 1998 95 7805). Methionine plays a vital role not only in proteins but also as a precursor for S-adenosylmethionine which is used for a range of important methylation reactions. Labelled glycerol has been used to investigate the biosynthesis of acaterin 14 from Pseudomonas sp.A92 (Y. Fujimoto and co-workers Tetrahedron Lett. 1998 39 6233). The results OH Formation of abasic site strand scission OH (Scheme 2) indicate that acaterin is formed from a C10 polyketide precursor condensing with a glycerol metabolite. The biosynthesis of cytrienin A 15 from Streptomyces sp. RK95-74 has been investigated and the results indicate that the 14 O O OH MeO MeO O D D iv Natural Product Reports 1998 OH O adenine of DNA NH N O N O HO O H 16 CD2H Scheme 3 1-aminocyclopropane-1-carboxylic acid moiety is derived from methionine (H. Osada and co-workers Tetrahedron Lett. OH H N HO O OH OMe H O O O 15 1998 39 6947).The biosynthesis of the prenyl chain of ubiquinone 16 in Escherichia coli has been studied using deuterium-labelled 1-deoxy-D-xylulose (Scheme 3) (D. Arigoni and co-workers Chem. Commun. 1998 1857). In contrast to the mevalonate pathway a diVerence in labelling patterns was observed in this non-mevalonate route. The label from [4-2H]- 1-deoxy-D-xylulose is only found in the unit corresponding to the starter dimethylallyl diphosphate unit whereas [3-2H]-1- deoxy-D-xylulose labels all prenyl units. The biosynthetic origin of the trichloromethyl group of barbamide 17 a metabolite of the marine cyanobacterium Lyngbya majuscula has been studied using [13C]- and [2H]-labelled leucines (W. H. Gerwick and co-workers J.Am. Chem. Soc. 1998 120 7131). The pro-S methyl group of leucine was shown to be the one that is trichlorinated and interestingly deuterium label is retained at C-2 of barbamide 17 indicating that no double bond activation of the methyl group occurs. A study of the biosynthesis of verrucosan-2‚-ol 18 in Chloroflexus aeratiacus using incorporation of singly and doubly labelled 13C acetate and comparison of the labelling patterns to those of intermediate metabolites deduced by ‘‘retrobiosynthetic’’ examination of labelled nucleotides and amino acids has shown that it occurs by the mevalonate pathway and that the formation of the unusual ring system involves a Wagner–Meerwein rearrangement a 1,5-hydride shift and a cyclopropylcarbenium to cyclopropylcarbenium rearrangement (Scheme 4) (C.Rieder et al. J. Biol. Chem. 1998 273 18 099). An in-depth study of every step of the reaction of the ‚-glycoside hydrolase celA5 from Bacillus agaradhaerens by a multidisciplinary analysis of the native substrate-bound covalent-intermediate and product-bound complexes has been reported (G. J. Davies et al. Biochemistry 1998 37 11707). Ranging from identifying the 1S3 skew-boat configuration of the bound substrate to the disordered and hence low aYnity product bound complex this gives an in depth view of the mechanism of this ‘‘textbook’’ enzyme. A Cys to Ser mutant of a UDP-glucose dehydrogenase has been used to trap a OH D HO N O OH MeO CCl Me 3 2 XH N OH H O N S O HOO H HO OUDP + X PPO O O HOO H HO OUDP H H + + X = S fast X = O slow XH OH O O HOO H HO + H H + H H H OUDP insight into the role of the two N-terminal arginines (D.C. Williams et al. Biochemistry 1998 37 12 213). Removal or mutation of one of the N-terminal arginines of this ‘‘pseudomature’’ protein gives an enzyme that cannot use geranyl pyrophosphate 19 but can use the later intermediate 3S-linalyl pyrophosphate 20 to synthesise limonene 21 (Scheme 6) suggesting that the arginines play an important role in the diphosphate migration. H + H H OPP H + 19 NAD+ NADH Scheme 5 OPP 20 Scheme 6 HO H H 17 Scheme 4 The Cu,Zn superoxide dismutase from Photobacterium leiognathi is a super-eYcient enzyme compared to the mammalian enzyme with a rate constant of 8.5#109 M"1 s"1 at pH 7 and low ionic strength (M.E. Stroppolo et al. Biochemistry 1998 37 12 287). Experiments suggest that this is due to a protonic channel greater than 4.4 Å in width giving a high degree of solvent exposure at the active site. The crystal structure of Bacillus cereus metallo ‚-lactamase at 1.9 Å including the active site waters shows an active site containing one strongly bound zinc with a water at 1.9 Å (eVectively an activated hydroxide) and a second loosely bound zinc that appears not to be involved in water activation (S. M. Fabiane et al. Biochemistry 1998 37 12 404). This enzyme appears to be an evolutionary intermediate between the one and two zinc ‚-lactamases.Using a concerted randomisation method to introduce a range of amino acids into certain positions of a 21 18 covalently bound intermediate (X. Ge et al. J. Am. Chem. Soc. 1998 120 6613). The enzyme is able to catalyse both of the NAD+-dependent reductions but release of the resulting ester as opposed to the thioester formed in the native enzyme is very slow (Scheme 5). Truncation of expressed limonene synthase by removal of the plastid target sequence not only gives a more catalytically eYcient enzyme but has given an Hill and Pitt Hot oV the press XH O O HOO H HO OUDP X HO O HOO H HO OUDP v DNA binding zinc finger array A. Klug and his group have identified the important underlying mechanism of DNA sequence recognition by these arrays which will point the way to improved designs (M.Isalan et al. Biochemistry 1998 37 12 026). The crystal structure of arginine kinase at 1.86 Å with ADP and nitrate ion mimicking the planar „-phosphoryl during the in-line transfer shows extreme precision in the alignment of reacting orbitals such that it would appear that orbital steering plays the key role in rate acceleration for two substrate enzymes (G. Zhou et al. Proc. Natl. Acad. Sci. USA 1998 95 8449). Polarisation strain and acid-base catalysis do contribute but compared to one substrate reactions these appear to play a secondary role to substrate alignment. O O N NH S O N O O 22 The crystal structure of a mutant E.coli phosphate binding protein with an N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)- coumarin-3-carboxamide 22 fluorophoric phosphate probe at the binding site gives a clear indication of why this probe works well and may help in the design of other environmentally sensitive fluorescent probes for enzymes (M. Hirshberg et al. Biochemistry 1998 37 10 381). A combined theoretical and mutagenesis study of the electron transfer process between cytochrome P450cam and its natural redox partner putidaredoxin has identified the amino acids in the contact area that are mainly responsible (A. E. Roitberg et al. J. Am. Chem. Soc. vi Natural Product Reports 1998 1998 120 8927). The dominant pathway appears to be from the Fe2S2 via Cys39 and Asp38 of the putidaredoxin across to Arg122 and thence through a haem propionate on to the iron in the haem of the cytochrome.The interpretation of EXAFS spectra to determine the ligands to zinc in enzymes has been improved by developing new methodology using model compounds (K. Clark-Baldwin et al. J. Am. Chem. Soc. 1998 120 8401). This enables O and N ligands to be identified in the presence of S ligands. In the following paper (K. Peariso et al. J. Am. Chem. Soc. 1998 120 8410) they put this to the test with E. coli methionine synthase. Electrospray ionisation coupled to Fourier transform ion-cyclotron resonance mass spectroscopy has been used to analyse the binding of T4regA to a number of RNA sequences (C.Liu et al. Anal. Biochem. 1998 262 67). Competitive assays using this method reveal a similar relative binding aYnity to that found by other methods. The recent proposal of a two covalent intermediate mechanism for 5-enoylpyruvylshikimate-3-phosphate (EPSP) synthase (as opposed to the more commonly identified one non-covalent intermediate mechanism) based on sub-zero entrapment and solid state REDOR NMR (Studelska et al. Biochemistry 1998 37 15 555) has been refuted by a re-examination of this system by the group of Jeremy Evans (D. L. Jakeman et al. Biochemistry 1998 37 12 012). The key NMR signals actually appear to be due to EPSP and EPSP ketal. A low specificity D-threonine aldolase isolated from Arthrobacter species DK-38 appears to be a new type of pyridoxal enzyme with no sequence similarity to known enzymes and a requirement for Mn2+ ions for maximal activity (J.-Q. Liu et al. J. Biol. Chem. 1998 16 678). The role that RNA may have played in prebiotic chemistry has been called into question by recent studies (M. Levey and S. L. Miller Proc. Natl. Acad. Sci. USA 1998 95 1933). Examination of the temperature stability of the RNA bases at 100 )C gives half lives for A and G of approximately 1 year and only 19 days for C too short for these to have accumulated on the geological timescale in suYcient quantities to be significant. Even at low temperatures the half lives are shown to be relatively short.

ISSN:0265-0568    

DOI:10.1039/a806hopy

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Jasmonates key players in the plant defence Michael H. Beale and Jane L. Ward IACR-Long Ashton Research Station Department of Agricultural Sciences University of Bristol Long Ashton Bristol UK BS41 9AF Reviewing the literature up to March 1998 1 Introduction 2 Structure nomenclature and occurrence 2.1 Jasmonic acid and methyl jasmonate 2.2 12-Oxophytodienoic acid and its relatives 2.3 Jasmonate metabolites and other related compounds 3 Biosynthesis 3.1 The pathway 3.2 Enzymology 3.3 Metabolism 4 Synthesis of jasmonates 4.1 Enantioselective syntheses of methyl jasmonate 4.2 Enantioselective syntheses of methyl 7-epijasmonate 4.3 Synthesis of 12-oxophytodienoic acid OPC 8:0 and homologues 4.4 Isotope-labelled jasmonates 5 Structure–activity relationships 5.1 Optical and cis–trans isomers 5.2 Derivatives with a stereochemical anchor at C-7 or C-3 5.3 Other changes to the cyclopentanoid ring 5.4 The pentenyl side-chain 5.5 The carboxymethyl side-chain and the importance of ‚-oxidation 5.6 Coronatine related bicyclic amides and jasmonate amino acid conjugates 6 Conclusions and future prospects 7 References 1 Introduction (") Methyl jasmonate [(3R),(7R)-(")-MeJA] 1 has been known1 since 1962 as a fragrant component in the essential oil from flowers of jasmine Jasminium grandiflorum.The corresponding free acid (")-jasmonic acid (JA) 2 was first isolated2 from culture filtrates of the fungus Botryodiplodia (syn.Lasidiplodia) theobromae in 1971. Subsequently both of these compounds as well as a number of derivatives and homologues have been shown to be widespread in the plant kingdom and in some fungi (see section 2.1).The generic term jasmonate is used loosely in the literature to describe jasmonic acid methyl jasmonate their isomers and sometimes biosynthetic precursors and metabolites. For some years jasmonates have been known to be involved in events in plant development in particular in the promotion of senescence and inhibition of growth. These eVects are comparable to and sometimes synergistic with those of other plant hormones such as abscisic acid and ethylene. More recently it has become obvious that jasmonates and related oxylipins play a signifi- cant cell-signalling role in the defence response of plants. The volatility of MeJA led to the suggestion3 that it may also be involved in interplant communication. Although we now know that this is unlikely in normal environments this report stimulated intense research interest in jasmonate signalling.Jasmonates in plants are formed via 12-oxophytodienoic acid (12-oxo-PDA) 5 the product of an oxidative cyclisation of ·-linolenic acid. The obvious similarities of 12-oxo-PDA and jasmonates both in structure and biogenesis to the animal prostaglandins allow parallels to be drawn between the molecular mechanisms of lipid oxidative cyclisation in plants and animals and the role of the resultant cyclopentanoid products as signals. In plants these oxylipins are involved in marshalling the response to outside attacks by herbivores insects and microbes. Thus jasmonates are involved at the molecular level in the wound response induced chemical defence as tuberisation signals and as messengers involved in the contact coiling of tendrils on climbing plants.In this review we report on recent research on biosynthetic and synthetic routes to jasmonates homologues and analogues and discuss current data on structure–activity relationships. There also exists a large body of literature on the biological eVects of jasmonates. Where relevant this is discussed briefly but this area has been extensively reviewed recently,4–13 and the reader is directed to these publications for in-depth discussions of the physiology and molecular biology of jasmonate action in plants. 2 Structure nomenclature and occurrence 2.1 Jasmonic acid and methyl jasmonate In this review we use the fatty acid based numbering system for jasmonates and their precursors as this reflects their biosynthetic origin.Many earlier papers use a numbering system based on the cyclopentane ring the side chains being attached at C-1 and C-2 and the ketone at C-3. A further diYculty with the earlier literature is that the configuration at C-3 (previously C-1) of natural (")-MeJA was wrongly assigned as (S). The correct configuration of (")-MeJA (3R,7R) was given in 1965 by Hill and Edwards.14 Hormonal CO2R O CO2R O 3 7 1 2 3 4 5 6 7 8 9 10 11 12 1 R = Me 2 R = H 3 R = Me 4 R = H (CH2)7CO2H O (CH2)7CO2H O O • O O CO2H (CH2)4CO2H 9 9 10 11 12 13 14 18 1 5 6 7 8 9 (CH2)4CO2H Beale and Ward Jasmonates key players in the plant defence 533 concentrations of MeJA and JA have been detected in many plant species. A recent compilation of sources and references has been provided by Hamberg and Gardner.9 Extraction of JA and/or MeJA from natural sources in both cases gives a mixture of 7-epimers (3R,7R)-(")-MeJA/JA 1,2 and (3R,7S)- (+)-7-epi-MeJA/JA 3,4.This arises because the thermodynamically less stable cis-isomers 3,4 which are the biosynthetic products readily epimerise at C-7 via the enol. The equilibrium ratio15 of trans:cis (1:3) appears to be 93:7 although figures of 90:10 and 95:5 are often quoted. It is not clear how much epimerisation occurs within the plant or whether most occurs during the extraction process. By modification of the extraction procedure Miersch et al.16 were able to isolate jasmonate with a 65:35 trans:cis ratio indicating that some isomerisation occurs during the original procedure. Similarly extraction of lemon peel yielded17 material consisting of 95% cis-isomer.Furthermore derivatisation as the dithioketal of de novo jasmonate induced by treatment of plant cell cultures with fungal elicitors gave18 material with a trans:cis ratio of 26:74. Unchallenged cells gave jasmonate dithioketals at the equilibrium ratio. Thus jasmonate is biosynthesised in the cis form and epimerisation occurs in resting plant cells. Similarly although the original isolation2 yielded JA from the fungus B. theobromae it has been shown19 more recently that this source also produces mainly cis-form (3R,7S)-(+)-7-epi-JA 4. Hormonal concentrations of jasmonate in unchallenged plants are generally in the range 1–50 ng/g fresh weight but some tissues such as fruits can contain more.16,20 Concentrations in resting plant cell cultures are somewhat lower.On wounding or elicitation concentrations in whole plants or cultures rapidly increase in some cases to Ïg/g levels.13,18,21 The biological activities of jasmonate are considered to be properties of (3R,7S)-(+)-7-epi-MeJA/JA 3,4. However most biological work has been carried out with synthetic racemic MeJA or JA. The acid can be resolved as the (")-borneol esters and further separated by HPLC of the methyl esters into pure cis- and trans-forms of each enantiomer.15,22 Interestingly the characteristic odour of jasmine is only shown by (+)-7-epi-MeJA 3 the same isomer that appears to be responsible for many of its biological activities in plants. 2.2 12-Oxophytodienoic acid and its relatives 12-Oxophyto-10,15(Z)-dienoic acid (12-oxo-PDA) 5 is an important biosynthetic precursor of jasmonate.It was first reported23,24 by Zimmerman et al. as a product of the action of an enzyme extract from flax seed on ·-linolenic acid (18:3). It was then shown25 to be endogenous in sunflower seedlings. Subsequent identifications have been made in a number of plant species during the course of routine natural product screening programs26–30 and as part of focussed studies31,32 on the occurrence of this compound and its isomers. As discussed above for JA 12-oxo-PDA can similarly equilibrate via the enol (at C-13) to the more stable trans-arrangement of the side chains. However the natural isomer is cis-orientated dextrotatory and has the (9S,13S)-configuration33. An early report34 indicated that the material produced from ·-linolenic acid by flax seed extracts was racemic.More recent work has shown31 that the flax system was variable but was capable of producing an asymmetric cis-product as had already been observed33 with a crude enzyme preparation from corn germ. It is of interest to note that we now know that the biosynthesis of (9S,13S)- (+) cis-12-oxo-PDA from (13S)-hydroperoxy intermediates derived from ·-linolenic acid is achieved by the co-ordinate action of two enzymes allene oxide synthase and allene oxide cyclase. However the allene oxide intermediate is unstable and in the absence of cyclase enzyme activity may spontaneously cyclise to give 12-oxo-PDA (amongst other products) which is racemic in composition (see section 3.2). As well as the tendency of the cis-isomer to equilibrate to the trans-isomer by enolisation 12-oxo-PDA on treatment with acid base or heating will also undergo an isomerisation reaction with the ring double bond migrating from 10,11 to give the analogue 6 containing a tetrasubstituted 9,13-double bond.A number of metabolites and analogues of 5 and 6 have also been reported. These include chromomoric acid (13-hydroxy-5),26,35 14-hydroxy-5,29 13,14-didehydro-5,26,27 9-hydroxy-13,14-didehydro-526 and 11-hydroxy-6.27 The acetylenic and allenic analogues dicranenones A and B1 7 and 8 are interesting compounds that have been isolated from Dicranium mosses.36 They appear to be formed by the action of the enzymes involved in 12-oxo-PDA biosynthesis operating on dicranin the 6-acetylene-analogue of ·-linolenic acid which is also present in these and other mosses.36–38 Dinoroxophytodienoic acid 9 a sixteen carbon analogue of 12-oxo-PDA has recently been isolated39 from Arabidopsis and potato.This compound apparently arises from a parallel biosynthetic pathway i.e. via the action of lipoxygenase and allene oxide synthase and cyclase on the 16:3 fatty acid homologue. The alternative route—via ‚-oxidation of 12-oxo- PDA—is ruled out39 by the absence of 9 from plants of the Arabidopsis fad5 mutant which is deficient in the 16:3 fatty acid substrate. 2.3 Jasmonate metabolites and other related compounds (3R,7R)-(")-9,10-Dihydrojasmonic acid 10 is a natural product in plants,40 while its 7-epimer (3R,7S)-(+)-7-epi- 9,10-dihydrojasmonic acid 11 has been isolated from B. theobromae.19 As with JA the trans-isomer 10 can be considered to be formed from equilibration of 11. Higher homologues of 7-epi-JA are 12 and 13 with C3 and C4-carboxylic acid side-chains.These have been reported41 to occur in the fungus B. theobromae. An alternate nomenclature for such compounds is based on the systematic naming; for example CO2H O CO2H O O CO2H O CO2H O CO2H OR O CO2H OH O C OH CO2H OH CO2H OH CO2H O CO O CO O CO2H O CO2H O CO2H O O Ile Ile 10 11 12 13 14 R = H 15 R = Glc 16 17 18 19 20 21 22 23 24 25 534 Natural Product Reports 1998 3-{3-oxo-2-cis-[(Z)-pent-2-enyl]cyclopentyl}propionic acid 12 is known as OPC-3:0 while 13 is OPC-4:0 and 7-epi-JA is OPC-2:0. The even chain length homologues OPC-4:0 OPC-6:0 and OPC-8:0 (10,11-dihydro-12-oxo-PDA) are anticipated to be intermediates in the biosynthesis of 7-epi-JA. These have not been detected in untreated plants but can be observed as metabolites of 12-oxo-PDA incubated with plant tissues.42,43,44 A number of hydroxylated derivatives of JA have been reported in the literature.Tuberonic acid (12-hydroxy-7-epi- JA) 14 and its glycoside 15 are tuber-inducing signals isolated from potato.45 12-Hydroxy-JA 16 has been reported46 from B. theobromae while the 1,12-lactone 17 derived from it is a constituent of jasmine absolute.47 8-Hydroxy-JA and 11-hydroxy-JA acid also occur46 in B. theobromae. (+)- Cucurbic acid 18 presumably derived by biosynthetic reduction of 7-epi-JA was originally isolated48 from seeds of pumpkin Cucurbita pepo. As the stereochemical assignment of the side-chains was based on the original erroneous jasmonate assignments the configuration at C3 was deduced as 3S (previously 1S). The correct configuration of natural (+)- cucurbic acid 18 is (3R,6S,7S).Other stereoisomers of 18 are also naturally occurring. The biological reduction of the 6-oxo group is not stereospecific in some plants giving rise49 to (+)-6-epi-cucurbic acid 19 [(3R,6R,7S)] as well as 18. To complicate this area further the (3R,6R,7R)-isomer (+)-6-epi- 7-epi-cucurbic acid 20 has also been found in plants.40,49 This compound presumably arises from the biosynthetic reduction of JA. In this case the reduction appears to be stereospecific as the corresponding (6S) isomer (7-epi-cucurbic acid) has not been found in plants. As well as hydroxylated jasmonates some dehydro-analogues have also been reported. 4,5- Dehydro-7-epi-JA 21 and its isomer 4,5-dehydro-JA 22 have been reported from B. theobromae41 and Equisetum sp.(horsetails) 50 respectively. 3,7-Dehydro-JA 23 has been reported40 from Vicia faba fruits although it may of course be formed from isomerisation of 22 as reported for 12-oxo-PDA above. Similarly 11,12-dehydro-JA was reported19 to be a constituent of B. theobromae but was later demonstrated46 to be formed from 11-hydroxy-JA during the isolation procedure. Amino acid conjugates of jasmonates are also naturally occurring and indeed now appear to have an active role in jasmonate action. The first conjugates to be reported51 were jasmonoyl-L-isoleucine 24 and the corresponding 9,10- dihydrojasmonoyl-L-isoleucine. These ironically are metabolites of Gibberella fujikuroi a fungus much better known for the production of gibberellic acid another important plant growth regulator.A later reinvestigation of culture filtrates of G. fujikuroi revealed52 that the 7-epi-jasmonoyl-L-isoleucine 25 was also present. The L-isoleucine conjugate 24 is also present in Vicia faba leaves.53 Similarly 24 and also the corresponding L-leucine and L-valine conjugates are the main stress inducible jasmonates in barley leaves.54 It has been suggested54 that leucine isoleucine and valine conjugates are produced in leaves while aromatic amino acid conjugates of jasmonate predominate in flowers and fruit. This is supported by reports of jasmonoyl-L-tryptophan -L-phenylalanine and -L-tyrosine conjugates in V. faba flowers.55,56 3 Biosynthesis 3.1 The pathway An excellent review of jasmonate biosynthesis has recently been provided by Mueller,57 and the reader is referred to this for additional detail.Here we provide an overview of the pathway drawing upon parallels with mammalian prostaglandin biosynthesis and highlight recent literature describing the cloning of genes encoding important enzymes of the pathway. In plants the basic linear pathway from ·-linolenic acid to jasmonate shown in Scheme 1 was identified in the early 1980s in a series of papers23,43,44 by Vick and Zimmerman and colleagues. With the recent discovery39 of dinor-12-oxo-PDA 9 and its putative formation from the 16:3 fatty acid the existence of a parallel pathway which converges at OPC 6:0 29 should be considered. Similarly there may be another parallel pathway from linoleic acid (18:2) leading to dihydrojasmonates 42 although this has not been studied in detail. In response to stress stimuli jasmonate biosynthesis appears to be initiated by release of the 18:3 fatty acid from the cell membrane by the action of a lipase (Scheme 2).This is followed by an oxidative cascade involving lipoxygenase and allene oxide synthase and cyclase to give 12-oxo-PDA 5. From 12-oxo- PDA the linear pathway to jasmonic acid proceeds by saturation of the cyclopentenone double bond followed by shortening of the carboxy side-chain via three cycles of ‚-oxidation. The cis-configuration of the side-chains appears to be maintained throughout these conversions in vivo although the reductase58 and ‚-oxidase44 are capable of turning over the trans-12-oxo-PDA in vitro. In mammals prostaglandin (PG) biosynthesis is initiated in the same way but here the fatty acid—arachidonic acid (20:4)—is converted to cyclopentanoids by a cycloxygenase via an endoperoxide as shown in Scheme 2.Arachidonic acid is also oxidised to the 5-hydroperoxide in mammals. This pathway leads to the leukotrienes. It is of interest to note that A-type PGs are present in quite large amounts in the soft corals Plexaura homomalla and Clavularia viridis. In these systems the cyclopentanoids are apparently biosynthesised from arachidonic acid by the action of an 8-lipoxygenase and allene oxide synthase.59–61 However although formation of the cis-disubstituted cyclopentanoid preclavulone A has been demonstrated59 in enzyme preparations from these corals there is no direct proof that this compound is a precursor of PGs.60,61 There are also a number of reports of the presence of PGs in plants,62 but only a few are backed up by spectroscopic evidence.However there are several firm identifications for example of PGA1 in onion63 and PGF2· in Kalanchoe blossfeldiana pollen.64 Thus the existence of a minor PG pathway in plants possibly based on lipoxygenase as in the soft corals must be considered. 3.2 Enzymology In mammalian cells standing concentrations of arachidonic acid in unactivated cells are low and thus the release of this fatty acid from membranes by the action of a phospholipase A2 is thought to be a controlling step in PG biosynthesis. In plants the observation of linolenic acid release upon wounding65 or elicitation66 indicates that a similar lipase is involved. An increase in phospholipase activity in tobacco cells67 and soybean cells68 after elicitation has also been determined.However it is not yet clear if this enzyme is regulatory as standing pools of free linolenic acid in most systems appear to be suYcient to maintain the level of jasmonate biosynthesis observed on elicitation.66 The cascade from linolenic acid to jasmonate begins with stereospecific lipoxygenase oxidation to the (13S)- hydroperoxide 26. Several lipoxygenase isozymes have been noted in plants. In soybean three distinct isozymes LOX-1 2 and 3 have been characterised.69 LOX 3 can be further subdivided into forms 3a and 3b but the diVerences appear to be due to post-transcriptional modification of the same gene product. Oxidation of the cis,cis-9,12-diene system of linolenic or linoleic acid by lipoxygenase is initiated by stereospecific removal of the pro-(11S) hydrogen atom.The resultant pentadienyl radical can react with oxygen at either end giving the (9S) and/or (13S)-hydroperoxide (Scheme 3). LOX-1 gives almost exclusively 13-hydroperoxide whereas LOX 2 and 3 both give approximately equal amounts of 9- and 13-hydroperoxides.70 The crystal structure of soybean LOX-1 has been determined.71 The enzyme contains a ferrous iron atom coordinated by three histidines and one carboxy group Beale and Ward Jasmonates key players in the plant defence 535 in a cavity lined with hydrophobic groups that can accommodate molecular oxygen and the fatty acid substrate of lengths up to at least 20 carbon atoms. The mechanism has been investigated72 with irreversible inactivators such as 9- and 12-acetylene derivatives of linoleic acid which confirm the initial loss of the 11-hydrogen.The conversion of (13S)-hydroperoxylinolenic acid 26 to the unstable allene oxide 27 is catalysed by an allene oxide synthase (also known as an hydroperoxide dehydrase). This enzyme has been purified and cloned from flax seed.73,74 Subsequently it has been cloned from Arabidopsis75 and from Parthenium argentatum (guayule) where it is the major protein in rubber particles.76 Even though the enzyme does not act as an oxygenase it is a 55 kDa cytochrome P450 now designated CYP74. It catalyses the conversion of (13S)-hydroperoxides of linolenic and linoleic acids to the respective allene oxides which can be isolated,77 but normally in vitro in the absence of allene oxide cyclase readily react with water to form ·- (route a) and „- (route b) ketols77 (Scheme 4).Spontaneous decomposition of allene oxide 27 also gives rise to small amounts of 12-oxophytodienoic acid 5. This chemical cyclisation lacks the stereospecificity of that catalysed by allene oxide cyclase and yields a racemic product.31,77 In vivo allene oxide cyclase operates in coordination with allene oxide synthase to produce (9S,13S)-12-oxo-PDA 5. The first indications that the cyclisation was enzyme catalysed came from Hamberg et al.33,78,79 who demonstrated that maize seeds contained an activity that produced optically pure 5 and that the enzyme responsible was a 45 kDa soluble protein that was separable from allene oxide synthase activity. Further developments have led to the purification to apparent homogeneity of a 47 kDa dimeric enzyme.80 It should be noted that the allene oxide derived from (13S)-hydroperoxylinoleic acid (i.e.from the 18:2 fatty acid) does not appear to be a substrate for this purified cyclase. This indicates that 15,16-dihydro-12-oxo- PDA (and hence 9,10-dihydrojasmonate 11) is not formed 9 13 (CH2)7CO2H (CH2)7CO2H (CH2)5CO2H (CH2)7CO2H HOO (CH2)7CO2H O O CO2H O CO2H O CO2H O CO2H O CO2H O CO2H O CO2H O CO2H O CO2Me O CO2Me Linoleic acid (18:2) a-Linolenic acid (18:3) (16:3) 26 (13 S )-Hydroperoxylinolenic acid 27 (12 S ,13 S ,9 Z,11 E,15 Z)-12,13-epoxyoctadeca-9,11,15-trienoic acid Dihydro 12-oxo-PDA 5 (9 S ,13 S )-12-Oxophytodienoic acid 9 Dinor-12-oxo-PDA 28 10,11-Dyhydro-12-oxophytodienoic acid OPC 8:0 29 OPC 6:0 13 OPC 4:0 2 11 Dihydroepijasmonic acid 4 OPC 2:0 3 1 O CO2H Lipoxygenase Allene oxide synthase Allene oxide cyclase 12-Oxo-PDA Reductase? b-Oxidase b-Oxidase b-Oxidase 12-Oxo-PDA Reductase Scheme 1 536 Natural Product Reports 1998 from a parallel pathway.This point has been discussed more recently by Gundlach and Zenk.42 12-Oxo-PDA reductase catalyses the conversion of 5 to OPC 8:0 28 Scheme 1. The activity was first characterised in extracts of maize seeds and seedlings by Vick and Zimmerman.81 The enzyme preferred NADPH (Km 13 ÏM) over NADH (Km 4.2 mM) and accepted both cis- and trans-isomers of 5 as well as the C20-homologues which it converted to OPC 10:0. Further work in this area led to the purification of this enzyme from cell cultures of Corydalis sempervirens58 and cloning of its homologue from Arabidopsis.82 The C. sempervirens enzyme is a 41 kDa protein which reduces the ring double bond of either cis- and trans-12- oxo-PDA with a preference for the cis-isomer (6:1).It should be noted that the enzyme is also capable of reducing cyclohexenone to cyclohexanone at a rate comparable to that of its reduction of trans-12-oxo-PDA. The sequence of the cloned enzyme from Arabidopsis revealed that it is a flavin-containing protein with significant sequence and functional similarities to yeast Old Yellow Enzyme one of the first flavoproteins to be (CH2)7CO2H (CH2)7CO2H HOO (CH2)7CO2H O CO2H O (CH2)3CO2H (CH2)3CO2H HOO (CH2)3CO2H O O O CO2H CO2H OH (CH2)3CO2H (CH2)4CH3 (CH2)3CO2H O OH (CH2)3CO2H O O OH HO (CH2)4CH3 (CH2)4CH3 (CH2)4CH3 Allene oxide synthase Allene oxide cyclase 8-LOX 5-LOX 15-LOX and Cyclooxygenase ? PLANTS SOFT CORALS MAMMALS Plexaura Clavularia Lipase a-Linolenic acid (18:3) Arachidonic acid (20:4) 12-Oxo-PDA JASMONATE Preclavulone A PGA2 Leukotriene A4 PGE2 Arachidonic acid (20:4) 13-LOX Lipase 15 OOH (CH2)3CO2H (CH2)3CO2H O Scheme 2 Lipoxygenase 13 13 (CH2)7COOH (CH2)7COOH HR HS HR (CH2)7COOH HR (CH2)7COOH (CH2)7COOH OOH HOO 9 9 11 • O2 H+ + 26 Scheme 3 Beale and Ward Jasmonates key players in the plant defence 537 discovered.82 This yeast enzyme also carries out a NADPHdependent reduction of cyclohexenone and furthermore is also capable of reducing 12-oxo-PDA.82 Thus it is possible that the role of the enzyme in yeast is also in oxylipin metabolism.The least studied steps in jasmonate biosynthesis are the three rounds of ‚-oxidation required to convert OPC 8:0 28 to jasmonic acid via OPC 6:0 29 and OPC 4:0 13 (Scheme 1).This conversion was first demonstrated by Vick and Zimmerman using [14C]-5 feeds to bean pericarps.43 Further investigations in several other plants demonstrated44 that the ‚-oxidase like the preceding reductase was relatively non-specific with regard to the stereochemistry of the side chains and the presence or absence of the 15,16 double bond in 5. Thus it is intriguing that the conversion of 5 to the highly biologically active jasmonic acid a processs which requires 15 chemical reactions is apparently carried out by two non-specific enzymes. In prostaglandin and leukotriene biosynthesis reduction and ‚-oxidation are associated with deactivation of active compounds. Intermediates between 5 and jasmonic acid have not been detected in unchallenged plant systems and concentrations are assumed to be below current detection limits.However labelled OPC 8:0 and 4:0 were detected from [18O]-5 feeds to oat and wheat plants,44 while OPC 6:0 is detectable in bean after feeding large amounts of unlabelled 5.43 More recently OPC 8:0 6:0 and 4:0 have been detected in plant cell cultures that have been treated with precursors such as linolenic acid and 12-oxo-PDA.42 The methylation of jasmonic acid and the relationship of the endogenous methyl ester and free acid in their roles as signals is another area where we have little information. Because 12-oxo-PDA methyl ester shows high biological activity in systems where ‚-oxidation is a requirement it is assumed that these cyclopentanoid methyl esters are readily hydrolysed in plant tissue prior to ‚-oxidation (see later).It would appear that jasmonic acid and methyl jasmonate are interconvertible within plants and that some sort of equilibrium exists. This presumably can be perturbed by losses of methyl jasmonate via evaporation. However there is no experimental data on this equilibrium or on the relative importance of endogenous free acid and methyl ester as signals. 3.3 Metabolism As well as methyl ester formation jasmonic acid is metabolised in a number of ways by plant tissue. Conjugation with amino acids also appears to be biologically relevant as some amino acid conjugates have strong bioactivity (see section 5.6). Whether amino acid conjugation is reversible in plants has not been determined. However an enzyme that will hydrolyse jasmonoyl-L-amino acid conjugates has been identified in the jasmonate producing fungus Botryodiplodia theobromae.83 As well as formation of hydroxylated and glycosylated metabolites already mentioned above (section 2.3) another biologically significant pathway of jasmonate removal has recently been described84 in Lima bean.Use of [2H5]-labelled jasmonate led to the identification of cis-jasmone 30 as a metabolite (Scheme 5). It was proposed that 30 is formed via the 3,7-enone 23 which undergoes decarboxylation. This pathway represents another method of removal of jasmonate as a volatile metabolite. 4 Synthesis of jasmonates Given the importance of methyl jasmonate to the fragrance industry it is not surprising that there are numerous syntheses in the literature.85,86 Most synthetic routes to jasmonate (CH2)7CO2H HOO (CH2)7CO2H O• (CH2)7CO2H O H (CH2)7CO2H HO (CH2)7CO2H (CH2)7CO2H O O O OH (CH2)7CO2H O (CH2)7CO2H O • 9 13 a b Allene oxide synthase a b 5 rac (9 R S 13 R S ) 5 (9 S 13 S ) a-ketol g-ketol 27 H2O b Allene oxide cyclase Non Enzymatic Cyclisations Scheme 4 CO2H O CO2H O Me O 2 23 30 [O] – CO2 Scheme 5 538 Natural Product Reports 1998 appear to be either via intramolecular cyclisation as shown in the example87 in Scheme 6 for racemic methyl 7-epijasmonate 3 or via conjugate addition–alkylation to the double bond of cyclopentenone as illustrated88 in Scheme 7 for racemic methyl jasmonate 1.Since we are concerned with the plant cell signalling function of natural jasmonates in this review we concentrate on enantioselective syntheses producing (")-methyl jasmonate 1 and (+)-methyl 7-epijasmonate 3.4.1 Enantioselective syntheses of methyl jasmonate There are several reports on the synthesis of enantiomerically pure (3R,7R)-(")-methyl jasmonate 1. The first synthesis by Quinkert et al.89 made use of bis(8-phenylmenthyl) malonate as a chiral starting point. This led to the cyclopropyl derivative 31 shown in Scheme 8 which was converted into the alkyne analogue 32 by alkylation and thermal rearrangement. Functional group manipulation provided asymmetric 33 which was converted to (")-methyl jasmonate by methods previously reported for racemic 33 by Buchi and Egger.90 A short asymmetric synthesis of (")-methyl jasmonate with 98% enantiomeric purity was subsequently presented by Posner and Asivatham.91 The critical step involved carbon– carbon bond formation by conjugate addition of an ·-lithioacetate unit to the doubly activated enantiomerically pure Michael acceptor (R)-(")-2-(p-tolylsulfinyl)cyclopent-2- enone to aVord the 2,3-disubstituted cyclopentanone sulfoxide 34 (Scheme 9).Sulfoxide deoxygenation followed by desilylation gave the ‚-keto sulfide 35 which was alkylated with (Z)-pent-2-enyl bromide to give 36. Reductive removal of the sulfinyl group with Raney nickel gave (")-methyl jasmonate in 45% yield with high enantiomeric purity ([·]D "93.0). A similar synthesis from the same group,92 based on dichloroketene addition to (R)-(")-2-(p-tolylsulfinyl)cyclopent-2- enone was less enantioselective and gave (")-methyl jasmonate with [·]D "18. An alternative synthesis of (")- methyl jasmonate with high enantiomeric purity by Weinges et al.,93 is outlined in Scheme 10.The chirality at C-3 arises from the iridoid natural product catalpol used as the starting material. This was converted to (")-methyl jasmonate in 16 steps with an overall yield of 7%.Montforts et al. described the synthesis of both the enantiomers of methyl jasmonate in their pure form.94 The synthesis of both enantiomers relied upon enantiodivergent functionalisation of cyclopent-2-ene-1,2-diol monoacetate using a palladium catalysed alkylation with malonate to aVord the chiral building blocks which were to be transformed into (")(3R,7R) and (+)(3S,7S)-methyl jasmonate according to Scheme 11. 4.2 Enantioselective syntheses of methyl 7-epijasmonate The vicinally cis-disubstituted cyclopentanone system of 3 is a more challenging synthetic target than its more stable transepimer.As a consequence there have been fewer syntheses of this compound. The first synthesis of enantiomerically pure (+)-(3R,7S)-methyl 7-epijasmonate was presented by CO2Me OTBDPS CO2Me TBDPSO CO2Me OTBDPS CO2Me O O OTHP OTBDPS OH SiMe3 OTBDPS SiMe3 CO2Me SiMe3 xi xii vii ix x 3 i–iv v vi viii Scheme 6 O CH2CO2Me O CH2CO2Me O OSnBu3 CH(SiMe3)CO2Me O CH(SiMe3)CO2Me i ii iii iv v 1 Scheme 7 R R O O R R O O R R O O MeO OMe O O O H O O C6H5 H H H H H O O OMe O OMe v vi x R = 31 33 1 (–)-Methyl jasmonate ii 32 iii iv i vii–ix Scheme 8 O S Tol O S Tol O O O OMe Me3Si SiMe3 O O S Tol OMe O CO2Me O CO2Me STol 34 35 36 1 (–)-Methyl jasmonate iv v i ii iii Scheme 9 Beale and Ward Jasmonates key players in the plant defence 539 Helmchen et al.95 The starting point for this synthesis was an asymmetric Diels–Alder reaction as shown in Scheme 12 using cyclopentadiene as the diene and the fumarate of ethyl (S)- lactate 37 as the chiral dienophile.Hydrolysis of the adduct followed by cyclisation to the iodolactone aVorded enantiomerically pure 38 in 70–80% yield. Conversion to the tricyclic lactone 39 and opening of the cyclopropyl ring gave the key intermediate 5-iodonorbornanecarboxylic acid 40. Functional group manipulation led to (+)-methyl 7-epijasmonate with [·]D +57.3 in agreement with the natural product. At about the same time Weinges and Lernhardt reported96 a synthesis of (+)-methyl 7-epijasmonate from catapol using a variation of their route93 to (")-methyl jasmonate shown in Scheme 10 and discussed in section 4.1. Stadtmuller and Knochel have described a procedure for the preparation of (+)-(3R,7S)-methyl 7-epijasmonate in which the key step is a nickel catalysed ring-closure which introduces three chiral centres with high diastereoselectivity.97 The synthesis outlined in Scheme 13 relies on the availability of the chiral building block 41 which was transformed to the iodo compound 42 setting up the key cyclisation/alkylation to give 43 which is readily converted to (+)-(3R,7S)-methyl 7-epijasmonate.The transformation of 43 to (+)-methyl epijasmonate was via methyl cucurbate 44. Curiously synthetic 44 was levorotatory whereas 44 derived from natural methyl 7-epijasmonate is known49 to be dextrorotatory ([·]D +14). A similar confusion arises in a synthesis of both enantiomers of methyl 7-epijasmonate described by Kitihara et al.98 The route starts from the resolved enantiomers of the dichloro ketone 45 which was manipulated as shown in Scheme 14 to give methyl 6-epicucurbate 46.Oxidation of 46 gave methyl 7-epijasmonate with the correct optical rotation. However the optical rotation given for synthetic methyl 6-epicucurbate 46 ([·]D "2.4) disagrees with that known for 46 derived from natural methyl 7-epijasmonate ([·]D +10.7).49 The most recent synthesis of (+)-methyl 7-epijasmonate has been presented by Roth et al.99 The short synthetic route depicted in Scheme 15 uses a Diels–Alder reaction to set up the required cis-orientated side-chains. The key step relies on condensation of the chiral cyclopentenone building block 47 with 2-methoxybutadiene to give the adduct 48. Epimerisation was avoided by the transformation of the carbonyl group into the TBS-ether.Ozonolysis of this product followed by an immediate Wittig reaction of the resulting aldehyde aVorded 49 with a cis arrangement of the side-chains. Deprotection of the 1,3-dioxolan moiety followed by deoxygenation of the resulting 1,2-diol aVorded the didehydro derivative 50. Removal of the silyl protecting group and subsequent oxidation followed by selected reduction of the conjugated double bond yielded (+)-methyl 7-epijasmonate with 80% cis selectivity. This synthesis was also used to prepare (")-methyl 7-epijasmonate. This unatural enantiomer is also available from a recent synthesis based on a commercially available derivative of the Corey lactone.100 4.3 Synthesis of 12-oxophytodienoic acid OPC 8:0 and homologues Compared to jasmonates there has been little synthetic work on the higher homologues.The first synthesis of 12-oxophytodienoic acid 5 was described by Crombie and Mistry.101,102 The route from cis-cyclopentenediacetic acid shown in Scheme 16 involves another variation on the Corey lactone procedure using a selenolactonisation to give 51 and subsequent oxidative elimination to introduce the ring double bond. Construction of the pentenyl side-chain by Wittig olefination of the lactol gave the dehydrocucurbate 52. The extended carboxy side chain was added by a Grignard O O O CH2CO2Me O CH2CO2Me O OMe CH2CO2Me O OBn OMe O OH SBn O BnO O CO2Me H H H H H HO Scheme 10 OAc O H H I HO HO HO CO2Me CO2Me CO2Me CO2Me CON(Me)2 O MeO2C O H H OH MeO2C O OH CO2Me O CO2Me O CO2Me OH AcO OCO2Et AcO AcO CO2Me CO2Me HO CO2Me CO2Me O CO2Me O ii i iii iv v vi vii (+)-Methyl jasmonate viii ix x xi xii xiii ii–x 1 (–)-Methyl jasmonate Scheme 11 540 Natural Product Reports 1998 addition followed by deoxygenation of the so formed secondary alcohol.For the synthesis of the methyl ester of OPC 8:0 and its lower homologues Crombie and Mistry102,103 used the same strategy employing the iodo compound 53 formed by iodolactonisation of the same cis-cyclopentenediacetic acid as shown in Scheme 16. In this synthesis the varying length carboxy side-chain was introduced by Kolbe electrolysis with adipic acid monomethyl ester (for OPC 8:0 methyl ester 54) or suberic acid monomethyl ester (for OPC 6:0 methyl ester 55). The side chain in OPC 4:0 methyl ester 56 was constructed using a double Arndt–Eistert homologation.The syntheses were then completed by olefination of the corresponding lactols as shown. More recently the Kolbe electrolysis method has been applied104 directly to jasmonic acid to give trans-OPC analogues with side-chains of 6–9 carbon atoms (Scheme 17). An alternative synthesis105 of trans-OPC homologues with 3–8 carbon atoms in the chain using the conjugate addition of a range of ¢-carboxyalkylcopper–zinc reagents to a cyclopentenone is shown in Scheme 17. The only enantioselective synthesis of 12-oxophytodienoic acid 5 has been achieved by Grieco and Abood.106 The route relies on a retro Diels–Alder reaction of a suitably substituted norbornene as shown in Scheme 18. Starting from the enantiomerically pure tricyclo[5.2.1.02,6]decadienone 57 the side-chains were added by conjugate addition– alkylation.Subsequent [4+2] cycloreversion was achieved at room temperature using ethylaluminium dichloride catalysis and fumaronitrile as the external dienophile to give (+)-12-oxophytodienoic acid with an optical rotation of [·]D +104 slightly higher than that determined for natural material.31 RO2C CO2R O CO2Me OH CO2Me O R O O CO2H O CO2H O CO2H O O O CO2H I O O X + i–iii iv 37 38 39 40 v vi vii ix X = I X = H viii R = CH=CHOMe R = CH2CHO R = CH2CH=CHCH2Me xiii xiv x–xii xv xvi xvii 3 (+)-Methyl-7-epi-jasmonate R = –CH(Me)CO2Et * Scheme 12 vii x CH2OPiv TMS OH CHO OBn OBn OBn CO2Me OH CO2Me O CO2Me CO2Me I i–iv v vi 41 42 43 44 3 viii ix Scheme 13 O OH CO2Me O CO2Me O O OMe O OH O O Cl Cl O CO2Me 45 46 3 i ii–v vi vii viii–xi xii Scheme 14 O CO2Me OH CO2Me OTBS CO2Me OTBS CO2Me OTBS CO2Me O O OTBS O O OMe O O O OMe OMe O O O HO HO + i 47 48 49 50 3 ii iii iv v vi vii viii ix x Scheme 15 Beale and Ward Jasmonates key players in the plant defence 541 4.4 Isotope-labelled jasmonates Stable isotope labelled plant hormones are essential for the accurate quantification of hormone concentrations by gas chromatography–mass spectrometry (GC–MS).For jasmonate several methods of labelling have been developed. Miersch has described107 the preparation of [10-2H,11-2H2,12- 2H3]jasmonate by ozonolysis of jasmonate and reconstruction of the pentenyl side-chain by a Wittig reaction with [2H5]propylidenetriphenylphosphorane. Semi-deuteriogenation of the 9,10 acetylene-analogue of jasmonate has been used to prepare [9,10-2H2]jasmonate.108 Methods for the incorporation of carbon-13 at C-2 or at C-1 and C-2 using 13C-labelled malonates in total syntheses have been published.109,110 [2-14C]Jasmonate has also been prepared by this method.111 Labelled precursors of jasmonate have been prepared.Crombie and Morgan112 have synthesised [14-2H2]linolenic acid. Use of [17-2H2,18-2H3]Linolenic acid and recombinant allene oxide synthase has enabled the preparation of [2H5]-12- oxophytodienoic acid which has been used eVectively in GC–MS quantification.113 5 Structure–activity relationships Jasmonates exert a variety of eVects when applied to plant tissue. Some of these give rise to obvious physiological changes such as promotion of senescence,4 inhibition of growth,4,5 promotion of coiling in tendrils of climbing plants such as Bryonia dioica,7 and the induction of tubers in potato stolons.5 Less externally obvious but of considerable importance are the roles of jasmonates as molecular messengers in the defence response.Wounding of some plants such as tomato and potato induces the biosynthesis of jasmonate and transcription of genes encoding defensive proteins such as proteinase inhibitors (PINs).3 Treatment of these plants with jasmonates also causes PIN gene expression indicating that endogenous jasmonate is involved in signalling the wound response. Similarly in soybean and barley leaves stress such as osmotic shock or jasmonate treatment causes the synthesis of a number of proteins known as jasmonate-induced proteins (JIPs).4 Some of these proteins have defensive roles while others are vegetative storage proteins.Lipoxygenase is also jasmonate-inducible and thus there is the potential for jasmonate to regulate its own biosynthesis by a feedback mechanism. An important component of the plant defence response is production of ’secondary’ metabolites aimed to deter herbivores or invading microorganisms. Jasmonate has been shown to be a regulator of secondary metabolite xii O O CO2H CO2H CO2Me H H H H H O O CO2Me H H H O OH CO2Me H H H OH CO2Me OSi(Me)2But OSi(Me)2But OTHP OH OH O CO2H O O CO2H H H H O O (CH2) nCO2Me O OH (CH2) nCO2Me O (CH2) nCO2Me PhSe O H H R i ii iii iv 51 52 5 v vi vii viii ix x xiii n = 7 xiv n = 5 xv n = 3 iii iv x xi 53 R = I R = H 54 n = 8 55 n = 6 56 n = 4 Scheme 16 O CO2H O (CH2) nCO2R O n = 2–8 i ii Scheme 17 O (CH2)8OTHP O (CH2)7CO2H H H O (CH2)7CO2H H H O H H (+) 5 57 v i ii iii iv Scheme 18 542 Natural Product Reports 1998 biosynthesis in a number of systems such as plant cell cultures producing alkaloids,114 nicotine accumulation in tobacco plants115 and in the production of volatile compounds in maize and Lima bean.116 The various jasmonate-inducible physiological and molecular events described above form the basis of a number of assay systems for jasmonate activity.Most early work was done using growth inhibition senescence and tuberisation assays which are conducted over relatively long time periods with the assessment being carried out days or even weeks after the application. EVects on tendril coiling PIN or JIP gene expression and secondary metabolite biosynthesis are faster and are generally more sensitive and thus form the basis of more recent molecular assays for jasmonate structure–activity studies.Most assays have been carried out using racemic MeJA (containing 7% Me 7-epiJA) as a standard. It is widely assumed that the methyl esters are hydrolysed rapidly in plant tissue. However there is very little experimental data demonstrating the rate of hydrolysis of applied methyl jasmonate. When applied to plants both free acids and methyl esters are active. However relative rates of uptake by plant tissue are rarely considered. It also may be dangerous to assume that the hydrolytic enzymes turnover all the isomers of applied MeJA at the same rate. This point is also relevant to assays of the longer chain OPCs some of which show considerable activity when assayed as methyl esters or free acids.Here we report on those structure–activity studies that have been carried out so far and try to draw some general conclusions. The number of bioassays in use does cause some diYculties in interpretation of the data as some compounds show diVerent bioactivities in diVerent systems and many synthetic analogues have only been tested in a few systems. 5.1 Optical and cis–trans isomers The biological activity of the natural (") and unnatural (+) enantiomers of MeJA was first compared by Yamane et al. using the inhibition of growth of rice seedlings as an assay.117 (")-MeJA was more active than the racemate or (+)-MeJA. However the (+)-enantiomer was active at higher doses. The same trend was also observed in an assay based on promotion of senescence of oat leaf tissue.118 In this system the (") enantiomer is more active at lower doses (0.1–2.5 Ïg ml"1) but at higher doses (>5 Ïg ml"1) both enantiomers and the racemate had equally strong activity.Thus it appears that although the natural enantiomer is as expected more active the unnatural compound can show activity at high doses. At equilibrium natural (")-MeJA contains 7% of the cis isomer (+)-Me 7-epiJA. The question of the relative activity of these isomers was first addressed for the corresponding free acids by Miersch et al.,119 who compared (+)-7-epiJA isolated from the fungus B. theobromae with (")-JA prepared by alkali-induced isomerisation of (+)-7-epiJA. In growth inhibition assays on rice and wheat seedlings the cis-isomer (+)-7- epiJA was more active than the trans-isomer (")-JA.In contrast there was no significant diVerence between (+)-7- epiJA and racemic JA on the inhibition of growth of oat or barley seedlings. These plants however are less sensitive than rice or wheat to jasmonate induced growth inhibition. Senescence promotion assays on oat and barley confirmed the higher activity of (+)-7-epiJA when compared with racemic JA. A similar comparison of all four stereoisomers of MeJA has been described by Koda et al.120 In oat senescence assays the order of activities was (+)-Me 7-epiJA>(")-MeJA=(+)- MeJA>(")-Me 7-epiJA; results broadly in agreement with those above. In an assay based on the inhibition of growth of soybean callus cultures the order of activity was (+)-Me 7-epiJA>(")-MeJA>(+)-MeJA=(")-Me 7-epiJA. However in an assay for the induction of potato tubers the stereoisomers behaved quite diVerently.(+)-Me 7-epiJA was the most active and the unnatural (")-Me 7-epiJA was also quite active although the nature of the tubers induced was diVerent to those from the (+)-enantiomer. (")-MeJA showed the lowest activity of the four stereoisomers and was less active than the unnatural (+)-MeJA. Thus for the assays based on plant growth and development it can be concluded that the natural cis-isomer (+)-Me 7-epiJA is more active than the natural trans-isomer (")-MeJA. Some of these assays are conducted over a long time period; for example the soybean callus growth is assessed four weeks after treatment and therefore cis–trans equilibration of applied compounds must be considered. It should also be noted that in some cases the unnatural enantiomers show moderate activity.There has been only one report of the assessment of the optical isomers of jasmonate and 7-epijasmonate in more rapid molecular assays based on wound-inducible genes.121 Transgenic tobacco plants containing tomato PIN II promoter-‚- glucuronidase reporter genes were assayed for ‚-glucuronidase activity and also for endogenous tobacco PIN II enzyme activity. (+)-Me 7-epiJA was the most active for the induction of both enzyme activities. It is diYcult to draw any further firm conclusions from the data presented but it appears that (")-MeJA is active in induction of both enzymes but that the free acids (")-JA and the unnatural (+)-JA are more active but only for ‚-glucuronidase expression not for endogenous PIN II expression. 5.2 Derivatives with a stereochemical anchor at C-7 or C-3 To address the problem of cis–trans isomerisation via enolisation during the assay process derivatives bearing a methyl group at C-7 have been synthesised by several groups.122,123 However both cis-locked compound 58 and trans-locked compound 59 are virtually inactive in a range of assays including tuberisation soybean callus growth oat leaf senescence,124 PIN gene expression121 and tendril coiling.123 It is probable that the methyl group at C-7 interferes with receptor binding although there are suggestions that these compounds can compete with jasmonate and are antagonists.125 Compounds 60 and 61 with the smaller fluorine atom at C-7 are also inactive,121,123 suggesting that steric interactions are not the cause of the inactivity of this type of compound.It could be that the ability to enolise to C-7 may be a factor in jasmonate receptor interactions. This would explain the inactivity of 58–61 and to some extent the activity of both natural cis- and trans-jasmonates in some bioassays. To explore this point further Ward et al. synthesised126 the C-3 methylated compound 62 which can still enolise to C-7. Molecular modelling had indicated that at equilibrium the compound 62 would contain an increased ratio of cis:trans side-chains as the change to methyl at C-3 increases steric interactions to the C-7 substituents. NMR analysis of synthesised 62 confirmed a much increased cis:trans ratio. However 62 was devoid of bioactivity in four assays including the induction of barley JIP gene expression and the promotion of oat leaf senescence.This result perhaps indicates that enolisation is not a requirement for receptor binding. On the other hand steric interactions due to the introduced C-3 methyl group may prevent any binding whether or not the compound is free to enolise. Interestingly methyl 3,7-dehydrojasmonate 63 a compound within which the oxygen and C-7 (and C-3) side-chains are co-planar thus resembling to some extent an enolate is also inactive in JIP gene expression senescence tuberisation and callus growth assays.126 It has been reported that 63 was very active in the inhibition of lettuce seed germination a system in which methyl jasmonate is barely active.127 Conversely 63 was not active in inhibition of rice or radish seed germination systems where methyl jasmonate is active.127 Thus in general 23 the naturally occurring40 free acid of 63 can be regarded as an inactive metabolite of jasmonate.Beale and Ward Jasmonates key players in the plant defence 543 5.3 Other changes to the cyclopentanoid ring A number of other modifications to the cyclopentanoid ring have been tested. Methyl 4,5-dehydrojasmonate 64 has been synthesised.126,127 This compound is of interest because it has the same ring structure as 12-oxo-PDA 5 which shows very high activity (see section 5.5). The biological activity of 64 was moderate in barley JIP gene expression and high in rice seed germination assays. The 5-diazo compound 65 synthesised126 as a potential photoaYnity label also has activity in induction of barley JIP gene expression indicating that changes to this side of the cyclopentane ring do not result in complete loss of activity.The role of the ketone in receptor binding has been investigated. The ethylene ketal 66 retains some activity in potato tuberisation.128 Similarly 66 and the corresponding dithioketal 67 have 60% of the activity of methyl jasmonate for the induction of nicotine biosynthesis in N. sylvestris.129 Reduction of the ketone group of 7-epijasmonic acid gives rise to the two C-6 stereoisomers of cucurbic acid 18 and 19. These compounds and their methyl esters are less active than the corresponding jasmonate for nicotine induction.129 Additionally 18 and particularly its methyl ester gave moderate activity in potato PIN gene expression.130 In potato tuberisation the acid 18 is more active than the corresponding methyl ester but both are less active than JA or MeJA.128 For the induction of alkaloid biosynthesis in cell cultures of Eschscholtsia californica and in Bryonia tendril coiling the methyl esters of 18 and 19 and also the 6-deoxy compound 68 are all inactive.131,132 Thus in summary it can be concluded that the ketone group is essential for high activity in all systems although those derivatives with sp3 centres at C-6 can show some activity they give little information on the question of enolisation.5.4 The pentenyl side-chain The double bond in the pentenyl side-chain is an absolute requirement in most assay systems. Dihydrojasmonic acid 11 and its methyl ester have been tested in many systems including tuberisation,128 nicotine production,129 potato PIN gene expression130 and tendril coiling.132 In all of these assays the 9,10-dihydro-derivatives are inactive.However this derivative does have some activity in the E. californica alkaloid system.131 The corresponding alkyne 9,10-dehydrojasmonate 69 has been shown to have moderate activity in the E. californica alkaloid system but no activity in tendril coiling.133 Conversely in a bioassay system based on inhibition of rice seedling growth dihydrojasmonic acid 11 was as active as JA while the alkyne 69 was much less active.134 The high activity of 11 in the rice system enabled Yamane et al.134 to test the eVects of the C-7 side-chain length. They found that the compound with no side-chain (70 R=H) was inactive. Thereafter increasing length [70 R=propyl butyl pentyl (=11) etc] gave increasing activity up to a maximum for the pentyl-derivative 11 falling oV again for R=hexyl or heptyl.The only other change to this chain that has been examined in structure–activity relationships is at C-12. The 12-hydroxycompound tuberonic acid 14 is regarded as a metabolite of epijasmonic acid. It and its glycoside 15 have activities as high as JA in the promotion of potato tuberisation.128 However 14 and 15 have no activity in oat leaf senescence soybean callus growth and lettuce seedling assays.5 Thus apart from producing an endogenous tuberisation signal in potato 12-hydroxylation appears to be a deactivating mechanism in most systems. This aspect has been partially explored by Kiyota et al.135 who synthesised the C-12 trifluoro analogue 71. This compound proved more active than jasmonate in tuberisation assays indicating that metabolism of jasmonate to tuberonate was not a requirement for activity.This trifluoro-compound was also as eVective as jasmonate in the inhibition of rice seedling growth and in seed germination assays. 5.5 The carboxymethyl side-chain and the importance of ‚-oxidation Biosynthetic precursors of jasmonate are biologically active. For example ·-linolenic acid and 13-hydroperoxylinolenic acid show activity in the Bryonia tendril coiling system.132,136 The activity is not high and can be attributed to conversion to jasmonates in vivo. In contrast 12-oxo-PDA 5 and particularly its methyl ester are more active than jasmonic acid and methyl jasmonate in the Bryonia assay.132 This result taken together with similar observations in the induction of secondary metabolites in parsley cell cultures137 and of genes encoding alkaloid biosynthetic enzymes in E.californica cell cultures138 have led to the suggestion131 that 12-oxo-PDA is active per se and that ‚-oxidation is not necessary for octadecanoid signalling. Support for this came from the observation that the 3-oxa-analogue of OPC 5:0 methyl ester 72 was highly active in induction of alkaloid biosynthesis genes in E. californica cell cultures.131 This derivative cannot be converted to jasmonate by ‚-oxidation and thus its activity supported the suggestion of a second perception mechanism O CO2Me O CO2Me O CO2Me O CO2Me O CO2Me O CO2Me CO2Me CO2Me O CO2Me CO2Me R O CO2Me CF3 O CO2Me O O O O O O N2 O O S S O CO2 Me O CO2 Me S CO2 Me S CO2 Me CO2Me Me CO2Me Me R R Me 58 R = Me 60 R = F 59 R = Me 61 R = F 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 544 Natural Product Reports 1998 based around longer homologues of jasmonate.The longer 3-oxa-OPC 8:0 methyl ester 73 the direct analogue of 12-oxo- PDA was reported to be toxic to these cells.131 However in a subsequent more detailed study by the same authors,139 both 72 and 73 were reported to be equally active for alkaloid elicitation in E. californica cells. These 3-oxa-analogues were however inactive for tendril coiling.139 The corresponding sulfur-containing analogues 3-thia-OPC 5:0 methyl ester 74 and 3-thia-OPC 7:0 methyl ester 75 have been prepared and are inactive for the induction of JIP genes in barley (J. L. Ward M. H. Beale C. Wasternack and O. Miersch unpublished results). It should be noted that compounds 72–75 are methyl esters and thus require hydrolysis by plant esterases before interaction with the ‚-oxidation system.A more direct analysis of the need for ‚-oxidation involves preparation and bioassay of odd and even chain-length OPCs as their free carboxylates. The rationale here is that even chains can give rise to jasmonate by ‚-oxidation and odd chains will not. These compounds have been examined in several systems. In tomato PIN II gene expression has been examined along with the expression of a large group of other jasmonate up- and down-regulated genes.140 A clear cut difference between odd and even OPCs was observed. OPC 3:0 5:0 and 7:0 were inactive while OPC 2:0 (JA) 4:0 6:0 and 8:0 were all active. The same pattern of activity was observed when these OPCs were assayed for JIP gene expression in barley.141 Furthermore even length branched OPC 4:0 and 6:0 analogues 76 and 77 are inactive in the barley system (J.L. Ward M. H. Beale C. Wasternack and O. Miersch unpublished results). Thus ‚-oxidation is clearly a requirement for activity in the tomato and barley systems. In contrast assay of OPC methyl esters for alkaloid induction in E. californica cells revealed high activity regardless of the odd or even nature of the chain-length.139 A similar experiment with Bryonia tendril coiling showed that in general even-chain OPC methyl esters were active and odd-chain OPC methyl esters were inactive.139 One exception to this was OPC 7:0 methyl ester which was active in tendril coiling. This result was interpreted as evidence for a second perception mechanism based on 12-oxo-PDA which OPC 7:0 can also activate.However the inactivity of 73 in tendril coiling does not support this hypothesis. Thus there appears to be quite significant system diVerences in the role of ‚-oxidation and more work needs to be done to clarify the situation. The use of transgenic plants with the 12-oxo-PDA reductase gene silenced by antisense expression should reveal whether 12-oxo-PDA is active per se. 5.6 Coronatine related bicyclic amides and jasmonate amino acid conjugates Coronatine 78 is a phytotoxin produced by strains of the pathogenic bacterium Pseudomonas syringae the causative agent of chocolate spot disease. In 1993 Greulich et al. working with tomato cell cultures reported142,143 that one of the secondary eVects of coronatine on plants–induction of ethylene biosynthesis—followed the same kinetics as that shown by methyl 7-epijasmonate treatment.This led to the suggestion that coronatine which bears some structural resemblance to jasmonate may act at the jasmonate receptor. This idea was further developed by several groups. Coronatine is very active often more active than jasmonate in jasmonate bioassays including tendril coiling alkaloid biosynthesis in E. californica cells,144 potato tuberisation senescence and soybean callus growth,145 production of volatiles,146 JIP gene expression in barley147 and PIN II gene expression in tomato.140 An exception is in whole tobacco plants where coronatine has growth inhibitory activity but no eVect on nicotine production.129 The parent free acid coronofacic acid 79 is structurally more similar to jasmonic acid.However this compound and its methyl ester are much less active than coronatine. In tendril coiling,144 barley JIP gene expression147 and tomato PIN II gene expression140 coronafacic acid is inactive while in potato tuberisation and soybean callus growth it has similar activity to jasmonic acid.145 Thus the amide part of coronatine is important for high activity making it less likely to be a direct structural analogue of jasmonic acid. It is possible that coronatine could act by induction of endogenous jasmonate biosynthesis perhaps via ethylene production. However the strong and rapid response seems to rule this out. Furthermore jasmonate levels have been shown to be unaltered in coronatine-treated E. californica and Agrostis tenuis cells.144 It has been suggested144 that coronatine is highly active because it is a structural analogue of 12-oxo-PDA 5.This tenet requires that 12-oxo-PDA is active per se. This does not appear to be the case for all the systems in which coronatine shows high activity (see above). Structural overlays of the cyclopentanone ring of coronatine onto that of 12-oxo- PDA (or 7-epijasmonate) reveal a good match for the pentenyl side chain of 12-oxo-PDA or JA onto part of the cyclohexene ring and ethyl substituent of coronatine. The amide side-chain of coronatine is less well matched. Synthetic analogues of coronatine are the amides of 1-oxoindane-4-carboxylic acid introduced by Krumm et al.145 These also show jasmonate-like activity. The L-isoleucine amide 80 was prepared and found to be active in the induction of volatiles in Lima bean.Unlike coronatine which is only active as the acid both the free acid 81 and methyl ester 80 are active. With the exception of L-leucine conjugate other amino acid conjugates of this indanone were inactive. It appears that isoleucine is a close mimic of the cyclopropyl amino acid coronamic acid that comprises the coronatine side-chain. Indeed the coronatine analogue coronafacic acid L-isoleucine amide 82 is naturally occurring in Pseudomonas syringae and has both phytotoxic and jasmonate-like activities.148 The indanone-L-isoleucine amide 80 is also active in tomato PIN II gene expression140 and nicotine production in tobacco.129 It is not active in tendril coiling and barley JIP gene expression.145 Thus 80 is not a mimic of coronatine in all systems.Reduction of the ketone group of 80 gives the alcohol 83. This compound is also active in volatile emission145 and O O N H CO2H O O OH O O N H HO2C O O N H RO2C OH O N H MeO2C O NH OH O O NH2 OH H 78 79 80 R = Me 81 R = H 82 83 84 Beale and Ward Jasmonates key players in the plant defence 545 nicotine production.129 It has been suggested that this alcohol is a mimic of the enol of jasmonate which may be the active form in receptor interactions (see section 5.2). It is clear that the amino acid part of coronatine and the indanoyl-isoleucine are very important in giving jasmonate responses. This suggests that the naturally occurring jasmonate amino acid conjugates are active molecules and not deactivated metabolites. Recent experiments seem to support this. In barley (")-JA-L-isoleucine conjugate 24 was as found to be as active as (")-JA for the induction of JIP gene expression.147 Quantification of JA in tissue treated with the conjugate indicated that the conjugate was active per se.A similar experiment measuring JA-L-isoleucine following JA treatment indicated that JA also was active per se. (")-JA-L-Isoleucine is also active in tomato PIN II gene expression.140 However in this system (+)-JA-L-isoleucine is also active. This appears to correlate with the coronatine and indanoyl conjugates in that it is the isoleucine part of the molecule that is important and that the lipophilic ‘jasmonate’ part can be varied. However it should be noted that the stereochemically anchored C-7 methyl derivatives (section 5.2) are not active as their L-isoleucine conjugates,145 indicating perhaps that an enolisable ketone is needed even in these amides.A recent paper on the induction of phytoalexins in rice leaves describes the activity of a number of amino acid conjugates of jasmonate.149 In this system JA-L-isoleucine JA-L-phenylalanine and JA-L-leucine conjugates are all as active if not more so than JA. 6 Conclusions and future prospects Over recent years our understanding of the role of jasmonates in plant growth and development has developed rapidly. As with many biological systems investigations have revealed a much greater complexity than was first expected. The notion of an attack on plants causing linear biosynthesis of jasmonate which then interacts with a receptor that universally activates defence responses and growth responses is too simple.It is now emerging that there are important diVerences between systems and that more than one jasmonate can be involved. With the cloning of some of the biosynthetic enzyme genes our knowledge of biosynthesis is advancing. Complexities that need to be understood here are a determination of the regulatory steps and the importance of parallel pathways starting from the 18:2 or 16:3 fatty acids. ‚-Oxidation and also amino acid conjugation appear to be crucial processes in some systems and thus warrant the attention of the molecular biologist. The use of mutants and transgenic plants with sense and antisense constructs of biosynthetic enzymes will be useful in determining the regulatory steps in the pathway. These will also aid in further investigations of the absolute activity of 12-oxo-PDA 7-epijasmonic acid and its isoleucine amide.Currently the picture emerging from jasmonate structure– activity studies has several facets—that 12-oxo-PDA may be active per se in a few systems while 7-epijasmonic acid and the isoleucine amide are active in all systems studied and the phytotoxin coronatine and its analogues are most likely mimics of JA-amino acid conjugates. It may be that a family of receptors each recognising diVerent naturally occurring jasmonates has evolved. However the experience with gibberellins a family of over 100 similar plant hormones has demonstrated that only a few compounds are active per se the rest being precursors or inactive metabolites. A similar unified theory for jasmonate structure–activity relationships is not possible at present.One of the major diYculties is the large variety of bioassay systems in use. 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ISSN:0265-0568    

DOI:10.1039/a815533y

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

The application of root cultures to problems of biological chemistry Richard J. Robins Laboratoire d’Analyse Isotopique et Electrochimique de Métabolismes, CNRS UPRES-A 6006, Faculté des Sciences, Université de Nantes, 2 rue de la Houssinière, F-44322 Nantes cedex 03, France 1 Introduction 2 The role of root cultures 3 Production, cultivation and properties of root cultures 4 The range of natural products in root cultures 4.1 General aromatic compounds 4.2 Anthraquinones 4.3 Alkaloids 4.3.1 Pyrrolidine alkaloids 4.3.2 Indole alkaloids 4.3.3 Pyrrolizidine alkaloids 4.3.4 Tropane and related alkaloids 4.3.5 Indolizidine alkaloids 4.3.6 Benzylisoquinoline alkaloids 4.4 Terpenoids and steroids 4.4.1 Sesquiterpenes 4.4.2 Diterpenes 4.4.3 Triterpenes and steroids 4.5 Miscellaneous nitrogen compounds 4.6 Miscellaneous sulfur compounds 5 Future prospects 6 References 1 Introduction The pursuit of the chemical steps involved in the formation of complex natural products has occupied numerous organic and biological chemists throughout the last 50 years.Major advances tend to have come in waves, often following the introduction of a new technique. Thus, the availability of radioactively-labelled precursors permitted the elucidation of many pathways in whole plants. The subsequent introduction of stable isotopes,1 coupled with analysis by NMR, had the major asset that site-specific information was readily obtained without the need to perform laborious and complex degradations. Indeed, NMR can provide detailed information on impure samples.As the power of NMR has increased, so has the ease of carrying out such studies. Once magnetic fields providing 300 MHz or more were routinely available, it became possible to study biosynthesis in plants in vivo,2 avoiding the additional complications of purification of intermediates and products. The other technique that has greatly aided biosynthetic studies is GC-MS, whereby isotopic incorporation into numerous compounds in complex mixtures can be obtained without prior purification.GC-MS oVers the additional advantage that experiments with small amounts of labelled precursor suYce to yield accurate quantitative data. The vast majority of such studies of natural product biosynthesis have used whole plants, precursors being added by a variety of techniques and products extracted some weeks later. Despite frequently finding the level of incorporation to be miserably low (0.008% for example) much of our present understanding of biosynthetic chemistry in plants was established by this approach.During the 1970s, however, a new tool came onto the scene, cell cultures. While the concept of growing deregulated plant cells in a defined medium was not novel, it took the pioneering studies of Zenk and co-workers3 to demonstrate the power of this approach to examining the biosynthetic steps to plant products. A particular strength of the cell culture method was that, by developing high-producing lines, it was possible to define the steps in the pathway at the enzymatic level, thus resolving a number of conflicts derived from feeding experiments alone.Thus, the pathways to the indole alkaloids and to the benzylisoquinoline alkaloids were largely defined by work in plant cell suspension cultures.3,4 Subsequently, cell cultures have proved an eVective means of studying the metabolic steps involved in the biosynthesis of complex plant products.They have many advantages: · the potential for fast growth rates, · high yields of metabolites, · the possibility of having continuous production of a metabolite normally only transiently made in the life cycle of the plant (e.g. flower colours), · the possibility to have actively synthetic tissue for a metabolite normally accumulated in a non-active tissue (e.g. bark), · the facility to cultivate cells on a very large scale.But they are also subject to limitations. First, it appears that certain pathways are not well expressed, or are not expressed at all, in supension cultures. This deficiency relates notably to products such as the volatile monoterpenes, typically biosynthesized in leaf-glands, and certain types of alkaloid, normally biosynthesized in the roots. Second, in some plants, products appear to be formed in one organ and transported to another for further modification. Therefore, it was recognised that for certain groups of products, it was necessary to establish organized cultures.Notable amongst these targets were the pyrrolidine and tropane alkaloids and mono- and sesqui-terpenes. The alkaloids became acessible to study through root organ cultures5 and the terpenes via shoot organ cultures.6 2 The role of root cultures Although root cultures of alkaloid-producing species were established more than 50 years ago7 it was not until the 1950s that alkaloid production was demonstrated in such systems.8,9 In the early 1980s the possibility of using root cultures for alkaloid production was reexamined and the direct establishment of cultures of Hyoscyamus niger10 producing tropane alkaloids and Senecio vulgaris11,12 producing pyrrolizidine alkaloids were reported.Such cultures proved eVective means of examining the biosynthesis in vitro of these alkaloids. Establishing and maintaining root cultures remains diY- cult, however, limiting severely the range of studies and the cultivation of roots has been restricted to a few examples.The demonstration by Hamill and coworkers13 that transformed root cultures of Nicotiana rustica produced nicotine and those of Beta vulgaris produced betaxanthin represented a significant advance in the technology. These transformed roots diVer from the normal cultures in being the result of the genetic alteration of the plant genome by genes carried on a plasmid of the pathogenic bacterium Agrobacterium rhizogenes.14 This plasmid system was already being developed for the insertion of genes into plants and, in 1990, the same authors showed that a plant pathway could be genetically engineered when they increased the level of nicotine accumulation in N.rustica transformed roots by gene insertion.15 The advantage of transformation is that transformed roots have no phytohormone requirement and, generally, show good stability in Robins: The application of root cultures to problems of biological chemistry 549culture. Furthermore, as shown now for a number of cases, genes can be inserted into the plant allowing the pathway to be altered, potentially providing new chemical species.Since these early reports, a wide range of root cultures, both transformed and untransformed, have been generated that can accumulate plant products (Table 1).5 In the majority of cases, these consist of straightforward reports of the basic characteristics of the culture and involve no or little biosynthetic analysis.In a minor number of cases, notably the pyrrolidine alkaloids of Nicotiana (Section 4.3.1), the pyrrolizidine alkaloids of Senecio (Section 4.3.3) and the tropane alkaloids of Datura and Hyoscyamus (Section 4.3.4), the use of root cultures has made a significant contribution to elucidating the ‘chemical reactions’ of biosynthesis. In another group of reports, root cultures have been used to address a particular problem of biosynthetic chemistry and the majority of the studies have been performed in whole plants or other sorts of cultures.Thus, it must be emphasized that the role of root cultures in biological chemistry cannot be considered in isolation but must be related to studies performed in other systems. 3 Production, cultivation and properties of root cultures Roots are organized plant organs involved in the absorption of mineral nutrients and water from the soil or a liquid medium (hydroponics) and supplied with sugars from the aerial parts of the plant.It might therefore be predicted that they should be readily grown in a defined medium containing mineral and organic nutrients. Most roots when excised will continue elongating in such conditions but generally fail to branch or to sustain growth for a prolonged period. The addition of phytohormones to stimulate growth can help but tends to induce the propagation of a disorganised culture.Thus, roots of Hyoscyamus species and Senecio species can be excised from sterile seedlings and grown freely and rapidly on a well-defined mineral medium containing vitamins, sucrose, auxin (IAA or IBA) and cytokinin. Such cultures are typically maintained in Erlenmeyer flasks with rotary agitation at about 60–100 rpm and at 25 )C. Healthy rapidly-growing material is passaged into fresh sterile medium every 2 weeks, a 1 g FM transfer leading to about 6 g FM in 14 days and an alkaloid content in H.niger of about 1% DM.16 To generate transformed root cultures,5,16 a part of the plant is treated with a suspension of the bacterium Agrobacterium rhizogenes. Sterile seedlings are also a good source of material but the crucial feature appears to be that the tissue is active in cell division. Transformation occurs when the plant is wounded and infected by a small length of DNA (T-DNA) copied from a plasmid carried by the bacterium. This DNA, which becomes integrated into the plant’s own genome, contains a number of genes, notably the loci rolA, rolB and rolC, that influence the balance of the plant’s endogenous phytohormones, stimulating the formation of root primordia and the proliferation of roots from the point of infection.These can be removed from the parent plant tissue and cultivated in vitro. Thanks to the presence of the T-DNA, they do not require phytohormones and growth is usually rapid. The fast growth is a typical feature of transformed roots and results from the abundant formation of new root primordia, leading to extensive branching of the root mass.There are, inevitably, exceptions. Some roots grow poorly and show minimal branching. Some species of plant are resistant to infection by A. rhizogenes, or have proved very diYcult to transform (e.g. Papaver somniferum). Monocotyledons, although the T-DNA can be inserted, do not respond and no phenotype results. Nevertheless, once established, transformed root cultures display many of the properties of normal roots in planta.5 They can be maintained apparently indefinitely without any major alteration in their phenotype or chemotype and frequently possess a chemotype the same as or closely similar to that of the parent plant.There are, however, a number of exceptions when novel compounds accummulate. Most likely these are minor (unidentified) components in the roots in planta but which accumulate to a higher level in the transformed roots due to an altered environment. 4 The range of natural products in root cultures Most classes of natural products found in plants have now been produced in root cultures. In Table 1 these are summarized, grouped loosely on their biosynthetic origin according to Robinson.17 4.1 General aromatic compounds In general, root cultures have not been used extensively for the study of the biosynthesis of general aromatic compounds. A major reason for this is undoubtedly the relative ease with which many aromatic compounds can be produced in high yield by suspension cultures.For example, much of the work leading to our understanding of the biosynthesis of flavonoids and their regulation at the genetic level was gained by studies using suspension culture cells.4 By feeding experiments in a transformed root culture of Coreopsis tinctoria, the conflicting hypothesies for the biosynthesis of the side chain of the allylphenols was resolved.18 By feeding L-[3*-13C]phenylalanine 1a it was unequivocally demonstrated that the 3* of 2 was enriched 15-fold, excluding the previous hypothesis that the side chain of phenylalanine underwent rearrangement or loss of the carbonyl group during biosynthesis of 2.Furthermore, L-[methyl-13C]methionine only enriched the methoxy group of 2. The isobutyroyl acid moieties were confirmed to originate from DL-valine. Root cultures of Ageratina riparia accumulate chromenes,19 notably ripariochromenes B and C, at levels up to those found in whole plants.An eYcient hydroxylation of the chromene demethoxyencecalin 3 to demethylacetovanillochromene 4 was found, although neither oxidation at the 7-position nor of 5 occurred. As the ripariochromenes are typified by both 7- and 8-hydroxymethyl groups, this activity would appear to be an important initial step in the elaboration of the ring system, and to be required prior to 7-hydroxylation. Why there is no further metabolism of 3, however, is not apparent.COOH NH2 (CH3)2CHCO2 O2CCH(CH3)2 MeO Val Met 1 1a = [3¢-13C]-1 1b = [1¢-14C]-1 2 Val * O O R1 R2 R3 3 R1 = R2 = R3 = H 4 R1 = R2 = H; R3 = OH 5 R1 = R3 = H; R2 = OCH3 550 Natural Product Reports, 1998Table 1 Species and products from root cultures Species Nature of root Type of product Major products Novel products References Aromatic compounds Coreopsis tinctoria t-R Phenylpropane 1*-Isobutyroyloxyeugenol-4-isobutyrate 18 Geranium thunbergii t-R Tannin 1,2,3,6-tetra-O-galloyl-‚-D-glucose, 1,2,3,4,6-penta-O-galloyl-‚-D-glucose, Geraniin 178 Lotus corniculatus t-R Tannin (condensed) 179 Sanguishorba oYcinalis n-R Tannin Sanguiin H-6 180 1,2,3,6-tetra-O-galloyl-‚-D-glucose Pimpinella major n-R Phenylpropanoid Epoxy-pseudoisoeugenol tiglate 181 Lithospermum erythrorhizon t-R Naphthoquinone Shikonin 182 Tanacetum parthenium t-R Coumarin Isofraxidin 9-epicatechachol B 183 Linum flavum n-R Lignan 5-Methylpodophylotoxin 184 Ageratina riparia n-R Chromene Ripariochromene B, C 19 Swertia japonica t-R Phenyl glucoside 1-Sinapoyl glucoside 5-(3*-glucosyl)benzoyloxygentisic acid 185 2,6-Dimethoxy-4-hydroxyphenol-1-glucoside Nicotiana tabacum t-R Hydroxycinnamic acid amide Hydroxycinnamoylputrescine Hydroxycinnamoylcadaverine 20 Miscellaneous unsaponifiable lipids Lobelia chinensis t-R Polyacetylene Lobetyolin, lobetyol 186 Lobelia inflata t-R Polyacetylene Lobeline Lobetyolin, lobetyol 187,188 Lobelia sessilifolia t-R Polyacetylene Lobetyolin, lobetyol 189 Platycodon grandiflorum t-R Polyacetylene Lobetyolin, lobetyol 190,191 Tanacetum parthenium t-R Spiroketal enol ether acetylene 192 Cassia obtusifolia t-R Anthraquinone Chrysophanol, emodin 1,8-Di-O-methylchrysophanol 21 Cassia occidentalis t-R Anthraquinone Germichrysone, pinselin 21 Cassia torosa t-R Anthraquinone Germichrysone 21 Rubia peregrina t-R Anthraquinone 22 Rubia tinctorum t-R Anthraquinone Nordamnacanthal, alizarin 193 Alkaloids Nicotiana alata n-R Pyrroline Nicotine, nornicotine 43 Nicotiana glauca t-R, n-R Pyrroline Nicotine, anabasine 25,37,194 Nicotiana hesperis t-R Pyrroline Nicotine, anabasine 27,28,195 Nicotiana rustica t-R, n-R Pyrroline Nicotine, anabasine 15,26,31,194,196 Nicotiana tabacum t-R, n-R Pyrroline Nicotine, nornicotine, anabasine 24,31,36,38,197,198 Amsonia elliptica t-R Indole 199 Catharanthus roseus t-R, n-R Indole Ajmalicine, serpentine, catharanthine O-Acetylvalesamine 47,48,49,200,201,202,203,204 Catharanthus trichophyllus t-R, n-R Indole Lochnericine, tabersonine, morhammericine, akuammicine Dimethoxyanthraserpine 205 Vinca minor t-R Indole Vincamine 206 Peganum harmala t-R ‚-Carboline Harmine, harmalol 5-Hydroxy- and 6-hydroxy-tetrahydronorharman 53,54,55,56 Cinchona ledgeriana n-R, t-R Quinoline Quinine, quinidine, quinamine 58,59,61,60 Eupatorium cannabinum n-R Pyrrolizidine 7,65 Seneco pleistocephalus t-R Pyrrolizidine Rosmarinine, senecionine 75 Seneco erucifolius n-R Pyrrolizidine Senecionine N-oxide 68 Robins: The application of root cultures to problems of biological chemistry 551Table 1 Continued Species Nature of root Type of product Major products Novel products References Seneco vulgaris n-R Pyrrolizidine Senecionine N-oxide 67,69,70,73,74 Spartium junceum t-R Quinolizidine Cytisine, N-methylcytisine 207 Atropa belladonna t-R, n-R Tropane Atropine, littorine, calystegines 103,208,209,210,211 Datura innoxia t-R Tropane Hyoscyamine 212,213 Datura candida#aurea t-R Tropane Hyoscyamine, scopolamine 87,208 Datura quercifolia t-R Tropane Hyoscyamine, apoatropine 214 Datura stramonium t-R Tropane Hyoscyamine, scopolamine 80,81,195,208,215,216,217,218,219,220,221 Datura wrightii t-R Tropane Hyoscyamine, scopolamine 208 Duboisia hopwoodii n-R Tropane Hyoscyamine, scopolamine, nicotine 222 Duboisia leichhardtii n-R, t-R Tropane Scopolamine 113,136,222,223,224 Duboisia myoporoides n-R, t-R Tropane Hyoscyamine, scopolamine 222,225,226,227 Hyoscyamus albus n-R, t-R Tropane Hyoscyamine, scopolamine 228,229,230 Hyoscyamus muticus n-R, t-R Tropane Scopolamine, littorine, hyoscyamine 208,228,231,232 Hyoscyamus niger n-R, t-R Tropane Scopolamine, hyoscyamine 228,231,233 Nicandra phsaloides t-R Tropane Hygrine, cuscohygrine 88 Scopolia carniolica t-R Tropane Hyoscyamine 234 Scopolia japonica t-R Tropane Hyoscyamine 235 Swainsonia galegifolia t-R, n-R Indolizine Swainsonine 139,140 Cephaelis ipecacuanha n-R Benzylisoquinoline Cephaelin, emetine 236,237 Menispermum dauricum n-R Benzylisoquinoline Dauricine, acutumine 144,145 Papaver bracteatum n-R Benzylisoquinoline Thebaine 238 Papaver somniferum n-R, t-R Benzylisoquinoline Codeine, morphine, thebaine 239,240 Stephania cepharantha n-R Benzylisoquinoline Aromoline, homoaromoline, berbamine 142,143 Terpenoids and steroids Artemisia absinthium t-R Monoterpene Linalyl-3-methylbutanoate, nerol 241 Coleus forskohlii n-R, t-R Diterpene Forskolin 242,243 Jatropha elliptica n-R Diterpene Jatrophone 244 Salvia militiorrhiza t-R Diterpene Cryptotanshinone, tanshinone II 245 Lippia dulcis t-R Diterpene glucoside Hernandulcin 246 Stevia rebaudiana n-R Diterpene glucoside Stevioside 149 Capsicum annuum n-R Sesquiterpene Capsidiol 146 Datura stramonium t-R Sesquiterpene Lubimin, 3-hydroxylubimin 247 Hyoscyamus muticus n-R, t-R Sesquiterpene Solavetivone 147,148,248,249 Leontopodium alpinum t-R Sesquiterpene n.d. 250 Perezia cuernavacana t-R Sesquiterpene Perezone 251 Tanacetum parthenium t-R Sesquiterpene Pathenolide 252 Glycyrrhiza uralensis t-R Triterpenoid Glycyrrhizin 155 Taraxacum oYcinale n-R Triterpenoid Taraxasterol, lupeol, ‚-amyrin 151 Taxus brevifolia t-R Taxane Taxol 253 Ajuga reptans t-R Sterol Clerosterol 165 Withania somnifera n-R, t-R Sterol Withanolide D 254 Trigonella foenum-graecum t-R Sterol Diosgenin 255 Astragalus membranaceus t-R Steroidal saponin Astrolagosides I, III Agroastrolagoside I 152 Astragalus mongholicus t-R Steroidal saponin Astrolagosides I, II, III 153 Panax ginseng t-R Steroidal saponin Ginsenosides 156,256 Saponaria oYcinalis n-R Steroidal saponin Saporin 257 552 Natural Product Reports, 1998Table 1 Continued Species Nature of root Type of product Major products Novel products References Solanum aculeatissimum t-R Steroidal saponin Aculeatiside A, B 154 Solanum aviculare t-R Steroidal alkaloid Solasodine 258 Solanum mauritianum t-R Steroidal alkaloid Solasodine 157 Ajuga reptans t-R, n-R Phytoecdysteroid 20-Hydroxyecdysone, 29-norcynasterone, 29-norsergasterone 158,159 Serratula tinctoria t-R Phytoecdysteroid 20-Hydroxyecdysone 160 Flavonoid Glycyrrhiza glabra t-R Flavonoid (prenylated) Glabrol, abssinone Licoagrochalcone A, licoagrocarpin 259 Fragaria#ananassa t-R Flavanol (+)-Catechin, procyanidine B-3 260 Fagopyrum esculentum t-R, n-R Flavanol (+)-Catechin, (")-epicatechin 261 Lotus corniculatus t-R Isoflavan Vestitol, satavan 262 Lupinus polyphyllus t-R Isoflavonoid 2-Hydroxygenistein-7,2*-di-O-glucoside 263 Lupinus hartwigii t-R Isoflavonoid 2-Hydroxygenistein-7,2*-di-O-glucoside 263 Leontopodium alpinum t-R Anthocyanin n.d. 250 Miscellaneous nitrogen compounds Beta vulgaris n-R, t-R Betaxanthin Portulaxanthin II, vulgaxanthin I Vulgaxanthin III, IV 13,166 Miscellaneous sulfur compounds Chaenactis douglasii t-R, n-R Thiophene Thiarubine A, B 264,265 Rudbeckia hirta t-R Thiophene 5-(but-3-en-1-ynyl)-2,2*-bithienyl, 5-(4-acetoxybut-1-ynyl)-2,2*-bithienyl 266 Tagetes patula n-R, t-R Thiophene 5-(but-3-en-1-ynyl)-2,2*-bithienyl, 5-(4-acetoxybut-1-ynyl)-2,2*-bithienyl 167,168,169,170,171,201 Tagetes laxa t-R Thiophene 5-(but-3-en-1-ynyl)-2,2*-bithienyl, 5-(4-acetoxybut-1-ynyl)-2,2*-bithienyl 267 Allium cepa n-R S-Allyl-cysteine S-Alk(en)yl-L-cysteine sulfoxides Propyl, prop-2-enyl-, ethyl- 172 Allium sativum n-R S-Allyl-cysteine Aliin 173 Notes: n.d.=Not determined.n-R=normal roots; t-R=transformed roots. References—this is not a comprehensive list but gives key and recent references. Robins: The application of root cultures to problems of biological chemistry 553While a number of root cultures have been found to accumulate compounds previously not identified from the parent plant (Table 1), Berlin20 has recently reported an exciting development, the creation of novel metabolites as a result of the insertion of a gene into a transformed root culture.Previously, this group have shown that expressing a bacterial gene coding for lysine 6 decarboxylase (LDC) would alter alkaloid production in N. glauca and N. tabacum roots (see Section 4.3.1). It is now demonstrated that, simultaneously, the unnaturally elevated level of cadaverine 7—the decarboxylation product of the action of LDC on lysine—is metabolized into the hydroxycinnamoylcadaverines 8, 9, 10 forming a series parallel to the normal hydroxycinnamoylputrescines identified many years ago in these species.This trait was not apparently recoverable in plants regenerated from root cultures, possibly due to toxicity caused by cadaverine overproduction. 4.2 Anthraquinones The incorporation of [1-13C]acetate into the anthraquinone germichrysone 11 at high levels (ca. 10%) by transformed root cultures of Cassia torosa confirmed the polyketide origin of this compound.21 The anthraquinones of Rubia, in contrast, are derived from the shikimate pathway via O-succinyl benzoate. Following the isolation of the gene for isochorismate synthase from the bacterium Escherichia coli, it was demonstrated22 that the overexpression of this gene in transgenic root cultures of R.peregrina enhanced the overall production of alizarin 12 by 30%. The eVect of this transgene on the yield of 12 strongly supports the previous chemical and biochemical evidence for the importance of isochorismate in anthraquinone biosynthesis in Rubia. 4.3 Alkaloids 4.3.1 Pyrrolidine alkaloids The demonstration by Dawson23 that nicotine 13 is synthesised in roots and transported to the shoots of tobacco plants established this alkaloid as an early target for biosynthetic studies in root cultures. Exploiting sterile root cultures of Nicotiana tabacum and N. glauca, this group performed a series of feeding experiments24,25 that were definitive in defining nicotinic acid 14 and ornithine 15 as the precursors of 13.The importance of 6 as a precursor of anabasine 16 was also confirmed.25 Furthermore, it was concluded that synthesis of 13 and 16 does not occur strictly in parallel, even though 14 was proved to be a common precursor. It was also shown that 13 turned over in the cultures, up to 40% of added [14C]-13 being degraded within 38 days. Nearly 25 years later, in 1987, it was again tobacco roots that were used to demonstrate that plant metabolites would eVectively be biosynthesised by transformed root cultures.13 These cultures have proved valuable in confirming from biochemical and genetic evidence the deductions made at the chemical level. Feeding 14 to roots of N.rustica enhanced both 13 and anatabine 17, with the eVect being much more marked for the latter product.26 The central role of 14 as precursor was confirmed and it was suggested that these products compete for this common intermediate, condensing with N-methylpyrrolinium 18 to form 13 or with ƒ1-piperideine 19 to form 16.Similarly, feeding 7 greatly stimulated the formation of 16 in transformed root cultures of both N. rustica27 and N. hesperis.28 This occurred at the expense of 13, again showing a strong metabolic linkage between the relevant pathways. Indeed, the kinetic properties of the diamine oxidase29 of N. tabacum, support the proposal that there is a common pathway of biosynthesis of the pyrrolyl and piperidyl moieties of 13 and 16 respectively. A major diVerence between the biosyntheses of 13 and 16, however, appears to be that, while formation of 13 involves a free diamine intermediate (putrescine 20), free 7 is not naturally an intermediate for 16.This conclusion is based on findings that label from [U-14C]-6 is not diluted by added 7.29 Previously, it had been reported in whole plants30 that a symmetrical intermediate was not involved in the biosynthesis of 16, all the radioactivity from fed [2-14C]-6 being recovered at the C-2* position.If this were the case, it might explain the apparent inability of 7 to dilute out 6 from the pathway. When, however, (R)-and (S)-[2-2H]-7 were fed31 to transformed cultures of N. rustica, the labelling patterns observed in the extracted anabasine clearly demonstrated that the H2N COOH H2N NH2 COOH R1 R2 C NH O H2N NH2 6 1 7 8 R1 = R2 = OH 9 R1 = OH; R2 = OMe 10 R1 = R2 = OMe LDC Scheme 1 HO O OH OH Me O O OH OH 11 12 H2N NH2 H2N NH2 COOH N N N Me COOH N N Me N NH N NH N N H N N Me O ODC + X– 4¢ 5¢ 2x 6 7 20 15 18 14 19 13 17 16 21 22 LDC Scheme 2 554 Natural Product Reports, 1998incorporation of 19 proceeds without stereoselectivity.With (S)-[2-2H]-7, the pro-S hydrogen is lost on oxidation of the primary amino group and only the C-6* hydrogen is labelled. With (R)-[2-2H]-7, the pro-R hydrogen is retained at C-2* and C-6*. Interpretation of the NMR data was complicated, as an equimolar chiral mixture of (R)- and (S)-16 accumulated.The same authors were also able to show31 that [2,2,4,4-2H4]-7 fed to N. tabacum transformed root cultures led to no 2H enrichment of the pyridine ring of 16, thus that 16 was made by the correct in vivo route and not via an aberrant mechanism demonstrated in pea and lupin extract wherein both rings appear to be derived from cadaverine. To show that added metabolites are correctly incorporated is an important experiment.Plants are notorious for their capacity to adapt pathways to metabolise exogenous substrates, potentially creating false ‘pathways’. This capacity can be exploited in the search for novel products. Transformed root cultures of N. rustica, when fed N-ethylputrescine, ef- ficiently converted this into (S)-N*-ethylnornicotine.32 Other substituted putrescines were also converted to the equivalent substituted nornicotine, although much less eYciently.33 Substituted pyridines have previously been shown to be incorporated in whole plants.34 In both untransformed25 and transformed root cultures27,28 it was found that feeding 6 to root cultures had little influence on the overall yield of 16.Similarly, feeding 15 to transformed root cultures of N. rustica failed to enhance accumulation of 13. In contrast, supplying the relevant diamine, 7 or 20, the products of decarboxylation of 7 or 15 respectively, enhanced the accumulation of the relevant alkaloids.This evidence indicated limitations to the pathway at the level of the activity of the decarboxylation enzymes. By expressing a gene for ornithine decarboxylase (ODC) isolated from a yeast, Hamill15 improved the yield of 13 in transformed root cultures of N. rustica by about 75%. Levels of intermediates were also enhanced. In a more complex series of experiments, the gene coding for LDC was isolated from the bacterium Hafnia alvei and used to alter alkaloid metabolism in Nicotiana roots.35–38 When the expression of the gene was directed into the plastidic compartment of the cells, Berlin35,36 showed a substantially altered alkaloid composition in transformed root cultures of N.glauca37 or N. tabacum,36,38 both the accumulation of 16 being enhanced two-fold and the 13:16 ratio being changed from 3:1 to 2:3. Furthermore, the regulation of the pathway was now altered so that the supply of 6 became limiting,36 as shown by the increased levels of 16 that accumulated after feeding 6.As observed by Solt,25 nicotine is not accumulated in a static manner but turns over. Several lines of evidence have indicated that nornicotine 21 is an important primary degradation product of 13, both in plants and in mammalian or microbial systems.39 Suspension culture cells of several Nicotiana spp. actively demethylate added nicotine,40–42 an eVect studied in detail in root cultures of N. alata.43 While the most logical route of degradation of 13 would involve direct oxidation of the N-methyl group, there was some evidence to indicate that demethylation could be initiated by an oxidation at the C-5* position.34 Using quantitative 2H NMR, it was shown that the molar ratios between the four enriched positions of [4*,4*,5*,5*-2H4]-13 were retained in the derived 21, ruling out the possibility of any oxidation at the C-5*.This report also showed that substantial ‘aberrant’ oxidation of the C-5* does occur, cotinine 22, a trace metabolite in control tissues, accumulating to high levels.Thus, two oxidative activities acting on nicotine which are already known from bacterial and mammalian metabolism39 are identified in these root cultures. The demethylation appears to be the primary activity in vivo but the C-5* oxidation becomes important in the presence of excess 13. The respective roles of these two primary degradation products in nicotine turnover remain to be established. The importance of intact roots in the biosynthesis of 13 has been shown.When root cultures of N. rustica were treated with phytohormones,44 biosynthetic capacity was lost, returning only after transfer of culture to fresh medium. Interestingly, the degradation of 13 did not appear to be stimulated. This is surprising as, although some carefully selected suspension cultures do make 13,45 they can also show a strong degradative capacity.41 Loss of biosynthetic activity in both types of culture44,46 has been shown to be due to the rapid disappearance of the enzyme for the N-methylation of 20, putrescine N-methyl transferase (PMT). 4.3.2 Indole alkaloids The definition of the biosynthetic routes to ajmalicine 23 and serpentine 24 in Catharanthus roseus and ajmaline in Rauwolfia serpentina represent the seminal studies of indole alkaloid biosynthesis using suspension cell cultures.3,4 Root cultures of C. roseus (Table 1) yield a similar range of products to suspension culture cells, dominated by 23 and 24, although some novel products have been identified47 and, significantly, levels of catharanthine 25 and vindoline 26 up to 103 times greater than in suspension cultures can be accumulated.48,49 The accumlation of 23 in at least some root cultures seemed to be directly related to the level of tryptophan 27 decarboxylase (TDC), in a similar way to the limiting eVect of ODC levels on the accumulation of 13 in N.rustica roots.15 This problem was directly addressed by expressing the gene for TDC in transformed suspension cultures of C.roseus (introduced via A. tumefaciens that generates a dispersed culture rather than an organized root culture).50 Although the cultures had enhanced TDC protein, enzyme activity and tryptamine 28, no increase of terpene indole alkaloid accumulation occurred, indicating that the pathway was further limited elsewhere. Following on from this work, the same authors51 introduced a double construct containing TDC and the gene for the next step, strictosidine 29 synthase (SS), into transformed tobacco suspension cultures.The advantage of working in the ‘non-natural’ context is that the background activities are eVectively zero. The culture accumulated 28 and produced 29 on feeding secologanin 30, the terpenoid moiety of the alkaloids. Thus, two key steps of this pathway are shown to be linked and transposable to other species. However, 30 is itself the product of a long pathway and its supply appears limiting in many indole alkaloid producing cultures.Genetic manipulation of key genes in the biosynthesis of this intermediate will probably prove crucial to the overall enhancement of the indole alkaloid pathway. Unlike cell suspension cultures,52 untransformed53 and transformed root cultures54,55 of Peganum harmala accumulate both ‚-carboline alkaloids and serotonin 31. While both types of root culture contain harmalol 32, harmol 33, harmaline 34 and harmine 35, the glucoside ruine 36 and the simple aromatic amine 5-hydroxytryptamine 31 also accumulate in the transformed root cultures.The biosynthetic route to these alkaloids is still poorly described, particularly at the enzymatic level. As with the indole alkaloids, the accumulation of 31 seems to be directly related to the level of TDC.55 Feeding 27 had little eVect on levels of either alkaloids or 31, whereas feeding 28 enhanced 20-fold the accumulation of 31, indicating a deficit in the supply of this precursor.In contrast, the alkaloid levels were unaltered. The limitation imposed by TDC expression was confirmed by expressing the gene for TDC in root cultures of P. harmala.56 The TDC activity was enhanced up to 40-fold and, while there was no increase in 28, this was because immediate hydroxylation to 31 occurred. Under these conditions the supply of 27 became limiting as, when high levels of 27 were fed the yield of 31 was as high as 5% of the dry matter.No eVect on harmaline alkaloids occurred, however, indicating that this pathway is regulated at a stage beyond 28. Attempts to define the critical 6-oxidation that distinguishes the harmaline alkaloids have so far proved unsuccessful. Thus, it remains to be established whether formation of the C-ring is prior to 6-oxidation or after. Aromatization, however, appears to be a late step in biosynthesis, as fed 32 and 34 were Robins: The application of root cultures to problems of biological chemistry 555converted to 33 and 35 respectively.55 Recently, the enzymatic O-methylation of 32 and 33 has been identified, as has a dehydrogenase converting 34 to 35.52 Interestingly, this enzyme was not active with 32 as substrate, indicating that a second specific activity may exist for the unmethylated forms.When [3-14C]-27 was fed, label was identified in two novel compounds, 5-hydroxytetrahydronorharmane and 6- hydroxytetrahydronorharmane.These apparently represent minor alkaloids and are not intermediates of the harmane alkaloids.55 Although quinine 37 and quinidine 38 contain no indole ring they are classified with the indole alkaloids on biosynthetic grounds as they are derived from 29. Quinoline alkaloids are not eVectively accumulated by dispersed cultures, although some alkaloid is found in aggregated disorganised cultures.57 Root cultures, both non-transformed58 and transformed,59 contain alkaloids of the 8R,9S and 8S,9R series, the proportions varying considerably with age, the line under study and environmental conditions.In addition, indole alkaloids, notably quinamine 39 and cinchonamine 40 were identified only in the transformed cultures.59 Root cultures incorporated L-[methylene-14C]-27, [2* -14C]-28 and [C5-3H]-29 into 36 and 37, confirming the biosynthetic origin of these alkaloids.60,61 Apparently, the tryptophan supply was limiting, as a five-fold increase in alkaloid occurred in response to exogenous 27.60 4.3.3 Pyrrolizidine alkaloids The pyrrolizidine alkaloids are biosynthetically esters between a necine base (e.g.retronecine 45), derived from arginine 41 or, in some cases 15, and a necic acid 47, frequently derived from isoleucine 46 or valine. They occur in several families of plants, notably the Asteraceae, Borginaceae and Fabaceae.62 Only root cultures, notably of Senecio, are competent in pyrrolizidine alkaloid biosynthesis, indicating the root to be the sole site of biosynthesis.63 In some genera, however, such as Crotalaria, this is not the case, the alkaloids apparently being synthesised in the shoots, root cultures being completely devoid of alkaloids.63 Although much of the biosynthetic groundwork came from stable-isotope tracer experiments in whole plants of Senecio spp., in particular the identification of homospermidine 44 as intermediate,62,64 root cultures have proved a valuable tool for defining specific details of the pathway of necine base formation and the enzymology involved.62,63 The intermediacy of (")-trachelanthamidine 49 and subsequently (")-supinindine 50 in the biosynthesis of 45 was established in Eupatorium cannabinum root cultures.65 Similarly, a direct conversion of the retronecine base into the otonecine base was shown in S.vernalis root cultures, which converted senecionine N-oxide 51 into senkirkine 52.66 The major product accumulated in root cultures of Senecio vulgaris is 51.67 This alkaloid is apparently made only in the actively growing parts of the root culture and is slowly transported throughout the root mass, as shown by pulse-feeding NH COOH NH2 NH NH2 NH NH O O NH N O NH N N Me H OH OAc CO2Me CO2Me N H O MeCO2 MeCO2 MeCO2 Oglc H H Oglc Me H H MeO 27 28 30 29 24 26 23 25 N+ Tryptophan decarboxylase Scheme 3 NH NH2 NH N NH NH2 NH N NH N MeO HO Me Me Me HO HO HO MeO GlcO 27 28 31 32 33 34 35 36 Scheme 4 N NH HO NH O MeO OH OH N N N H H 37 8 S,9 R 38 8 R,9 S 39 40 556 Natural Product Reports, 1998experiments.62,68 There appears to be very little turnover of 51, although there is some limited metabolism, primarily oxidation and acetylation.Feeding 14C-labelled precursors and inhibitors of metabolism, notably ·-difluoromethylornithine (DFMO) or ·- difluoromethylarginine (DFMA), that inhibit ODC and ADC respectively, showed both 15 and 41 to be incorporated into 51.69 Unexpectedly, however, DFMA prevented labelling from both precursors, whereas DFMO had no eVect, label from 15 being incorporated via 41.ODC was found to be absent from these roots and the enzymes involved in converting 41 to 20 have been characterised. The mechanism for the eYcient conversion of 15 to 41 is, however, not clear. Both of the polyamines spermine and spermidine 43 reduced the incorporation of label from 41, suggesting that there is feedback control of agmatine 42 biosynthesis by the polyamines.Spermidine 43 is as eVectively incorporated as 20, a finding initially interpreted as showing a rapid interconversion of these two metabolites. When the enzyme homospermine 44 synthase (HSS) was first isolated from roots of S. vulgaris it was considered that 20 was the exclusive substrate.70 It was shown that pyrroline was not incorporated into 44.70 However, studies70,71 of the HSS from roots of Eupatorium cannabinum showed that, while 44 could be formed from two molecules of 20, 43 also acted as an eYcient substrate of the enzyme, provided 20 was also present.This situation is quite the opposite to that described for Nicotiana, where 43 is only incorporated into the pyrrolidine ring of nicotine via 20.72 The aYnity of HSS for 43 is comparable to that for 20 indicating that, at the steady-state concentrations of these substrates in the roots (ca. 50–100 ÏM), incorporation would be roughly 1:1.Subsequent tracer experiments73 in roots of S. vulgaris with [3H]-20 and [14C]-43 confirmed 50 to 70% of the aminobutyl group of 44 to be derived in vivo directly from 43. This finding led to the resolution of a previous contradiction between experimental and theoretical data. Although in theory 50% of 2H label should be retained from (S)-[1-2H]-20, only 34% was found in the isolated retronecine.64 If this proceeded via a diamine oxidase, in which the stereochemistry is known, then the pro-R hydrogen would be retained, giving 25% at each labelled carbon atom in 45.However, the enzyme HSS is intriguing in that it carries out two sequential steps the first of which is a deaminative oxidation that generates NADH and the second of which is a reduction, utilising NADH and restoring the abstracted hydride to the product.73 Thus, as the formation of 44 does not involve diamine oxidase activity but HSS, the predicted incorporation should now be 50%, higher than found experimentally.The discrepancy was resolved74 by showing that (S)-[1-2H]-20 is incorporated into both spermidine and homospermine with retention but that loss of label occurs during the conversion of spermidine into putrescine, leading to the lower overall incorporation. The biosynthesis of the necic acid moiety 47, in contrast, is relatively poorly described.64 Root cultures of S. vulgaris eVectively incorporate label from [14C]-46 into 51.69 By feeding75 (&)-[3,4-13C2]-2-aminobutanoic acid 53 and (&)- [3,4-2H2]-2-aminobutanoic acid 54 to root cultures and plants of S.isatideus and S. pleistocephalus respectively, the complete labelling patterns in senicic acid 47 were obtained as 55 and 56. 4.3.4 Tropane and related alkaloids The tropane alkaloids occur in a subsection of the Solanaceae,76 notably species of Datura, Atropa, Hyoscyamus and Duboisia. Root cultures competent in alkaloid formation have been made from all these genera (Table 1).Tropane alkaloids also occur in the Erythroxylaceae, Convolvulaceae, Proteaceae and Rhizophoraceae76 but their synthesis remains relatively poorly investigated in these families, with the exception of cocaine 57 in Erythroxylon coca.77 This alkaloid appears to be biosynthesized in the leaf and, although root cultures of E. coca have yet to be established, there are no studies on this important alkaloid in root (or shoot) cultures. HN NH2 H2N H N N N HOOC Me COOH HO Me Me NH2 NH2 NH2 NH2 NH2 NH2 OH HO H O Me O O H HO Me O 41 Arginine 42 Agmatine 46 Isoleucine 43 20 20 44 45 47 48 ADC + HSS HSS Scheme 5 N OH H OH N+ O Me O O H HO Me O N O O– 49 50 51 52 CO2 – CO D3C CO NH3 + HO Me CO D CO D CD3 HO Me CD3CD2CH(NH3)CO2 – 53 54 55 56 Robins: The application of root cultures to problems of biological chemistry 557In Datura and related species the dominant alkaloids are the aromatic esters of tropine [endo-(·)-tropine] 58, hyoscyamine 59 and scopolamine 60, although a wide range of esters with aliphatic short chain acids also occur.76 A number of esters of the exo-(‚)-isomer, pseudotropine 61, also accumulate, although aromatic esters are not found.The basic details of the pathway to these alkaloids have been established in plants, although many details relating to the biosynthesis of 59 and 60 remained controversial or unresolved until the advent of root cultures competent in their synthesis.Studies in root cultures have been of major value in defining the pathway and in correcting a number of errors in previous results, notably relating to the intermediacy of hygrine 62, the pathway by which the phenylalanine-derived tropic acid moiety is made and in defining the nature of the esterification reaction. The biosynthesis of these alkaloids was reviewed in 199378 and only key features of the contribution of root cultures prior to this review will be repeated here.The accumulation of 60 was described79 in root cultures derived from rediVerentiated callus of Hyoscyamus niger in 1983, since when a number of other untransformed cultures have been generated (Table 1).78 Transformed cultures of Datura stramonium were reported in 1987 and also shown to be stable producers of tropane alkaloids, notably accumulating 59.80 Nearly all the biosynthetic work in root cultures has used these species. As with the pyrrolidine alkaloids, the first dedicated product of the pathway to 59 is N-methylputrescine 63, which can be derived directly from either 15 or 41.Evidence from feeding experiments in D. stramonium transformed root cultures81 with [U-14C]-15, [U-14C]-41 and [U-14C]-42 indicated, however, that 42 was the most direct precursor, implying that 41 is normally the principal source of 63.82 This was confirmed by experiments with DFMA and DFMO.83 Roots grown in the presence of DFMO contained amounts of 59 equivalent to the controls: those grown with DFMA were severely depleted.Feeding experiments in plants with [2-14C]-15 had lead to the hypothesis84 that 5-N-methylornithine 64 might be an intermediate in the formation of 63, as the extracted 59 appeared not to be derived from a symmetrical intermediate such as 20. No support for this could be found in root cultures, however, as neither of the required enzyme activites for the N-methylation of 15 nor for the decarboxylation of 64 could be found82 in roots of D.stramonium, while high levels of ODC and PMT were present. Curiously, however, unlabelled 20, even at a 2000:1 ratio, failed to dilute by more than 50% the incorporation of [5-14C]-15. This led to the proposal that there is a degree of metabolic tunnelling in the pathway, although this remains hard to prove. The enzymatic data for the intermediacy of 20 and 63 are now very strong and the enzyme PMT has been purified from roots of D. stramonium.85 While it remained improbable on the HN N Me Me NH2 C O N Me C O C OH O N Me C O N N Me N Me N Me N Me N Me N H N Me N Me Me O HO OH O C O O C O OH OH OH O O C O C H 15 64 42 41 63 18 62 66 72 65 58 61 57 79 75 59 60 20 67 Various tropine aliphatic esters Various pseudotropine aliphatic esters OH TR I TR II 70 OH CO2Me Scheme 6 558 Natural Product Reports, 1998basis of previous evidence84 that methylation was subsequent to pyrrolinium ion formation, the intermediacy of 63 was further strengthened by studies of the oxidase enzyme catalysing the conversion of 63 to 18, partially purified and characterised from H.niger roots.86 The kinetic properties of this enzyme showed a strong preference for N-methylputrescine over putrescine as substrate, much more marked than that for MPO from N. tabacum roots.29 The classical pathway from 18 to tropinone 65 involves the addition of three carbons ‘‘probably derived from acetate 67’’.78,84 Central to this proposed pathway was the intermediacy of 62 prior to closure to form the second ring of the azabicyclo[3.2.1]octane skeleton.Certainly, 62 occurs in root cultures of Datura81 and many other tropane-alkaloidproducing species.87,88 When (R,S)-[N-methyl-14C,2*-14C]-62 was fed to Datura stramonium plants,89 incorporation into 59 of intact precursor was reported. Crucially, incorporation was found90 to be stereoselective, (2R)-[2*-14C]-62 showing a 2.5 to 10-fold greater incorporation into the tropane alkaloids in Datura innoxia plants than (2S)-[2*-14C]-62. In contrast, in H.niger and A. belladonna plants both isomers were equally well incorporated,91 even though the unsymmetrical incorporation of 15 into (")-59 and (")-60 requires that only (2R)-62 serve as a precursor for the tropane ring. A series of experiments77 in E. coca led, however, to the conclusion that 62 was not a direct precursor of 57 and subsequent experiments92 in D. inoxia plants reached the same conclusion for 59.These experiments used stable isotope labelling and NMR analysis of incorporation at specific carbon centers, a more precise approach than the earlier experiments with radiolabelled molecules. The identification of 4-(1-methylpyrrolidin-2-yl)-3-oxobutanoate 66 as an intermediate in the biosynthesis of (")-5777 initiated a review of the role of 62 in the biosynthesis of 59 and 60.93 (R,S)-[2*,3*-13C2]-62 was not incorporated into 59 by root cultures of D.stramonium, confirming the improbable intermediacy of this molecule. In contrast, ethyl (R,S)-[2,3-13C2,3- 14C]-66, already shown to be incorporated into 60 in whole plants of D. innoxia,92 showed high incorporation into 59 in root cultures of D. stramonium.93 In contrast, neither plants92 nor root cultures93 incorporated ethyl (R,S)-[1,2-13C2,2-14C]- 2-(1-methylpyrrolidin-2-yl)acetate 71, as evidenced by stable isotope analysis. This latter finding was not compatible with the proposal that 65 was derived from 18 by the sequential addition of two units of 67, as initially suggested.77,92 The previous controversy over the mechanism for the incorporation of 67 into the C-2, C-3 and C-4 was partially resolved by feeding sodium [1,2-13C2]acetate 68 to root cultures of H.albus94 and D. stramonium.93 The 13C-NMR spectra of the isolated (")-59 showed high incorporations and both the C-4 and C-2 resonances to have a pair of satellites. It was unequivocally demonstrated that the C-2 and C-4 of 59 became equally labelled from 68, in contrast to previous suggestions95 that the C-2 is preferentially labelled.Crucially, in the NMR spectrum of 59 extracted from D. stramonium,93 the C-3 resonance was observed as a quintet. In view of the clear incorporation at both C-2 and C-4, this quintet can be interpreted as composed of a doublet symmetrically overlapped with a triplet. This triplet arises from (")-59 (chirality shown by Mosher acid analysis) labelled at C-2, C-3 and C-4 within the same molecule. The specific incorporation for the doubly-labelled species was 7.2% and the specific incorporation for the triply-labelled species was 2.9%.While the evidence from H. albus was interpreted94 as indicating the probable use of both isomers of hygrine (but see above), the C-3 resonance can also be interpreted as a doublet plus a triplet, due to the same labelling pattern. On the strength of this evidence and the lack of incorporation from ethyl (R,S)-[1,2-13C2,2-14C]-7193,95 it was proposed that the C-2, C-3 and C-4 are incorporated in a single step, probably by the condensation with 18 of acetoacetate 69 via the C-4 position.While the C-4 of 69 is less favoured than the C-2, the first condensation can take advantage of the activated state of the C-2 of 18, leaving the C-2 of 66 to react with the C-5* of the heterocycle. The stereochemistry of the reaction remains to be established, however, as does the incorporation of 69 which was not a satisfactory precursor in D.stramonium plants,96 due to rapid hydrolysis. The methyl ester of the proposed intermediate 70 has recently been identified in low amounts in root cultures of D. stramonium.97 This structure is retained intact in 57. Although not incorporated into 59, (R,S)-[2*,3*-13C2]-62 efficiently enriched a number of metabolically related pyrroline alkaloids in root cultures of Nicandra physaloides88 and D.stramonium.93 In D. stramonium, cuscohygrine 72 was enriched up to 22% in the M+1 and 14% in the M+2 ions, indicating that 62 is a proximal precursor for this dicyclic condensation product. A similar enrichment occurred with 68, confirming the origin of the bridge in cuscohygrine. Furthermore, ethyl (R,S)-[2,3-13C2,3-14C]-66 strongly enriched 72 in the M+2 (29%) but very little in the M+1 (3%) and not at all in the M+3 ion. The C-6 and C-6* were enriched although to an unequal extent, indicating the incorporation of both (R) and (S) isomers of the precursor, forming both (R,R)-72 and (R,S)-72, both enriched in the C-7 position also, as found in E.coca.98 As can be seen from the 13C–13C couplings, no triply labelled species occurred in either isomer, confirming that 72 probably originates by the condensation of 18 with 62 or 66. In E. coca, 66 proved98 a much better precursor for 72 than did 62, in contrast to in D. stramonium roots,93 wherein 62 was strongly enriched (46% at M+2) from ethyl (R,S)-[2,3-13C2, 3-14C]-66, presumably via the activity of a decarboxylase.Unfortunately the role, if any, of ethyl (R,S)-[1,2-13C2,2-14C]- 71 in the biosynthesis of 72 was not examined. Tropinone 65 is certainly an intermediate for the biosynthesis of 59. It is reduced by two stereospecific reductases, TRI and TRII, to form 58 or 61 respectively. The kinetic properties of these enzymes have been extensively examined and are summarized in ref. 78. The D. stramonium enzymes have been cloned99 and shown to be derived from separate genes. The two enzymes show 64% homology at the amino acid level.99,100 Stereospecificity appears to be conferred by the orientation of 65 within the active site, as determined by chimeric enzymes generated by fusing the substrate and cofactor binding O N Me TropateO N Me TropateO N Me TropateO O– Na+ 68 Scheme 7 O OR O O OR O N Me N Me O OR O OR O N Me N Me OR¢ O OR¢ O + 18 66 70 71 65 via 62 69 Scheme 8 Robins: The application of root cultures to problems of biological chemistry 559domains in diVerent combinations.100 While TRI activity is clearly important for the biosynthesis of 59, the role in D.stramonium root cultures of the TRII activity, which is present at about 20% of the TRI activity,101 is less clear. This species, however, accumulates a number of minor alkaloids of the exo-series, esters of 61 and aliphatic acids.When cultures are treated with 8-thiabicyclo[3.2.1]octan-3-one 73 this is reduced only by TRI to specifically the 3·-ol 74, which then inhibits TRI activity.102 The consequence is that alkaloids of the exo-series accumulate to a much higher level. In A. belladonna root cultures, however, the TRII activity is apparently involved in the biosynthesis of the highly watersoluble polyhydroxynortropane alkaloids, the calystegines (e.g. calystegine A3 75).103 These products show strong activity as glycosidase inhibitors.104 A number of lines of evidence103 suggest them to be derived from 65, with the first step of the pathway catalysed by TRII, and not to originate by a parallel pathway from pyrrolinium.Firstly, the cultures are rich in TRII. Secondly, when treated with 73, the level of 59 is decreased while simultaneously the level of 75 increased, a finding compatible with the previously described eVects of 73 on the formation of 61 in D. stramonium.102 Thirdly, 15N-65 enriched the M+1 of 75 isolated from fed cultures.Many steps in this pathway remain to be elucidated, however, notably the N-demethylation and hydroxylation reactions. The role of (S)-1 in the biosynthesis of 59 was proposed105 in 1955 and studies in a number of species of plant and root cultures, have confirmed this origin for the tropic acid moiety 76a in 59 and 60. Much of the detail of this metabolism has been established in a series of experiments in root cultures, which have recently been reviewed.106 Crucial to this pathway is the rearrangement of the C6–n-C3 of phenylalanine into the C6–iso-C3 of 76.Clear evidence for this process came from experiments107 in Datura innoxia plants fed (R,S)-[1,3-13C2]-1, wherefrom label was incorporated intact into the tropate moiety of 59 by an intramolecular rearrangement, the two isotopes becoming contiguous in the derived tropate. The substrate for the rearrangement remained undefined, however. 14C-Labelled phenylpyruvate 77 and phenyllactate 78 were shown to be intermediates in the formation of 76 by feeding experiments108 with plants of D. innoxia. The intermediacy of 78 was confirmed by feeding (R,S)-[1,3-13C2]-78 to D. stramonium transformed root cultures109 or plants,110 and the high incorporation into 59 and 60 observed. The 13C–13C spin–spin coupling shown in the extracted 59 and 60 confirmed the intramolecular rearrangement shown previously. The more direct intermediacy of 78 was demonstrated111 by feeding (R,S)-[2-13C, 2H]-78 to D.stramonium root cultures. Intact incorporation of the 13C–2H bond was found (17% retention), indicating 77 not to have intervened. However, significant 59 only containing the 13C label was also found. This was subsequently shown to be due to the incorporation of the fed (S)-[2-13C, 2H]-78 via [2-13C]-77 as when (R)-[2-13C, 2H]-78 and (S)-[2-13C, 2H]-78 were separately exhibited112 to the cultures, intact 13C–2H in 59 was only found with the (R)- isomer, demonstrating this to be the true substrate.Rearrangement has been shown to be subsequent to esteri- fication and not prior, as previously proposed.84 Indirect evidence for this was obtained109 by feeding root cultures of D. stramonium with (R,S)-[1,3-13C2]-78. The high incorporation found was not diluted out with unlabelled 76b, suggesting the free acid not to be an intermediate. Furthermore, [14C]-76b proved113 to be a very poor precursor for 59 compared with 1b in root cultures of Duboisia leichhardtii.Thus, rearrangement must occur either at the level of an activated form of 78 (e.g. a Co-A thioester) or after esterification with 58. That the latter is the case was unequivocally demonstrated114 by feeding (R,S)- phenyl-[1,3-13C2]lactoyl[methyl-2H3]-58 (littorine) to root cultures of D. stramonium. Littorine 79 is a natural alkaloid, co-occurring with 59 in many species. This enriched putative precursor was eYciently (4.5–6%) incorporated into quintuplylabelled 59, confirming the direct intermediacy of 79 in the biosynthesis of 59 and 60.While limited hydrolysis occurred, incorporation from the liberated 58 and 78 could not account for more than 0.2% enrichment and, furthermore, unenriched 58 or 78 did not diminish incorporation. That the rearrangement was intramolecular was shown by the 13C–13C coupling in the derived 59. The mechanism for the formation of 79, however, is yet to be described.To date little information on the activating enzymes or the enzyme that esterifies 58 with activated 78 has been published. A few early reports can be dismissed (see ref. 78). A very low activity with 58 and phenylacetoyl-CoA can be detected in some root cultures,115 although no activity could be found with 78-CoA. Enzyme activities responsible for the formation of the aliphatic esters of both 58 and 61 have, however, been found in D.stramonium and many other species115 and the acyl-CoA:pseudotropine acyltransferase purified to homogeneity.116 This enzyme shows a restricted activity for the alcohol receptor, notably being inactive with 58. In contrast, it accepts a wide range of alkyl-CoA thioesters, indicating that the range of minor alkaloids found may reflect the in vivo availability of substrates more than the selectivity of the enzyme. The equivalent enzyme that esterifies 58 is much less stable115 and has not been purified.Curiously, extracts of several species contain high levels of an enzyme that esterifies 61 with phenylacetoyl-CoA.115 The product, phenylacetoylpseudotropine is not found naturally in these cultures. This is an important area of tropane alkaloid metabolism that S O S HO H 73 74 O– O– H O O O O OR OH OH 1 77 78 76a 76b R = H Scheme 9 O O OH O O OH N N CD3 CD3 • • • • D. stramonium Scheme 10 560 Natural Product Reports, 1998requires further investigation: root cultures provide excellent material for such studies.In contrast, the mechanism of rearangement of 79 to 59 in root cultures is starting to be unravelled. The conversion of 78 to 76 necessitates the breaking of two bonds and the formation of two new bonds. Feeding experiments with specifically labelled 78 have shown that both of the bonds are broken/ formed with inversion of configuration. The stereochemistry of the C-2 migration terminus was investigated117 by feeding [2-3H]-78 to D.stramonium root cultures. The resultant 59 was shown to have 3H at the 3-pro-R position of 76 by enzymatic analysis of released chiral acetic acid. The stereochemistry at the C-3 migration terminus was probed in feeding experiments with (2R,3R)-[3-2H]-78 and (2R,3S)-[3-2H]-78. Only from 3-pro-S (2R)-78 was 2H retained, but as this 2H–C bond is configurationally inverted in the resultant 59, this demonstrated that the bond breaking/forming at the C-2 proceeds with an inversion of configuration.In the converse experiment the 3-pro-R hydrogen was lost. Such rearrangements are relatively rare in biological systems and the mechanism oVers a challenge to the biological chemist. Similar reactions,106 such as the rearrangement of (R)- methylmalonyl-CoA to succinyl-CoA have been shown to involve vitamin B12 but the absence of this cofactor in plants makes its role improbable. As the experiments with labelled 78 have demonstrated that there is not a vicinal interchange, this type of mechanism can be disregarded.An attractive working hypothesis is that the rearrangement involves a P450 type system, with oxygen rebound catalysed by the active Fe centre, as apparently is the case in isoflavanoid biosynthesis.119 Two lines of evidence obtained with root cultures of D. stramonium support this proposal. Firstly, inhibitors of P450 activity, such as chlotrimazole appears to inhibit the conversion of littorine to hyoscyamine.120 Secondly, some exchange of the oxygen at C-2 of 78 can be demonstrated.After cultures were fed (R,S)-[2-2H,18O]-78,121 both 79 and 59 had enriched M+3 ions showing that the 18O and 2H atoms were both incorporated, but to diVerent extents in each of the metabolites. The finding that retention of the 18O label is intermediate (71–75%), neither 50% (full exchange via a diol intermediate) nor 100% (no oxygen rebound mechanism) is intriguing and leaves open a number of possibilities.If only the (R)-isomer were incorporated, the data would imply a 50% loss of the 18O. As, however, it is known that the (S)-isomer is also incorporated, albeit with loss of the 2H, other explanations are required. Most probably there is a two step concerted reaction initiated by the abstraction of an hydrogen. The radical or carbocation is proposed106,121 to be quenched by the active iron centre either in a partially stereospecific oxygen rebound wherein oxygen exchange occurs in the diol or disproportionation, directly yielding an aldehyde.Partial exchange at this level is possible, prior to reduction to the hydroxymethyl of 59. Crucially, however, it still cannot be stated whether this is a radicular (one electron) or carbocationic (two elctron) process. Indeed, it cannot be ruled out that the enzyme is a Fe2+/ ascorbate dioxygenase rather than having a P450 iron centre. In many species, root cultures accumulate 60 in addition to 59.The mechanism of the introduction of the 6‚,7‚-epoxide was controversial78 prior to the isolation of the enzyme activity from root cultures of H. niger.122 The epoxide can only be inserted late in the biosynthesis as the pure enzyme is inactive with 58. However, the intermediacy of 6,7- dehydroxyhyoscyamine 80 had been implicated in earlier feeding experiments with plants. This route was ruled out, however, by showing that the same enzyme catalyses two sequential steps, the formation of 6‚-hydroxylhyoscyamine 81 followed by epoxide formation.123 The essential intermediacy of 81 was shown124 by the retention of label from 6‚-[6-18O]-81 in 60 in H.niger root cultures. The stereochemistry of elimination was probed with [7‚-2H]-81 which, when fed to shoot cultures of D. myoporoides, demonstrated the specific elimination of the 7‚-proton.125 In a more detailed study126 using 2H NMR, it was shown that (S)-[2-2H]-20 and (R)-[2-2H]-20 labelled the H-7 and H-6 protons of 60 respectively.These patterns confirmed that 59 is converted to 81 with retention of configuration and that it is exclusively the 7‚-proton that is eliminated during epoxidation. That the 6‚,7‚-epoxidase is responsible for both activities was uequivocally demonstrated by cloning the gene and expressing this in Nicotiana tabacum.127 Transgenic plants eVectively converted added 59 to 60. The apparent intermediacy of 80 was explained when it was shown that the pure enzyme accepts this non-natural alkaloid as substrate.122 OR H* O RO O OH HR HS HS HO H* Inversion Inversion HR Scheme 11 O– O O D O O OH D HO18 Me N HO18 D Me N O D.stramonium D. stramonium 79 59 25–29% loss oxygen-18 71–75% retention Scheme 12 Scheme 13 Robins: The application of root cultures to problems of biological chemistry 561Analogues of 65 (8-azabicyclo[3.2.1]octan-3-one), 58 and 61 altered in the N-substituent and a series of higher homologues based on the 9-azabicyclo[3.3.1]octane ring were fed128 to transformed root cultures of a Brugmansia (ex.Datura) candida#aurea hybrid129 which is capable of forming a large number of tropane alkaloids. The resultant alkaloid profile showed that, for N-ethyl- 82, N-fluoroethyl- 84 and N-isopropyl-83 nortropinone, homologous series of non-natural alkaloids were made. These included analogues of the tropoyl esters in which the methyl group has been replaced by the ethyl, fluoroethyl or isopropyl group respectively.In some cases, the aberrant products constituted the major alkaloids present. In all three cases, the analogue of 79 accumulated to a high level, indicating that the enzymes involved in esterification were able freely to cope with this substitution. The rearrangement, however, was found to have much more rigid substrate requirements, only 82 being converted to the analogue of 59, at about 10% the eYciency of forming the analogue of 79.This product was also metabolised at a low level to the analogue of 60. Expansion of the [3.2.1] to a [3.3.1] ring prevented ester formation with aromatic acids, indicating the enzymes to have rigid structural requirements in this part of the molecule. Analogues of early precursors, notably 7 or N-substituted-7, were hardly incorporated into novel tropane alkaloids. These compounds were, however, metabolised, to a variable extent, to form analogues of 62 and 72. Clearly, the root cultures can eVectively metabolise analogues of intermediates through a series of reactions to form novel tropane alkaloids and the extent of metabolism observed provides insights into the structure–function properties of the enzymes involved.These analogues were also tested130 in vitro as substrates for the reductases TRI and TRII and the aliphatic-CoA acyl transferase activities of B. candida#aurea and D. stramonium. It was shown that they are substrates for the 58- and 61-forming TR activities and that the reduced forms are substrates for the tropine: or pseudotropine:acyl-coenzyme A transferases.The observed in vitro metabolism correlated well with the range of products isolated when the compounds were fed to transformed root cultures of B. candida#aurea. Tropane alkaloid metabolism in root cultures of D. stramonium has also been studied by 15N NMR. Roots grown on K15NO3 accumulated label in a range of primary and secondary products, amongst which 65, 58 and 59 could be distinguished.131 Hydroxycinnamoylputrescines, compounds that may act as a reservoir of 20 for tropane alkaloid biosynthesis, 132 were also strongly labelled, making it feasible to conduct dynamic studies on biological flux.For example, it was possible to follow133 the reduction of 15N-65 to 15N-58 by D. stramonium root cultures. Within 2 to 4 h, labelled product could be detected, confirming that these enzymes are very active in vivo, as previously could only be deduced by their in vitro activities.101 Note that, as shown with the acyl- CoA:pseudotropine acyl transferase,116 in vitro and in vivo activities do not necessarily correlate.It was further demonstrated133 that 15N-65 was reduced to 15N-58 in root-derived suspension cultures that are incompetent in de novo alkaloid biosynthesis,134 showing that the loss of overall biosynthesis need not imply the loss of the whole pathway. Degradation and turnover of alkaloids is important when trying to assess overall flux or maximize the accumulation of desirable products.Inducing de-diVerentiation in the roots by phytohormone treatment134 causes a rapid degradation of accumulated alkaloid and a loss of biosynthetic capacity. The enzyme atropine esterase, which hydrolyses 59 to form 76a and 58 has been shown present in roots of a number of species,78 although this degradation was apparently absent in root cultures of D. myoporoides derived from these plants.135 Some evidence that 76 might be recycled into 59 has recently been presented,136 suggesting that either there is a minor esterifying activity using this acidic moiety or that 76a can be recycled to 78.[1-14C]-76a is, however, very poorly incorporated (1% of the level of 1b) by root cultures of D. leichhardtii.113 4.3.5 Indolizidine alkaloids The polyhydroxylated indolizidine alkaloids swainsonine 85 and castanospermine 86 are of great interest as glycosidase inhibitors, exhibiting antiviral activity against a range of viruses including HIV.137 Little is known, however, of the details of the biosynthesis in plants of this group of alkaloids, which also occur in the black fungus Rhizoctinia leguminicola.138 Root cultures of Swainsonia galegifolia, both transformed and untransformed, produce low levels of 85.139 In transformed roots the production (80 Ïg per gDW) was enhanced140 by the addition of malonic acid 87 (180 Ïg per gDW) or pipecolic acid 88 (220 Ïg per gDW), indicating that the biosynthesis in higher plants may be the same as that proposed in R.leguminicola.138 Much remains to be established for this pathway and these cultures, despite the low yields of alkaloid, oVer potentially useful N Me O C O C H OH N Me O C O C H OH N Me O C O C H OH N Me O C O C H OH H18O 2H 18O 59 81 60 80 Scheme 14 N R N R O 65 R = Me 82 R = Et 83 R = CH(Me)2 84 R = CH2CH2F HO OH N NH O O OH O OH H OH N OH H OH OH HO HO 87 85 88 86 Scheme 15 562 Natural Product Reports, 1998material for labelling experiments.Attempts to generate root cultures of Castanospermum for production of 86 have so far failed. 4.3.6 Benzylisoquinoline alkaloids The biosynthetic routes from tyrosine 89 to alkaloids of the benzylisoquinoline class were largely elucidated from studies in suspension culture cells.4,141 The role of tyramine 90 in their biosynthesis, however, remained unclear. Root cultures of Stephania cepharantha have proved142 to be a good source of bisbenzylisoquinoline alkaloids, including aromoline 91.When [U-14C]-89 or [7-14C]-90 were fed143 to these cultures, the former was much more eVectively incorporated into 91, in contrast to previous data. However, it was found that fed 89 accumulated as 90 that was then steadily incorporated into 91. Tracer experiments with [3-13C]-89 showed the incorporation of four molecules of 89 per molecule of 91 and the similar enrichments of the C-4 and C-· in the (R)- and (S)-moieties of 91 confirmed that the bis-alkaloid is formed from two molecules of (S)-coclaurine 92 derived from the same biosynthetic origin.A similar pattern of incorporation into dauricine 93 was shown from [3-13C]-89 in root cultures of Menispermum dauricum.144 However, only the isoquinoline portions of 93 were labelled from [2-13C]-90, showing that the incorporation of 89 into the benzyl portion does not proceed via 90 but probably via 4-hydroxyphenylpyruvic acid 94.M. dauricum root cultures also accumulated the unusual chlorinated alkaloid acutumine 95. Although this structure was proposed to be a benzylisoquinoline-type alkaloid over 30 years ago, this was only recently144 shown by the incorporation of [U-14C]-89 in the root cultures. The role of P450 type enzymes in alkaloid biosynthesis in both these species was investigated.145 While none of the desired monomeric precursors were found to accumulate in roots of M. dauricum treated with inhibitors, a number of compounds, including 90, did accumulate. 4.4 Terpenoids and steroids A broad range of terpenes and steroids have been reported from root cultures (Table 1).The biosynthesis of these products is often diYcult to study due to, for example, their localisation in gland tissue. Terpenes, notably sesquiterpenes, are often produced in high yield in response to stimulae received from the environment. Such systems are excellent for studying biosynthesis, as large amounts of product can accumulate in a short period of time, making it facile to achieve high incorporations of labelled precursors.Leaf monoterpenes, such as occur in Mentha spp. can be examined in shooty cultures.6 4.4.1 Sesquiterpenes Despite the identification of a range of sesquiterpenes in root cultures (Table 1) and their inducibility, root cultures have been little exploited to study these compounds. Capsidiol 96 was elicited146 in roots of Capsicum annuum by added cellulase, the product being primarily secreted into the medium.The key enzyme sesquiterpene cyclase showed induction, maximal activity being 6 to 8 h after elicitation. Similarly, solavetivone 97 is synthesized de novo and released from transformed root cultures of Hyoscyamus muticus treated with fungal elicitor.147 The amount may be further stimulated by adding neutral adsorbants such as XAD-7 to the medium,148 which aid recovery and diminish feed-back down-regulation of sesquiterpene biosynthesis. 4.4.2 Diterpenes Diterpene glucosides such as stevioside 98 are of interest for their intensely sweet taste.Neither root nor shoot cultures149 of Stevia rebaudiana, derived from callus, were able to accumulate 98. Rooted shoots, however, tasted sweet and accumulated 98, indicating that biosynthesis required intact plants, presumably due to an organ specific distribution of diVerent parts of the pathway.This was confirmed by showing that label from [2-14C]-67 was incorporated into steviol 99 only by rooted shoots. Both shoots and roots, however, could triglycosylate added [methyl-3H(N)]-99. As free 99 is not found in the cultures it appears that the glycosylation is important for storage and stability, as indicated by the inhibitory eVect of added 99 on [2-14C]-67 incorporation. These experiments leave open the intriguing question as to the role of roots in the biosynthesis of 98.In intact plants, 98 accumulates exclusively in the leaves, suggesting that either an essential precursor is derived from the roots or that a root-derived stimulatory factor is required. The former hypothesis is more probable but requires confirmation. NMe COOH NH2 COOH NH NMe MeN OMe OH O HO O MeO NH2 HO HO HO O MeO HO HO O OMe OMe O MeO OH Cl NMe MeN OMe OMe O HO MeO MeO 89 90 94 92 95 91 93 4¢ 4 3 S a¢ R a 4 S S R 4¢ 4 a¢ a Scheme 16 O CH2 OH H H HO CO2R1 OR2 96 97 98 R1 = Glc; R2 = Sophorose 99 R1 = R2 = H Robins: The application of root cultures to problems of biological chemistry 5634.4.3 Triterpenes and steroids A range of triterpenes occur in root cultures, both as the free products and as the glycones (saponins). While the biosynthesis of the terpenoid skeleton from mevalonic acid 100 is quite well described,150 there are numerous modifications yet to be defined. In addition, the recent description of the alternative pathway via 1-deoxyxylulose 5-phosphate 101150 requires that the origin of triterpenes in many species be redefined.In an interesting study, the incorporation of [2-14C]-100 into triterpenes by diVerent types of cultures of Taraxacum oYcinale was examined.151 Callus cultures labelled ·-amyrin 102 and ‚-amyrin 103, with little incorporation into taraxasterol 104 and lupeol 105. Root and shoot cultures, in contrast accumulated label primarily in 104, 105 and 106. Cycloartane-type steroidal saponins of the astragaloside (e.g.astragaloside I 107) and aculeatiside (e.g. aculeatiside A 108) types accumulate in root and transformed root cultures of Astralagus spp.152,153 and Solanum aculeatissium154 respectively, reaching levels of 8 to 10 mg l"1. Biosynthesis of aculeatisides required illumination and green cells, indicating a plastidic component in the biosynthetic pathway. Unfortunately, transformed roots of Glycyrrhiza uralensis did not produce the pharmaceutically important triterpene glycyrrhizin 109 in more than trace quantities.155 Curiously, the accumulation of ginsenosides 110 by Panax ginseng transformed roots was inhibited by added 67 but accumulation was enhanced with a yeast-derived elicitor.156 Steroidal alkaloids of Solanum spp.are also generally considered to be synthesised in leaf tissue. Solasodine 111 will, however, accumulate in root cultures, but, again, only at low levels.157 In contrast, the phytoecdysteroids are readily accumulated in both normal158 and transformed159 root cultures of Ajuga reptans and normal root cultures of Serratula tinctoria.160 In A.reptans, shoot cultures were shown unable to accumulate phytoecdysteroids.158 The cultures of S. tinctoria160 accumulated high levels (0.1–0.2%DW) of 20-hydroxyecdysone 112 with lower amounts of the 3-acetate. This ratio is the inverse of that found in whole roots. The accumulation of 112 in A. reptans could be enhanced by growing the roots in phosphatedepleted medium.161 These roots showed an eYcient incorporation of [2-14C]-100 and [1·,2·(n)-3H]-113 into 112.A. reptans non-transformed cultures, in contrast, accumulated a wider range of products, the composition of the mixture depending on the medium used.158 In transformed A. reptans,159 the major product was 112, with lesser amounts of 29-norcyasterone 114 and cyasterone 115. While 112 was labelled from both [2-13C]-67 and [26,27-13C2]-113, 114 and 115 were only labelled from [2-13C]-67.The high level of OP Me OP CO2 – O OH HO OH 100 101 HO H H HO H HO H H H H H H H H 105 106 104 103 102 Ac2GlcO O H CO2H R2 R1O H O OH OH RO OGlc OH O O OH HO2C OH O O OH HO OH CO2H 107 108 Aculeatiside A R = b-chacotriose 110 R1 = sugar; R2 = various C-8 groups 109 H O O O O OH OH HO OH HO H O N H Me 111 564 Natural Product Reports, 1998labelling from [2-13C]-67 in all three products conclusively confirmed that phytoecdysterols are formed from the classical steroid backbone.The route for the incorporation of 113 into 112 has been further elucidated.162–164 The protons at 3·, 4· and 4‚ of 113 were all shown to be retained and the metabolite 3‚-hydroxy- 5‚-cholest-7-en-6-one 116 was shown to be incorporated, confirming this to be an intermediate. The formation of 116 was shown, by feeding [6-2H]-113, to involve the migration of the H-6 of 113 to the C-5 during bioconversion.162 A feed with a 1:1 mixture of [4‚-2H]-113 and [6-2H]-113 confirmed the C-6 to C-5 migration but also introduced the unexpected result that 1/3 of the 5‚-hydrogen arose from another source.While the observed 1,2 hydrogen shift is compatible with a probable 5·,6·-epoxide intermediate, the partial loss of the proton could indicate an alternative as yet unelucidated mechanism. Since several mechanisms for the cis-A/B ring synthesis have already been described, this remains an intriguing question.The stereochemistry of the insertion of the C-25-OH of 112 was neatly followed using [26-13C]-113 and [27-13C]-113 as precursors.164 Following hydroxylation, the C-25 becomes a chiral centre, allowing the stereochemistry to be deduced. 13C NMR analysis of the isolated 112 indicated clearly that the pro-R methyl of 113 becomes incorporated into both pro-S and pro-R methyl groups in a 3:1 ratio. The pro-S of 113 showed the opposite result. Thus, hydroxylation proceeds with both retention and inversion in a 3:1 ratio.Hydroxylation with inversion at an isopropyl cryptic stereocentre is apparently unprecedented. Following the feeding159 of [2-13C ]-67, 114 and 115 were not enriched at the C-28 and C-29 positions, confirming the non-acetate origin of these carbons. The lack of incorporation of label from 113 into 114 and 115 indicated that a 24-alkyl sterol, rather than 113, is the more probable precursor.159 The intermediacy of 24-methylenecholesterol 117 in clerosterol 118 biosynthesis was confirmed165 by demonstrating the predicted incorporation into the C-28 of label from [28-13C]ergosta-5,24(28)-dien-3‚-ol 117a.In a further series of experiments the incorporation of [26,27-13C2]desmosterol 119a into the ƒ25-sterols 118 and codisterol 120 was shown. By using [26-13C]-119 or [27-13C]-119, it was possible to show the stereospecific incorporations into the C-26 (vinyl methyl) and C-27 (exo-methylene) respectively of 118 and 120, in agreement with previous reports for sterol biosynthesis in plants.Label from [24-2H]-119 was retained at the C-24 of 118 and 120, consistent with a concerted dehydrogenation to introduce the ƒ25-bond and suggesting that a ƒ24(25)-sterol is not involved in this transformation. The possibility remains, however, of a ƒ24(28)-sterol intermediate, with migration of the proton to and back from the C-25, as previously proposed. The proven incorporation of 117 into 118 strongly supports a mechanism involving a ƒ24(28)-sterol intermediate even though 117 was not identified as a product in the root cultures. 4.5 Miscellaneous nitrogen compounds Betacyanins (e.g. betanidine 121) and betaxanthins (e.g. vulgaxanthin I 122) from Beta vulgaris were the first secondary products shown to be made in transformed root cultures.13 The betaxanthins were seen to accumulate earlier in the maturing root tissue, showing as a yellow zone between the colourless meristem and the red roots containing both pigments.The profile of betaxanthins accumulated by B. vulgaris var. lutea was strongly influenced166 by feeding the L-amino HO H HO H HO H R O O OH O O O O OH OH HO HO 113 116 112 R = 114 R = 115 R = 3 Scheme 17 R1 R S SR2 S S HO S = 117 R = CH2 117a R = 13CH2 119 R1 = R2 = Me 119a R1 = R2 = 13Me 118 120 D D CH2 D D CH2 Me S S H S S H H S D Me–S+–Ad 117 118 120 Me–S+–Ad • • • • • + a b a b pro-R Scheme 18 Robins: The application of root cultures to problems of biological chemistry 565acids Val, Leu, Asn, Glu, Gln, Phe, Pro, Hyp or His, each of which stimulated the accumulation of the appropriate betalamic acid 123 derivative.Interestingly, feeding D-Phe stimulated the synthesis of a novel non-natural betalamic acid derivatized with D-Phe. The formation of this compound was as eYcient as with the L-isomer, implying that the in vivo condensation of 123 with an amino acid is probably not an enzymatic process. 4.6 Miscellaneous sulfur compounds Thiophenes, found in the roots of many members of the Asteraceae, are characterized by the presence of one or more heterocyclic sulfur-containing rings. Root cultures of several genera are easily established and, as they appear to accumulate higher amounts of these metabolites than the roots of whole plants, oVer excellent experimental material to study the metabolism of these nematocidal metabolites. Highest accumulation is in cultures which show limited root branching.167 Sulfur is derived from SO4 2" but appears to be inserted in a reduced form, possibly from cysteine.While the biosynthetic relationship of the thiophenes isolated from the root cultures can readily be proposed, direct experimental evidence is only recently becoming available. An important intermediate appeares to be 2-(but-3-en-1- ynyl)-5-(penta-1,3-diynyl)thiophene 124. Thiophene accumulation is diminished by SO4 2" starvation, as shown168 by the decrease of formation of 5-(but-3-en-1-ynyl)-2,2*-bithienyl, 125 from [35S]-124, indicating an as yet unelucidated enzymic process for heterocycle formation.Hydroxylation of 125 to 126 and acetylation of 126 to 127 were unaVected by SO4 2" starvation, suggesting these to be subsequent steps. As 126 and 126a were labelled from 125, it is apparent that the formation of the biheterocyclic occurs prior to the hydroxylation of the terminal methylene group.169 The formation of these more soluble derivatives of 125 was stimulated by added fungal filtrate,170 indicating an environmental control of metabolism.The proposed intermediacy of an epoxide in this hydroxylation remains, however, to be proven. It has also been shown that 124 is the precursor of the methyl-125 derivatives, minor metabolites in Tagetes.171 Neither 5*-methyl-5-(but-3-en-1- ynyl)-2,2*-bithienyl 128 nor 5*-(hydroxymethyl)-5-(but-3-en- 1-ynyl)-2,2*-bithienyl 129 were labelled by feeding 5*- (acetoxymethyl)-5-(but-3-en-1-ynyl)-2,2*-bithienyl 130 labelled with [35S], indicating 130 to be an end product rather than an intermediate of biosynthesis.Significantly, no labelling occurred in 125 or derivatives, confirming that the demethylation probably occurs at the level of 124. A mutant, apparently deficient in the demethylating enzyme, accumulates high levels of methyl derivatives,171 confirming this conclusion. Roots of Allium cepa172 and Allium sativum173 accumulate (+)-S-alk(en)yl-L-cysteine sulfoxides, which act as defense compounds against microbial pathogens but which make them agreeable to humans, being the precursors of the flavour compounds.Rooty cultures of A. sativum accumulate173 S-alkyl-L-cysteine sulfoxide, alliin, 131. S-Alkyl-L-[35S]cysteine 132a and S-alkyl-L-[1,2,3- 14C3]cysteine 132b were both oxidised to (+)-131, none of the (") isomer being identified in extracts, indicating this reaction to involve a specific oxidase.It remains to clarify the putative role of „-glutamyl precursors in (+)-S-alk(en)yl sulfoxide biosynthesis. The profile of flavour precursors accumulated by root cultures of A. cepa can be strongly influenced172 by the addition of thiol precursors, a range of novel nonnatural products being made. 5 Future prospects A particularly interesting aspect of transformed roots for the ‘chemical genetic engineer’ is the facility to introduce genes during the transformation process.This can be done to increase the amount of a known product15,36 or to create ‘novel’ products as a result of exposing the naturally accumulating chemicals to enzyme activities not normally present in that species. A number of examples of the way in which genetic engineering can be valuable to the natural products chemist are elegantly described by Scott.174 Chemical profiles can be altered, as in the manipulation of fatty acid composition.175 Novel products can be made from a defined pathway by the insertion of a specific gene.176 The introduced genes need not be of plant origin.Pathways or parts of pathways (e.g. an interesting biotransformation) might be moved from a ‘diYcult’ species to an ‘easy’ one. The regulation of secondary pathways might be probed in a similar manner to primary metabolism.177 Furthermore, it is possible either to exploit the cultures in fermenters or to regenerate plants to cultivate by conventional agriculture.Plant cultures can also potentially be used to study the activity and mechanism of NH COOH O H N N H COO– NH2 O HO HO COO– HOOC + + L-DOPA Cyclodopa Amino acid e.g. L-glutamine 121 122 123 Scheme 19 S S S S S S S HO R S S AcO S S S S OAc OH 124 125 127 130 126 R = H 126a R = OH 129 128 Scheme 20 S CO2H NH2 S CO2H NH2 O 131 132a S = 35S 132b 1,2,3-[14C] 566 Natural Product Reports, 1998non-plant-derived genes within the culture environment. 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ISSN:0265-0568    

DOI:10.1039/a815549y

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Indolizidine and quinolizidine alkaloids Joseph P. Michael Centre for Molecular Design, Department of Chemistry, University of the Witwatersrand, Wits 2050, South Africa Covering: July 1996 to June 1997 Previous review: 1997, 14, 619 1 General reviews 2 Slaframine 3 Hydroxylated indolizidine alkaloids 3.1 Lentiginosine and related compounds 3.2 Swainsonine and related compounds 3.3 Castanospermine and related compounds 4 Alkaloids from ants 4.1 3,5-Dialkylindolizidine alkaloids 4.2 Polycyclic alkaloids 5 Alkaloids from amphibians 5.1 Occurrence 5.2 Synthesis of amphibian alkylindolizidine alkaloids 5.3 Synthesis of alkaloids of the pumiliotoxin class 6 Elaeocarpus alkaloids 7 Ipalbidine 8 Phenanthroindolizidine and phenanthroquinolizidine alkaloids and seco analogues 9 Nuphar alkaloids 10 Alkaloids of the lupinine–cytisine–sparteine–matrine– Ormosia group 10.1 Occurrence, analysis and biological studies 10.2 Chemotaxonomic studies 10.3 Structural and spectroscopic studies 10.4 Synthesis and other chemical studies 10.5 Enantioselective transformations mediated by (")- sparteine 11 Alkaloids from marine sources 12 Alkaloids from coccinellid beetles 13 References 1 General reviews Bioactive indolizidine alkaloids isolated from fungal and plant sources have been surveyed in an important new review that constitutes the opening chapter in a larger work on natural toxins.1 Included in the chapter are brief accounts of several classes of indolizidine alkaloids customarily included in the present series of reviews (slaframine and other fungal indolizidine metabolites, polyhydroxylated indolizidines such as swainsonine and castanospermine, indolizidines from the genera Ipomoea, Elaeocarpus and Astragalus, phenanthroindolizidines and lupin alkaloids), as well as metabolites in which the indolizidine unit is embedded in a more complex polycyclic system (steroidal alkaloids, indoloindolizidines, Glochidion alkaloids).Another important review dealing with polyhydroxylated alkaloids that inhibit glycosidases describes not only the isolation and distribution of the well-known hydroxyindolizidines, but also summarises important results relating to their biosynthesis, toxicity and ecological signifi- cance, potential therapeutic value and chemical synthesis.2 2 Slaframine The weak step in Gallagher’s 1995 synthesis of slaframine 1 was the problematic aldol condensation 2]3, which was eventually achieved in 31% yield with piperidine as base.3 The yield of this step has now been improved to 68% by using 2,2,6,6-tetramethylpiperidine as base, following which the mixture was adsorbed on to, and eluted from, silica gel.4 It is possible that cyclisation actually occurs by Mannich reaction, with the acidic adsorbent promoting the formation of an iminium ion intermediate between the secondary amine and the aldehyde. 3 Hydroxylated indolizidine alkaloids 3.1 Lentiginosine and related compounds Brandi, Goti and their co-workers have previously reported syntheses of (+)-lentiginosine 45 and the hydroxylated lentiginosine analogue 5.6 These routes suVered from poor selectivities in the dipolar cycloaddition between methylenecyclopropane and the enantiomerically pure nitrone 6, and in the rearrangement of the resulting isoxazolidines (cf.ref. 7a,b). They have now improved the syntheses by choosing diVerent substrates for the initial cycloaddition.8 Reaction between but-3-en-1-ol and the tert-butyl-protected nitrone 7, which is derived from L-tartaric acid, quantitatively gave an easily separated mixture of three diastereoisomers in the ratio 10:2:1 (Scheme 1).The dominant isomer 8 was isolated in 79% yield. Its mesylate readily rearranged via salt 9 to give hydroxyindolizidine 10, which aVorded the lentiginosine analogue (+)-(1S,2S,7R,8aS)-trihydroxyindolizidine 5 after removal of the protecting groups. The total yield from L-tartaric acid over eight steps was 37%.The synthesis of (+)-lentiginosine 4 itself was completed by deoxygenation of 10 by way of the thiocarbonylimidazolide 11. The new route to lentiginosine required ten steps from L-tartaric acid and gave an overall yield of 25%; by contrast, the previous synthesis5 took nine steps, and the overall yield was 2.4%. (+)-Lentiginosine is a potent inhibitor of amyloglucosidases, especially glucoamylase, an enzyme that is widely used in industry for the conversion of starch into glucose.Starting from published X-ray crystallographic data for the complex formed between a simpler glycosidase inhibitor, deoxynojirimycin, and glucoamylase II from Aspergillus awamori var. X100, the Brandi team used molecular dynamics methods to simulate the interaction between lentiginosine and the active site of glycoamylase II. The alkaloid’s ability to inhibit the enzyme appears to be a consequence of strong hydrogen bonding between the trans-disposed OH groups and the enzyme’s key Arg 54 and Asp 55 residues.9 3.2 Swainsonine and related compounds The (&)-pipecolic acid derivative 1210 was the starting material in the simple synthesis of racemic swainsonine rac-13 shown in Scheme 2.11 cis-Dihydroxylation of the unsaturated lactam 14 took place preferentially on the concave face (2:1), CHO N O CbzNH O N O CbzNH O N H OAc H2N 3 2 1 (–)-Slaframine Michael: Indolizidine and quinolizidine alkaloids 571yielding acetonide 15 after chromatographic separation and protection. The central reaction in this route was the removal of the bridgehead ester group, which was achieved in 75% yield by heating the isolable acid chloride 16 in a mixture of xylene and 1,2-dichloroethane.Hydroboration and oxidation of the resulting enamide 17 completed the synthesis of (&)-swainsonine. The previously communicated synthesis of (")-swainsonine 13 by Hunt and Roush12 (cf. ref. 7c) has now been published as a full paper that includes further examples of their tartratemodified (menthofuryldimethylsilyl)allylboronate reagent for making anti-3,4-dihydroxylated but-1-enes from aldehydes.13 Another short synthesis of (")-swainsonine 13, by Pearson and Hembre,14 commenced with the three-step conversion of the protected D-erythronolactone derivative 18 into a diastereoisomeric mixture (97:3) of allylic alcohols 19 (Scheme 3).Separation of the epimers was possible but unnecessary, as both alcohols yielded the same product 20 after Johnson orthoester Claisen rearrangement.The major product of the subsequent Sharpless asymmetric dihydroxylation in the presence of AD-Mix ‚> was lactone 21, a product that has all of the carbon atoms and three of the four stereogenic centres of the target alkaloid in place. The simple transformations shown in the Scheme completed a straightforward synthesis of (")- swainsonine 13 via known intermediates 22 and 23.This route is notable in yielding a comparatively large amount (4.5 g) of the alkaloid in eleven steps and an overall yield of 20%. Will the structural features that cause (")-swainsonine to inhibit D-mannosidases permit its enantiomer to inhibit enzymes whose substrates have absolute configurations mirroring that of D-mannopyranose, 24, for example, L-rhamnopyranose, 25? Fleet and co-workers, hypothesising that L-(+)-swainsonine ent-13 might prove to be a good N O HO H OBut OBut +N OR OR –O N OBut OBut H HO N+ OBut OBut H O N OH OH H HO N OBut OBut H O S N N N OBut OBut H N OH OH H MsO– 93% iv v 99% iv 93% 11 5 86% from 8 iii 10 9 6 R = TBDPS 7 R = Bu t 79% + isomers i 8 ii vi 68% 4 (+)-Lentiginosine Scheme 1 Reagents: i, but-3-en-1-ol, 60 )C, 2 d; ii, MsCl, Et3 N, CH2Cl2; iii, H2 (50 psi), 10% Pd/C, MeOH; iv, TFA; v, Im2CS, THF, reflux; vi, Bu3SnH, toluene, reflux N O ButO2C N O ButO2C N O O O ButO2C N O O O ClOC N O O O N H HO OH OH 66% ix, x 17 96% vi, vii 16 rel-15 70% i–iii 14 12 75% viii 70% + isomer (2:1) iv, v rac-13 (±)-Swainsonine Scheme 2 Reagents: i, LDA, THF, "78 )C; ii, PhSeCl; iii, H2O2, AcOH; iv, OsO4 (cat.), NMO, acetone, H2O, ButOH; v, Me2C(OMe)2, PPTS, CH2Cl2, then chromatography; vi, TFA, CH2Cl2, 0 )C; vii, (COCl)2; viii, ClCH2CH2Cl–xylene (1:2), reflux; ix, B2H6, THF, then H2O2, NaOH; x, 6 M HCl, THF, rt, then ion exchange with Dowex 1X8 (OH") O O O O O O OTBDMS HO O O OTBDMS O OMe HO O O O OTBDMS O MsO O O O N3 O N O O H HO O N O O H HO N H HO OH OH vi–viii 57% 13 (–)-Swainsonine 23 22 96% xii 94% xi 75% ix, x 21 20 19 18 70% from 19 + isomer (9%) v iv 71% + isomer (2%) i–iii Scheme 3 Reagents: i, DIBAL, toluene–CH2Cl2, "78 )C, then MeOH; ii, H2C=CHMgBr, THF, "78 )C to 0 )C; iii, TBDMSCl, THF–DMF (3:1), imidazole, 0 )C; iv, MeC(OMe)3, C2H5CO2H, toluene, reflux; v, AD-Mix ‚, MeSO2NH2, H2O, ButOH, 0 )C to rt, then Na2SO3, then chromatography; vi, Bu4NF, THF, 0 )C; vii, MsCl, DMAP, pyridine, 0 )C; viii, NaN3, DMSO, 80 )C; ix, H2 (1 atm), Pd(OH)2/C, MeOH, rt; x, NaOMe, MeOH, reflux; xi, BH3·Me2S, THF, 0 )C to rt, then EtOH, reflux; xii, 6 M HCl, THF, rt, then ion exchange with Dowex 1X8 (OH") 572 Natural Product Reports, 1998inhibitor of L-rhamnosidase (naringinase), used the cheap heptonolactone 26 as the starting material in a new synthesis of ent-13 and related hydroxyindolizidines.15 Conversion into the octonolactone 2716 followed by a further eight steps17 yielded the protected pentahydroxyindolizidine 28, a product that can also be viewed as a hydroxylated castanospermine derivative (see Section 3.3).A sequence of standard functional group transformations on 28 provided access not only to L-(+)- swainsonine (five steps, 31% yield), but also to dehydro-Lswainsonine 29 (four steps, 37% yield) and the two dextrorotatory 7-hydroxyswainsonines 30 (three steps, 32% yield) and 31 (four steps, 39% yield).In tests with naringinase obtained from Penicillium decumbens, (+)-swainsonine indeed proved to be a very potent and highly specific inhibitor, showing a Ki of 0.45 ÏM. By contrast, natural (")-swainsonine was indiVerent to the enzyme. Compounds 29–31 were only weakly inhibitory. New synthetic analogues of swainsonine continue to be spawned in the search for novel or improved glycosidase inhibitors based on the structural motifs present in the parent alkaloid. A new synthesis of the triacetate of 8-epi-swainsonine 32 has appeared,18 and previously reported syntheses19 of related 1,2,7-trihydroxyindolizidines have been included in a published conference report.20 Swainsonine has been tethered to an agarose matrix, as shown in 33, for evaluation as an aYnity material for mannosidases.21 The sulfonium species 34 represents a more unusual variation on the swainsonine theme; a key intermediate en route to it has recently been synthesised,22 and the methoxy analogue 35 has been prepared as the tosylate salt.23 When one compares the structures of swainsonine and D-mannopyranose 24, the former can be seen to lack the sugar’s C-4 hydroxymethylene group.Pearson and Hembre have now synthesised the tetrahydroxyquinolizidines 36–39,24 which contain the ‘missing’ CHOH group between positions C-8 and C-8a of the indolizidine. The products can also be viewed as ring-expanded castanospermine derivatives (see Section 3.3). Ironically, the new analogues had little or no activity as glycosidase inhibitors, and insignificant anticancer and anti-HIV activity.However, a diVerent range of synthetic swainsonine analogues 40–43 having a hydroxymethyl substituent at C-3 inhibited jack bean ·-mannosidase, although about two orders of magnitude less strongly than swainsonine itself.25 More interestingly, these compounds were only about ten times weaker as inhibitors of amyloglucosidase from Aspergillus niger than the pyrrolizidine alkaloids alexine 44 and australine 45. Like the pyrrolizidines, 40–43 were not active towards ·-glucosidase from bakers’ yeast and almond ‚-glucosidase, which provides further support for viewing them as homoalexines and homoaustralines rather than as swainsonine surrogates. 3.3 Castanospermine and related compounds Aldehyde 46, which is readily prepared from methyl ·-Dglucopyranoside, has been converted into (+)-castanospermine 47 in nine steps and 22% overall yield by the short reaction sequence shown in Scheme 4.26 Ultrasound-promoted allylation of 46 with allyl bromide and tin was diastereoselective (9:1), aVording alcohol 48 as the major product.Four of the target alkaloid’s five stereogenic centres are present in this early intermediate. The most interesting step was the triple reductive amination of the tricarbonyl compound 49 (which appears to exist entirely as the lactol tautomer illustrated) with ammonium formate and sodium cyanoborohydride.The major product 50 (53% yield) had the correct bridgehead stereochemistry for (+)-castanospermine, and less than 5% of the epimer at C-8a was observed. Work in progress towards the synthesis of castanospermine that has so far appeared only in a published conference paper21 exploits the intramolecular dipolar cycloaddition of nitrone 51. Product 52, formed in 75% yield, was transformed in several steps into indolizidinone 53, Baeyer–Villiger oxidation of which is expected to introduce the hydroxy group required at C-8.The completion of this unusual route to the target alkaloid is awaited with interest. The potentially valuable antitumour agent 1-Obutanoylcastanospermine 54 has been prepared by acylation of the parent alkaloid with 2,2,2-trifluoroethyl butyrate in the presence of cross-linked enzyme-crystals (CLECs) of subtilisin in pyridine.27 Selective chemical derivatisations of castanospermine have allowed the synthesis of analogues selectively modified at C-1 (the esters, ethers and fluoride 55–59, 1-epicastanospermine 60 and its fluoro derivative 61, and the 1-C-methyl analogue 62) or C-7 (the pivalate ester 63, 7-epicastanospermine 64 and its fluoro analogue 65, and the two 7-C-methyl compounds 66 and 67).28 None of the new compounds was as eVective as castanospermine itself in inhibiting ·- and ‚-glucosidases from human liver; configurational inversion or esterification at C-1 abolished activity towards ent-13 (+)-Swainsonine 34 R = H 35 R = Me 32 33 29 28 30 R1 = OH; R2 = H 31 R1 = H; R2 = OH 27 26 24 25 + N H HO OH OH O OH OH OH OH O OH HO HO OH OH O H HO OH OH O HO HO O H O O O O O O O N H HO OH OH R2 R1 N H O O O O HO N H HO OH OH N H HO OH OH O(CH2)4 N H HO OH OH S H RO OH OH N OH OH OH H R1 R2 N OH OH OH H R1 R2 N H OH OH OH R2 R1 N H OH OH OH R2 R1 N H OH OH OH HO N H OH OH OH HO 44 Alexine 42 R1 = H; R2 = OH 43 R1 = OH; R2 = H 40 R1 = H; R2 = OH 41 R1 = OH; R2 = H 38 R1 = H; R2 = OH 39 R1 = OH; R2 = H 36 R1 = H; R2 = OH 37 R1 = OH; R2 = H 45 Australine Michael: Indolizidine and quinolizidine alkaloids 573both glucosidases, while modifications at C-7 severely curtailed or destroyed the ability to inhibit ‚-glucosidase but only weakly impaired the eVects on ·-glucosidase.Other new analogues of castanospermine reported during the period under review include the three 1,2,6,7,8- pentahydroxyindolizidines 68–70, the ability of the first two to inhibit naringinase probably relating to their structural resemblance to L-(+)-swainsonine (see Section 3.2).17 Several previously reported29 1,2,3,7-tetrahydroxyquinolizidines, which may be viewed as ring-expanded analogues of castanospermine, have been included in a published conference paper.20 Castanospermine itself has been used as the starting material from which to prepare the deoxynojirimycin analogue 71.30 The published X-ray crystallographic structures of a glucoamylase and its complex with 1-deoxynojirimycin were the starting points for a molecular dynamics study of the interaction between the enzyme and a range of inhibitors that included castanospermine.31 Experimentally determined biological consequences of castanospermine’s ability to inhibit glycosidases also continue to be probed; for example, the alkaloid was included in a survey of inhibitors of gut glycosidases in herbivorous insects.32 Standing out in this year’s selection of papers are several relating to the alkaloid’s potential as an immunosuppressant for prolonging the survival of heart transplants in rats, a feature that directly relates to its ability to inhibit oligosaccharide processing and hence to reduce the expression of adhesion molecules involved in cell–cell interaction during allorejection.In grafted rats, the alkaloid appears to inhibit both lymphocyte-endothelial cell binding and lymphocyte activation; its principal eVect is apparently to downregulate a specific ligand-receptor adhesion molecule pair (LFA-1·«ICAM-1), though there are certainly subtle eVects on other adhesion molecules (class I and II MHC, CD4 and CD8).33,34 With limited testing, the alkaloid was relatively non-toxic, and its eVects were dose-dependent. Castanospermine also operated synergistically with tacrolimus, a potent but toxic immunosuppressant, in prolonging allograft survival, suggesting that it may be possible to combine the two drugs clinically in order to reduce the dosage of the more toxic component.35 4 Alkaloids from ants 4.1 3,5-Dialkylindolizidine alkaloids The major constituent of the venom produced by queen ants of a species of Solenopsis (Diplorhoptrum) collected on Mona Island, Puerto Rico, is the known metabolite (5Z,9Z)-3-hexyl- 5-methylindolizidine 72.36 A characteristic range of Bohlmann bands in its FTIR spectrum supported the stereochemical assignment, which was in any case confirmed by direct comparison with an available synthetic sample.Workers from the same colony did not produce 72, but instead secreted a 1:4 mixture of 2,6-cis- and trans-disubstituted piperidines 73, the former of which is a plausible biosynthetic precursor of the indolizidine. The piperidines were trace components of the queens’ venom. Even more interestingly, while the venom of queens of a species collected in Cabo Rojo contained indolizidine 72 as the major component, workers produced two structurally isomeric indolizidines, 74 and 75, which were present only in trace amounts in the queens.The ratios of these two isomers varied between 6:1 and 1.7:1 depending on the population examined. This is the first report of 3-butyl-5- propylindolizidines as ant metabolites; they have previously been found only in amphibians. Compound 74 is, in fact, identical with the well-known and much-synthesised dendrobatid frog alkaloid indolizidine 223AB, while 75 has previously been found in a range of frogs and toads.Their discovery OH BnO BnO BnO OBn MeO OMe O BnO BnO BnO OH O OBn OH O BnO BnO BnO CHO OMe O BnO BnO BnO OMe OH N OBn BnO H BnO BnO N OH HO H HO HO isomer (8%) 75% + 47 (+)-Castanospermine 50 70% ix 48 46 i 49 75% v–vii viii 53% ii–iv 72% Scheme 4 Reagents: i, H2C=CHCH2Br, Sn, MeCN–H2O (10:1), ultrasound; ii, BnBr, NaH, Bu4NI, DMF, 0 )C; iii, iodonium dicollidine perchlorate, CH2Cl2, MeOH, rt; iv, Zn, EtOH, reflux; v, Swern oxidation; vi, O3, CH2Cl2, MeOH, "78 )C, then Ph3P; vii, 9 M HCl, THF, rt; viii, NH4HCO2 (1.3 equiv.), NaBH3CN (30 equiv.), MeOH; ix, 10% Pd/C, HCO2H, MeOH, rt TBDPSO OBn N –O CO2Me BnO N BnO TBDPSO O CO2Me BnO N R HO H HO HO N R HO H HO HO N OBn HO H BnO O O N OH R H HO HO N OH HO H HO HO Me N OH Me3CCO2 H HO HO N OH HO H HO HO OH N OH H HO HO R1 R2 N OH HO H HO HO R1 R2 N OH HO H HO HO H 6 8 69 R1 = OH; R2 = Me 70 R1 = Me; R2 = OH 66 R1 = OH; R2 = Me 67 R1 = Me; R2 = OH 63 62 64 R = OH 65 R = F 53 54 R = OCO(CH2)2Me 55 R = OCOCMe3 56 R = OCOPh 57 R = OSiMe2But 58 R = OMe 59 R = F 60 R = OH 61 R = F 52 51 + 71 574 Natural Product Reports, 1998in ants adds fuel to the growing belief that the skin alkaloids of frogs are probably sequestered from dietary sources (cf.ref. 7d). Momose et al. have previously communicated a synthesis of (+)-monomorine I 76 by a route involving the use of a chiral lithium amide base to desymmetrise the 8-azabicyclo[3.2.1]octan-3-one 77.7e,37 Full experimental details of this work have now been reported in two publications, the first dealing with aspects of the desymmetrisation and cleavage of azabicyclo[3.n.1]alkan-3-ones38 and the second with applications to the synthesis of alkaloids39 (also see Section 5.2).Similar ideas underlie a formal synthesis of (+)-monomorine I by Muraoka and co-workers,40 who took advantage of asymmetric deprotonation and alkylation to prepare the bridged bicyclic ketone (+)-78.Beckmann rearrangement of the oxime derivative 7941 followed by Huisgen– White rearrangement of lactam 80 with nitrogen peroxide and cleavage with base yielded the 2,6-cis-disubstituted piperidine 81 as a 15:1 E/Z mixture after esterification (Scheme 5). Four standard steps completed the transformation of 81 into aldehyde 82, a compound that Takahata et al. have previously converted into (+)-monomorine I.42 4.2 Polycyclic alkaloids Following their recent isolation of myrmicarins 237A and 237B 83 from the African ant Myrmicaria eumenoides,43 Francke and co-workers have now found the same basic C15N indolizidine motif in three entirely new families of alkaloids present in poison gland secretions of M.opaciventris collected from two diVerent African sources.44 The secretions, which also contained simple monoterpenes, displayed a very high degree of intraspecific variability. GC-MS analysis showed that specimens collected in Kenya were rich in compounds representing variants of the basic C15N theme, while some late-eluting components appeared to be ‘dimers’ containing thirty carbons and two nitrogens.Specimens from Cameroon, on the other hand, contained only trace amounts of the C15N and C30N2 compounds, but instead were dominated by ‘trimers’, especially myrmicarin 663, a compound of nominal mass 663 with a molecular composition of C45H65N3O. Other West African colonies showed intermediate alkaloid composition.Preliminary bioassays showed that the toxicity of the poison gland secretion rested with the new alkaloids, the function of the monoterpenes apparently being to lower the viscosity and improve the spreading properties. Both spectroscopic studies and chemical interconversions were used to ascertain the structures of the new metabolites, which were isolated in milligram quantities from 40–100 ants. The C15N alkaloids from the Kenyan collection were dominated by myrmicarin 215A, which was isolated as a 2:1 mixture with an isomer, myrmicarin 215B.Hydrogenation of the mixture at atmospheric pressure yielded a single product identical with myrmicarin 217, another alkaloid isolated from the venom, thus demonstrating 215A and 215B to be unsaturated analogues of myrmicarin 217. Prolonged hydrogenation at 50 bar yielded a compound of nominal mass 221, indicating the presence of two further sites of unsaturation in the latter.Extensive NMR experiments were necessary to elucidate the full structures of the three alkaloids, which turned out to be 84, 85 and 86 respectively – the first examples of hexahydropyrrolo[2,1,5-cd]indolizines from a natural source. When these compounds were exposed to air or deliberately treated with oxygen, they were slowly transformed into the unsaturated analogues 87, 88 and 89, which proved to be identical to three minor venom alkaloids named myrmicarins 213A, 213B and 215C respectively.It is uncertain whether these compounds are genuine enzymically-produced metabolites of the ants, or merely secondary oxidation products produced during storage in the ants’ poison gland reservoirs. The main dimeric component, myrmicarin 430A, could not be isolated because exposure to silica or alumina led to virtually instantaneous decomposition; even fresh secretions exposed to air showed more than 90% decomposition of the alkaloid within one hour.However, direct analysis of unpurified freshly collected secretion by two-dimensional NMR techniques permitted assignment of peaks due to monoterpenes and myrmicarins 215A, 215B and 217; remarkably, the remaining cross-peaks could be analysed with little ambiguity to reveal the unique heptacyclic structure shown in 90, complete with stereochemistry except at C-4*a.45 The moieties of the dimer are clearly a hexahydropyrrolo[2,1,5- cd]indolizine akin to myrmicarin 217, and a related but nonetheless unique tetracyclic unit.The structure of a minor isomer, myrmicarin 430B, has not been elucidated. The dominant ‘trimer’, myrmicarin 663, has also been isolated from a diVerent myrmecine ant, M. striata.46 All operations on this highly temperature- and air-sensitive compound had to be carried out under argon. Characterisation was performed on a sample obtained in quantity (14 mg) from the secretions of over 300 M. striata ants. Carbonyl and enamine functions were indicated by IR absorptions at 1712 N H (CH2)5CH3 N H (CH2)3CH3 N H (CH2)2CH3 N H (CH2)3CH3 N O Cbz N H (CH2)3CH3 77 76 (+)-Monomorine I 73 74 72 75 N CHO Cbz N OHC Cbz N CO2Me Cbz N N Cbz O H N O Cbz N N Cbz OH 81 81% + Z isomer (12%) 79 78 vi, vii 93% ii 91% 80 54% iii–v (E/Z 15:1) 82 63% viii, ix i Scheme 5 Reagents: i, NH2OH·HCl, NaOAc, EtOH, H2O, reflux; ii, p-TsCl, K2CO3, MeOCH2CH2OMe, H2O, 80 )C; iii, N2O4, MeOCH2CH2OMe, NaOAc, 0 )C; iv, NaOH (5% aq.), "10 )C; v, MeOH, HCl; vi, DIBAL, toluene, "10 )C; vii, (COCl)2, DMSO, Et3N, CH2Cl2, "55 )C; viii, (Ph3P)3RhCl, BuCN, 140 )C; ix, O3, CH2Cl2, "60 )C, then Ph3P Michael: Indolizidine and quinolizidine alkaloids 575and 1651 cm"1 respectively, and hydrogenation to a product of nominal mass 671 indicated four double bonds.A monumental set of NMR experiments, including HMBC and NOE experiments to unravel the enormously complex connectivities and stereochemical relationships, revealed the astonishing decacyclic structure shown in 91; one sole stereochemical ambiguity was resolved by molecular modelling.The structure of a related alkaloid, myrmicarin 661, from M. opaciventris has not been elucidated, but the gross structure 92 has been proposed for myrmicarin 645 from M. striata. The inclusion of these polycyclic alkaloids in this review is justified in view of the obvious structural relationships to ‘simple’ indolizidines such as myrmicarin 237A/B 83.All contain unbranched C15 chains joined at three sites to a nitrogen atom, and the basic indolizidine unit is easily traced. Additional carbon–carbon bonds are formed only at welldefined positions along the C15 chain, for example at the carbonyl position of myrmicarin 237A/B, which remains undisguised in myrmicarin 663. The points of junction in the ‘dimeric’ and ‘trimeric’ alkaloids can be regarded as arising from suitable condensations on precursors such as those shown in the disconnection Scheme 6, in which the (presumably) acetate-derived C15 chains are highlighted.These precursors have a nominal molecular mass of 233. Intriguingly, a trace component of the Kenyan population of M. opaciventris, myrmicarin 233A, remains uncharacterised other than by mass spectrometry, which supports a structure consistent with one of the proposed unsaturated indolizidine precursors 93 shown in the Scheme. 5 Alkaloids from amphibians 5.1 Occurrence Previous suspicions7d,47 that the conjectural 1,4-dialkylquinolizidine alkaloid 223A is in fact an indolizidine have now been substantiated.48 Although the indolizidine core had been suspected for some time, FTIR evidence for a 5,8-disubstituted indolizidine structure, as evinced by a characteristic set of Bohlmann bands, was apparently contradicted by the observed mass spectral fragmentation pattern.It was only when HPLC purification of a sample of 223A isolated from the skin extracts of a Panamanian population of Dendrobates pumilio yielded enough material (1 mg) for 1H NMR characterisation that the puzzle was solved: the compound proved to belong to a new class of amphibian alkaloids, the 5,6,8-trisubstituted indolizidines.The structure was established as rel-(5R,6S,8R,8aS)-6,8- diethyl-5-propylindolizidine 94, the stereochemistry being assigned after analysis of Bohlmann bands and coupling constants. The gross structures of three minor alkaloids from other Central American populations of D.pumilio could also be tentatively assigned as trisubstituted indolizidines on the basis of mass spectrometric comparisons with alkaloid 223A. Indolizidines 237L and 251M are the 5-butyl and 5-pentyl analogues 95 and 96 of 223A, while alkaloid 267J appears to bear an additional hydroxy group as shown in 97. FTIR spectra could not be recorded on the very limited quantities of alkaloids 237L and 251M present in the skin extracts.However, a 5,9-trans stereochemical relationship has been mooted for the attached hydrogen atoms in indolizidine 267J, since the pattern of Bohlmann bands in its FTIR spectrum diVered from those of 223A. N O H N N N N N H N 4¢a 90 Myrmicarin 430A 89 Myrmicarin 215C 87 Myrmicarin 213A ( Z) 88 Myrmicarin 213B ( E) 86 Myrmicarin 217 84 Myrmicarin 215A ( Z) 85 Myrmicarin 215B ( E) 83 Myrmicarins 237A,B N N N H N N H O H H 92 Myrmicarin 645 91 Myrmicarin 663 N N N N N O N O N O NH O O N N O N 93 Myrmicarin 233? 84 Myrmicarin 215A ( Z) 85 Myrmicarin 215B ( E) 90 Myrmicarin 430A 91 Myrmicarin 663 Scheme 6 576 Natural Product Reports, 19981,4-Disubstituted quinolizidine alkaloids were first revealed as amphibian natural products in 1993,49 but full details of their structural elucidation were not reported at the time.Extracts from fifteen skins of the Madagascan frog Mantella baroni have recently been purified by HPLC to yield enough of the putative quinolizidine 217A, slightly contaminated by a diastereoisomer, for comprehensive 1H NMR analysis.50 The structure and relative stereochemistry have been assigned as shown in 98.The assignment of the 4,10-cis relative stereochemistry of the attached hydrogen atoms in 217A and several congeners was based on analysis of Bohlmann bands, while the 1,10-trans relationship was deduced from analysis of coupling constants, which showed that the methyl group had to be equatorially disposed.The tentative structures 99–102 were proposed for the minor quinolizidine alkaloids 207I, 231A, 233A and 235E* respectively on the basis of FTIR and MS studies. While the IR studies on these alkaloids served to establish the relative configurations between C-4 and C-10, the 1,10 relationship remains uncertain. Furthermore, in the light of the discoveries mentioned in the preceding paragraph, the M. baroni metabolites coded as 231B and 273A and previously thought to be 1,4-dialkylquinolizidines7f,49 are more likely to be (5,9-cis)-6-methyl trisubstituted indolizidines.Alkaloids 249C and 263A could either be (4,10-trans)-1,4-dialkylquinolizidines or (5,9-trans)-5,6,8-trisubstituted indolizidines, while alkaloids 275A, 277A and 289A, originally thought to be 1,4- disubstituted quinolizidines, require further investigation as they lack the expected mass spectrometric fragments. 5.2 Synthesis of amphibian alkylindolizidine alkaloids A concise synthesis of (+)-indolizidine 209D 103 from the Comins group illustrates a new modification of their elegant generalised approach to alkaloid synthesis based on the reactivity of 2,3-dihydro-4-pyridones (Scheme 7).51 Starting in the usual way by converting the 4-methoxypyridine 104 into the optically active enaminone 105, they set the scene for a novel anionic cyclisation by transforming 105 into the Z-vinyl iodide 106.Transmetallation with tert-butyllithium produced a transient organolithium species that underwent stereospecific intramolecular conjugate addition to the enaminone.The intermediate enolate was trapped as the vinyl triflate 107, which was isolated in 80% yield. Catalytic reduction aVorded the target alkaloid 103 directly. The overall yield of this brief but eVective reaction sequence was 35% based on 104. This synthesis has also been highlighted in a published conference paper that includes details of a short stereoselective synthesis of another frog alkaloid, (")-indolizidine 235B 108.52 The synthesis of racemic indolizidine 209B 109 shown in Scheme 8 incorporates an Eschenmoser sulfide contraction to form the vinylogous urethane 110, the enamine-like nucleophilicity of which was exploited in a subsequent cyclisation step to create the unsaturated indolizidine nucleus of 111.53 Stereoselective reduction of this compound, itself a bicyclic vinylogous urethane, was achieved in two complementary modes.The less satisfactory route, involving reduction of the C=C bond with sodium cyanoborohydride at pH 4, yielded the diastereoisomeric products 112 and 113 in 33% and 14% yields respectively.The more stereoselective reduction, accomplished with hydrogen and Adams catalyst, showed reversed selectivity, giving the same two products in 6% and 71% yields respectively. In both products, hydrogen was delivered to the bridgehead position on the face remote from the pendent pentyl chain.Isomer 112 was transformed into the target alkaloid 109 as shown, while similar reactions on the epimer 113 yielded the previously unreported 209B diastereoisomer 114. This synthesis has also been described in the published proceedings of a recent conference;54 the article includes details of an enantioselective modification of the route, and a related synthesis of the frog indolizidine 167B 115. Momose et al. have reported a synthesis of (")-indolizidine 223AB 74 by a route that is very similar to the one by which they prepared (+)-monomorine I (see Section 4.1).In a nutshell, desymmetrisation of the azabicyclo[3.3.1]nonan-3- one 116 by enantioselective deprotonation with the chiral base 117 followed by ozonolysis of the silyl enol ether 118 yielded the 2,6-cis-disubstituted piperidine 119 (Scheme 9).38 Chain extensions on both sides of the ring were readily accomplished via the important intermediates 120 and 121 as illustrated, and culminated in the synthesis of the N-protected piperidine 122 in which the fragment destined to form the five-membered ring 101 Quinolizidine 233A 98 Quinolizidine 217A 99 Quinolizidine 207I 95 Indolizidine 237L R = Me 96 Indolizidine 251M R = Et 97 Indolizidine 267J 94 Indolizidine 223A 102 Quinolizidine 235E¢ 100 Quinolizidine 231A N H N R N H HO N H N H N H N H C6H11 N H 80% 104 87% (92% de) i, ii iii–vi 64% 106 107 vii, viii 108 (–)-Indolizidine 235B 103 (+)-Indolizidine 209D ix 79% 105 N H N H N O I TfO N H N MeO TIPS O Ph O N TIPS O Scheme 7 Reagents: i, (1S,2R,4S)-2-(1-methyl-1-phenylethyl)-4- isopropylcyclohexyl chloroformate; ii, CH3(CH2)5MgCl; iii, NaOMe, MeOH; iv, 10% HCl; v, NaHDMS, THF, "78 )C; vi, (Z)-1,3- diiodopropene, THF, "78 )C to 0)C; vii, ButLi, THF, "78 )C; viii, N-(5-chloro-2-pyridyl)triflimide; ix, H2, Pt/C, Li2CO3, EtOAc Michael: Indolizidine and quinolizidine alkaloids 577was developed from 120’s substituent at C-6.The bicyclic nucleus itself was formed by a striking reaction rarely seen in indolizidine alkaloid chemistry: the stereoselective cyclisation of an aminyl radical (derived from the N-chloropiperidine 123) on to a nearby alkene.Dechlorination of the resulting intermediate yielded the target alkaloid (")-74.39 Furthermore, by creating the precursor fragment of the five-membered ring from the C-2 substituent of 120, these workers could also access the enantiomer of the target alkaloid.By making the 2-iodoethyl piperidine ent-121, they eVectively accomplished a formal synthesis of (+)-indolizidine 223AB, ent-74, thereby providing a fascinating example of an enantiodivergent strategy in alkaloid synthesis. Lhommet and his co-workers have over the past few years reported several syntheses of indolizidine alkaloids in which enaminones derived from condensation of Meldrum’s acid with (S)-pyroglutamic acid derivatives are stereoselectively reduced to give 2,5-trans-disubstituted pyrrolidines as key building blocks.A recent extension of this methodology proceeds via vinylogous urethane 124, which was prepared in six steps and 42% overall yield from 125 (Scheme 10).55 In this case, reduction of 124 with sodium borohydride in acidic medium yielded a mixture of cis- and trans-disubstituted pyrrolidines. Kinetically controlled N-carbamoylation of the dominant trans product permitted chromatographic separation of 126, which was transformed in three steps into the aldehyde 127.Wittig reaction with the acetal-containing phosphorane 128 eVected chain extension to 129. Once the double bond had been reduced and the carbamoyl functionality removed, the indolizidine nucleus was created by spontaneous iminium ion formation between the secondary amine and the ketone group. A highly diastereoselective hydrogenation of this ionic intermediate 130 (de 95%) was followed by acidic work-up to remove the acetal protecting group.The final product was (")-indolizidine 239AB 131, which was obtained over 15 steps in an overall yield of 5.6%. This strategy is potentially applicable to the synthesis of all the natural 3,5-disubstituted hydroxyalkyl indolizidine alkaloids. 5.3 Synthesis of alkaloids of the pumiliotoxin class The synthesis of pumiliotoxin 251D 132 shown in Scheme 11 commenced with L-proline 133, which was transformed in five standard steps (42% yield) into the tert-butyloxycarbonyl (Boc)-protected (S)-2-ethynylpyrrolidine 134.56,57 The centrepiece of the route is the 6-exo-dig radical cyclisation of the 3-bromopropionamide 135.When the reaction was performed with tributyltin hydride, indolizidinone 136 was obtained in 40% yield together with 10% of the debrominated compound 137. The reaction also proceeded photochemically, but yields of the products were lower (10% each). Oxymercuration of 136 followed by reductive demercuration yielded a 5:1 mixture of tertiary alcohols (95%), from which the major 8S,8aR isomer 138 could be obtained pure by recrystallisation.The preparation of this isomer, which has previously been transformed into the target alkaloid in three steps,58 completes a formal synthesis of pumiliotoxin 251D. The entire route, accomplished in twelve steps, is the shortest enantioselective synthesis of this alkaloid to date. Pioneering syntheses of the pumiliotoxins and allopumiliotoxins by Overman and his co-workers were published in the early 1980s, and incorporated stereospecific iminium ion–vinylsilane cyclisations for making the (Z)-alkylidenesubstituted indolizidine ring.However, Overman’s substantially improved ‘second-generation’ syntheses make use of iodide-promoted iminium ion–alkyne cyclisations. A synthesis of (+)-pumiliotoxin A 139 based on this methodology was reported in preliminary form in 1988;59 it has now appeared with full experimental details together with related syntheses of the model compound nor-11-methylpumiliotoxin 237A 140 and (+)-pumiliotoxin B 141.60 The synthesis of pumiliotoxin A was highlighted in an earlier review in this series (cf.ref. 7g) and will not be repeated here. Important late steps in the analogous synthesis of pumiliotoxin B are shown in Scheme 12. Alkyne 142 was prepared in eight steps (46% overall yield) from optically pure (2R)-2-methylpent-4-enol 143, itself made in four steps from commercially available (4S,5R)-4-methyl-5-phenyl-2-oxazolidinone 144.Coupling of alkyne 142 with the proline-derived epoxide 145 followed by hydrolysis of the N-Boc protecting group yielded pyrrolidine 146 in 73% yield. The iminium ion intermediate 147 was generated in situ from 146 by treatment with paraformaldehyde in aqueous acidic medium containing a large excess of sodium iodide. Although alkynes are normally unreactive towards iminium ions, the presence of iodide, which is a strong nucleophile for carbon, induced the highly stereoselective cyclisation illustrated.The choice of acid also proved to be critical; the best yields were obtained with pyridinium toluenep- sulfonate, which also brought about cleavage of the acetonide protecting group. The vinyl iodide 148 was isolated in 68% yield as a single isomer. In view of its sensitivity to light, 148 was immediately deiodinated by lithium–iodine exchange with tert-butyllithium followed by protonation with degassed aqueous ammonium chloride to give the target alkaloid (+)- 141 in 89% yield.The synthesis is comparatively short and eYcient [4 steps and 44% overall yield based on 142 and 145; 10% based on (4S,5R)-4-methyl-5-phenyl-2-oxazolidinone 144; 8% based on N-Boc-L-proline, the precursor of 145]. It also lends itself to comparatively large-scale manipulations – in this case, 500 mg of 141 could be prepared for biological and structural investigations. N CH2OH C5H11 H N C5H11 H CH2OMs N C5H11 H H EtO2C N Me C5H11 H N C5H11 H Me N C3H7 H HO C5H11 N CO2Et N CO2Et C5H11 N CO2Et C5H11 H N S H C5H11 EtO2C CO2Et N EtO2C C5H11 C5H11 EtO2C S N ii, iii i 110 85% 74% iv 91% 112 v 85% vi 33% (+ 14% 113) 111 71% (+6% 112) iv 92% ix 115 Indolizidine 167B 109 Indolizidine 209B 114 x 65% vii, viii 40% 113 88% iv, vii Scheme 8 Reagents: i, NaOH (cat.), THF, rt; ii, BrCH2CO2Et, MeCN, rt; iii, Ph3P, Et3N, MeCN, rt; iv, LiAlH4, THF, 0 )C to rt; v, CBr4, Ph3P, Et3N, MeCN, 0 )C to rt, then rt to reflux; vi, NaBH3CN, HCl (pH 4), EtOH, rt; vii, MeSO2Cl, Et3N, CH2Cl2, 0 )C; viii, LiEt3BH (1 M in THF), THF, 0 )C; ix, H2 (1 atm), PtO2, AcOH, rt; x, Raney Ni W-2, EtOH, reflux 578 Natural Product Reports, 1998The iodide-promoted iminium ion–alkyne cyclisation methodology has also been applied by the Overman group to the synthesis of alkaloids of the allopumiliotoxin class.61 This work, which includes full experimental details for the synthesis of the alkaloids (+)-allopumiliotoxin 267A 149, (+)-allopumiliotoxin 323B* 150 and (+)-allopumiliotoxin 339A 151 as well as the model compound nor-11- methylallopumiliotoxin 253A 152, complements an earlier communication on the synthesis of 151.62 The syntheses all commenced with optically pure aldehyde 153, prepared in nine steps and about 40% overall yield from the L-proline derivative 154.Reaction with alkyne anions 155 bearing appropriate substituents was reasonably diastereoselective (between 3:1 and 4:1), and yielded propargylic alcohols in which the major isomer 156, possessing R stereochemistry at the newly created stereogenic centre, was formed in accordance with the expected chelation control (Scheme 13).The N-cyanomethyl substituent is essentially a disguised iminium ion; treatment with silver(I) or copper(II) salts removed cyanide ion and yielded the bicyclic oxazines 157, which are themselves masked iminium ions. The key cyclisation step, involving in all cases treatment with sodium iodide, camphorsulfonic acid and additional paraformaldehyde, is similar to that previously illustrated in Scheme 12.Immediate deiodination of the ensuing vinyl iodides 158 by lithium–halogen exchange and protonation, followed where necessary by removal of protecting groups, yielded the target alkaloids. These syntheses rigorously establish the full stereostructure of the previously unsynthesised alkaloids 150 and 151. Homopumiliotoxin alkaloids (e.g., homopumiliotoxin 233F 159) have not been synthesised to date.However, a recent model study opens up a comparatively simple route to indolizidine and quinolizidine analogues of these alkaloids (Scheme 14).63 The pivotal reaction was a palladium-induced cyclisation of a vinyl bromide onto an alkyne, followed by reduction of the intermediate vinylpalladium species with ammonium formate as the source of hydride ion. The substrates for this cyclisation were the dibromoalkenes 160, which yielded exclusively the (Z)-alkylidene bicyclic compounds 161 in 61% yield (for the indolizidine system) or 54% yield (for the quinolizidine system).The products always retained one bromine atom even when an excess of reducing agent was used. A problem to be solved before the route can be applied to alkaloidal targets is the low-yielding Corey–Fuchs dibromomethylenation of ketones 162. 6 Elaeocarpus alkaloids When the diene 163, prepared in six steps from the pyroglutamate derivative 164, was treated with triethylsilane and a nickel(0) complex, the �-allylnickel intermediate initially generated reacted with the nearby aldehyde group to produce the diastereoisomeric azabicyclic products 165 and 166 in yields of 36% and 37% respectively (Scheme 15).64 Compound 166 was readily transformed into 165 in 81% yield by a four-step reaction sequence involving Mitsunobu inversion.This product possesses the carbon skeleton as well as the correct relative and absolute stereochemistry of the alkaloid (")-elaeokanine C 167.A further eight steps, mainly entailing functional group manipulations in the side chain, served to convert 165 into the bicyclic lactam (")-168. Unnatural (+)-elaeokanine C, ent-167, has previously been obtained from (+)-168,65 so the illustrated route represents a formal synthesis of the naturally occurring alkaloid – the first on record. The authors have also applied the new cyclisation strategy to the creation of related indolizidine systems as well as pyrrolizidines. 7 Ipalbidine Several interesting new transformations are to be found in a recent communication by Sheehan and Padwa outlining a N O MeO2C N OTMS MeO2C N N –N Me Ph H N CO2Me HO CO2Me N CO2Me TBDMSO OH N CO2Me OMOM N CO2Me I N CO2Me N Cl N H N CO2Me HO N CO2Me I N H 122 121 120 119 118 117 116 ent-121 ent-74 74 (–)-Indolizidine 223AB 27% viii, xxi–xxiii, xii, xiii 18% xviii, xix 43% viii, xxii, x, vii 69% xv–xvii 98% xiv 52% x–xiii 73% vi–ix v 95% 60% ii–iv 94% i, Li+ 123 Scheme 9 Reagents: i, Base 117, THF, "100 )C, then TMSCl; ii, O3, CH2Cl2–MeOH (10:1), "78 )C; iii, NaBH4, "78 )C; iv, CH2N2, Et2O, 0 )C to rt; v, Superhydride, THF, 0 )C to rt; vi, MOM-Cl, CH2Cl2, EtNPr2 i, 0 )C to rt; vii, Bu4NF, THF, 0 )C to rt; viii, (COCl)2, DMSO, CH2Cl2, Et3N, "78 )C to 0 )C; ix, Ph3P=CHMe, THF, 0 )C to rt; x, H2 (1 atm), 5% Pd/C, MeOH, rt; xi, conc.HCl, MeOH, 60 )C; xii, MsCl, pyridine, CH2Cl2, rt; xiii, NaI, Me2CO, rt; xiv, Me(CH2)2C/CLi, THF, 0 )C to rt; xv, Na, NH3, "50 )C, then NH4Cl (aq.); xvi, PrSLi, HMPA, THF, 0 )C to rt; xvii, NCS, Et2O, 0 )C; xviii, CuCl, CuCl2, THF, AcOH, "45 )C; xix, Bu3SnH, AIBN, C6H6, reflux; xx, Ph3P=CH2, THF, 0 )C to rt; xxi, Ph3P=CHOMe, THF, 0 )C to rt; xxii, conc. HCl, CH2Cl2, rt; xxiii, NaBH4, MeOH, 0 )C to rt Michael: Indolizidine and quinolizidine alkaloids 579short synthesis of the Ipomoea alkaloid ipalbidine 169.66 When the diazoimide 170 was heated with cis-1-phenylsulfonylprop- 1-ene 171 and a catalytic quantity of rhodium(II) acetate, the isomu�nchnone intermediate 172 underwent a [3+2] dipolar cycloaddition to yield the 3-hydroxy-2-pyridone 173 (Scheme 16).Judging from other examples cited, this appears to be a reasonably general new strategy for making 3-hydroxy- 2-pyridones. Another novel step is the palladium-catalysed Stille coupling between the triflate derived from 173 and tributyl(p-methoxyphenyl)tin to yield 174.Although cross couplings with simple vinyl triflates and organotin compounds are well known, there are no precedents for coupling with pyridone-derived triflates; several related examples presented in the communication suggest that this might also be a generally useful transformation. The synthesis of racemic ipalbidine 169 was readily completed as shown in an overall yield of 23% based on the diazoimide 170. 8 Phenanthroindolizidine and phenanthroquinolizidine alkaloids and seco analogues Methodology previously devised by Ciufolini and co-workers for making sterically congested pyridines has been adapted O OEt N+ C4H9 N H HO N CO2H O H N H MeO2C OH N C4H9 OHC Cbz N OH MeO2C Cbz PPh3 O O OEt N C4H9 Cbz O O OEt 129 128 vi 76% 126 70% iii–v 127 125 124 42% 6 steps i, ii 80% vii–ix 131 (–)-Indolizidine 239AB 130 (de 95%) Scheme 10 Reagents: i, NaBH4, AcOH, MeCN, 0 )C; ii, BnO2CCl, NaHCO3, H2O, 0 )C; iii, p-TsCl, Et3N; iv, Pr2CuLi, Et2O, "80 )C; v, DIBAL, toluene, "78 )C; vi, toluene, 80 )C; vii, H2, PtO2 , MeOH: viii, H2, Pd/C, MeOH; ix, HCl (0.5 M) in CH2Cl2 (2.5:1), 20 )C N O H HO N H HO H N O N O H N O Br N HO2C H N BOC N+ H H Cl– 5 steps 42% 134 133 135 + 137 136 or iv (10% + 10%) iii (40% + 35%) 68% from 134 ii 95% (5:1 with C-8 epimer) v 132 Pumiliotoxin 251D 3 steps 138 i Ref. 58 Scheme 11 Reagents: i, HCl (8 M), EtOH, reflux; ii, Br(CH2)2COCl, Et3N, 0 )C; iii, Bu3SnH, AIBN, C6H6, reflux; iv, hÌ (254 nm), Et3N, MeCN; v, Hg(OAc)2, H2O, THF, rt, then NaBH4, aq.NaOH N H HO R R¢ OH OH OH O N H Ph O HO O O N H HO2C H N Cbz H O R N H H HO N H HO I R R HO H N+ iv, v 68% I– 147 148 (+)-141 146 145 L-proline 142 143 144 4 steps 46% i, ii 73% iii 139 Pumiliotoxin A R = Me; R¢ = 140 R = H; R¢ = CH2CH3 141 Pumiliotoxin B R = Me; R¢ = 89% 8 steps Scheme 12 Reagents: i, BuLi (1 equiv.), Et2AlCl (1 equiv.), toluenehexane, 0 )C; ii, Ba(OH)2, H2O–dioxane (1:1.5), 100 )C; iii, NaI (10 eq), (CH2O)n (5 equiv.), PPTS (3 equiv.), H2O, 105 )C; iv, ButLi, THF, "78 )C; v, NH4Cl (aq.)- 580 Natural Product Reports, 1998for the synthesis of several phenanthroindolizidine alkaloids and their seco analogues.67 Syntheses of two representative alkaloids, septicine 175 and tylophorine 176, are shown in Scheme 17.On the basis of FMO arguments, the unsaturated diketone 177 and enol ether 178 were chosen as starting materials for a lanthanide-catalysed ‘cycloaddition’ reaction, which turned out to be slow but completely stereoselective.The all-trans adduct 179 was isolated in 96% yield. The pivotal pyridine synthesis involved conversion of the benzoate derivative 180 into 181 on heating with hydroxylamine hydrochloride (80% yield). Thereafter, the construction of the indolizidine nucleus proceeded by a three-step transformation of 181 into the 2-vinylpyridine 182, which underwent eYcient conjugate addition with lithium cyanide to aVord nitrile 183 (99%).The synthesis of racemic septicine 175 was completed by common functional group transformations in the side chain of 183, cyclisation to give the bicyclic pyridinium salt 184, and reduction of the latter with sodium borohydride. Tylophorine 176 was obtained from septicine in 74% yield by an oxidative coupling of the two aromatic rings with vanadium(V) oxyfluoride under acidic conditions. A similar sequence of reactions was used to prepare the diarylindolizidine 185, which underwent oxidative cyclisation to yield racemic antofine 186 in 51% yield.Finally, it was also shown that the diarylquinolizidine alkaloid julandine 187 could be prepared by essentially the same route, the major diVerence being the N H HO R R¢ HO OH OH OH N Cbz H N H CN BnO O H R Li R N H BnO CN HO N H BnO O R N H BnO R HO N H BnO I R HO 66%–94% Ag+ or Cu2+ 158 76%–86% iii 76%–92% ii 149–152 i 66%-81% + epimer 157 156 + 155 153 154 149 Alloumiliotoxin 267A R = Me; R¢ = n-C3H7 150 Allopumiliotoxin 323B¢ R = Me; R¢ = 151 Allopumiliotoxin 339A R = Me; R¢ = 152 R = H; R¢ = CH2CH3 Scheme 13 Reagents: i, NaI (10 equiv.), (CH2O)n (2–3 equiv.), camphorsulfonic acid, H2O or H2O-acetone, 100 )C; ii, BunLi or BusLi, THF, "78 )C, then MeOH; iii, Li, NH3 ii, iii v 161 162 160 iv, iii i vi N O N O H EtO2C N O EtO2C (CH2)4CH3 N O (CH2)4CH3 H O N O (CH2)4CH3 O N O (CH2)4CH3 Br Br N O Br (CH2)4CH3 ( ) n 159 Homopumiliotoxin 233F ( ) n ( ) n ( ) n ( ) n ( ) n Scheme 14 Reagents: i, NaH, Me(CH2)4C/CCH2OMs; ii, NaBH4, EtOH; iii, (COCl)2, Et3N, DMF, "40 )C; iv, MeMgBr; v, Ph3P, CBr4; vi, Pd(OAc)2 (2 mol %), Ph3P (4 mol %), NH4OCHO (2 M in MeCN) N O H HO O N H HO O N O H TBDMSO N O O H N O H Et3SiO N O H Et3SiO N O H Et3SiO O N O H AcO OH 79% iv - vii ii (4 steps) iii 99% i 36% + 37% viii - x 56% 81% 165 166 + 163 164 31% 6 steps 168 Ref. 65 167 (–)-Elaeokanine C Scheme 15 Reagents: i, Ni(COD)2 (20 %), Ph3P (40%), Et3SiH (5 equiv.), THF, rt; ii, Mitsunobu inversion; iii, MCPBA, CH2Cl2; iv, Bu4NF, THF; v, Ac2O, pyridine; vi, Me3SiI, DBU, MeCN; vii, H3O+; viii, NaOH (10%), MeOH; ix, MnO2, CH2Cl2; x, H2, Pd/C, EtOAc Michael: Indolizidine and quinolizidine alkaloids 581use of acetonitrile anion rather than cyanide in the conjugate addition step in order to produce the higher homologue of the appropriately substituted pyridine 182.Since cryptopleurine 188 has previously been made from julandine 187 by oxidative coupling,68 a formal synthesis of the phenanthroquinolizidine alkaloid has eVectively been achieved as well. 9 Nuphar alkaloids Nuphar Rhizoma, the dried rhizomes of the waterlily Nuphar pumilum, are used in Japanese and Chinese traditional medicines.Although the species is known to contain sesquiterpene alkaloids, few pharmacological studies of these constituents have hitherto been undertaken. However, after finding potent immunosuppressant activity in an alkaloidal fraction of a rhizome extract, Yamahara and co-workers undertook a bioassay-guided fractionation.69 The assay, based on inhibition of anti-sheep erythrocyte (STBC)-plaque forming cell (PFC) formation in mice spleen cells, indicated that the activity resided in four known dimeric thioalkaloids, 6-hydroxythiobinupharidine 189, 6,6*-dihydroxythiobinupharidine 190, 6-hydroxythionuphlutine B 191 and 6*-hydroxythionuphlutine B 192.Also isolated were five biologically inactive alkaloids, neothiobinupharidine 193, nupharidine 194, deoxynupharidine 195, 7-epideoxynupharidine 196 and nupharolutine 197.In order to determine the structural requirements for immunosuppressive activity, the nine alkaloids and three synthetic analogues 198–200 were separately assayed in vitro for PFC inhibition. Only dimeric compounds possessing hydroxy groups in the quinolizidine ring showed significant activity. The eVect of hydroxy groups appears to be cumulative, since the two compounds with two hydroxy groups, 190 and 200, were also more cytotoxic to mice spleen cells at a concentration of 10"6 mol dm"3.Other workers have demonstrated an insecticidal eVect for (")-7-epideoxynupharidine 196 against both larvae and adults of the fly Drosophila melanogaster.70 6,6*-Dihydroxythiobinupharidine 190 and nupharolutine 197 have also been isolated as major alkaloids from N. lutea ssp. macrophyllum.71 Both compounds were fully characterised by means of spectroscopic techniques, and NMR spectroscopic studies in particular were used for confirming the relative stereochemistries and preferred conformations as shown in the diagrams 210 and 202.The crude alkaloidal mixture from the plant strongly inhibited the growth of lettuce seedling radicles and hypocotyls. The eVect was due principally to 6,6*- dihydroxythiobinupharidine, which, when tested on its own, reduced radicle growth to more than 30% of the control at a concentration of about 2 ppm.Pure nupharolutine showed no allelopathic activity. 10 Alkaloids of the lupinine–cytisine–sparteine–matrine– Ormosia group 10.1 Occurrence, analysis and biological studies Several new lupin alkaloids were isolated and characterised during the period covered by this review. However, an even greater number of ostensibly new alkaloids were tentatively identified in plant extracts by means of GC-MS analysis only. Table 1 contains a list of the new compounds detected (with varying levels of confidence) in, or isolated from, plants belonging to the Leguminosae.72–84 New sources of known alkaloids are also listed; as usual, however, alkaloids previously recorded in a species are not included in the Table, even though they are frequently the major metabolites.Lupanine 203 and other alkaloids of L. albus can be extracted from plant material with supercritical carbon dioxide.85 A simple new procedure for the isolation of angustifoline 204 from extracts of Lupinus angustifolius involves partial precipitation of (+)-lupanine perchlorate, formation of N-trichloroacetyl derivatives of the residual secondary amine bases, crystallisation of N-trichloroacetylangustifoline from the mixture, and basic hydrolysis to yield the pure crystalline alkaloid.77 Solvent extraction from pH-controlled solutions (pH range 7 to 13) also yielded relatively pure angustifoline, and could be applied to the isolation of epilupinine 205 and multiflorine 206 from extracts of L.atlanticus. Both reversed-phase HPLC and normal-phase TLC/ densitometry methods for simultaneously assaying the principal alkaloids of Caulophyllum thalictroides (anagyrine 207, baptifoline 208 and N-methylcytisine 209, and the aporphine alkaloid magnoflorine) have proved to be simple and reliable. 86 The two methods gave comparable results, though the HPLC method was about 100 times more sensitive. EYcient electrochemical analysis of amines, including sparteine, with graphite electrodes has been shown to depend on prior chemical modification of the surface.87 A decrease in quinolizidine alkaloid production by seedlings of Lupinus albus has been observed, especially in the roots, in the presence of a biotic elicitor (a fungal cell wall extract); with an abiotic elicitor (2 mM CuCl2), alkaloid production was increased slightly.88 Chromium ion, however, inhibited in vivo acylation of 13-hydroxylupanine 210.89 Lupanine can function as the sole carbon and energy source for a range of Gramnegative bacterial strains, a finding that has implications for the microbial debittering of lupin crops used as dietary sources of protein.90 A study on the subcellular localisation of two acyltransferases responsible for the esterification of hydroxylated lupin alkaloids has shown that they are not located in chloroplasts, where de novo synthesis of the quinolizidine alkaloid skeleton is supposed to occur.91 13·-Hydroxymultiflorine/13·- hydroxylupanine O-tigloylase activity was found in mitochondrial fractions from hypocotyls of Lupinus albus and L.hirsutus seedlings, while epilupinine O-p-coumaroyltransferase was concentrated in yet a diVerent subcellular compartment. A hypothesis for the intracellular transport of quinolizidine alkaloids was presented on the basis of the findings. The discovery that (")-sparteine 211 can act as a scavenger of superoxide may indicate a role for this alkaloid in the protection of plants against oxidative damage.92 However, O N N2 O PhSO2 N +O O PhSO2 SO2Ph N O HO Me SO2Ph N O Me SO2Ph MeO N O Me HO N Me HO 84% vi 62% ii, iii iv, v 87% i 51% 169 Ipalbidine 174 173 171 – 172 170 Scheme 16 Reagents: i, Rh2(OAc)4, C6H6, 80 )C; ii, Tf2NPh, Et3N, CH2Cl2, 0)C to rt; iii, p-MeOC6H4SnBu3, Pd(Ph3P)4, LiCl, N-methylpyrrolidin-2-one, 150 )C; iv, Raney Ni, EtOH, 65 )C; v, HBr (48%), reflux; v, LiAlH4, AlCl3, THF, reflux 582 Natural Product Reports, 1998neither it nor cytisine were substrates for peroxidases, which, like the alkaloids, are located in plant epidermal tissue.Although the cytochrome P4502D6-induced oxidation of (")- sparteine in man has been well explored in the past, a new investigation with [17,17-3H2]-sparteine as substrate has shown that 17-oxosparteine 212, previously overlooked as a urinary metabolite, is produced along with the previously detected 2- and 5-dehydrosparteines.93 10.2 Chemotaxonomic studies A survey of the alkaloidal constituents of the American genera Brongniartia and Harpalyce, undertaken to provide further evidence for the concept of the tribe Brongniartieae in the family Leguminosae (Fabaceae), has revealed significant qualitative diVerences in the alkaloidal profiles of the various species examined.74 All but two of the six Brongniartia species included in the survey and all three Harpalyce species showed a typical pattern of ·-pyridone alkaloids, with cytisine 213 and related alkaloids such as anagyrine 207 and baptifoline 208 as major constituents.Three species (B. discolor, B. lupinoides and B. sousae) also accumulated ormosanine-type alkaloids, while H. formosa was unusual in producing epilupinine and a suspected dehydroepilupinine as additional major products. In the two anomalous species (B. flava and B. vazquezii), lupanine, hydroxylated lupanines and esterified derivatives of the latter (especially the rare alkaloid oroboidine 214) were predominant, raising doubts about what the authors termed the ‘generic circumscription’ of the genus.Dicraeopetalum stipulare, a leguminous tree that grows in south-eastern Ethiopia and neighbouring parts of Somalia and Kenya, belongs to a monotypic genus of the tribe Sophoreae. A study of the chemical constituents of its leaves76 – erroneously claimed to be the first on this species (cf. ref. 94) – was performed with the aid of GLC-MS.The alkaloid profile, dominated by lupanine derivatives and ·-pyridone alkaloids, proved to be similar to that of the genus Acosmium (in which the species was formerly included), but diVered in not containing pentacyclic alkaloids. Two new alkaloids detected in trace amounts were tentatively identified from O MeO MeO OCOPh OMe OMe OMe O MeO MeO O OMe OMe OMe O MeO O OMe MeO OMe OMe N MeO MeO CN OMe OMe N MeO MeO OMe OMe N MeO MeO OCOPh OMe OMe N MeO MeO OMe OMe N MeO MeO OMe OMe N+ MeO MeO OMe OMe N MeO MeO OMe N MeO MeO OMe N MeO MeO OMe N MeO MeO OMe 74% xiii 47% from 182 xii ix–xi –OMs 99% viii 65% v–vii iv 80% 77% ii, iii 91% i 188 Cryptopleurine 187 Julandine 186 Antofine 185 176 Tylophorine 175 Septicine 184 183 182 181 180 179 178 177 Scheme 17 Reagents: i, Yb(fod)3, ClCH2CH2Cl, reflux; ii, DIBAL, CH2Cl2, "78 )C, then MeOH, aq.NaHCO3; iii, PhCOCl, pyridine, CH2Cl2, rt; iv, NH2OH·HCl,MeCN, reflux; v, aq.NaOH (1%), MeOH, rt; vi, SOCl2, pyridine, C6H6, reflux; vii, ButOK, THF, reflux; viii, LiCN, AcOH, DMF, 140 )C; ix, conc. HCl, dioxan, 40 )C; x, LiAlH4, THF, rt to reflux; xi, MsCl, Et3N, CH2Cl2, 0 )C; xii, NaBH4, EtOH, rt to reflux; xiii, VOF3, CH2Cl2, MeCN, 0 )C, then TFA Michael: Indolizidine and quinolizidine alkaloids 583their MS fragmentation patterns as isomers of 10-oxolupanine 215 and 17-oxolupanine 216. The rare alkaloid 6‚-hydroxylupanine 217, a putative precursor of the ·-pyridone alkaloids, was detected as a trace component in both D.stipulare and in Platycelphium vöense,82 a representative of another monotypic genus of the Sophoreae from a similar geographical region. The dominant alkaloids in the latter species were also ·-pyridones and lupanine derivatives. Systematic surveys of the alkaloid contents of plant species as a function of geographical origin, seasonal changes and organs of localisation are uncommon. A good example of this type of study is devoted to the flowering shrub Cytisus scoparius, specimens of which were harvested at diVerent stages of growth from diVerent localities in Germany, Russia, Italy and France and analysed for their quinolizidine alkaloids by means of capillary GLC.75 Of the 34 alkaloids detected, 20 could be identified unambiguously.The remainder, apparently derivatives of lupanine 203, sparteine 211 and rhombifoline 218 (some of them indubitably new alkaloids), were present only as trace components.Sparteine and its derivatives tended to be concentrated in shoots and flowers, while lupanine-type alkaloids were accumulated mainly in roots, pods and seeds. Geographical origin made little diVerence to the observed alkaloid patterns; far more significant was the state of maturity of the plants. Seeds, for example, invariably showed more complex alkaloidal profiles than young shoots, and the total alkaloid content increased substantially at the end of the vegetation period.The seed alkaloids of Lupinus tassilicus, which is found in the Central Sahara of Algeria, are qualitatively similar to those of related Old World rough-seeded species.79 The major difference was that species originating in desert and arid regions of Africa (L. tassilicus, L. atlanticus and L. digitatus) accumulated epilupinine 205 as the major alkaloid, while those distributed in the Mediterranean region (L. pilosus, L. palaestinus and a questionable specimen of L.digitatus) were richer in multiflorine 206. In other respects the findings support the accepted view that rough-seeded lupins originate from a common ancestral genetic pool. However, it should be mentioned that the taxonomy of many Lupinus species appears to be in disarray; some of the above-named plants, which were collected from diVerent geographical locations, may well be identical. The inclusion of Templetonia incana, a shrub widely found in the deserts of Western Australia, in the genus Templetonia has been in dispute since it was first described.Its recently determined alkaloidal profile84 has now provided added support for excluding it from the genus. It lacks ormosanine 219 and ormosanine-type alkaloids, which are typical Templetonia constituents, but contains pyridone alkaloids such as cytisine as major constituents, as well as smaller amounts of bicyclic alkaloids. This pattern is reminiscent of that found in the genera Lamprolobium, Harpalyce and (more recently) Plagiocarpus, and gives support to the tribal concept of Brongniartieae, in which these genera appear to belong.However, unlike another disputed Templetonia species, T. biloba (cf. ref. 7h), T. incana contains no tetrahydrocytisine derivatives, thereby adding to the taxonomic ambiguity. 10.3 Structural and spectroscopic studies Naturally occurring methoxylated quinolizidine alkaloids are suYciently uncommon that the isolation of no fewer than three such compounds from the bark of the Central and South American tree Acosmium panamense72 is noteworthy. 4·-Hydroxy-13‚-methoxylupanine 220 and 3‚,4·-dihydroxy- 13‚-methoxylupanine 221 are new natural products whose structures were established by means of spectroscopic measurements, notably a suite of NMR spectroscopic experiments that confirmed the carbon and heteronuclear connectivities. The third compound, 13‚-methoxylupanine 222 (also known as 13-epimethoxylupanine), has previously been isolated from other plant sources as well as in an earlier phytochemical study on A.panamense; however, this is the first time that full NMR spectroscopic data have been reported for it. Since Acosmium is acknowledged to be the most primitive taxon of the Leguminosae to biosynthesise quinolizidine alkaloids, the 13-methoxy group may prove to be a valuable marker for chemosystematic studies. A new alkaloid isolated from the seeds of Lupinus termis has been tentatively identified as ƒ3-8-oxo-13-propionyloxyspartalupine 223 on the basis of spectroscopic data.80 The salient features were the presence of Bohlmann bands in the IR spectrum (indicating a trans-fused quinolizidine ring), ketonic (1720 cm"1, ‰C 207) and ester (1690 cm"1, ‰C 167) carbonyl groups, and olefinic carbons (‰C 131, 129; ‰H 7.65, 7.48).If the proposed structure is indeed correct, it establishes at least two precedents, since neither 8-oxosparteines nor ƒ3-sparteines in which the alkene unit is unconjugated with a carbonyl group at C-2 have been found previously as natural products. The choice of the parent name ‘spartalupine’ (a synonym for ‚-isosparteine) is also misleading, since the stereochemistry of the new alkaloid could not be established.(+)-Sparteine N(16)-oxide 224, isolated as a crystalline compound from aerial parts of Lygos raetam var. sarcocarpa, has been claimed as a new natural product.81 In fact, it had previously been obtained from Ammodendron karelinii, but 201 194 193 191 R = H; R¢ = OH 192 R = OH; R¢ = H 199 R = R¢ = H 200 R = R¢ = OH 189 R = H; R¢ = OH 190 R = R¢ = OH 198 R = R¢ = H 195 R = H; R¢ = Me 196 R = Me; R¢ = H 197 R = OH; R¢ = Me 202 N S N H H Me Me R R¢ N S H Me R N O R¢ H Me S N O H Me N O H Me N+ H Me Me O– N H Me R¢ R N OH HO S N Fur Fur N Fur OH O O O O O 584 Natural Product Reports, 1998Table 1 Isolation and detection of alkaloids of the lupinine–cytisine–sparteine–matrine–Ormosia groupa Species Alkaloid Ref.Acosmium panamense 3‚,4·-Dihydroxy-13‚-methoxylupanineb 221 4·-Hydroxy-13‚-methoxylupanineb 220 13‚-Methoxylupanine 222 72 Argyrolobium uniflorum N-Acetylcytisine 11-Allylcytisine Baptifoline 208 Camoensidine isomerb (tentative) Dehydrocytisine I and II 5,6-Dehydrolupanine 11,12-Dehydrosparteine and isomers N-Formylcytisine ·-Isolupanine ‚-Iso-17-oxosparteine ‚-Isosparteine 252 Lupanine 203 Lupinine 249 Lusitanine N-Methoxycarbonylcytisine 17-Oxosparteine 212 Retamine Rhombifoline 218 Sparteine 211 Thermopsine Tinctorine 73 Brongniartia spp.c N-Acetylcytisine Anagyrine 207 Aphylline Baptifoline Cytisine 213 Dehydroaphyllineb (tentative) 5,6-Dehydrolupanine 11,12-Dehydrosparteine Epibaptifoline N-Formylcytisine Homo-6-epipodopetaline (tentative) Homopiptanthine 13-Hydroxylupanine 210 Hydroxyoroboidineb (tentative, position uncertain) ·-Isolupanine ·-Isosparteine ‚-Isosparteine Lupanine Lusitanine N-Methylcytisine 209 Nuttalline Ormosanine 219 Oroboidine 214 Panamine and isomer (tentative) Rhombifoline 218 Sparteine 74 Cytisus scoparius (=Sarothamnus scoparius) 13· -Angeloyloxylupanine Aphylline 11,12-Dehydro-·-isolupanineb (tentative) 11,12-Dehydrolupanine 11,12-Dehydrosparteine and isomers Dihydrorhombifolineb (tentative) Dihydroxyoxolupanineb (tentative, position uncertain) 10,17-Dioxosparteine 3‚-Hydroxy-·-isolupanineb (tentative) 13-Hydroxy-·-isolupanine (tentative) Hydroxylupanineb (tentative, position uncertain) 3‚-Hydroxylupanine Hydroxyoxolupanineb (tentative, position uncertain) Hydroxysparteines (tentative, position uncertain) Hydroxytetrahydrorhombifoline (tentative, position uncertain) Multiflorine 206 13-Oxolupanineb (tentative) 75 Michael: Indolizidine and quinolizidine alkaloids 585Table 1 Continued Species Alkaloid Ref. 17-Oxolupanine Tetrahydrorhombifoline 75 Dicraeopetalum stipulare 6‚-Hydroxylupanine 10-Oxolupanineb (tentative) 215 17-Oxolupanine (tentative) 216 76 Harpalyce spp.d N-Acetylcytisine 11-Allylcytisine Anagyrine Baptifoline Cytisine Dehydroepilupinine (tentative) 5,6-Dehydrolupanine Epibaptifoline Epilupinine N-Formylcytisine ·-Isolupanine Lupanine N-Methoxycarbonylcytisine N-Methylcytisine Rhombifoline Tetrahydrocytisine Tinctorine 74 Lupinus angustifolius cv.Fest Angustifoline 204 (+)-13-Hydroxylupanine Isoangustifoline (tentative) (+)-·-Isolupanine (+)-Lupanine 77 Lupinus princei 11·-Allylcytisine Epilupinine 205 ‚-Isosparteine Lupanine Lupinine N-Methylcytisine 78 Lupinus tassilicus and related speciese Anagyrine 5,6-Dehydrolupanine 11,12-Dehydrosparteine Epilupinine 13-Hydroxylupanine 13·-Hydroxymultiflorine ‚-Isosparteine Lupanine Multiflorine 11,12-Seco-12,13-didehydromultiflorine Sparteine 79 Lupinus termis ƒ3-8-Oxo-13-propionyloxyspartalupineb 223 17-Oxosparteine 212 80 Lygos raetam var.sarcocarpa (+)-Aphylline (")-5,6-Dehydrolupanine (")-N-Formylcytisine (+)-Sparteine N(16)-oxide 224 81 Platycelphium vo�ense N-Ethylcytisine 6‚-Hydroxylupanine 217 Thermopsine 82 Sophora tonkinensis (+)-Allomatrine (")-N-Formylcytisine (")-14‚-Hydroxymatrineb 225 (+)-5·-Hydroxysophocarpine (+)-Lehmannine (+)-Leontalbinine (")-N-Methylcytisine (")-Sophocarpine 83 Templetonia incana N-Acetylcytisine Acetylepibaptifolineb? Aloperine 231 Anagyrine Baptifoline Cytisine 84 586 Natural Product Reports, 1998was reported under an alternative name, pachycarpine N(16)- oxide.95 Its structure in the present case was confirmed by analysis of spectroscopic data, by catalytic hydrogenation over platinum oxide to yield (+)-sparteine ent-211, and by comparison with a specimen made by oxidation of (")-lupanine ent-203 to its N-oxide followed by reduction of the amide group with sodium borohydride.An extract from the dried roots of Sophora tonkinensis (the Chinese drug ‘San-Zi-Gong’) has yielded 11 known lupin alkaloids together with a minor new compound, (")-14‚- hydroxymatrine 225.83 Once the gross structure and relative stereochemistry had been inferred from the spectroscopic data, a direct comparison was undertaken with a synthetic sample made by hydroxylation of (+)-matrine 226.This reaction involved treating the enolate of matrine (LDA, THF, 0 )C) with gaseous oxygen followed by reductive work-up with sodium bisulfite, acetylation of the resulting mixture of diastereoisomers, and chromatographic separation.The orientations of the acetoxy groups in the two products were readily established by analysis of coupling constants. Deacetylation of the equatorial (14‚) acetoxy isomer yielded a product identical with natural (")-14‚-hydroxymatrine. Sophoridine N-oxide 227, a related alkaloid isolated from Mongolian Sophora alopecuroides, was characterised by NMR spectroscopy, by deoxygenation to sophoridine, and by comparison with two isomeric synthetic N-oxides prepared by oxidation of sophoridine.96 The pharmaceutical properties of many matrine alkaloids are often reported in the inaccessible Chinese literature; much of this research has now been summarised in a brief review, also in Chinese.97 A general strategy for determining the structures of lupin alkaloids by means of NMR spectroscopy has been exempli- fied with 3‚-hydroxylupanine 228, a major alkaloid of Lupinumutabilis.98 The recommended sequence of experiments is a natural abundance 13C–13C correlation to reconstruct the carbon backbone, followed by C–H correlation for unequivocal assignment of protons; H–H double-quantum correlation experiments provide a final check of the selfconsistency of the assignments.While demanding in terms of Table 1 Continued Species Alkaloid Ref. 5,6-Dehydrolupanine 11,12-Dehydrosparteine Epibaptifoline 13-Epihydroxysparteine Epilupinine N-Formylcytisine ·-Isolupanine ‚-Isosparteine Lupanine Lupinine Lusitanine N-Methoxycarbonylcytisine N-Methylcytisine Rhombifoline Sparteine 84 aOnly new alkaloids and new records for a given species are listed in the Table.Structures of most known alkaloids may be found in previous reviews in this series. bNew alkaloids. cBrongniartia discolor, B. flava, B. intermedia, B. lupinoides, B. sousae, B. vazquezii. dHarpalyce brasiliana, H. formosa, H. pringlei. eLupinus atlanticus, L. digitatus, L. palaestinus, L. pilosus, L. pilosus ssp. tassilicus, L.tassilicus. 209 R = Me 213 Cytisine R = H 206 Multiflorine 207 (–)-Anagyrine R = H 208 Baptifoline R = OH 204 Angustifoline 205 Epilupinine 211 (–)-Sparteine X = 2H 212 X = O 203 (+)-Lupanine R = H 210 R = OH N OH H N N H H H O N N H H O R N N H O R N N H H O N N R O N N H H X N N OH H O N N O N N H H O O N O H N N H H O X Y N N H H H H NH H 219 Ormosanine 215 X = O; Y = 2H 216 X = 2H; Y = O 214 Oroboidine 218 Rhombifoline 217 Michael: Indolizidine and quinolizidine alkaloids 587availability of material and spectrometer specifications, the general approach is claimed to be error-free, and should have value for compiling reliable data bases for this large family of alkaloids.Continuing 1H and 13C NMR spectroscopic investigations of the conformational equilibria in tetracyclic bisquinolizidine lupin alkaloids have resulted in the quantitative determination of the solution conformations of twelve alkaloids and two of their salts.99 By analysing the 13C signals for C-12 and C-14 as well as 3J7-17‚ proton–proton coupling constants, the authors were able to quantify the boat–chair equilibrium in ring C (as shown in 229&230 for sparteine), and to calculate equilibrium constants and free energies of conformational equilibrium for the systems.The analysis of 3J7-17‚ proton–proton coupling constants was also useful for assessing the conformational equilibrium in related tricyclic alkaloids. This article contains useful speculations on the structural factors influencing the equilibria, the most important of which appears to be the trans or cis ring fusion of the quinolizidine units.Related studies on the stereochemistry of 2-(p-tolyl)sparteine, its perchlorate salt and some deuterated analogues (determined with the aid of IR and 13C NMR spectroscopy),100 and on 2,13-dioxo-11·- sparteine101 have also been published recently. The structures of the complexes formed between (")-sparteine 211 and lithium hexamethyldisilazide102 and lithium diisopropylamide103 in hydrocarbon solvents have been investigated by means of NMR spectroscopy.X-Ray diVraction studies have shown that the four piperidine rings of 17-oxolupanine 216 have half-chair, chair, ‘sofa’ and chair conformations respectively, with the ring junction between rings A and B being quasi-trans and that between rings C and D quasi-cis.104 Crystallographic studies have also been undertaken on several synthetic analogues of lupin alkaloids, including the perchlorates of 17‚- isopropylsparteine and 17‚-isopropyllupanine105 and episparteine N(16)-oxide.106 Single crystal X-ray analysis of the dihydrochloride monohydrate salt of the rare lupin alkaloid aloperine has been used to establish the 6R,7R,9R,11S absolute stereochemistry depicted in 231;107 the report also presented interesting syntheses of the two aloperine stereoisomers 232 and 233. 10.4 Synthesis and other chemical studies New derivatives of cytisine 213 prepared by simple chemical transformations of the parent alkaloid include the ester 234, the corresponding acid 235, the hydrazide 236 and the amide 237.The crystal structure of ester 234 was also reported.108 An interesting organoyttrium-mediated cyclisation of a 1,6- diene forms the centrepiece of a new synthesis of (&)- epilupinine, rac-205, byMolander and Nichols109 (Scheme 18). Five simple steps suYced to convert (&)-piperidine-2- methanol 238 into 1-allyl-2-vinylpiperidine 239.Treating the tetrahydrofuran complex of methylbis(tetramethylcyclopentadienyl) yttrium (Cp*2YMe·THF) with methylphenylsilane produced the active organometallic hydride (‘Cp*2YH’) in situ. This reagent, used catalytically (5 mol %), presumably acts on diene 239 by initially hydrometallating the less hindered allylic double bond, after which cyclisation occurs by intramolecular carbometallation of the vinyl unit. The catalytic cycle is completed by replacing the organometallic fragment in the probable intermediate 240 by the silyl moiety to yield 241. It is noteworthy that complexation of the metal by the tertiary amine does not inhibit the insertion of the second alkene unit.Furthermore, the exclusive production of the equatorial metallomethyl substituent in 240 is presumably a consequence of the steric bulk of the cyclopentadienyl ligands. The reaction was completed in an hour at ambient temperature, and silane 241 could be isolated in 84% yield.However, it proved preferable to oxidise it directly to the target alcohol 205 by using a recently developed procedure110 involving gentle warming with tert-butyl hydroperoxide, caesium fluoride and excess potassium hydride in dimethylformamide. The yield from diene 239 to epilupinine 205 was on average about 57%. A readily prepared dimer of 1,2-dehydropiperidine 242, tetrahydroanabasine 243, has been used as the starting material in syntheses of several lupin alkaloids.111 When its O-methyl oxime 244 was treated with ortho-quinone 245 – a model for topaquinone, which is the cofactor present in copper-containing amine oxidases – the enamine-containing quinolizidine 246 could be isolated in 45% yield (Scheme 19).This rather unstable compound proved to be a versatile intermediate from which to prepare several target alkaloids. For instance, reduction with sodium borohydride yielded oxime ether 247 (89%), after which reduction with lithium aluminium hydride followed by acetylation aVorded N-acetyllupinamine 248 (84%).Alternatively, mild hydrolysis of 247 with ozone and trifluoroacetic acid followed by further reduction gave lupinine 249 (54%) or lupinine–epilupinine mixtures if the hydrolysis was performed under more vigorous conditions with titanium trichloride. Treating the quinolizidine enamine 246 with dehydropiperidine gave a tricyclic oxime ether 250 (80%), mild hydrolysis of which was followed by reduction with sodium cyanoborohydride to aVord sparteine 211 in 21% yield, presumably via the bis(iminium) system 251. Once again, more vigorous hydrolysis conditions brought N N O H H H H O N N O H R H H H N N H H O HO N N H H O N N H H O OMe R1 R2 N O O O N 220 R1 = H; R2 = OH 221 R1 = R2 = OH 222 R1 = R2 = H 224 (+)-Sparteine N-16-oxide 228 225 R = OH 226 Matrine R = H 227 223 N N H N N N N H H H N N H H H N N CH2COR O N N H H H 234 R = OMe 235 R = OH 236 R = NHNH2 237 R = NHCH2CO2Me 233 232 231 Aloperine 230 229 588 Natural Product Reports, 1998about some epimerisation, and led to the isolation of a mixture of sparteine (9%) and ‚-isosparteine 252 (11%).These syntheses have been reprised in a published conference paper together with related syntheses of some indoloquinolizidine alkaloids.112 When the imidosulfoxide 253 was treated with acetic anhydride, the ensuing thionium ion that resulted from Pummerer rearrangement was captured intramolecularly to give the mesoionic dipolar intermediate 254.By trapping this intermediate with methyl acrylate, Kuethe and Padwa devised a short new formal synthesis of lupinine as shown in Scheme 20.113 The cycloadduct 255 was readily cleaved with boron trifluoride to yield the ethylthio-substituted bicyclic pyridone 256 (62%). Hydrogenolysis with Raney nickel aVorded 257 (85%), conversion of which into racemic lupinine rac-249 was reported nearly half a century ago.114 Alternatively, oxidation of cycloadduct 255 gave sulfone 258, which was converted into the triflate 259 in two steps.The tricyclic derivative 260 resulted from Stille cross-coupling of 259 with 2-tributylstannylpyridine. Subsequent catalytic hydrogenation on platinum dioxide followed by base-induced equilibration yielded the known quinolizidinone 261 (85%). This alternative sequence of reactions converged with a classic synthesis of (&)-anagyrine rac-207 published in 1972.115 10.5 Enantioselective transformations mediated by (")-sparteine A timely review by Beak and his co-workers on synthetic transformations involving lithiation followed by electrophilic substitution gives a good summary of enantioselective reactions induced by (")-sparteine–alkyllithium complexes and the mechanisms by which these processes occur.116 In brief, asymmetric deprotonation appears to be the operative pathway when lithiation/substitution reactions are performed · to nitrogen in Boc-protected amines.However, with ‚-lithiated carboxamides in which the deprotonated site is also benzylic, the alkaloid 211 can coordinate to the configurationally labile and essentially racemic lithiated intermediates to give diastereoisomeric complexes that subsequently undergo asymmetric electrophilic substitution in post-deprotonation processes entailing either dynamic thermodynamic or kinetic resolution under carefully controlled conditions.Evidence for the latter pathway has been presented in an article that also introduces improvements in enantioselectivity by the novel (but logically rationalised) expedient of using much less than stoichiometric amounts of electrophile in combination with strict temperature control.117 An interesting application of dynamic kinetic resolution is shown in Scheme 21, in which a more reactive diastereoisomeric (")-sparteine–amide complex is selectively captured by one electrophile, after which a diVerent electrophile is used to trap the less reactive complex in 241 51%-62% (2 steps) vii 240 vi 239 238 59% i–v rac-205 Epilupinine N H OH N H Y N N H Si Ph Me H N H OH Scheme 18 Reagents: i, (Boc)2O, Et3N, CH2Cl2, rt; ii, pyridine·SO3, DMSO, Et3N, CH2Cl2, 0 )C; iii, Ph3P=CH2, Et2O, rt; iv, CF3CO2H, CH2Cl2, rt; v, H2C=CHCH2Br, K2CO3, THF, reflux; vi, (Me4Cp)2YMe·THF (5 mol %), MePhSiH2, cyclohexane, rt; vii, ButOOH, DMF, KH, CsF, 0 )C to 45 )C N N N H H N N OMe H H NH2 O O N NHCOMe H N N OMe H N N OMe H N N OMe H N H N+ H N+ H N H N H N H N H N OH H 252 b-Isosparteine 211 Sparteine 251 21% ix 250 246 247 248 54% 85% iv, v 80% vii 89% iii 245 244 243 242 98% i 45% ii viii vi, iii 249 Lupinine Scheme 19 Reagents: i, MeONH2, H2O, MeOH, rt; ii, MeOH, THF, 0 )C to rt; iii, NaBH4, MeOH, rt; iv, LiAlH4, THF, reflux; v, Ac2O, EtOAc, reflux; vi, CF3CO2H, MeOH, "60 )C, then O3; vii, compound 242, NaOAc, MeOH, rt; viii, conc.HCl, MeOH, "50 )C, then O3, then Me2S; ix, NaBH3CN, NaOAc, AcOH, rt Michael: Indolizidine and quinolizidine alkaloids 589a one-flask sequence. Dynamic thermodynamic resolution has been expanded upon in another article,118 which also details 6Li, 13C and 15N NMR spectroscopic experiments showing that the benzylic lithium is complexed by the amide’s nitrogen atom rather than oxygen, as depicted in 262 and 263. The spectroscopic experiments proved that while (")-sparteine can complex with 262 in solution, 263 is too crowded to be complexed by the sterically demanding ligand – a finding that ties in with the complete lack of asymmetric induction in (")-sparteine-mediated substitution reactions of 263.Further research from the Beak group shows how the changes can be rung in the enantioselective synthesis of ·-, ‚- and „-aryl amino acids and esters from N-Boc-N-benzylanilines,119 and in the (")-sparteine-promoted lithiation–substitution reactions of N-Boc-N-(p-methoxyphenyl)allylamines120 and N-Boc-Nalkylcyclopropylamines. 121 A short review by Snieckus ‘published’ as part of an electronic conference includes the use of (")-sparteinemediated lithiation–substitution for enantioselective reaction at the benzylic site in carbamates derived from o-ethylphenols, and for enantioselective ring metallation of ferrocenylcarboxamides possessing planar chirality.122 Other recent examples of lithiation–substitution reactions assisted by (")-sparteine include the enantioselective synthesis of ‚-amino alcohols from 2-[N-(diphenylmethyleneamino)]alkyl carbamates;123 transmetallation of ·-lithiated N-Boc amines with copper(I) followed by acylation (the enantioselectivity of the reaction was, surprisingly, not specified);124 synthesis of enantioenriched (Z,E)-1,2,5-triphenylphospholane oxide by deprotonation of the meso-(E,E) isomer followed by reprotonation;125 and diastereoselective or enantioselective synthesis of ferrocenyl mono- and di-phosphines by asymmetric o-lithiation of 1,1*- diamidoferrocenes followed by treatment with chlorodiphenylphosphane. 126 (")-Sparteine was a good enough base to induce the formation of a 2H-azirinecarboxylate from the O-tosyloxime of ethyl acetoacetate (a Neber reaction), but the reaction was not enantioselective.127 Examples of (")-sparteine-mediated asymmetric polymerisation of acrylates and other activated alkenes continue to accumulate, and do not merit individual mention here.However, since it is still unusual to encounter other enantioselective nucleophilic additions performed in the presence of the alkaloid, a brief selection follows. Enantiomerically enriched 2-arylalkanoic acids were produced by adding alkyllithium reagents to styrenes and trapping the chelated benzyllithium intermediates with carbon dioxide; the best enantiomeric excesses (ca. 72%) were obtained at "94 )C with o-methoxystyrene as substrate and cumene as solvent.128 The addition of organolithium reagents to chromium tricarbonyl complexes of 2-phenyloxazolines, however, was less enantioselective with (")-sparteine as ligand than with simple chiral ethers.129 The (")-sparteine–ethylmagnesium bromide complex catalysed a Tishchenko reaction of racemic 2-phenylpropanal (presumably initiated by addition to the carbonyl group followed by oligomerisation of the aldehyde); (S)-2-phenylpropyl (R)-2-phenylpropanoate, the major N O O CO2Me SO2Et N O O CO2Me SEt N O+ O SEt N S Et O O O N O CO2Me OTf N O CO2Me SEt N O CO2Me N H OH N O CO2Me N N O CO2Me N H N H N O rac-207 Anagyrine 261 260 Ref. 115 85% vii, viii rac-249 Lupinine 257 256 259 Ref. 114 85% iii 253 254 – 255 258 91% iv 61% i ii 62% ii, v 81% vi 70% Scheme 20 Reagents: i, Ac2O, H2C=CHCO2Me, p-TsOH, toluene, 90 )C; ii, BF3·Et2O, CH2Cl2, rt; iii, Raney Ni, EtOH, reflux; iv, RuCl3 (cat.), NaIO4, MeCN–dioxan, rt; v, Tf2NPh, Et3N, CH2Cl2, rt; vi, 2-tributylstannylpyridine, Pd2(dba)3, LiCl, (2-furyl)3P, THF, reflux, then KF, H2O; vii, H2 (75 psi), PtO2, MeOH, rt; viii, NaOMe, MeOH 63% yield 44% ee 78% yield 52% ee i–iv + NH O NH O NH O TMS Scheme 21 Reagents: i, BusLi, "25 )C, 2 h, then "78 )C, 15 min; ii, (")-sparteine 211, precooled to "78 )C; iii, TMSCl (0.5 equiv.), 90 min, "78 )C; iv, H2C=CHCH2Br (excess), 2 h, "78 )C 262 R = Me 263 R = Pri OLi N R Li L L 590 Natural Product Reports, 1998product, was obtained in an enantiomeric excess of 67%.130 A similar reaction on terephthalaldehyde yielded random estercontaining copolymers.131 Finally, 3,3-dialkylcyclopropenes underwent polymerisation with (Á3-allyl)[(")-sparteine]- palladium(II) hexafluoroantimonate as catalyst to give polycyclopropyls with 1,2-cis-linked repeating units and a reasonable degree of tacticity (estimated from 13C NMR spectroscopic signals as 65% meso triads).132 (")-Sparteine has served as a chiral modifier in various oxidation processes.Moderate to good levels of enantioselectivity (12–73% ee) were observed when 2,2-dimethylchromenes were epoxidised with iodosobenzene in the presence of the alkaloid and catalytic amounts of achiral manganese(III)- salen.133 Far more noteworthy are two reports on the electrochemical oxidation of alcohols on graphite felt electrodes modified with the stable free radical TEMPO (2,2,6,6- tetramethylpiperidine-1-oxyl); the reactions showed remarkable enantioselectivity and current eYciency when (")- sparteine was present in the electrolyte (0.2 M sodium perchlorate in acetonitrile).134,135 Only the S enantiomers of racemic secondary alcohols (including 1-phenylethanol, 2-phenylcyclohexanol, octan-2-ol, and cyclohex-2-enol) were converted into the corresponding ketones, while the unconsumed (R)-alcohols were recovered in enantiomeric excesses of between 99.4 and 99.8%.134 Selective mono-oxidation of three methyl-substituted diols [pentane-1,4-diol, (S)-2- methylbutane-1,4-diol and 3-methylpentane-1,5-diol] was equally eVective, yielding lactones [(S)-5-methyl-„- butyrolactone, (S)-4-methyl-„-butyrolactone and (S)-4- methyl-‰-valerolactone, respectively] in better than 98% ee.135 Sparteine N-oxides have been used to bring about kinetic resolution of the racemic iron tricarbonyl complexes of eucarvone by inducing an oxidative ligand substitution in only one of the enantiomers (CO replaced by triphenylphosphine); the most eVective alkaloid for this process was 17-oxosparteine N(16)-oxide, the enantiomeric excess for the resolution being estimated as 70%.136 11 Alkaloids from marine sources An unidentified sponge of the genus Stelletta collected oV Keomun Island, Korea yielded an extract exhibiting moderate antifungal activity and toxicity in the brine shrimp assay.137 One of the components isolated after reversed-phase chromatography was the new indolizidine alkaloid (")-stellettamide B 264, the nucleus of which appears to be the same as in stellettamide A 265, previously isolated from a Japanese specimen of Stelletta sp.138 Two-dimensional and long-range NMR spectroscopic experiments served to define the bicyclic system fully, and revealed the relative configuration as 1S*,4S*,8aR*.The spin system along the length of the chain in the norsesquiterpene unit was determined with the aid of TOCSY data, and the 2*E,4*E geometry was assigned with NOESY correlations.Oxidation with sodium periodate in the presence of ruthenium trichloride yielded (S)-(+)-2- methylglutaric acid, thus proving the absolute stereochemistry at C-13*. Curiously, the authors have incorrectly drawn the alkaloid with 13*R absolute configuration; the correct sidechain absolute stereochemistry is shown in 264, although obviously the indolizidine stereochemistry is only relative. The counter-ion was determined as chloride by an energydispersive spectroscopic experiment performed on a scanning electron microscope.Stellettamide B was fairly active against Candida albicans at a concentration of 25 Ïg ml"1, and it was also found to cleave both single- and double-stranded RNA at a concentration of 50 Ïg ml"1. A further unusual macrocyclic diamine alkaloid, (")- isosaraine-3 266, has been isolated from the Mediterranean sponge Reniera sarai.139 The structures and relative stereochemistries of this compound and the related alkaloid (")- saraine-3 267, until now only partially characterised (cf.ref. 7i), were elucidated with the aid of NMR spectroscopic measurements. In particular, the position of the double bond in the chain linking the two heterocyclic moieties was unequivocally determined by means of long-range HETCOR and TOCSY experiments, and its Z configuration was consistent with the observed 13C chemical shifts of the vinyl carbon atoms. The chain lengths were deduced with reasonable certainty from EIMS fragmentation patterns.Bohlmann bands at about 2760 and 2805 cm"1 provided evidence for a transfused quinolizidinone nucleus. The only residual uncertainty is in the relative stereochemistry at C-3 in the unsaturated piperidine ring; the proton at this position, however, is axially orientated. The absolute configurations of two related alkaloids, saraine-1 268 and saraine-2 269, have been determined by means of the Mosher method.139 Reduction of the alkaloids with sodium borohydride aVorded separable mixtures of epimeric 8-OH alcohols, the equatorial or axial orientations of the hydroxy groups being assigned with the aid of NMR experiments.Both R and S Mosher’s esters were then prepared N N H O H N N H O H N N H O H N N H O H N H O H N+ N H O H N+ Cl– H2PO4 – 265 264 13 S 268 Saraine-1 3¢ 269 Saraine-2 3¢ 267 Saraine-3 3¢ 266 Isosaraine-3 3¢ 271 chilocorine A 270 exochomine 272 chilocorine B N Me Me O H N N Me O H N N Me N H O Michael: Indolizidine and quinolizidine alkaloids 591from each alcohol. Full spectroscopic analysis of all eight compounds allowed the assignment of 1S,2S,9R,10R absolute configuration for both saraine-1 268 and saraine-2 269. It is to be noted that the conventional representations of these alkaloids have hitherto shown the opposite configuration (cf.ref. 7i). Of course, the stereochemistry at C-3* remains elusive. 12 Alkaloids from coccinellid beetles The structurally complex ‘dimeric’ coccinellid alkaloids exochomine 270, chilocorine A 271 and chilocorine B 272 contain a 3,4-disubstituted octahydro-8b-azaacenaphthylene moiety that is unique in natural product chemistry.In preparation for more ambitious syntheses of these alkaloids, Shattuck and Meinwald have devised syntheses of several functionalised representatives of the new tricyclic system (Scheme 22).140 Diethyl 4-oxopimelate 273 was easily converted into the pyrrole 274, which underwent Friedel–Crafts acylation with boron tribromide to yield the oxoindolizidine 275.Because this compound is a 2-acylpyrrole, it is deactivated for a second Friedel–Crafts acylation at C-5. The ketone group had to be protected as a thioketal before the second acylation took place to give 276. Straightforward transformations were then used to convert 275 and 276 into products 277–280, all of which might in time prove to be suitable precursors for making the more complex alkaloids. 13 References 1 S. M. Colegate and P. R. Dorling, in Handbook of Plant and Fungal Toxicants, ed. J. P. F. D’Mello, CRC Press, Boca Raton, 1997, ch. 1, pp. 1–18. 2 R. J. Nash, A. A. Watson and N. Asano, in Alkaloids: Chemical and Biological Perspectives, ed. S. W. Pelletier, Pergamon Press, Oxford, 1996, vol. 11, ch. 5, pp. 345–375. 3 P. Szeto, D. C. Lathbury and T. Gallagher, Tetrahedron Lett., 1995, 36, 6957. 4 R. A. Stockman, P. Szeto, S. H. J. Thompson, M. S. Hadley, D.C. Lathbury and T. Gallagher, Synlett, 1996, 853. 5 (a) F. M. Cordero, S. Cicchi, A. Goti and A. Brandi, Tetrahedron Lett., 1994, 35, 949; (b) A. Brandi, S. Cicchi, F. M. Cordero, R.Frignoli, A. Goti, S. Picasso and P. 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ISSN:0265-0568    

DOI:10.1039/a815571y

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Quinoline, quinazoline and acridone alkaloids Joseph P. Michael Centre for Molecular Design, Department of Chemistry, University of the Witwatersrand, Wits 2050, South Africa Covering: July 1996 to June 1997 Previous review: 1997, 14, 605 1 Quinoline alkaloids 1.1 Occurrence 1.2 Non-terpenoid quinoline and quinolinone alkaloids from higher plants 1.3 Hemiterpenoid quinoline alkaloids, tricyclic derivatives and furoquinoline alkaloids 1.4 Quinoline alkaloids from microbial sources 1.5 Quinoline alkaloids from animals 2 Quinazoline alkaloids 2.1 Occurrence and biological activity 2.2 Synthesis 3 Acridone alkaloids 3.1 Occurrence 3.2 Synthesis and biological studies 4 References 1 Quinoline alkaloids 1.1 Occurrence The new alkaloids reported in the period covered by this review are listed in Table 1 together with their sources.1–16 The Table also includes a list of known alkaloids isolated from new sources.Since the characterisation of new compounds by spectroscopic methods is usually a matter of routine, only significant spectroscopic details will be mentioned in the ensuing discussion. 1.2 Non-terpenoid quinoline and quinolinone alkaloids from higher plants The known compound 6-methoxykynurenic acid 1 has been found for the first time as a natural product in Ephedra pachyclada ssp. sinaica, where it occurs together with kynurenic acid 2 and 6-hydroxykynurenic acid 3.7 The compounds undoubtedly exist as a mixture of the 4-keto and 4-hydroxy tautomers.The structure of 1 was confirmed spectroscopically, by direct comparison with an authentic sample, and by conversion (with CH3I–K2CO3–DMF) into methyl 4,6-dimethoxyquinoline-2-carboxylate 4, which was also formed by methylation of 3. Separation of the quinolin-4-one and indoloquinazoline alkaloids from Chinese herbal preparations containing Evodia fruits has been achieved with the aid of capillary electrophoresis17,18 and liquid chromatography.19 A recent investigation of Evodia rutaecarpa, long known as a source of 2-alkylquinolin-4-ones, has yielded a suite of twelve alkaloids, five of which were claimed to be new.8,9 The novel compounds include 1-methyl-2-dodecylquinolin-4-one 5 and the three alkaloids 6–8 bearing unsaturated side chains.The fifth compound, 2-tridecylquinolin-4-one 9, has in fact been identified previously along with lower and higher homologues in an unseparated mixture of alkaloids from Ruta graveolens.20 The representation of its structure in the quinolin-4-one form as illustrated is done for convenience; the amount of 4-hydroxyquinoline tautomer present depended on the medium in which spectra were recorded.The unsaturated compounds were more diYcult to characterise because they were isolated as mixtures of positional isomers. For example, 8 was inseparable from the known alkaloid 10; however, the Z geometry of both components was deduced from the 13C NMR chemical shifts of the allylic carbon atoms (‰ 26.9), and hydrogenation of the mixture yielded a single compound, the known alkaloid 1-methyl-2-undecylquinolin-4-one 11.The positions of the double bonds were eventually determined by Lemieux–Johnson oxidation with osmium tetroxide and sodium periodate followed by HPLC detection of the resulting aldehydes (hexanal and pentanal, in the ratio 3:1) as 2,4-dinitrophenylhydrazone derivatives. Similarly, the new alkaloid 6 accompanied the known pentadec-10-enyl isomer 12 (1:1.2), and 7 was inseparable from evocarpine 13 (1:10).A taxonomically problematic plant formally belonging to the family Simaroubaceae – probably the same as an eastern Australian species that has been placed at diVerent times in at least four diVerent genera and now accepted as showing an aYnity to the species Samadera bidwillii – is the source of a unique combination of metabolites comprising a new quinolone alkaloid, two acridone alkaloids (see Section 3.1), a quassinoid, a limonoid and seven lignans.14 Some of these are typical metabolites of the Simaroubaceae, while the alkaloids in particular are typical of the Rutaceae.Does the new species represent a primitive member of the order Rutales prior to the evolutionary separation into the two named families? The new quinolone is 1-acetoxymethyl-2-(10-acetoxyundecyl)quinolin- 4-one 14; the extremely rare substituent on nitrogen has previously been found only in the rutaceous genera Boronia and Eriostemon (cf.ref. 21a). Further investigations on the eVects of pure alkaloids and alkaloid mixtures from Galipea longiflora in the treatment of tropical diseases (cf. ref. 21b) have now been performed with BALB/c mice infected with Leishmania amazonensis and L. venezuelensis.22 Both oral treatment and intralesional injection of chimanine B 15 reduced lesion weight and parasite loads substantially, showing improved performance over the reference drug Glucantime (N-methylglucamine antimonate).Intermediate antileishmanial activity resulted from oral administration of crude alkaloidal extracts from G. longiflora stem and bark. In addition, the purified alkaloids 2-propylquinoline 16 and 2-phenylquinoline 17 were reasonably eVective when administered orally, as were 2-propylquinoline and 2- pentylquinoline 18 when administered by injection. The results suggest that chimanine B in particular would make a promising lead compound for the development of oral therapy against leishmaniasis.An elaborate new synthesis of 2-substituted quinolines involved ortho-specific hydroxyalkylation of secondary anilines via N-alkylanilinochlorophenylboranes 19, pyrolysis of the products 20 to give dihydroquinolines 21 (presumably by electrocyclisation of quinomethane imine intermediates), and finally deallylation of 21 induced by hydridotetrakis- (triphenylphosphine)rhodium (Scheme 1).23 Amongst the many products obtained were the three simple natural products 2-methylquinoline, 2-propylquinoline 16 and 2-phenylquinoline 17.Other new routes to 2-phenylquinoline, which is frequently included amongst the targets when new methods for the synthesis of substituted quinolines are developed, include the Suzuki coupling of 2-chloroquinoline 22 and Michael: Quinoline, quinazoline and acridone alkaloids 595phenylboronic acid 23,24 and the titanium-mediated reduction of the nitrochalcone 24a.25 Ruthenium-catalysed carbonylation of the same nitrochalcone in the presence of a rigid diimine ligand yielded a mixture of 2-phenylquinoline (57%) and 2-benzoylindole (43%).26 An alternative reduction of nitrochalcone 24a with bakers’ yeast stopped at the quinoline N-oxide stage 25a; with the methyl analogue 24b, further reduction of the N-oxide 25b to 2-methylquinoline took place.27 The recently described28 Eriostemon alkaloid 3,4,8- trimethoxy-2-quinolone 26 has been synthesised as shown in Scheme 2.29 Eaton’s reagent (methanesulfonic acid– phosphorus pentoxide) provided a superior medium for cyclising the malonic acid-derived dianilide 27 to the 4-hydroxyquinolin-2-one 28.However, cyclisation of the methoxy-substituted analogue 29 to give the target alkaloid 26 directly failed, necessitating the less direct approach illustrated in the Scheme. Table 1 Isolation and detection of quinoline alkaloids from plant, microbial and animal sources Species Alkaloida Ref.Acronychia baeurlenii Kokusaginine Pteleine 1 Acronychia pubescens Dictamnine Evolitrine Kokusaginine 1 Aegle marmelos Integriquinolone 4-Methoxy-N-methylquinolin-2-one 2,3 Agathosma barosmaefolia N-Methylhaplamine N-Methyl-4,6-dimethoxyquinolin-2-one Skimmianine 53 4 Arthrobacter sp. strain YL-02729S YM-30059b 55 5 Chorilaena quercifolia Skimmianine 6 Ephedra pachyclada ssp. sinaica 6-Hydroxykynurenic acid 3 Kynurenic acid 2 6-Methoxykynurenic acidb 1 7 Evodia rutaecarpa 1-Methyl-2-dodecylquinolin-4-oneb 5 (Z)-1-Methyl-2-(pentadec-9-enyl)quinolin-4-oneb 6 (Z)-1-Methyl-2-(tridec-7-enyl)quinolin-4-oneb 7 (Z)-1-Methyl-2-(undec-5-enyl)quinolin-4-oneb 8 (Z)-2-Tridecylquinolin-4-oneb 9 8 8,9 8 Melicope erromangensis Dutadrupine Flindersiamine Kokusaginine Skimmianine 10 Penicillium sp. no. 410 (+)-Penigequinolones A and Bb 56a,b 11 Penicillium sp. NTC-47 (")-3-Methoxy-4,5-dihydroxy-4-(4-methoxyphenyl)quinolin-2-oneb 57 (")-3-Methoxy-4-hydroxy-4-(4-methoxyphenyl)quinolin-2-oneb 58 (+)-Penigequinolones A and Bb 12,13 12 aV.Samadera SAC-2825 2-(10-Acetoxyundecyl)-1-acetoxymethylquinolin-4-oneb 14 14 Scolopendra subspinipes mutilans Jineolb 15 Ticorea longiflora Dictamnine Evolitrine „-Fagarine 54 4-Methoxy-N-methylquinolin-2-one Skimmianine 16 aOnly new alkaloids and new records for a given species are listed in the Table. Structures of most known alkaloids may be found in previous reviews in this series. bNew alkaloids.N CO2 R3 R1 OR2 N (CH2 ) nCH3 O R N O Me (CH2)mCH=CH(CH2) nCH3 6 m = 8; n = 4 7 m = 6; n = 4 8 m = 4; n = 4 10 m = 5; n = 3 12 m = 9; n = 3 13 m = 7; n = 3 5 R = Me; n = 11 9 R = H; n = 12 11 R = Me; n = 10 1 R1 = OMe; R2 = R3 = H 2 R1 = R2 = R3 = H 3 R1 = OH; R2 = R3 =H 4 R1 = OMe; R2 = R3 = Me Z N O CH2OAc OAc N R N 15 16 R = n-propyl 17 R = Ph 18 R = n-pentyl 14 596 Natural Product Reports, 1998A versatile new route to 2-substituted quinolin-4-one alkaloids exploited regioselective addition of Grignard reagents to the N-Cbz-4-triisopropylsilyloxyquinolinium triflate 31, prepared in situ from quinolin-4-one 32 and triisopropylsilyl triflate (Scheme 3).30 Removal of the benzyloxycarbonyl protecting group from the adducts 33 by hydrogenolysis over a palladium–carbon catalyst simultaneously eVected dehydrogenation of the heterocyclic ring.This short sequence provided easy access to five representative alkaloids 34, including norgraveoline 34e.A strategy for the synthesis of the decahydroquinoline Lycopodium alkaloid phlegmarine 35 has been described in a published conference contribution by Comins and co-workers.31 Applications of this team’s dihydropyridone methodology have so far culminated in the preparation of the advanced intermediate 36. Completion of what promises to be the first asymmetric synthesis of this type of Lycopodium alkaloid is awaited with interest. 1.3 Hemiterpenoid quinoline alkaloids, tricyclic derivatives and furoquinoline alkaloids When (")-preorixine, a metabolite of Orixa japonica, was first reported some years ago,32 its absolute configuration was assigned as S (i.e., ent-37) after its conversion into (R)-(+)- orixine 38 by hydrolysis of the epoxide ring with putative inversion (cf.ref. 21c). However, NMR analysis of (R)- and (S)-·-methoxy-·-trifluoromethylphenylacetic acid esters (Mosher esters) of various alcohols derived from (")- preorixine has now served to prove that this assignment was incorrect.33 Two hydrolysis products, (+)-orixine and (+)- isoptelefolidine 39 – which, incidentally, accompanied (")- preorixine when it was isolated – were unambiguously shown to possess the R configuration with the aid of this technique.(+)-Orixine could also be derived from (")-preorixine by opening the epoxide ring at the more substituted site with formic acid followed by cleavage of the formate ester intermediate, a process that does not involve an inversion of configuration.Moreover, the stereogenic centre of (")- preorixine is not aVected when it is dehydrated to give (+)- isoptelefolidine. The accumulated evidence strongly suggests that (")-preorixine must also have R configuration as shown in 37. Additional evidence was obtained by reducing (")-preorixine with diborane and lithium borohydride to yield 40, the R configuration of which was again proved by NMR analysis of its two Mosher esters.(R)-(+)-3*-OMethylpreorixine 41, a minor constituent of the O. orixa extracts, was suspected to be an artefact of the isolation procedure, since it was easily prepared from (")-preorixine and methanol in the presence of acid. N R O NO2 R O N R N Cl (HO)2B N R NH OH R N B Ph Cl ii 46-94% i 19 20 21 R = Me, Pr, Ph 45-68% iii v 67% iv R = Ph 23 22 + vii 34% R = Me 24 R = Ph 26 R = Me 50% (R = Ph) 89% (R = Me) vi 25a R = Ph 25b R = Me R = Ph 54% 37-68% Scheme 1 Reagents: i, RCH=CHCHO, ClCH2CH2Cl, Pri 2NEt, "20 )C to 0)C; ii, p-xylene, reflux; iii, Rh(PPh3)4H, TFA, EtOH, reflux; iv, Pd(PPh3)4, BHT, Ba(OH)2·3H2O, THF, 75 )C; v, TiCl4, Zn, THF, reflux, then add 24, rt; vi, bakers’ yeast, NaOH, H2O–EtOH (7:3), reflux, 1 h; vii, bakers’ yeast, NaOH, H2O–EtOH (7:3), reflux, 24 h OMe OMe O N H OMe N OMe H O OH OMe N OMe H O O OMe OMe N OMe H O N O H OMe R N OMe H O OH 28 60% i, R = H 27 R = H 29 R = OMe 30 60% iv 26 73% v 66% ii, iii i, R = OMe Scheme 2 Reagents: i, P2O5 (10%) in MeSO3H, 150–155 )C; ii, SO2Cl2, dioxane, rt; ii, NaOMe, MeOH, reflux, 5 min; iv, Zn, AcOH, EtOH, reflux; v, (MeO)2SO2, NaHCO3, H2O, rt N O H R N OSiPri 3 Cbz R N O Cbz N OSiPri 3 Cbz OTf– + ii i 31 32 iii 33a R = n-propyl 56% 33b R = n-pentyl 72% 33c R = n-heptyl 70% 33d R = phenyl 93% 33e R = 3,4-methylene- dioxyphenyl 38% 34a R = n-propyl 95% 34b R = n-pentyl 97% 34c R = n-heptyl 98% 34d R = phenyl 96% 34e R = 3,4-methylene- dioxyphenyl 92% Scheme 3 Reagents: i, Pri 3SiOTf, 20 )C; ii, 2,6-lutidine (2.0 equiv.), CH2Cl2, RMgBr (1 M in THF; 2.0 equiv.), rt; iii, H2 (1 atm), 10% Pd/C, MeOH, rt Michael: Quinoline, quinazoline and acridone alkaloids 597Flindersine 42, N-methylflindersine 43 and veprisine 44, isolated from the bark of the medicinally important East African tree Fagara chalybea, are active as SRS-A antagonists. 34 In this work, veprisine was prepared in two steps (21% yield) from 2,3-dimethoxy-N-methylaniline 45 and diethyl prenylmalonate 46 (Scheme 4).Other workers have recently made veprisine from 2,3-dimethoxyaniline 47 and malonic acid; heating the intermediate quinolinone 48 with 3-methylbut-2- enal in pyridine followed by N-methylation yielded the target alkaloid in an eYcient 51% overall yield.35 Adaptations of this procedure also resulted in syntheses of the related alkaloids 49, 50 and 51 (zanthobungeanine); oxidation of the latter with osmium tetroxide aVorded the cis-diol 52 in 42% yield.It is of interest that the two hydroxy groups in the naturally occurring diol from Zanthoxylum simulans have recently been shown to be trans.36 The accumulation of the furoquinoline alkaloids skimmianine 53 and „-fagarine 54 in cell suspension cultures of Fagara zanthoxyloides showed a marked dependence on exogenous growth regulators.37 Removal of the auxin ·-naphthylacetic acid from the growth medium slightly stimulated the accumulation of the alkaloids (by 1.2 and 1.9 times respectively) relative to a control culture. However, the absence of the cytokinin benzylaminopurine brought about a ninefold decrease in alkaloid production.Removal of both phytohormones decreased alkaloid accumulation by 3.5 and 2.1 times respectively. The results show that the cytokinin plays an essential role in alkaloid biosynthesis within the cells, but the auxin inhibits biosynthesis. 1.4 Quinoline alkaloids from microbial sources YM-30059, an antibacterial and cytotoxic compound isolated from Arthrobacter sp.YL-02729S, has been formulated as the N-hydroxyquinolin-4-one 55 on the basis of its spectroscopic features.5 It showed moderate activity against Gram-positive bacteria, including Bacillus subtilis and multiple-drug resistant Staphylococcus aureus and S. epidermidis. The metabolite was also a potent inhibitor of lipoxygenase. Two active inhibitors of pollen-growth have been located by bioassay in the mycelial mats of Penicillium sp.No. 410.11 The active fraction was obtained as an amorphous yellow powder by repeated chromatography of the acetone extract of the mats. This dextrorotatory product proved to be an inseparable mixture of two diastereoisomers 56a,b in the ratio 2:1, though it was not possible to tell which was the major isomer. The structures and relative configurations of penigequinolones A and B, as the compounds were named, were assigned after extensive NMR work in which the ROESY technique proved crucial in permitting the complete assignment of the relative stereochemistry and the chair conformation of the tetrahydropyran ring.The styryl substituent on this ring is axial. The absolute configurations remain undetermined. The N O O R N O O Me OMe MeO N N H H H COMe Me N N H H H CO2R* Me O O O N OMe OMe O O O N OMe OMe OH R O O N OMe OMe OH 38 ( R)-(+)-Orixine R = OH 40 R = H 41 R = OMe 37 ( R)-(–)-Preorixine 36 35 44 Veprisine 42 Flindersine R = H 43 R = Me 39 ( R)-(+)-Isoptelefolidine N OH O Me MeO OMe NHMe OMe MeO EtO2C CO2Et N O O H OMe MeO N OH O H MeO OMe NH2 OMe MeO 52% ii 90% v 48 47 iv 69% iii 82% 46 45 + 40% i 44 Veprisine Scheme 4 Reagents: i, Ph2O, 230–255 )C; ii, DDQ, C6H6, reflux; iii, malonic acid, POCl3; iv, 3-methylbut-2-enal, pyridine, MgSO4, reflux; v, NaH, THF, rt, then MeI N O O R2 OR1 N O O Me OMe OH OH N O OMe R OMe rac-52 49 R1 = Me; R2 = H 50 R1 = prenyl; R2 = Me 51 R1 = R2 = Me 53 Skimmianine R = OMe 54 g-Fagarine R = H 598 Natural Product Reports, 1998demonstrable inhibition of the growth of tea pollen tubes by the isolated penigequinolones, complete at 100 mg l"1, oVers the possibility of developing these and related compounds as herbicides.Interestingly enough, an independent group of workers has isolated the same two diastereoisomers 56a,b, albeit in a diVerent but unspecified ratio, from a culture of the fungus Penicillium sp. NTC-47 grown on an okara medium (the insoluble residue of whole soybean).12 The compounds, unnamed in this work, were detected by a bioassay based on toxicity to the brine shrimp Artemia salina; when purified, they displayed an LC50 of 0.90 Ïg ml"1.The results were reported in a published conference proceeding that also contains information on two related metabolites, the dihydroquinolinones 57 and 58. Full details of the isolation and spectroscopic characterisation of the latter two compounds have since been published.13 X-Ray crystallography complemented the usual battery of NMR studies on 57, and confirmed its relative, but not its absolute, stereostructure.Dihydroquinolinone 57 was less toxic in the brine shrimp assay than the diastereoisomeric penigequinolone mixture (LC50 20.0 Ïg ml"1), but its deoxy analogue 58 was inactive. A major review on alkaloids containing quinolinequinone and quinolinequinonimine units includes a substantial section giving the most complete account to date of the isolation, characterisation, synthesis, biosynthesis and biological activity of the microbial metabolites lavendamycin, streptonigrin, streptonigrone and related compounds.38 At the heart of the most recent synthesis of lavendamycin methyl ester 59 is a new and eYcient five-step synthesis of 7-acetamidoquinolinequinone 60 from the commercially available 8- hydroxy-2-methylquinoline 61 (Scheme 5).39 Compound 60 was readily converted into the target as shown, thereby making available gram quantities of the antibiotic in overall yields of 37–43%.The sequence was easily adapted for the preparation of aminoquinolinequinone derivatives related to 60, which are interesting in their own right as potent antitumour agents. An important synthesis of racemic virantmycin 62 that established its previously disputed relative stereochemistry once and for all was reported in a communication in 1991 by Morimoto et al.40 (cf. ref. 21d). Full details of this synthesis and those of the racemic diastereoisomer 63 and the model compounds 64–67 have now been published together with particulars of the significant NOE studies on which the relative stereochemical assignments were based.41 Moreover, an enantioselective synthesis of unnatural (+)-(2S,3S)-virantmycin, ent-62, has also appeared recently.42 In this route, the key to stereocontrol lay in the Sharpless asymmetric epoxidation of allylic alcohol 68, the product 69 in turn being transformed into another epoxide 70, which underwent cyclisation to yield the tetrahydroquinoline 71 (Scheme 6).A further nine steps aVorded alcohol 72, the absolute configuration of which was confirmed by applying the exciton chirality method to its 4-dimethylaminobenzoate ester. The synthesis of (+)- virantmycin was completed as illustrated. Natural (")- (2R,3R)-virantmycin and the synthetic racemate showed excellent antiviral activity in a test involving the growth inhibition of influenza virus.However, the antiviral activities of the unnatural (+)-enantiomer and the racemic diastereoisomer 63 were negligible. 1.5 Quinoline alkaloids from animals The centipede Scolopendra subspinipes mutilans has traditionally been used in Korea for the treatment of various disorders, including convulsions and seizures. Bioactivity-guided fractionation of the ethanolic extract of this animal has now yielded a very simple new alkaloid, 3,8-dihydroxyquinoline 73, which has been given the trivial name jineol.15 Its structure was deduced from a thorough spectroscopic study of the native metabolite and its methyl and acetyl derivatives.The 3-hydroxyquinoline moiety, rare in nature, is apparently unique in a metabolite from an animal source. Jineol proved to be moderately cytotoxic in five human tumour cell models; it was less eVective than cisplatin, but more eVective than carboplatin, 3,8-dimethoxyquinoline and 3,8-diacetoxyquinoline.Interest in the synthesis of the important frog skin alkaloid decahydroquinoline 195A (pumiliotoxin C) continues N OH O N O H OH O OH OMe OMe N O H R OH OMe OMe 57 R = OH 58 R = H 56a,b Penigequinolines A, B 55 vi 91% + 60 73% i 61 ii–iv 85% 71% v vii, viii 79% 59 N OH NHAc AcHN N O O AcHN N OH N OH NO2 O2N N O CHO O AcHN H2N HN CO2Me N O O H2N N HN CO2Me Scheme 5 Reagents: i, HNO3–H2SO4 (7:3 v/v), 0 )C; ii, H2 (30 psi), 5% Pd/C, HCl (10% aq.), rt; iii, Na2SO3, NaOAc, H2O, then Ac2O, rt; iv, MeOH–H2O (10:1), reflux; v, K2Cr2O7, HOAc, H2O, rt; vi, SeO2, dioxane, H2O, reflux; vii, xylene, reflux; viii, H2SO4, H2O (3:4), 0 )C to 60 )C Michael: Quinoline, quinazoline and acridone alkaloids 599unabated.No fewer than four routes to this pharmacologically interesting compound, all involving new methodological developments, were reported during the period under review. In the approach of Back and Nakajima43 (Scheme 7), the amino ester 74, derived in several steps from the Diels–Alder adduct of piperylene and maleic anhydride, underwent conjugate addition to the alkynyl sulfone 75 to yield a 2:1 mixture of the Z and E isomers of 76.Ring closure of 76 with LDA did not proceed to completion (33% of the substrate was recovered), but conversion to the octahydroquinolin-4-one 77 was eYcient (94%). Reduction of the enol triflate 78 removed both the superfluous functionality at C-4 as well as the C2–C3 double bond, although the latter process aVorded a mixture of diastereoisomers 79 and 80 in a 5:1 ratio.After chromatographic separation, these isomers were converted as shown into (&)-pumiliotoxin C 81 and (&)-2-epipumiliotoxin C 82. The approach of Kuethe and Padwa44 (Scheme 8) employed a tandem Pummerer rearrangement–isomu�nchnone dipolar cycloaddition on substrate 83 via intermediates 84 and 85 to create the tetrahydroquinolinone nucleus of 86a in an eYcient 73% yield. A minor product, 86b, was also isolated (13%).Both products were defunctionalised to the common intermediate 87, which was readily reduced to the bicyclic lactam 88 with the required cis stereochemistry of ring fusion. Although the authors asserted that the preparation of lactam 88 completed a formal synthesis of racemic pumiliotoxin C, the references they cited45,46 describe the N-debenzyl analogue of 88 rather than 88 itself. A paper by Fukumoto and co-workers47 gives full details of, and additional information on, a synthesis of (+)-pumiliotoxin C (the unnatural enantiomer, as shown in 81) that was previously reported in a communication48 (cf.ref. 21e). A key step was cyclisation of enyne 89, made in fourteen steps from the chiral acrylate 90 and butadiene via optically pure cyclohexene alcohol 91 (Scheme 9). Under free radical conditions, cyclisation of 89 gave the bicyclic compound 92 in 99% yield. However, diYculties in the operating conditions prompted a search for an alternative cyclisation, which was achieved much more easily, though in poorer yield (61%), by a palladium-induced hydrosilylation.After several functional group manipulations, bicyclic ketone 93 was obtained. Beckmann rearrangement of a second bicyclic ketone, 94, gave lactam 95, which has the requisite decadroquinoline skeleton of the target alkaloid. A more recent approach by the same research team49 also proceeded by way of cyclohexene alcohol 91, which was transformed in three steps and 34% yield NH Ts TIPSO OH NH Ts TIPSO OH O N Ts OH OH TIPSO N MeO2C H OH OMe N HO2C H Cl OMe N MeO2C OMe H N HO2C H Cl OMe N HO2C H Cl OMe N H R2 OMe R1 N R2 O O R1 NAc Ts OH TIPSO NH2 EtO2C NAc Ts OH TIPSO O 72 71 70 69 68 55% 98% i 9 steps 66 R1 = H; R2 = Cl 67 R1 = Cl; R2 = H 64 R1 = H; R2 = Cl 65 R1 = Cl; R2 = H 62 Virantmycin 63 ent-62 81% viii, ix 89% vii 59% 9 steps 67% vi 96% v 81% ii–iv Scheme 6 Reagents: i, L-(+)-diethyl tartrate, Ti(OPri)4, But OOH, CH2Cl2, "20 )C; ii, MsCl, NEt3, CH2Cl2, 0 )C; iii, NaI (5 equiv.), Zn (2 equiv.), DMF, 100 )C; iv, DIBAL, toluene, "15 )C; v, ButOOH, VO(acac)2, CH2Cl2, 0 )C; vi, TFA (2 equiv.), toluene, rt; vii, DEAD, PPh3, THF, rt; viii, NaOH, MeOH, reflux; ix, NEt4Cl (20 equiv.), TFA (4 equiv.), CH2Cl2, "15 )C 73 Jineol N OH OH 600 Natural Product Reports, 1998into the nitro compound 96.Treatment with p-chlorophenyl isocyanate induced intramolecular nitrile oxide cycloaddition, giving a tricyclic isoxazoline 97 that incorporated several of the stereochemical relationships required for the target alkaloid. Cleavage of the heterocyclic ring followed by functional group interconversions aVorded 93, at which point the two routes converged.Since both ketone 94 and lactam 95 have been prepared in racemic form by previous workers and converted into (&)-pumiliotoxin C,45,46,50 the enantioselective synthesis of these two compounds in the present investigations can be viewed as completing a formal synthesis of (+)-pumiliotoxin C 81. 2 Quinazoline alkaloids 2.1 Occurrence and biological activity 1,3-Dimethylquinazoline-2,4-dione 98 has been identified as the sex pheromone of the pale-brown chafer beetle Phyllopertha diversa, a devastating turf pest in Japan.51 Released in picogram quantities by females, this pheromone has long eluded detection. In this study, it was an almost unnoticed component when a biologically active ether extract was analysed by gas chromatography with an electroantennographic detector.Male beetles were successfully lured in significant numbers to field traps baited with a synthetic sample of the pheromone, made by methylating 99 with iodomethane and sodium hydroxide in DMSO. This is the first time that this simple alkaloid, which has antiinflammatory, anticonvulsant and analgesic properties, has been found as a natural product. Quinazoline-2,4-dione 99 has itself been isolated from dyer’s woad, Isatis tinctoria, and shown to have antiinflammatory and antihypertensive properties.52 Although this was claimed to be the first isolation of 99 from a natural source, its isolation from Strobilanthes cusia has previously been reported in this series of reviews (cf.ref. 21f). The related plant Isatis indigotica, the principal source of the antipyretic and antiviral Chinese drug ‘Ban-Lan-Gen’, yielded the known alkaloid qingdainone (candidine) 100 and several new natural products, amongst them 3-(2-carboxyphenyl)-4(3H)-quinazolinone 10153 and 3-(2-hydroxyphenyl)-4(3H)-quinazolinone 102.54 Both were previously known as synthetic compounds.The carboxylic acid 101 showed endotoxic activity in vitro in the limulus amoebocyte lysate test. The most interesting new metabolite in the present group is the pyrrolo[2,1-b]quinazolinone isaindigotone 103,54 the tricyclic core of which is reminiscent of the medicinally important vasicine group of alkaloids.The HMBC spectrum of 103 unambiguously indicated the location of the arylidene unit at C-3, while NOE diVerence spectra established the (E) configuration and the positions of the hydroxy and methoxy substituents. Tryptanthrin 104 has been obtained from the Indian medicinal plant Wrightia tinctoria.55 The better-known Ayurvedic medicinal plant Adhatoda vasica has yielded the new pyrroloquinazolinone alkaloids desmethoxyaniflorine 105 and 7-methoxyvasicinone 106, as well as 5-methoxyvasicinone 107, which appears to be new as a natural product.56 The metabolites were characterised by means of spectroscopic studies; in addition, the structure of desmethoxyaniflorine was confirmed by X-ray crystallography.As a matter of interest, the crystal structure of 7-methoxyvasicinone57 was reported in the previous review in this series (cf. ref. 21g). The present work assigns the R absolute configuration at C-3 to both 106 and 107 without presenting convincing evidence for the assignment.It appears that the configurational assignment may have been assumed on the basis of a widely accepted 3R assignment 94% (67% conversion) ii 75 + 76 74 i iii + 77 78 + 80 79 iv (5 : 1) 46% (based on 77) v–vii 46% (based on 77) v–vii 82 rac-81 93% N H H H H H N H N H H H SO2- p-Tol H H N H SO2- p-Tol N H H H SO2- p-Tol O H H N H SO2- p-Tol OTf N H CO2Me SO2- p-Tol H NH2 CO2Me SO2- p-Tol OTf– Scheme 7 Reagents: i, THF, rt; ii, LDA, THF; iii, Tf2O, CH2Cl2, reflux; iv, H2 (100 atm), PtO2, MeOH; v, CbzCl, K2CO3, H2O, CHCl3, reflux; vi, 5% Na–Hg, Na2HPO4, MeOH-THF (1:1); vii, H2, Pd/C, EtOH 87 66% vi, vii 74% v 81% ii–iv 86b 86a + 85 84 83 i – 13% 73% 88 N Bn O O SEt N O O Bn S Et N Bn +O O SEt N O Bn OAc N O Bn SEt N O Bn N O Bn H H O Scheme 8 Reagents: i, Ac2O, p-TsOH (cat.), reflux; ii, K2CO3, MeOH; iii, Tf2NPh, NEt3; iv, Pd(OAc)2, Ph3 P, HCO2H, NEt3; v, Raney Ni, EtOH, 65 )C; vi, LiB(Bui 3)3H; vii, H2, PtO2 Michael: Quinoline, quinazoline and acridone alkaloids 601for vasicine alkaloids. However, as was pointed out in the previous review, there is excellent recent evidence58 to suggest that the traditional 3R assignment for alkaloids in the vasicine/ vasicinone manifold is wrong (cf.ref. 21g; see also Section 2.2 below). The known fungal metabolite fiscalin B 108 and two new tremorgenic principles have been isolated from the ethyl acetate extract of the ascomycete Corynascus setosus cultivated on sterilised rice.59 Extensive NMR data were reported for all three compounds.The spectroscopic properties of the new compounds were very similar to those of the known Aspergillus metabolites tryptoquivaline 109 and nortryptoquivaline 110 respectively, the major diVerences in chemical shift being observed for C-26 and C-27. The CD spectra were also similar to those of the tryptoquivalines except for extra positive Cotton eVects near 280 nm.The implication was that the new compounds were probably diastereoisomers of the tryptoquivalines. This hypothesis was confirmed for the tryptoquivaline analogue, the relative stereochemistry of which was established by means of X-ray crystallography. On the reasonable assumption that the absolute configurations of the new alkaloids are the same as those of tryptoquivaline and nortryptoquivaline except at C-27, the compounds were identified as 27-epi-tryptoquivaline 111 and 27-epinortryptoquivaline 112, both having 2S,3S,12R,15S,27R con- figuration.On intraperitoneal injection of 50 mg kg"1 of the metabolites in mice, 111 and 112 caused weak tremor with paralysis, which appeared at about 30 and 120 min after injection and lasted for about 120 and 30 min respectively. In comparison, after about 60 min tryptoquivaline 109 induced a similar response that lasted for about 90 min, while nortryptoquivaline 110 was inactive. 2.2 Synthesis The recent reversal of the accepted stereochemistry for vasicine and related alkaloids58 has already been mentioned in this review (see Section 2.1).An important report describing the first asymmetric syntheses of both enantiomers of vasicinone H H O H H N O H O O O O BnO OH BnO H H BnO H H HO O BnO NO2 H H BnO O N H BnO O xii, xiii 34% iv–vi 99% ix 97 96 x, xi 68% or viii 61% vii 99% 75% xv, xvi 93 92 89 77% xiv 70% i–iii 28% 11 steps 90 91 95 94 41% xvii, xviii (+)-81 35% Scheme 9 Reagents: i, CBr4, Ph3 P, CH2Cl2, 0)C to rt; ii, LiC/CSiMe3, HMPA, THF, "78 )C to rt; iii, NaOH (1 M), MeOH, rt; iv, TPAP, NMO, 4 Å molecular sieves, CH2Cl2, rt; v, MeNO2, KF, Bu4NCl, toluene, rt; vi, Ac2O, pyridine, rt, then NaBH4, EtOH, 0 )C; vii, Bu3SnH, AIBN, C6H6, reflux, then SiO2, CH2 Cl2, rt; viii, (dba)3Pd2, CHCl3, N,N*-bis(benzylidene)ethylenediamine, polymethylhydrosiloxane, AcOH, ClCH2CH2Cl, rt; ix, p-chlorophenyl isocyanate, NEt3, toluene, sealed tube, 60 )C; x, Na, NH3, THF, "78 )C; xi, O3, MeOH–CH2Cl2 (3:1), "78 )C, then Me2S; xii, H2, Raney Ni(W2), (MeO)3B, MeOH–H2O (15:1), rt; xiii, p-TsOH, C6H6, 60 )C; xiv, H2, 10% Pd/C, EtOAc, rt; xv, (imid)2CS, DMAP, CH2Cl2, reflux; xvi, Bu3SnH, AIBN, C6H6, reflux; xvii, NH2OH·HCl, NaOAc, MeOH, rt; xviii, p-TsCl, NaOH, THF–H2O (2:3), 0 )C to rt N N R R O O N N O N O H N N O R N N O O N N O OH OMe OMe 103 Isaindigotone 104 Tryptanthrin 101 R = CO2H 102 R = OH 98 R = Me 99 R = H 100 N N O OH R1 R2 N N O HO Me2N N N O O N N H O O H R HO OCOMe N H N H O N O N 108 Fiscalin B 109 Tryptoquivaline R = Me; 27 S 110 Nortryptoquivaline R = H; 27 S 111 R = Me; 27 R 112 R = H; 27 R 27 105 Desmethoxyaniflorine 106 R1 = OMe; R2 = H 107 R1 = H; R2 = OMe 602 Natural Product Reports, 1998now provides indisputable evidence that the long-standing assignment of 3R configuration to (")-vasicinone 113 is wrong.60 In this work, Eguchi and co-workers first prepared (&)-vasicinone, rac-113, to establish the viability of their synthetic approach (Scheme 10, with racemic substrates) before embarking on the illustrated synthesis with the TBDMS-protected (3S)-3-hydroxypyrrolidin-2-one 114, made in six steps from L-aspartic acid.The key step was the aza-Wittig cyclisation of 115, after which the tricyclic product was deprotected to yield (S)-(")-vasicinone 113 ([·]D 28 "58, c 0.45, CHCl3) in 97% enantiomeric excess and 52% overall yield based on 2-azidobenzoic acid.With this reference compound in hand, the authors investigated the asymmetric oxidation of the anion derived from deoxyvasicinone 116 with (1R)-(")-(10-camphorsulfonyl)oxaziridine and its enantiomer (Davis reagents) as sources of electropositive oxygen. The best enantiomeric excess (71%) of (R)-(+)-vasicinone, ent-113, was obtained in THF at "78 )C with sodium hexamethyldisilazide as base and only half an equivalent of (")-oxaziridine, although the isolated yield of the product was only 39%.Intramolecular aza-Wittig reaction on the azidolactams 117 has also been used to prepare the deoxyvasicinone derivatives 118.61 More conventional routes were employed to make the halogenated deoxyvasicine derivatives 119, all of which showed good activity as bronchodilators.62 The synthesis of (S)-(")-chrysogine 120 highlighted in last year’s review (cf. ref. 21g) was claimed by the authors to be the first asymmetric synthesis of this mould metabolite. 63 Bergman has now pointed out64 that his research group had already published essentially the same route to (S)-chrysogine in 1990.65 The only diVerence was the base used in the final cyclisation step (Na2CO3 instead of NaOH). This earlier synthesis had, in fact, also been reviewed in these pages (cf. ref. 21h). The low optical rotation reported for the target in the more recent synthesis ([·]D "27, vs. "41 in Bergman’s work) may have been due to partial racemisation of the chiral starting material, (S)-2-acetoxypropanoyl chloride.For well over a century it has been assumed that the structure of ‘isatin chloride’, formed by heating isatin 121 with phosphorus pentachloride, is 122. Cornforth and his co-workers have now proved by spectroscopy and crystallography that the product is actually the dimer 123.66 Treatment of this dimer with dry methanol gave a mixture of indoloquinazolinones 124a, 124b and 125. Compound 125 was photolabile, and deposited tryptanthrin 104 when kept for several days in deuterated chloroform.Several routes to the reduced tryptanthrin analogue 126 were also explored in this work, and persuasive evidence was adduced for the complex mechanisms involved in the transformations. Coincidentally, tryptanthrin (also known as couropitine A) 104 was recently prepared in 97% yield by heating isatin 121 with phosphoryl chloride and working up the reaction mixture with ice.67 The first synthesis of (+)-fumiquinazoline G 127, the enantiomer of the naturally occurring fungal metabolite, has been accomplished by He and Snider.68 With the protected L-tryptophan derivative 128 as starting material, the diketopiperazine 129 was prepared in 45% yield over six steps as illustrated in Scheme 11.The diastereofacially selective hydrogenation of this product set up the second stereogenic centre with the correct relative stereochemistry. Acylation of the resulting compound 130 with 2-azidobenzoyl chloride yielded intermediate 131, deprotection and aza-Wittig reaction of which created the quinazolin-4(3H)-one ring present in the final target.The synthesis, completed as shown, contains twelve steps in total and was achieved in an overall yield of 11%. Comparatively easy oxidation of fumiquinazoline G with manganese dioxide aVorded the dehydro product 132, thereby introducing useful functionality for the synthesis of more complex fumiquinazolines. 3 Acridone alkaloids 3.1 Occurrence The five new acridone alkaloids reported during the review period were isolated from rutaceous plants.They are listed in 113 (–)-Vasicinone 62% iii, iv i 83% over two steps ii 116 39% (71% ee) v ent-113 (+)-Vasicinone 114 115 N N O OH N N O N3 O Cl N3 O OH HN O OTBDMS N N O OH N O OTBDMS O N3 Scheme 10 Reagents: i, SOCl2, reflux; ii, NaH, THF, 0 )C to rt; iii, Bu3P, toluene, rt to reflux; iv, Bu4NF, THF, 0 )C to rt; v, NaHDMS, THF, "78 )C, 1 h, then (1R)-(")-(10-camphorsulfonyl)oxaziridine, THF, "78 )C, 30 min 124a R = H 124b R = Me 123 122 121 120 ( S)-(–)-Chrysogine 117 X = OMe, NEt2 118 X = OMe, NEt2 119 X = F, Cl, Br, I 126 125 N N O OH N N X HO OMe N N O COX N O N3 O COX N NH O OH N O O H N O Cl N O N Cl Cl O N N O MeO2C OR N N O (MeO)3C OH Michael: Quinoline, quinazoline and acridone alkaloids 603Table 2 together with known alkaloids from new plant sources.14,69–72 Two of the new alkaloids have surprisingly simple structures. 1-Hydroxy-3-methoxyacridone 133 was found with several related compounds by bioactivity-guided fractionation (brine shrimp test) of a bark extract from the African medicinal plant Fagara macrophylla, which is also known as Zanthoxylum gillettii.71 1-Methoxy-N-methylacridone 134, which was isolated from the taxonomically puzzling Samadera specimen described previously (see Section 1.2),14 is one of a mere handful of acridone alkaloids lacking the strongly hydrogen-bonding hydroxy substituent at C-1.It was accompanied by 1,8-dihydroxy-N-methylacridone 135, an atypically substituted acridone alkaloid that was very recently found for the first time as a natural product in Boronia lanceolata.73 (")-Margrapines A and B, 136 and 137, two alkaloids of unknown relative and absolute stereochemistry isolated from the roots of the Marsh grapefruit, Citrus paradisi, are highly oxidised 4-prenylacridones.70 The gross structures were assigned on the basis of comprehensive NMR data.The bizarre new alkaloid fareanine 138 has been isolated from the leaves of the Australian tree Medicosma fareana.72 Its inclusion amongst the acridone alkaloids is justified on the grounds of the postulated biogenesis for this compound, which the authors suggest is initiated by oxidative cleavage of the electron-rich aromatic ring of an acridone precursor such as normelicopicine 139. Indeed, normelicopicine and related compounds were also isolated in this study.If one envisages the oxidation proceeding through a quinone ketal such as 140 – a type of intermediate for which precedent exists74 – then cleavage of the C3–C4 bond followed by recyclisation between C2 and C4 will give rise to the new alkaloid’s unique cyclopenta[b]quinoline skeleton. 3.2 Synthesis and biological studies Clinical trials on the potent anticancer agent acronycine 141 have hitherto been hampered by the alkaloid’s poor solubility in water. Modifications in the pyran ring have now been studied as a means of gaining access to more soluble derivatives. 75 Mild nitration (nitric acid, acetic acid, 0 )C) yielded 142 (90%), a variety of reductions on which provided access to the oxime 143 and the dihydroacronycine derivatives 144 and 145. Further derivatives 146–148 could be prepared in turn from 145. The nitro and oxime compounds 142 and 143 proved to be 300 and 10 times more active respectively than acronycine in inhibiting the proliferation of L1210 leukaemia cells, but 142 was inactive against P388 leukaemia and C38 colon adenocarcinoma in mice.Condensation of synthetic 2-hydroxy-1,2-dihydroacronycine 149 with various glycoside donors has yielded a wide range of ·-hexopyranosides, the antiproliferative activity of which has been tested in murine L1210 leukaemia cells.76 While most of the products were about as active as acronycine itself, the azides 150 and 151 (as R/S mixtures at C-2 of the acronycine moiety) were approximately ten times more potent than the parent alkaloid in inhibiting cell proliferation.Since glycosides N F3C O N CbzNH H OMe OMe N H O OH CbzNH OMe OMe O N H N O O F3C N N F3C O N H O N O N OMe OMe O N H N O O F3C N OMe OMe O N O N O N3 O F3C N N H N N O N O H N H N H O N O N 80% xii 132 127 (+)-Fumiquinazoline G 100% iv 42% viii, ix 85% vii 131 130 129 81% i–iii 128 56% iv–vi 68% x, xi O Scheme 11 Reagents: i, 2,4-dimethoxybenzylamine (DMBn-NH2), DCC; ii, BH3, THF, TFA; iii, NEt3, TFAA; iv, H2, Pd/C; v, MeCOCOCl; vi, TFA, toluene, reflux; vi, H2, Pd/C; vii, 2-azidobenzoyl chloride; viii, CAN, MeCN, H2O; ix, Bu3P, benzene; x, NH3, MeOH; xi, MnO2, EtOAc, 1 d; xii, excess MnO2, EtOAc, several d 604 Natural Product Reports, 1998bearing amine substituents were only marginally active, it appears that activity may be related to lipophilicity of the sugar unit.Synthetic 1,2-dihydroxy-1,2-dihydroacronycine 152, made by treating acronycine with a catalytic quantity of osmium tetroxide and N-methylmorpholine N-oxide as reoxidant, has also been converted into a variety of derivatives for cytotoxic and antitumour investigations.77 In this case, all of the products 153–157 were more active than acronycine itself when tested against L1210 leukaemia cells in vitro.However, removal of the methoxy group at C-6 rendered the compounds inactive. In addition, compounds 153–157 were active in vivo against murine P388 at doses four to sixteen times lower than with acronycine itself, while 154, 156 and 157 were highly eYcient in reducing the volume of colon 38 adenocarcinoma, which is normally a highly resistant solid tumour.Compound 154 in particular was remarkably active; all the treated mice were free of tumours after 23 days of treatment. These are the most promising results to date in the search for antitumour drugs based on acronycine as a lead compound. 4 References 1 A. Nouga Bissoue, F.Muyard, A.Regnier, F. Bevalot, J. Vaquette, T. G. Hartley and P. G. Waterman, Biochem. Syst. Ecol., 1996, 24, 805. 2 X. Yang, H. Masao and N. Tsuneo, J. Chin. Pharm. Sci., 1996, 5, 68 (Chem. Abstr., 1997, 126, 328 058). 3 X. Yang, H. Masao and N. Tsuneo, J. Chin. Pharm. Sci., 1996, 5, 132 (Chem. Abstr., 1997, 126, 312 214). 4 W. E. Campbell and A. Bean, Biochem. Syst. Ecol., 1996, 24, 591. 5 K. Kamigiri, T. Tokunaga, M. Shibazaki, B. Setiawan, R. M. Rantiatmodjo, M.Morioka and K.-I. Suzuki, J. Antibiot., 1996, 49, 823. 6 A. Nouga Bissoue, F. Muyard, F. Bevalot, F. Tillequin, M.-F. Mercier, J. A. Armstrong, J. Vaquette and P. G. Waterman, Phytochemistry, 1996, 43, 877. 7 A. N. Starratt and S. Caveney, Phytochemistry, 1996, 42, 1477. 8 Y.-Q. Tang, X.-Z. Feng and L. Huang, Phytochemistry, 1996, 43, 719. 9 Y. Q. Tang, X. Z. Feng and L. Huang, Chin. Chem. Lett., 1995, 6, 877 (Chem. Abstr., 1995, 123, 334 995). Table 2 Isolation and detection of acridone alkaloids Species Alkaloida Ref.Bosistoa selwynii Bosistine Citrusamine Junosine N-Methylataphylline N-Methylataphyllinine Yukocitrine 69 Citrus paradisi (")-Margrapine Ab 136 (")-Margrapine Bb 137 70 Fagara macrophylla (=Zanthoxylum gillettii) Arborinine 1-Hydroxy-3- methoxyacridoneb 133 1-Hydroxy-3-methoxy-Nmethylacridone Xanthoxoline 71 Medicosma fareana Fareanineb 138 Melicopidine Normelicopicine 139 1,3,4-Trimethoxy-Nmethylacridone 72 aV. Samadera SAC-2825 1,8-Dihydroxy-Nmethylacridone 135 1-Methoxy-Nmethylacridoneb 134 14 aOnly new alkaloids and new records for a given species are listed in the Table.Structures of most known alkaloids may be found in previous reviews in this series. bNew alkaloids. 136 Margrapine-A R = H 137 Margrapine-B R = Me 134 133 138 Fareanine 139 Normelicopicine 140 135 N Me O O OR OR RO OR N Me O OH OMe OMe OMe N Me O O OMe OMe CO2Me OH N O O H H OMe N O OMe Me N O O H Me OMe OMe RO OH O N O O H Me OH 150 R1 = H; R2 = OAc 151 R1 = OAc; R2 = H 144 R = NO2 145 R = NH2 146 R = NMe2 147 R = NHCOMe 148 R = NHCOPh 149 R = OH 143 6 1 2 141 Acronycine R = H 142 R = NO2 152 R1 = R2 = H 153 R1 = H; R2 = COMe 154 R1 = R2 = COMe 155 R1-R2 = (C=O) 156 R1 = H; R2 = COPh 157 R1 = COMe; R2 = COPh N O O Me OMe R N O O Me OMe NOH N O O Me OMe R O O OMe N Me O O N3 R1 R2 N O O Me OMe OR2 R1O Michael: Quinoline, quinazoline and acridone alkaloids 60510 F. Muyard, A.Nouga Bissoue, F. Bevalot, F.Tillequin, P. 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ISSN:0265-0568    

DOI:10.1039/a815595y

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Fatty acids, fatty acid analogues and their derivatives Marcel S. F. Lie Ken Jie and Mohammed Khysar Pasha Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong Covering: 1996 and 1997 Previous review: 1997, 14, 163 1 Periodicals, books and reviews 2 Natural compounds: occurrence and structure 2.1 Unbranched fatty acids 2.2 Branched-chain and cyclic fatty acids 2.3 Oxygenated fatty acids 2.4 Other fatty acid derivatives 2.5 Conjugated linoleic acids (CLAs) 3 Synthetic fatty acids 3.1 Synthesis of olefinic and acetylenic fatty acids 3.2 Synthesis of methyl-branched fatty acids 3.3 Synthesis of hydroxy and other oxygenated fatty derivatives 3.4 Fluorinated analogues of fatty acids 3.5 Synthesis of cyclic fatty acids 3.6 Synthesis of unusual fatty acids 3.7 Miscellaneous reactions of fatty acids and derivatives 3.8 Synthesis and reactions of glycerides 4 Antioxidants 5 Hydrogenation 6 Polymorphism, lipid membranes and lipid vesicles 7 Physical properties and methodology 7.1 Thin layer chromatography 7.2 High pressure liquid chromatography 7.3 Gas liquid chromatography 7.4 Supercritical fluid chromatography 7.5 Infrared and Raman spectroscopy 7.6 Nuclear magnetic resonance spectroscopy 7.7 Mass spectrometry 8 Biosynthesis and biotechnology 8.1 Biosynthesis 8.2 Lipase-catalyzed reactions 8.3 Sources and biological eVects of n-3 polyunsaturated fatty acids 8.4 Lipid peroxidation and autoxidation 9 References 1 Periodicals, books and reviews One newsletter on polyunsaturated fatty acids and their relation to human health1 and another on lipid technology2 have appeared in circulation.Handbooks and manuals published include topics on physical and chemical characteristics of fats, oils, waxes,3–6 nutrition7 and fat replacers,8 biology of essential fatty acids,9 testing of lipoproteins10 and membrane lipids.11 Books on specific oils and lipids include: olive oil,12 saZower oil13 and fungal lipids.14 A new edition of the classic Fatty Acid and Lipid Chemistry by F.D. Gunstone has been published.15 Books on methodology, technology and analysis of lipids include: Advances in Lipid Methodology – Three and Four,16,17 techniques and applications in lipid analysis,18–20 lipid technology and application,21 technology for extracting oilseeds22 and supercritical fluid extraction technology,23 applied lipid research24 and oleochemicals.25 Books on the following topics include: synthesis of glycerides, fatty acids, phospholipids and glycolipids,26 a guide to phospholipids,27 biochemistry of lipids,28 metabolism of „-linolenic acid,29 molecular biology of plant lipids,30 lipid messenger,31 lipoxygenase pathway enzymes,32 fats and oils related to nutrition,33–37 and on health and lipid related diseases.38–42 Books on antioxidants43–45 and on environmental issues facing the edible oil industry have also been published.46 Reviews have appeared that deal with the following topics: trans-unsaturated fatty acids in bacteria,47 bacterial lipopolysaccharides, 48 bacterial polyesters,49 positively charged lipids, 50 medium-chain lipids,51 n-3 fatty acids and lipoproteins,52 micellar solutions as reaction media,53 lipids and biopackaging, 54 ether lipids,55 betaine ether-linked glycerolipids,56 synthesis of phospholipids,57 synthesis of perdeuterated phospholipids,58 assessment of prooxidant and antioxidant actions,59 tocopherols and tocotrienols,60 nuclear magnetic resonance spectroscopy of fatty acids and triacylglycerols61 and mass spectrometric analysis of fatty acid derivatives.62–64 Reviews on the biochemical, metabolic and physiological aspects of lipid molecules include: metabolism of thia fatty acids,65 import of lipids into mitochondria,66 role of essential fatty acids in calcium metabolism and osteoporosis,67 fatty acid activation,68 cellular fatty acid-binding proteins,69 dietary polyunsaturated fatty acid relation of gene transcription,70 regulation of fatty acid synthase,71 synthesis and biological activity of oxygenated unsaturated acids,72 hydroxylation and epoxidation of polyunsaturated fatty acids by cytochrome P450,73 phospholipid metabolism in the mammalian heart,74 phospholipid membranes,75 isoprostanes from lipid peroxidation,76 lipids and membrane function in green algae.77 Reviews on biotechnology of lipids include: application of lipase,78 catalytic activity of lipases towards hydroxy fatty acids,79 uses of biotechnology in modifying plant lipids,80 lipase specificities,81 arachidonates 12-lipoxygenases82 and phosphotidate phosphotases.83 Reviews on the biosynthesis of fatty acids and related metabolites,84 hydroperoxy unsaturated fatty acids,85 triacylglycerols,86 plant fatty acids,87 inhibitors of glycosphingolipid biosynthesis88 and marine oxylipids89 have been published.Reviews on dietary fat include: n-6/n-3 fatty acid balance and chronic elderly diseases,90 eVect on plasma cholesterol,91 lipids in human milk,92 applications of milk-fat fraction in confectionary products,93 usefulness of dietary medium-chain triglycerides,94 phospholipids in fat emulsion95 and oxysterols in tissues and foods.96 2 Natural compounds: occurrence and structure 2.1 Unbranched fatty acids A novel fatty acid, 16:4(4Z,7Z,10Z,13Z), was identified in the extract of the sponge Callyspongia sp.found oV the coast of New South Wales, Australia.97 Another new fatty acid, 17:3(5Z,9Z,12Z), was isolated from the cellular slime mold Polysphondylium pallidum.98 Spitzer has isolated two unusual fatty acids, 16:2(9Z,12Z) and 20:2(11Z,14Z), as minor components in the seed oils of several species of the Sapindaceae family which contain 18:1(11Z) and 20:1(13Z) as the main components of the monoenoic acid fraction.99 Substantial amounts of „-linolenic acid, 18:3(6Z,9Z,12Z) (6.6–13.0%) and stearidonic acid, 18:4(6Z,9Z,12Z,15Z) (2.4–21.4%) were found in the seed oils of nine Central Asian species of the Boraginaceae family.100 Stearidonic acid was also identified in the extract of leaves of the plant Borago oYcinalis.101 The level of ƒ5-olefinic fatty acids, namely 18:2(5Z,9Z), 18:3(5Z,9Z,12Z) and 20:3(5Z,11Z,14Z), in edible pine seeds (Pinus cembroides edulis) was found to be exceptionally low Lie Ken Jie and Pasha: Fatty acids, fatty acid analogues and their derivatives 607(ranging from 0.1–0.4% of the total fatty acid pool) as compared to other conifer species.102 The positional distribution of ƒ5-olefinic acid in triacylglycerols from conifer seed oils appeared to be mainly acylated to the sn-3 position of the glycerol backbone.103 The seed oil of Caesalpinia bonduc (Cesalpiniaceae), which is used in the treatment of filariasis, contains two unusual fatty acids 18:1(4) and 18:2(2,4).The configurations of the olefinic bonds are not indicated.104 The presence of ·-parinaric acid, 18:4(9Z,11E,13E,15Z), in the seed oil of Sebastiana brasiliensis (Euphorbiaceae) was confirmed by a combination of mass spectroscopic and 1H and 13C nuclear magnetic resonance spectroscopic analyses.105 A new triacylglycerol containing two punicyl, 18:3(9Z,11E,13Z), acyl groups and one 18:3(8Z,11Z,13E) acyl group was isolated from the seed of pomegranate (Punica granatum).106 An unusual polyunsaturated fatty acid, 18:5(3Z,6Z,9Z, 12Z,15Z), was isolated from a raphidophyte alga, Heterosigma akashiwo.107 A number of novel di-, tri- and tetra-enoic fatty acids (ranging from C16 to C34 in chain-length) with bismethylene- interrupted double bond systems were isolated from the sponge Haliclona cinerea.The structures of these fatty acid components were elucidated by gas chromatography–mass spectrometric analysis of the picolinyl ester derivatives.108 The presence of bis-homopinolenic acid, 20:3(7Z,11Z,14Z), was identified in the oil of pine (Pinus contorta) seeds.109 An extract of the marine chloromonad Heterosigma carterae (Raphidophyceae) contained a complex mixture of sulfoquinovosyl diacylglycerols.The main fatty acyl groups consisted of 16:0, 16:1(9Z), 16:1(11Z), 16:1(13Z) and 20:5 (5Z,8Z,11Z,14Z, 17Z).110 The latter compound (20:5) was also identified as an allelopathic substance, which displayed growthinhibitory and spore-settlement suppressive activities in the lipid extract of the red alga, Neodilsea yendoana.111 Two new isomers of eicosapentaenoic acids, namely 20:5(5Z,8Z,11E,14Z,17Z) and 20:5(5Z,8Z,11E,14Z,17E), were isolated from the extract of liver of rats fed heated vegetable oils.112 High levels of monoenoic fatty acids [16:1(9Z), 18:1(9Z), 20:1(9Z), 20:1(11Z), 22:1(13Z)] in some midwater fishes (Myctophidae) were found in the form of wax esters and triacylglycerols. 113 The phospholipids found in the membrane of the marine ciliated protozoan Parauronema acutum consist primarily of palmitic, oleic, linoleic, ·-linolenic acid and n-3 polyunsaturated fatty acids 18:4, 20:5 and 22:6.114 A high amount of 22:2(13Z,16Z) (57%) and some 22:3(5Z,13Z,16Z) were isolated from the seed oil Eranthis hyemalis (Ranunculaceae). 115 An unidentified bacterial strain SCRC-21406, isolated from the intestine of a marine fish Glossanodon semifasciatus was found to produce 22:6 (28% of total fatty acids) when cultured in a medium containing peptone and yeast extract.116 Several new mono and polyunsaturated long chain fatty acids [20:1(16Z), 24:1(11Z), 25:1(5Z), 26:1(11Z), 28:1(11Z), 30:1(23Z), 26:2(17Z,21Z), 28:2(19Z,23Z), 30:2 (9Z,23Z), 26:3(5Z,9Z,21Z) and 30:3(5Z,9Z,25Z)] were identi- fied from the phospholipids isolated from two Senegalese marine sponges Trikentrion loeve and Pseudaxinella cf.lunaecharta (order: Axinellida).117 A novel diunsaturated C10 diacid (2E,4Z-deca-2,4-dienedioic acid) was identified as one of the many acid metabolites isolated from cultures of the zygomycete Phycomyces blakesleeanus.118 An usual .-fluoro- 9,10-epoxy C18 fatty acid was identified together with other .-fluoro acids of 16:1, 18:0, 18:1, 18:2, 20:0 and 20:1 in the seed oil of Dichapetalum toxicarium (Dichapetalaceae).119 The distribution of crepenynic acid 18:2(9Z,12A)† and dehydrocrepenynic acid 18:3(9Z,12A,14Z) (65% of total fatty acids) in triacylglycerols of the aril and cotyledon oils of Afzelia cuanzensis (Caesalpinaceae) was studied by 13C nuclear magnetic resonance spectroscopy.120 Ximenynic acid [also known as santalbic acid, 18:2(9A,11E)] (40%) was isolated from sandalwood seeds (Santalum spicatum)121 and the structure confirmed by 1H, 13C nuclear magnetic resonance spectroscopic and mass spectrometric analysis.122 A novel acetylenicdiolefinic fatty acid, 18:2(7Z,9A,11E) (22.6%), has been isolated from the seed oil Heisteria silvanii (Olacaceae) and two conjugated diacetylenic fatty acids, namely 18:2(9A,11A) (0.1%) and 18:3(9A,11A,13Z) (0.4%), have been tentatively characterized by their mass spectra.123 A new very-longchain acetylenic fatty acid metabolite, 31-hydroxy-33:5 (2A,4Z,18Z,29E,32A), has been isolated from the marine sponge Pellina triangulata.124 An acetylene-containing natural product consisting of a taurine and two fatty acid residues has been isolated from the Okinawan marine sponge Hippospongia sp.One of the fatty acid residues is 25:2(4A,8Z).125 2.2 Branched-chain and cyclic fatty acids 14-Methyl-16:0 has been identified in Ginkgo biloba126 and pine (Pinus contorta) seeds.127 A new methyl-branched fatty acid, 9-methyl-16:1(10Z) and the uncommon 11-methyl- 18:1(12Z) were found in the lipid extract of a new strain of bacterium Vibrio alginolyticus associated with the alga Cladophora coelothrix.128 Another novel methyl-branched fatty acid, 10-methyl-18:1(9Z), was found in the lipid extract of the marine fungus Microsphaeropis olivacea.129 Two very unusual phytyl esters were obtained from the extract of the hornwort Megaceros flagellaris.The fatty acid moiety comprises 3,7,11,15-tetramethyl-16:1(2E) or 3,7,11,15-tetramethyl-16:0, which is esterified to the corresponding tetramethyl unsaturated (16:1) alcohol.130 Two unsaturated cyclopropyl C27 fatty acids, viz. 10,11-methylene-27:2(5Z,9E) and 10,11-methylene- 27:3(5Z,9E,20Z) were isolated from an Australian sponge Amphimedon sp., and were shown to be DNA topoisomerase inhibitors.131,132 A new bicyclo eicosanoid 1 from the Mediterranean sponge Reniera mucosa, which contains an unusual bicyclo[4.3.0]nonane system, has been identified.133 An unusual cyclic ether lipid 2, which inhibits gastrulation of starfish embryos, has been isolated from the lipid extract of a marine sponge Hippospongia sp.134 2.3 Oxygenated fatty acids 10-Hydroxystearic acid was identified in the phosphatidylethanolamine fraction of the lipid isolated from Cryptosporidium parvum oocysts.135 The triacylglycerols from the sclerotia of the rye ergot Claviceps purpurea contain high levels of ricinoleic acid [12-hydroxy-18:1(9Z)].136 A novel branched medium chain hydroxytrienoic C12 acid, 11-hydroxy-4-methyl 12:3(2E,4E,6E), was isolated from the fermentation broth of Mucor sp., strain KL 94-42 aq.137 A new hydroxy fatty acid, 15(R)-hydroxy-18:2(9Z,12Z), was isolated from oat seeds in quantities of 0.6–0.7 mg g"1.138 17(R)-Hydroxy-20:4(5Z,8Z, 11Z,14Z) and 17(R)-hydroxy-20:5(5Z,8Z,11Z,14Z,17Z) were obtained by alkaline treatment of the fatty acyl phloroglucinol isolated from the brown alga Zonaria diesingiana (Dictyota). 139 Three new oxylipins 3–5 were identified from the lipid extract of the temperate red alga Polyneura latissima.140 ‚-Dimorphecolic acid, (9S)-(+)-hydroxy-18:2(10E,12E), was isolated in relatively pure state by supercritical carbon dioxide extraction of Dimorphotheca pluvialis oil seeds.141 Very-long-chain fatty acids (C28–C34) containing a hydroxy †A=Acetylenic.COOH O COOH H H 1 2 608 Natural Product Reports, 1998group at the ¢-18 position have been identified in the microalgae from the genus Nannochloropsis. Two dihydroxy fatty acids, 15,16-dihydroxy-30:0 and 16,17-dihydroxy-33:0, have also been identified from the acid hydrolysis of the cell residue of the same algae.142 A new monoglyceride containing 26-hydroxy-26:0 fatty acid has been isolated from the root bark of Pentaclethra eetveldeana, which is used in Zairian traditional medicine for the treatment of haemorrhoids, malaria and epilepsy.143 The seed oil of Bernardia pulchella (Euphorbiaceae) contains 91% of vernolic acid, 9,10-epoxy- 18:1(12Z).144 Small but significant quantities (as high as 2.5%) of 9,10-epoxy-18:0, 12,13-epoxy-18:1(9Z) and 9,10- epoxy-18:1(12Z) were found in peanut (Arachis hypogaea) germplasm.145 A novel triacylglycerol containing a transepoxy diacetylenic-diolefinic C18 acid, viz. 8,9-epoxy- 18:4(4A,6A,13Z,15Z), was isolated from the slime mould Lycogala epidendrum.146 A new keto fatty acid, 9-keto- 18:1(13Z) (26%), was isolated from the seed oil of Smilax macrophylla (Liliaceae).147 The biosynthesis of furan fatty acids by a marine bacterium Shewanella putrefaciens was studied. The mutant derived from this species, when treated with N-methyl-N*-nitro-N-nitrosoguanidine, produced a range of C14, C16 and C18 furan fatty acids with 10,13-epoxy-11- methyloctadeca-10,12-dienoic acid occurring in 10–15% of the total fatty acid pool.148 The presence of furanoid fatty acids, namely 10,13-epoxy-11,12-dimethyl-18:2(10,12) and 12,15-epoxy-13,14-dimethyl-20:2(12,14), in soybean oil was reconfirmed and these unusual fatty acids were found to be preferentially esterified at the sn-1 position of the triacylglycerols.149,150 2.4 Other fatty acid derivatives The methyl ester of 10:3(2E,4Z,6Z) was identified as a sexspecific compound from the stink bug Thyanta pallidovirens.The ester was found to be thermally unstable.151 The methanolic extract of the marine sponge Ircinia felix from the Colombian coast has yielded nine novel fatty acid esters with the acyl groups esterified to variabilin 6. Two methyl branched fatty acids, namely 22-methyl-30:2(5Z,9Z) and 2,11-dimethyl- 18:0, in this series of esters were reported for the first time.152 The nuclear magnetic resonance spectroscopic and mass spectrometric properties of the bixin family of apocarotenoids 7 have been studied.153 The structure of an apocarotenoid (1% of total carotenoid) isolated from the seed coat of Bixa orellana fruit was established as compound 8.154 Four other new carotenoids were also isolated and identified from this seed coat.155 Two new marine prostanoids, preclavulone lactone I 9 and lactone II 10 were isolated from the Okinawan soft coral Clavularia viridis.The structures were determined by spectroscopic analysis and confirmed by chemical synthesis starting from (S)-malic acid.156 Several betaine lipids, diacylglycerylhydroxymethyltrimethyl-‚- alanine containing C18, C20 and C22 polyunsaturated fatty acid moieties, were identified in the extract of the marine algae Chroomonas salina 157 and Pavlova lutheri.158 An endogenous sleep-inducing lipid was identified as cis-octadec- 9-enamide (oleamide) and the synthetic oleamide was found to induce sleep at nanomolar quantities.Hydrolysis of oleamide with oleamide hydrolase furnished oleic acid.159,160 Two new amides, N-isobutyl amide of 14:5(2E,4E,8E,10E,12E) from the fruit of Zanthoxylum sp. (a commercially available fruit in Japan)161 and N-isobutyl amide of 18:2(2E,4Z) from the dried crushed fruits of Piper nigrum were isolated and identified.162 Ten unsaturated alkylamides were isolated from the pericarps of Zanthoxylum bungeanum.163 2.5 Conjugated linoleic acids (CLAs) Conjugated linoleic acids [CLAs, a mixture of conjugated 18:2(9,11) geometric isomers] have attracted great interest among nutritionists, as these compounds are natural fat components which appear to induce a number of healthgiving properties.Animal studies have shown that CLAs display anticarcinogenic eVects and reduce the risk of atherosclerosis. Methods of analysis of CLAs have been reviewed by Christie.164 The potentials of CLA research have been discussed. 165,166 Gas chromatography–mass spectrometric analysis of methyltriazolinedione adducts of commercial CLAs showed the composition as a mixture of 18:2(8,10), 18:2(9,11), 18:2(10,12) and 18:2(11,13) isomers.167 The synthesis and nuclear magnetic resonance spectroscopic properties of all four geometric isomers of 18:2(9,11) have been described.168 A large scale synthesis of methyl 18:2(9Z,11E) from methyl ricinoleate [12-hydroxy-18:1(9Z)]169 and the preparation of deuterium labeled methyl 18:2(9Z,11E)- and 18:2(9E,11E)-[17,17,18,18- 2H4] have been reported.170 The oxidative stability of CLAs relative to other polyunsaturated fatty acids was investigated.The results showed that CLAs were readily decomposed due to the formation of unstable free-radical intermediates during oxidation.171 CLAs were also found to modulate the hepatic lipid composition in mice.172 The eVect of CLAs on the body and body fat composition in mice has also been reported.173 Rats fed commercial CLAs were shown to produce significant levels of 20:3(8,12,14), 20:4(5,8,12,14) and 20:4(5,8,11,13) in the liver by chain elongation and desaturation of 18:2(10,12) and 18:2(9,11).174 A specific method for the methylation of milk and rumen fatty acids with emphasis on the presence of CLAs and trans fatty acids has been developed.175 CLAs were found O COOH COOH O OH H H COOH O OH H H 3 4 5 O O OR O HOOC COOH MeO2C O O O O O O 6 R = fatty acyl 7 8 9 10 Lie Ken Jie and Pasha: Fatty acids, fatty acid analogues and their derivatives 609to be neither antioxidants nor prooxidants of linoleic and ·-linolenic acids when the oxygen uptake in these substrates was monitored.176 CLAs were shown to inhibit phorbol ester skin tumor promotion in mice.177 3.Synthetic fatty acids 3.1 Synthesis of olefinic and acetylenic fatty acids A facile method to prepare symmetrical Z-olefins was reported, which involved phosphonium salts via an autoxidation process in salt free conditions (modified Wittig reaction).Application of this method allowed for the synthesis of (7Z)- tetradecenedioic acid and dimethyl (10Z)-eicosenedioate from 8-bromooctanoic acid and methyl 11-bromoundecanoate, respectively, by reaction of the respective alkyltriphenylphosphonium bromide with oxygen.178 A similar approach for the preparation of symmetric cis-skipped polyenes, which combines the controlled classical Wittig reaction or the oxidative dimerization of phosphoranes procedure, has been described.179 The following radiolabelled fatty acids, (9Z,12E)-, (9E,12Z)-[1-14C]linoleic acid, (9Z,12Z,15E)-, (9E,12Z,15Z)-[1-14C]linolenic acid and (5Z,8Z,11Z,14E)-[1- 14C]arachidonic acid were synthesized by chain elongation and through cis-olefination via stereospecific Wittig reactions.The radioactive carbon atom was introduced from potassium [14C]cyanide, where the resulting [14C]nitrile intermediates were hydrolyzed to the requisite radiolabelled carboxylic acids.180 The synthesis of ‚-parinaric acid [18:4(9E,11E,13E,15E)] was reported.181 Santalbic acid or xymenynic acid [18:2(9A,11E)] was prepared from ricinoleic acid [12-hydroxy- 18:1(9Z)]. The first steps involved the bromination and ultrasound-assisted dehydrobromination of ricinoleic acid, which gave the corresponding hydroxyacetylenic intermediate. The latter compound was mesylated and ultrasound-assisted demesylation furnished the requisite santalbic acid and its Z-isomer.Santalbic acid was subsequently isolated by urea fractionation.182 A new stereocontrolled route to conjugated dienynes was developed, which involved Wittig homologation and dehydrohalogenation. This method was applied to the synthesis of 23:4(4A,6Z,8E,19A).183 3.2 Synthesis of methyl-branched fatty acids The synthesis of racemic 2,4-dimethyltetradecanoic acid was described starting from 3,5-dimethyl-6-triphenylmethyloxyhexanal, which was chain extended via the Wittig reaction.Trityl deprotection and hydrogenation of the resulting intermediate gave 2,4-dimethyltetradecanol, which was oxidized to the requisite carboxylic acid.184 The synthesis of 15-methyl-16:1(11Z) was achieved via the Wittig reaction of (4-methyl-1-pentyl)triphenylphosphonium bromide and 10-bromodecanal.185 4(R)-Methyl-17:2(7Z,11Z) was synthesized from enantiomerically pure (S)-citronellol as the starting material.The synthesis involved the alkylation of pent-4-yn- 1-ol with (S)-citronellyl bromide, followed by depyranylation, semi-hydrogenation over P-2 Ni in ethanol to the (Z)-alcohol and oxidation to the aldehyde intermediate. Wittig olefination of the latter with hexylidenephosphorane gave 9Z,13Z-2,6- dimethylnonadeca-2,9,13-triene, which was regioselectively epoxidized at the 2-position. Cleavage of the epoxide with HIO4 followed by oxidation of the aldehyde intermediate gave the requisite (4R)-methyl-17:2(7Z,11Z) fatty acid.186 Racemic 3-hydroxy-2,4,6-trimethyl-24:0 was synthesized from 2,4- dimethyldocosanal, which was reacted with methyl propionate to give methyl 3-hydroxy-2,4,6-trimethyl-24:0. Reaction of 2,4-dimethyldocosanal with 1-ethoxycarbonylethylidenetriphenylphosphorane furnished 3-hydroxy-2,4,6-trimethyl- 24:1(2E).187 The synthesis of phomenoic acid 11 was achieved by application of the Wittig reaction for the homologation of aldehyde intermediates and coupling of iodo intermediates with the lithiated derivative of 1,3-dithiane via the Corey– Seebach umpolung method.188 The synthesis of [1-14C]-nordolichoic acid (12) was achieved by coupling of polyprenol (isolated from the leaves of Ginkgo biloba) with ethyl acetoacetate followed by hydrolysis, decarboxylation and reduction to yield the 2-polyprenyl-1- methylethanol intermediate.The latter was converted to the mesylate and subjected to one-carbon elongation with K14CN to give 12 on hydrolysis with KOH in toluene–ethanol.189 The syntheses of the following archaebacterial lipids have been reported: archaebacterial lipid C20 chirons,190 archaeal 36-membered macrocyclic diether lipids191 and 72-membered macrocyclic tetraether lipids.192 3.3 Synthesis of hydroxy and other oxygenated fatty derivatives The relative and absolute configuration of 3S,12Sdihydroxypalmitic acid, a constituent of the Ipomea operculata M.resin, was confirmed by synthesis starting with dimethyl L-malate.193 An eYcient synthesis of 5(S)-hydroxy- 20:4(6E,8Z,11Z,14Z) was accomplished by coupling of methyl 5(S)-hydroxy-7-iodoheptanoate with 4Z,7Z-tridecadien-1- yne.194 A highly stereoselective synthesis of ‚-dimorphecolic acid [9(S)-hydroxy-18:2(10E,12E)] was reported.The procedure featured a diastereoselective reduction of a keto intermediate, where a tricarbonyliron lactone tether induced a 1,5-transfer of chirality. This was followed by a stereoselective decarboxylation to create all the stereochemical elements of ‚-dimorphecolic acid.195 The total synthesis of 5(S)-hydroxy-12-oxo-20:3(6Z,8E,14Z) involving Wittig coupling reactions of tailored intermediates was described.196 10(S)-Hydroxy-20:4(5Z,8Z,11Z,14Z) was synthesized in eight steps starting from enantiomerically pure (R)-glyceraldehyde acetonide.197 Chiral adducts from Grignard or allylsilane additions to 1,3-dioxan or 1,3- dioxolan-4-ones were exploited for the total synthesis of the (R)- and (S)-isomers of 16-, 17- and 18-hydroxy-20:4 (5Z,8Z,11Z,14Z).198 The chemoenzymatic synthesis of 13(S)-hydroxy-18:2 (9Z,11E) was achieved in nine steps starting from (E)-oct-2- enal.Of importance was the enzymatic conversion of (E)-oct- 2-enal to the (S)-cyanohydrin 13 with (S)-hydroxynitrile lyase cloned from Hevea brasiliensis.199 13-Hydroxy-10-oxo- 18:1(11E) was synthesized via a Knoevenagel-type reaction of isopropyl 11-phenylsulfinyl-10-oxoundecanoate with heptanal to form „-hydroxyenone functionality with carbon chain elongation.200 HOOC OH OH OH OH OH OH COOH n 11 12 CN OH 13 610 Natural Product Reports, 1998The regiospecific oxidation of a number of substituted unsaturated fatty esters with p-benzoquinone in the presence of palladium(II) chloride under concomitant ultrasonic irradiation was reported.For example, methyl 9-hydroxy- 18:1(12Z) furnished methyl 9-hydroxy-12-keto-18:0 exclusively. 201 The synthesis of symmetrically and asymmetrically substituted 1,2-dialkyl polyoxyethylene glycerol ethers has been reported.202 The stereoselective synthesis of (2R,2*S)-1- O-(2*-hydroxy-hexadecyl)glycerol and its oxo analogue are described.203 Several antifungal fatty acids containing hydroxy and amino groups have been synthesized: viz. fumonisin B2 14,204 sphingofungin B 15205 and sphingofungin F 16.206 3.4 Fluorinated analogues of fatty acids A series of fluorine-containing unsaturated fatty acids 17, which are potential fungicides, have been prepared by consecutive bromofluorination and dehydrobromination of ¢-alkenoic acids.207 Treatment of 18:1(6Z) with perfluorooctyl iodide in the presence of lead powder–CuII acetate gave a mixture of positional isomers of iodoperfluorooctyl-18:0 derivatives.208 The fluoro analogue of coriolic acid [13-fluoro-18:2(9Z,11E)] was prepared by a Wittig coupling reaction of methyl 9-triphenylphosphonobromononanoate and (E)-4-fluoronon- 2-enal.209 The synthesis of 5,6-difluoroarachidonic acid 18 has been described.210 3.5 Synthesis of cyclic fatty acids Three olefinic fatty acids containing a cyclopropenyl system (for example, compound 19) have been prepared by dibromocyclopropanation of 2-bromoalk-1-ene to give 1,1,2-tribromo- 2-alkylcyclopropane, which was then treated with butyllithium and followed by reaction with lithiopropene to yield the requisite olefinic cyclopropenyl fatty acids.211 EYcient methods for the enantiomeric synthesis of (")-methyl jasmonate 20 and (+)-methyl epijasmonate 21 have been reported.The procedure makes use of a chiral cyclopentanoid building block, which can be prepared from tartaric acid by way of a phosphorus ylide.212 Jasmonic acid was also prepared by mixed Kolbe electrolysis. 213 Methyl 3-methyljasmonate was synthesized from methyl jasmonate via methyl 3,7-dehydrojasmonate.214 The synthesis of methyl 3,7-didehydrojasmonate was also reported.215 The Friedel–Crafts adducts of methyl oleate, olefinic C6 hydrocarbons and esters with benzene and toluene have been described.The products are a mixture of monomer, dimer and trimer esters together with adducts containing one, two or three molecules of ester per mole of aromatic compound.216,217 The synthesis of polyunsaturated constituents of phenolic lipids, such as 2-hydroxy-6-(pentadeca-8Z,11Z-dienyl)benzoic acid, has been described.218 The synthesis of lurlenic acid 22 and some analogues starting from D-xylose, 2,3-dimethyl-phydroquinone and geranylgeraniol or farnesol has been described.219,220 Monocyclic fatty acids formed from oleic acid in heated sunflower oils have been identified as 4-(2*-nonylcyclopentyl) butanoate and 3-(2*-nonylcyclohexyl)propanoate by mass spectral analysis.221 The saturated bicyclic fatty acids were also detected in heated sunflower oils.222 A study of the eVects of such cyclic fatty acid monomers on cultured porcine aortic endothelial cells showed a significant reduction in Ca2+ ATPase activity and significant increase in prostacyclin synthesis and secretion.223 The nature of cyclic fatty acids and the gas chromatographic properties of cyclic fatty acids from heated polyunsaturated vegetable oils have been reported.224,225 The identification of traces of cyclobutane fatty acid from fatty acids in food by gamma irradation has been reported.226 3.6 Synthesis of unusual fatty acids The synthesis and the study of nuclear magnetic resonance spectroscopic properties have been reported for acetylenic tellura fatty esters where the number of methylene groups between the acetylenic bond and the tellurium atom varied from 0–4.227 Five positional isomers of thiaoleic acid, where the number of methylene groups between the olefinic bond and the sulfur atom ranged from 1–5, have been prepared as models for studies on f12-desaturation.228 A novel analogue of hepoxilin A3 23 containing a thiirano group at the C-11/C-12 position was accomplished through allylic rearrangement of (11R,12R)-hepoxilin B3 under Mitsunobu conditions followed by conversion of the epoxy group of the intermediate to the requisite thiirano analogue.229 A long chain conjugated diacetylenic fatty ester containing a tetrathiafulvalene group in the ester group has been prepared, which polymerizes in the solid state to yield polydiacetylenes. 230 12-Aminododecanoic acid was obtained by hydrogenation of vernolic acid [12,13-epoxy-18:1(9Z)] to give epoxystearic acid.The latter compound was oxidized by HIO4 to yield 12-oxododecanoic acid, which was treated with O O COOH O O COOH COOH OH NH2 COOH OH COOH COOH O OH OH OH OH OH OH OH Me NH2 NH2 15 14 16 COOH F COOH F F n 17 n = 5–15 18 COOH COOMe O COOMe O 19 20 21 22 O CO2H OH O OH HO HO Lie Ken Jie and Pasha: Fatty acids, fatty acid analogues and their derivatives 611hydroxylamine to form the oxime.The oxime was then catalytically reduced to yield the requisite 12-aminododecanoic acid.231 Reactions induced by ultrasonic irradiation of methyl 9,12-dioxostearate with hydrazines in water in the presence of acidic alumina gave high yields of pyridazine fatty esters directly.232 The synthesis of novel piperideine- (24) and pyridine- (25) containing long-chain fatty ester derivatives has been reported starting from methyl isoricinoleate [9-hydroxy-18:1(12Z)].233 Stereoisomers of flavolipins (e.g.compound 26) which contain an amido function have been synthesized from D-glucose in a stereocontrolled manner. However, none of the four stereoisomers prepared was identical with the reported structure of a natural product isolated from Flavobacterium meningosepticum. 234 The preparation of a number of interesting adducts of [60]-fullerene containing ester functions have been reported. Reaction of [60]-fullerene with ethyl bromoacetate and zinc followed by quenching with trifluoroacetic acid gives 1-ethoxycarbonylmethyl-1,2-dihydro-[60]-fullerene.235 Direct treatment of [60]-fullerene with ethyl or octadecyl malonate in the presence of CBr4 and diazabicyclo[5.4.0]undec-7-ene (DBU) furnished the corresponding methanofullerenes 27.236 The following fullerene adducts have been synthesized: fullerene esters containing cholesterol 28,237 D2h-symmetrical tetrakis(methano)fullerenes 29,238 tethered tris-adduct 30,239 trans-enediyne 31,240 and a triester derivative 32.241 3.7 Miscellaneous reactions of fatty acids and derivatives Oxidative cleavage of acetylenic and olefinic fatty acids with potassium permanganate was readily achieved under concomitant ultrasonic irradiation, which allowed the position of the unsaturated centre of the unsaturated fatty ester substrate to be determined.242 Ultrasonically stimulated oxidation reactions of 2,5-disubstituted C18 furanoid fatty ester with magnesium monoperoxyphthalate or m-chloroperoxybenzoic acid gave methyl 9,12-dioxo-18:1(10Z) and 10,11-epoxy-9,12- dioxo-18:0, respectively.243 4,5-Epoxy-14:1(2E) was synthesized starting with two sequential additions of acetate units to 10:0 followed by epoxidation of resulting 14:2(2E,4E) with Oxone (potassium peroxomonosulfate).244 Reaction of the seed oil of Vernonia anthelmintica [which contains vernolic acid, 12-13-epoxy-18:1(9Z)] with diol-ethers in the presence of BF3–diethyl ether caused ring opening of the epoxy function to yield oligoethylene glycol ethers.245 Oxidation of olefinic fatty esters to the corresponding keto derivatives was accomplished when the substrates were treated with palladium(II) chloride and p-benzoquinone in aqueous tetrahydrofuran under ultrasonic irradiation.For example, methyl oleate furnished a mixture of methyl 9(10)-oxostearate, while methyl 12-hydroxy-18:1(9Z) furnished methyl 12- hydroxy-9-oxostearate exclusively.246 The synthesis of ketoand hydroxy-dienoic acids from linoleic acid in a four-step procedure has been described.247,248 Oxidation reactions of acetylenic fatty esters with selenium dioxide–tert-butyl hydroperoxide gave mixtures of hydroxy- and oxo-acetylenic derivatives, but in the case of 18:2(9A,11E) the major product isolated was 8-hydroxy-18:2(9A,11E) (70%).249 Lipoxygenasecatalyzed asymmetric oxygenation of linoleic acid followed by cobalt porphyrin-catalyzed reduction-oxygenation furnished (E)-10-oxooctadec-11-en-13-olide and 13-hydroxy-10- oxo-18:1(11Z).250 A facile method for the synthesis of macrocyclic lactones was achieved from ¢-hydroxy fatty acids using di-tert-butyl pyrocarbonate in the presence of triethylamine.251 Reaction of 20:1(5Z) from meadowfoam seed oil (Limnanthes) with perchloric and sulfuric acid in chlorinated solvents gave the corresponding ‰-lactone.252 Epoxidation of oleic and linoleic acid was readily achieved by treatment with the acetonitrile complex of hypofluorous acid.253 Phase-transfer catalyzed biphasic epoxidation of unsaturated triglycerides was accomplished with ethylmethyldioxirane in butan-2-one.254 The enantioselective formation of an ·,‚-epoxy alcohol by reaction of methyl 13(S)- hydroperoxy-18:2(9Z,11E) with titanium isopropoxide has been reported.255 Four new octadecatrienoic acid ethyl esters [18:3(5Z,11Z,13E), 18:3(5Z,10Z,12E), 18:3(5Z,9E,11Z) and 18:3(5Z,10Z,12Z)] were synthesized, which on heating underwent cyclization via an intramolecular Diels–Alder reaction.256 Heat treatment of linolenic acid [18:3(9Z,12Z,15Z)] showed that the 12Z-double bond was not readily isomerized, however, this double bond appeared essential to induce cis–trans isomerization of the adjacent double bonds at the 9- and 15-positions.257 Homoallyl ethers were obtained from olefinic fatty esters by the ethylaluminium induced reactions with dimethyl acetals of formaldehyde, acetaldehyde, isobutyraldehyde and pivaldehyde.258 Esterification of oleic acid with glycerol can be achieved in the presence of sulfated iron oxide catalyst at 180–240 )C.259 A fluorescent analogue of cholesteryl oleate, namely 22-[ethyl(2*-naphthyl)amino]-23,24-dinorchol- 5-en-3‚-yl oleate, has been prepared from a steroidal carboxylic acid, 2-naphthylamine and oleic acid.260 Reaction of 18:2(9Z,12Z) with 50% BF3–methanol gave monomethoxy and dimethoxy derivatives.261 Allylic amination of methyl oleate with bis(N-p-toluenesulfonyl)sulfodiimide gave a mixture of methyl 11-amino-(N-p-toluenesulfonyl)-18:1(9E) and methyl 8-amino-(N-p-toluenesulfonyl)-18:1(9E).262 Thermal reactions of fatty acids with diethylene triamine gave 1,3-diamides in vacuo, but when carried out in a solvent (xylene) 1,2-diamide was produced which formed imidazolines. 263 The dehydration reaction kinetics of castor oil [with 12-hydroxy-18:1(9Z) as the major component] was investigated with sodium bisulfate–sodium bisulfite mixture or toluene-p-sulfonic acid as catalyst.264 Radical addition of alkyl 2-iodoalkanoates and 2-iodoalkanenitriles to alkenes initiated by electron transfer from copper in solvent-free systems gave rise to „-lactones and 4-iodoalkanenitriles.265 Pseudohalogenation of methyl 9-hydroxy-18:1(12Z) and methyl 12-hydroxy- 18:1(9Z) with N,N-dibromobenzenesulfonamide caused addition of bromine across the double bond and also the formation of a bromo substituted tetrahydrofuranyl fatty ester derivative.266 A bulky phosphite-modified rhodium catalyst was developed for the hydroformylation of methyl 18:1(9Z) 23 COOMe HO S 24 25 N COOMe N COOMe 26 HO HN (CH2)11CHMe2 HOOC O OCO(CH2)11CHMe2 612 Natural Product Reports, 1998and 18:1(9E), which furnished mixtures of formylstearate and diformylstearate.267 3.8 Synthesis and reactions of glycerides A number of triacylglycerols with branched acyl groups were prepared via 1,2-isopropylidene glycerol.268 Monoacylglycerols were synthesized by heterogeneous catalysis using MCM-41 type silicas (micelle-templated silica) functionalized with primary and tertiary amino groups as catalysts for glycidol ring-opening with fatty acids under mild reaction conditions.269 The synthesis of polymerizable 1-mono- and 1,2-di-acyl-sn-glycerols is reported through acylation of protected glycerol in the presence of 4-(dimethylamino)pyridine and dicyclohexylcarbodiimide.270 Transesterification of soybean oil with methanol was investigated to form products for use as biodiesel.271 A study of the kinetics of acyl migration in monoglycerides showed that the rate of rearrangement of ‚- to ·-monoglycerides depended on the fatty acyl chain length in the monoglyceride.272 4 Antioxidants Two brief reports have been dedicated to the role of natural antioxidants in metabolic and physiological activities in humans.273,274 High levels of tocopherols and tocotrienols are found in the seed oil of Indian pulses Bengal gram (Cicer arietinum), black gram (Vigna mungo), green gram (Vigna radiata) and horse gram (Dolichos biflorus).275 Diets supplemented with vitamin E (tocopherols) and selenium were shown to provide maximal protection against liver peroxidation in rat liver homogenate.276 ·-Tocopherol and trolox (2-carboxy-2,5,7,8-tetramethylchroman-6-ol) inhibit the formation of volatile decomposition products of methyl linoleate hydroperoxide.277 ·-Tocopherol has also been shown to exert a stabilizing eVect towards hydroperoxides of erythrocyte phospholipid species, limiting their further degradation into peroxyl radicals.278 Vitamin E inhibits fish oilinduced hyperlipidemia and tissue lipid peroxidation in hamsters279 and also against lipid peroxidation in the guinea pig liver.280 Vitamin E supplemenation significantly lowers blood lipoprotein and lipid levels in diabetic patients.281 ·-Tocopherol and ‚-tocopherol provide oxidative destruction of ‚-carotene when the latter compound is used in fat emulsions. 282 The antioxidant eVects of hydrophilic phenols and tocopherols on the oxidative stability in virgin olive oils and in purified olive oil have been evaluated.283 The structure of an antioxidant from fermented soybeans (tempeh) has been identified as 5-(‰-tocopheroxy)-‰-tocopherol.284 „-Tocopherol at very low levels (11 Ïg g"1)in vegetable and butter oils was found to decrease hydroperoxide and secondary product formation to 49 and 39%, respectively.285 Comparative studies of antioxidant activities of natural prenyllipids as plastoquinol-9, ·-tocopherol quinol, ubiquinol-10 and ·-tocopherol in egg yolk lecithin liposomes have been studied, where pastoquinol and ·-tocopherol quinol are found to be more active than ubiquinol and ·-tocopherol in the inhibition of lipid peroxidation.286 A novel derivative of vitamin E (compound 33) was synthesized from 2-hydroxymethyl-2,5,7,8- tetramethylchroman-6-ol and maltose. The new derivative was found to have very similar antioxidant activities to those of ·-tocopherol, trolox and ascorbic acid (vitamin C).287 Remarkable enhancement of antioxidant activity of vitamin C in an artificial bilayer has been achieved by making it into lipophilic ascorbyl-6-palmitate.288 The quenching mechanism and kinetics of ascorbyl palmitate for the reduction of O O R O O R O (CH2)10 CO2 O O O O O O O O O O O O O O O O 28 R = n n n 27 R = (CH2)17Me n n n n n 16 n = 2 (14%) 17 n = 0 (16%) 29 Lie Ken Jie and Pasha: Fatty acids, fatty acid analogues and their derivatives 613photosensitized oxidation of oils was studied, which showed the eVective singlet oxygen quenching ability by the antioxidant.289 The autoxidation of 3,5-di-tert-butyl-4- hydroxytoluene (BHT) was investigated with ion spray tandem mass spectrometric and on-line gas chromatographic–mass spectrometric methods.The results showed that BHT generates an excited phenol radical, which dimerises to compound 34.290 The antioxidant eVect of several flavonoids of Anthriscus sylvestris in lard was investigated with the result showing superior antioxidative activities over ·-tocopherol.291 Moderate antioxidative eYciencies of flavonoids during peroxidation of methyl linoleate in homogeneous and micellar solutions were studied. The results demonstrated that flavonoids did not behave as classic phenolic antioxidants such as ·-tocopherol, but showed only moderate chain breaking activities.292 Also, antioxidant activity of natural flavonoids appears to be governed by the number and location of their aromatic hydroxy groups.For example, quercetin was most protective against lipid oxidation.293 The antioxidant activity of grape extracts inhibited the formation of conjugated diene hydroperoxides by 25–68% and hexanal formation by 49–98% in a lecithin liposome system.294 The antioxidant activity of lipid-soluble phenolic diterpenes from rosemary (Rosmarinus oYcinalis) was several times greater than BHT (butylated hydroxytoluene) and BHA butylated hydroxyanisole), but less than TBHQ (tertbutylhydroquinone). 295 The antioxidative activity and phenolic composition of pilot-plant and commercial extracts of sage and rosemary and those in macadamia nuts have been examined.296,297 Capelin (Mallotus villosus) protein hydrolyzate was shown to possess antioxidant activities in a O O O O O O O O O O R R O O O O O O CH2N NC Me(CH2)14C O OCH2 C OCH2 Me(CH2)14C OCH2 Me(CH2)14C O O O 30 31 H 32 33 O HO CH2 O O CH2OH HO OH HO 34 Me3C OH CMe3 CH2 CH2 Me3C OH 614 Natural Product Reports, 1998‚-carotene–linoleate model system, but the protein hydrolyzate of seal (Phoca groenlandica) tissues displayed a signifi- cant prooxidative eVect.298 Antioxidant components of methanolic extracts of groats and hulls from Ogle oats were identified and quantified, which consist of phenolics such as ferulic, p-coumaric, p-hydroxybenzoic, 4-hydroxyphenylacetic acids and vanillin and catechol.299 The antioxidant activities of magnolol, honokiol and related phenolic compounds have been reported.300 Singlet oxygen was generated using the photosensitizer rose bengal, which was used in the study of the scavenging ability of various antioxidants.301 The antioxidative activity of tea extracts in vegetable and marine oils has been investigated.302–305 Antioxidant activities of the extract of mung bean hull (Phaseolus aureus)306 and phytosterol oxides have been reported.307,308 A novel oxygen-scavenging film that contained polyfuryloxirane (PFO) shows improved oxidative stability of sunflower oil when stored in sealed transparent packages containing PFO film.309 2+-O-Glycosyl isovitexin inhibited malonaldehyde formation from 20:5 and 22:6 fatty acids by 56 and 43%, respectively, showing the second greatest inhibitory activity after BHT.310 5 Hydrogenation The catalytic activity and selectivity for hydrogenation of linoleic acid were studied for Ni, Cu and Pd catalysts with results indicating double bond migration, cis–trans isomerization and formation of positional isomers of 18:1 acids.311 Catalytic hydrogenation of linoleic acid over Pt, Pd, Ru and Ir supported on alumina gave similar results.Conjugated octadecadienoic acids were also detected in the hydrogenated products.312 The catalytic properties of copper chromite and its activity and selectivity in the hydrogenation of fatty methyl esters to yield fatty alcohols have been reported.313,314 The eVect of a magnetic field during nickel-catalyzed hydrogenation of sesame and soybean oil showed a decrease of hydrogen uptake on the catalyst surface, which permitted higher selectivity ratios to be achieved in the stepwise reduction of polyunsaturated fatty acids.315 6 Polymorphism, lipid membranes and lipid vesicles A study on the melting processes in fatty acids and oil mixtures has been carried out by photopyroelectric and diVerential scanning calorimetry (DSC).316 The ·-melt-mediated crystallization of 1-palmitolyl-2-oleoyl-3-stearyl-sn-glycerols has been investigated by DSC combined with polarized-light microscopy.317 The phase diagram of mixtures of 1-mono- and 2-mono-stearoylglycerol from DSC analysis showed that the transition from the sub-·1-form to the ·-form and the melting point of the ·-form depended on the amount of the 2-monostearoylglycerol present.318 Direct measurement of thermal fat crystal properties by DSC makes it possible to detect polymorphic changes and co-crystallization in the crystals of milk-fat fractions.319 The chain-melting phase transition in dipalmitoylphosphatidylcholine foam bilayers have been studied by microinterferometric methods.320 The viscosities of fatty acids, triglycerides and their binary mixtures321 and its relationship to crystallization in a binary system have been determined.322 The thermal and structural properties of some triglycerides have been studied by the synchrotron radiation X-ray diVraction method.323,324 The relationship between the structure of monoalkyl branched saturated triacylglycerols and some physical properties (gel point, refractive index and density) are reported.325 The crystallization behaviour of cocoa butter was investigated by means of real-time X-ray powder diVraction. It appeared that all polymorphic forms of cocoa butter, with the exception of the ‚-form, could be formed from the liquid form.326 The melting behaviour of 12 diVerent cocoa butter samples, in the ‚-phase, has been studied.327 The influence of the thermal history of cocoa butter on its phase behaviour during ‚-crystallization of diVerent samples of cocoa butter has been studied by real-time X-ray powder diVraction methods.328 The following studies have been carried out on the physical characteristics of monomolecular films (bilayers and membranes): interaction of a poly(dimethylsiloxane) with triglyceride films,329 metal chelating lipids for protein targeting to membranes,330 mixed diacetylene lipid films on air/water interface,331 long-range electron transfer through a lipid monolayer at liquid/liquid interface,332 stabilization of selfassembled monolayers of alkanoic acid to an alkoxide of zirconium,333 conductance and fluorescence studies of phospholipid bilayers,334 and the influence of anchor lipids on the homogeneity and mobility of lipid bilayers on thin polymer films.335 Novel two-dimensional magic-angle spinning NMR experiments have been designed to measure the magnitude and signs of 13C–1H dipolar interactions in fluid phase lipid bilayers.336 Evidence has been obtained for the existence of lateral heterogeneity in fluid bilayers composed of mixtures of saturated and unsaturated phospholipids by use of nearest-neighbour recognition methods.337 The polymorphic and phase behaviour of the following lipid mixtures have been reported: binary mixture of 20:1 (11Z) with 18:1(11Z) or 18:1(9Z),338 mixtures of diacyl phosphatidylserine–fatty acids,339 stabilized and nonstabilized tristearin in the presence of emulsifiers,340 eVect of lanthanum ions on lipid polymorphism of phosphatidylethanolamines,341 curvature stress and polymorphism in lipid membranes,342 phase diagrams for oil–methanol–ether mixtures,343 pressure eVect on phase behaviour of binary mixture of (Z)-unsaturated fatty acids,344 phase diagram of various solvents used for the extraction of oleic acid from soybean oil and jojoba oil,345 non-equilibrium transitions in thermotropic phases of 20:5 methyl esters,346 mixing behaviour of saturated short-chain phosphatidylcholines and fatty acids,347 liquid crystalline phase metastability of phosphatidylglycerols,348 spreading properties of dimyristoyl phosphatidylcholine at the air/water interface,349 and the phase transition sequence between fluid liquid-crystalline and interdigitated lamellar gel phases in mixed-chain diacyl phosphatidylcholine.350 Regioselective photolabelling in 1,2-dimyristoyl-sn-glycerol- 3-phosphocholine vesicles to study the topography of biomembranes has been reported.351 Unilamellar vesicles, obtained from mixtures of egg lecithin and lipids containing hydrogen bonding head-groups (barbituric acid and triaminopyrimidine) were found to aggregate and fuse to form larger vesicles.352 The self-reproduction of vesicles formed by optically active 2-methyldodecanoic acid was investigated in order to relate the autocatalytic increase of the vesicle concentration with enantioselectivity.353 The construction and characterization of multi-heme ensembles in phospholipid vesicles have been described.354 Aggregation of stilbene derivatized fatty acids and phospholipids in monolayers and vesicles has been studied.355 A number of bis-cationic dimeric lipids have been synthesized and the vesicles formed show widely diVerent membrane organizations with exceptional thermotropic properties.356 Unusual dynamic behaviour of micellized radical pairs generated from photochemical active amphiphiles has been reported.357 A metal-chelating lipid (iminodiacetate–copper complex) has been designed to target proteins to Langmuir monolayers and to promote their two-dimensional crystallization based on histidine coordination.358 The polymerization of hydrated polar natural and synthetic lipids yields a variety of lipid phases including various inverted cubic phases and the inverted hexagonal phase.359 Selective polymerization of double-diene lipid assemblies gives ladder-like polymers.360 Polymerizable monoacylglycerols and 1,2-diacyl-sn-glycerols have been synthesized.361 Lie Ken Jie and Pasha: Fatty acids, fatty acid analogues and their derivatives 6157 Physical properties and methodology 7.1 Thin layer chromatography A second edition of the Handbook of Thin Layer Chromatography has been published, which contains an updated chapter on lipid analysis.362 Determination of 18:1(6Z), 18:1(9Z) and 18:1(11Z) in some seed oils of the Umbelliferae family was achieved by silver ion thin layer chromatography of their phenacyl esters.363 Separation and quantification of mono-, di- and tri-acylglycerols and free oleic acid were achieved by thin layer chromatography with flame ionization detection (TLC-FID) on chromarods using a mixture of hexane–diethyl ether–formic acid, 65:35:0.04 by vol, as the developer.364 Separation of polyunsaturated fatty acids (PUFA) containing three or more double bonds from PUFA containing two or fewer double bonds was achieved by silica gel TLC by using a mixture of hexane–diethyl ether–acetic acid, 95:5:1 by volume. 365 A simple, accurate and fast procedure for quantitative analysis of fatty acids in simple lipid subclasses after subfractionation by TLC has been developed.Bands co-migrating with authentic lipid standards were scraped oV and subjected to direct, in situ, transesterification with BF3–methanol complex in the presence of the TLC (silica gel 60) adsorbant. The fatty acid methyl esters were subsequently quantified by capillary gas chromatography.366 Quantification in the subnanomolar range of phospholipids and neutral lipids by TLC and image analysis was reported using EDTA-impregnated chromatoplates with step-wise developments.An image was acquired and the integrated optical densities of the individual spots were quantified by a camera-equipped image analyzer against an internal standard of cholesteryl formate.367 Separation of ceramide and diacylglycerol species was achieved by two-dimensional TLC technique.368 7.2 High pressure liquid chromatography Separation of triacylglycerols containing ·- and/or „-linolenic acid moieties was achieved by ordinary reversed-phase high performance liquid chromatography (HPLC)369 or by silverion HPLC technique, which shows that silver ions formed weaker complexes with triacylglycerols containing „-linolenic acid than with those containing ·-linolenic acid moieties.370 cis and trans Isomers of fatty acid methyl esters, fatty alcohols and triacylglycerols were separated on a silver-ion HPLC column using gradients of n-heptane and acetonitrile as eluent. 371 The mechanistic aspects of fatty acid retention in silver-ion HPLC have been described.372 An industrial HPLC purification method for the isolation of docosahexaenoic acid ethyl ester and docosapentaenoic acid ethyl ester from singlecell oil in 99+% purity has been developed.The system consists of two columns packed with octadecylsilica (reversedphase ODS) and the eluent was methyl alcohol–water, 98:2 by volume.373 The quantitative and qualitative determination of isomeric lipid hydroperoxides was achieved by HPLC method using post-column detection with diphenyl-1- pyrenylphosphine (DPPP).The method proved to be useful for the determination of the hydroperoxides containing both conjugated and non-conjugated diene structures.374 Saponified fats and oils can be readily derivatized with 2-nitrophenylhydrazine hydrochloride to yield the corresponding fatty acid hydrazides, which can be directly injected into a liquid chromatograph and successfully separated.375 Molecular species of digalactosyldiacylglycerol have been separated by HPLC in the order of increasing unsaturation using mixtures of alcohols and water.376 Quantitative HPLC analysis of plant phospholipids, glycolipids and cholesteryl oxides using light-scattering detection has been studied.377,378 Tocopherols (vitamin E) are readily separated by HPLC using a normal-phase diol column.379 A brief review of the HPLC separations of soy phospholipid has been published.380 Phosphatidylglycerol molecular species of photosynthetic membranes were separated and identified by HPLC analysis of the dinitrobenzoyl derivatives of diacylglycerols produced by hydrolysis of phosphatidylglycerols with phospholipase C.381 Separation of gangliosides by microbore HPLC was achieved on a column packed with Spherisorb-NH2 phase.382 7.3 Gas liquid chromatography An improved method for rapid analysis of the fatty acids of glycerolipids has been reported.The procedure includes KOHcatalyzed transesterification and gas chromatographic (GC) analysis on SP-2340 at 240 )C.383 Base-catalyzed transesterifi- cation of acyl lipids with trimethylsulfonium hydroxide to furnish methyl esters for GC analysis is suitable for nonhydroxylated triacylglycerols.384 Fatty acids containing an alcohol function are found to be converted to the O-methyl ether derivatives, which may interfere with the methyl esters during GC separation.384 A collaborative study to determine total, saturated/unsaturated fats in cereal products and foodstu Vs by GC analysis of the fatty acid methyl esters has been conducted with statistical evaluation of the results.385,386 Diacylglycerols in high density lipoprotein were identified as trimethylsilyl ethers by GC analysis.387 Methods for the identification and quantification of cis and trans fatty acid isomers in hydrogenated and refined vegetable oils and infant formulas by capillary GC analysis have been described.388–390 GC properties of positional isomers of methyl thia-, selena- and tellura-laurate analogues have been studied.391 7.4 Supercritical fluid chromatography A detailed triacylglycerol analysis of Lesquerella fendleri seed oil was carried out first by column chromatographic fractionation followed by supercritical fluid chromatography (SFC).The analysis showed that hydroxylated C18 acyl groups are only found in the sn-2 position of the triacylglycerols.392 Capillary SFC analysis of shark liver oils allowed the direct quantification of squalene and cholesterol, while quantification of triacylglycerols, cholesteryl esters and diacylglycerol ethers required TLC fractionation prior to SFC analysis.393 The content of vernolic acid, 12,13-epoxy-18:1(9Z), could be accurately determined by SFC of the whole oil.394 Ricinoleic acid, 12-hydroxy-18:1(9Z), contained in castor oil (Ricinus communis) and dimorphecolic acid, 9-hydroxy-18:2(10E,12E), found in Dimorphoteca pluvialis seed oil, were readily identified and quantified by SFC analysis of the fatty acid without further derivatization.395 7.5 Infrared and Raman spectroscopy An infrared spectrophotometric procedure, based on the fatty acid methyl ester mixture derived from a partially hydrogenated vegetable oil as the calibration standard, provided an accurate means of assessing the total trans content of hydrogenated fat.396 Fourier transform infrared (FTIR) spectroscopy has been used for the quantification of total trans triacylglycerols in hydrogenated oils and food products.397–399 Vegetable oils and lard have been characterized by FTIR spectroscopic analysis to show the relationships between composition and frequency of bands in the fingerprint region.400 Fat, protein and lactose in raw milk were determined by FTIR analysis and the results compared to the conventional filterbased milk analyzer.401 DiVused reflectance FTIR was used to observe adsorption complexes of triacylglycerol and oleic acid on silica gel and magnesium silicate.402 FTIR studies on microscopic structures and conformations of triacylglycerols containing palmitoyl and oleoyl acyl groups in diVerent combinations have been conducted.403 A unique and rapid FTIR method for the determination of the solid fat index of fats and oils was developed.404 Near-infrared transmission spectroscopy (NITS) was used as a nondestructive method of predicting the oil content of individual meadowfoam (Limnanthes sp.) seeds which contain „-linolenic acid.405 NITS technique was also used to determine 616 Natural Product Reports, 1998the fatty acid composition of the oil in intact seed mustard (Brassica carinata) samples.406 Stoichiometric determination of hydroperoxides in fats and oils was achieved by Fourier transform NITS.407,408 FTIR spectroscopic analyses were carried out on dioleoylphosphatidylethanolamine to show lyotropic phase transition,409 on the eVect of daptomycin on the barotropic behaviour of dioleoylphosphatidylglycerol,410 eVect of dehydration and hydrostatic pressure on phosphatidylinositol bilayers,411 and on the hydrated dispersions of N-acylethanolamine phospholipids by studying the ester and amide bands.412 The Raman and infrared spectral studies of methyl selena-, sulfinyl-, sulfonyl- and tellura-laurates are reported.413 Fourier transform Raman spectroscopy and diVerential scanning calorimetry (DSC) were used to study the thermotropic phase behaviour of mixtures of ceramides type IV, stearic acid and cholesterol, which are components of stratum corneum lipids.414,415 7.6 Nuclear magnetic resonance spectroscopy 13C Nuclear magnetic resonance spectroscopy (NMR) has been successfully used to determine the position of the double bonds in unsaturated fatty acids. The presence of 18:1(6E) and 18:1(7E) in partially hydrogenated soybean oil was determined by this method and confirmed by mass spectrometric analysis after derivatization to 2-alkenyl-4,4-dimethyloxazoline.416 Fatty esters containing an allylic hydroxy group [for example: 8-hydroxy-18:1(9E), 11-hydroxy-18:1(9E) and 8,11-dihydroxy- 18:1(9E)] were readily characterized by 13C and 1H NMR techniques.417,418 Lie Ken Jie et al.have synthesized all geometric isomers of conjugated linoleic acids [CLAs, 18:2(9,11)] and have studied the NMR properties of these isomers in detail.419 Some acetylenic tellura fatty acid esters were also prepared to study the 13C and 1H shift eVects of the tellurium atom on the acetylenic carbon atoms and on the shifts of the protons of the methylene groups adjacent to the hetero atom in the alkyl chain.420 trans Fatty acids occur in margarines and shortenings, a result from the partial hydrogenation of vegetable and marine oils.421 13C NMR spectroscopic analyses of some margarines have been carried out, which allow the diVerent positional isomers of cis- and trans-monoenoic moieties to be identified. 422 Similar 13C NMR analyses were conducted on the triacylglycerols of Biota orientalis and carrot seed oil. The presence of the unusual ƒ5-polyunsaturated fatty acids and 18:1(6Z) contained in these oils, respectively, was con- firmed.423 A quantitative method was developed to assess the quality of olive oil by examining the composition of di- and tri-glycerides present in the oil sample by 13C NMR analysis. 424 The distribution of ƒ5-unsaturated fatty acid in conifer seed oil was studied by 13C NMR spectroscopy, which showed that these unusual fatty acids were only acylated to the sn-1 position of the glycerol.425 Structure determination of longchain polyunsaturated triacylglycerols of borage and evening primrose oil was also carried out by 13C NMR spectroscopy. 426,427 The shifts of the critical carbon nuclei of sterculate and malvalate (C16 and C18 cyclopropenyl fatty acids) in the seed oil from Sterculia foetida have been studied.428 The presence of cyclopentenyl fatty acids (gorlic, chaulmoogric and hydnocarpic acid) in the seed oil of the genus Hydnocarpus sp.was confirmed by 13C and 1H NMR analysis.429 The positional distribution of butyryl groups in milk-fat triglycerides could be determined by 1H NMR spectroscopy from the distinct diVerences in the shifts of the methylene and methine protons of the glycerol backbone.430 A high field 1H NMR study was carried out on the minor components (including unsaturated, saturated aldehydes, phenols and sterols) of olive oil, which constituted a useful method for the assessment of the quality of virgin olive oil.431 A brief report on the use of in vivo 13C NMR spectroscopy on the applications and limitations for noninvasive method assessment of fatty acids in adipose tissues has been published. 432 Details of an in vivo 13C NMR study of the relationship between diet and adipose tissue composition revealed clear diVerences in the adipose tissue composition of vegans, omnivores and vegetarians in terms of the amount of saturated and unsaturated fatty acids present.433 Similar application of this technique has been applied to neonatal adipose tissue in full-term newborn infants to monitor the eVects of developmental changes due to gestational age and oral feeding.434 To study the metabolism of „-linolenic acid, in vivo experiments were conducted on suckling rat pups fed [3-13C]-„-linolenic acid.435 Identification of an ornithine-containing lipid from Cytophaga johnsonae Stanier strain C21 was achieved by 1H NMR spectroscopic analysis.436 Two-dimensional 1H NMR spectroscopy was used to compare changes in the concentration of isotropically-tumbling neutral lipid during the activation of splenic and thymic T lymphocytes, which showed that the synthesis of NMR-visible mobile neutral lipid in activated lymphocytes was linked to the phosphatidylcholine cycle.437 Phospholipids of control and lipid-modified Tetrahymena thermophila were identified and quantified using one- and two-dimensional proton NMR spectroscopy on intact lipids.This technique allowed the study of enzyme pathways and other metabolic processes involving phospholipids in Tetrahymena and related protozoa.438 31P NMR spectroscopic analysis of erythrocyte membranes using detergents allowed the identification and accurate quantitative determination of the various classes of phospholipids present.439 Glycerophospholipids and tissue phospholipids can also be quantified directly by 31P NMR spectroscopy.440,441 The binding of ethyl oleate to low density lipoprotein, phospholipid vesicles and albumin was investigated by 13C NMR spectroscopy, where no measurable transfer of ethyl oleate to low density lipoprotein or small unilamellar phospholipid vesicles was observed.442 A13C NMR technique was also used for the determination of the solubility and molecular dynamics of cholesteryl oleate in dimyristoylphosphatidylcholine (DMPC) vesicles.443 The interaction of chloroform with bilayers of DMPC has been studied by 2H and 31P NMR spectroscopy. The result showed that chloroform was localized principally in an ordered environment in the vicinity of the choline head group at lower temperatures and solute concentrations.Increasing either of these parameters favoured the penetration of the chloroform into the centre of the bilayer.444 The phase behaviour of vesicles consisting of binary mixtures of 1,2-dipalmitoyl-d62-sn-glycero-3-phosphocholine and dioctadecyldimethylammonium bromide has been investigated using 2H NMR spectroscopy.445 2H NMR investigations of non-lamellar phase promoters in the lamellar phase state have also been carried out,446 and also on the eVect of 1-palmitoyl lysophosphatidylcholine on phase properties of 1,2-dipalmitoyl phosphatidylethanolamine.447 The crosspolarization and magic-angle spinning NMR technique has been used to analyze the polymorphic forms of three triacylglycerols (viz. 1,3-dipalmitoyl-2-oleyl glycerol, 1,3-racpalmitoylstearoyl- 2-oleyl glycerol and 1,3-distearoyl-2-oleoyl glycerol).448 A very useful high resolution magic-angle spinning 1H NMR method for studying lipid dispersions (water interactions with zwitterionic phosphatidylcholines) was achieved by using spherical glass ampoules.449 19F NMR spectroscopy was used as a direct probe to monitor the relative eYciency with which various fluorinated aromatic sulfides were oxidized by the ƒ9-desaturating system of Saccharomyces cerevisiae (yeast).The results provided evidence that yeast ƒ9-desaturase initiated the oxidation of stearoyl CoA at C-9.450 7.7 Mass spectrometry Gas chromatography–mass spectrometric (GCMS) analysis has been carried out on the 4,4-dimethyloxazoline derivatives Lie Ken Jie and Pasha: Fatty acids, fatty acid analogues and their derivatives 617of ƒ5-unsaturated fatty acids from conifer seed oil.451 The methyl esters of petroselinic, 18:1(6Z), and oleic acid, 18:1(9Z), were epoxidized, then ring opened with hydrochloric acid and the resulting chlorohydrin was silanized.The mixture was analysed by GCMS which allowed these positional isomers to be identified.452 Nine trans positional monoene isomers of fatty esters were transformed to the 2-alkenyl-4,4- dimethyloxazoline derivatives and analyzed by GCMS to determine the position of the trans double bond in the alkyl chain.453 The dimethyl disulfide derivatization of ethyl 18:2(9Z,12Z) and 18:2(9E,12E) allowed each configurational isomer to be identified by GCMS analysis.454 The mass spectra of 4,4-dimethyloxazoline derivatives of conjugated hydroxy enyne C17 and C18 fatty acids have been studied.The position of the hydroxy group was unequivocally proven by characteristic odd-numbered fragment peaks found in the mass spectra. 455 Gas chromatography–combustion isotope ratio mass spectrometry was used to study the in vivo compartmental metabolism of 13C-labelled docosahexaenoic acid.456 The electron impact mass spectra of four tert-butyldimethylsilyl ether derivatives of the major metabolites of prostaglandins F1· and F2· were investigated.The spectra showed fragment ions suitable for identification and quantification of the metabolites obtained.457 Cyclic fatty acid monomers found in heat-abused edible oils have been confirmed by mass spectral analysis.458 Electron impact and chemical ionization mass spectral analyses of methyl ester species of free mycolic acid from Rhodococcus lentifragmentus have been compared.459 The presence of unsaturation in the meroaldehyde subunit of methyl mycolate was reflected by the appearance of dehydration fragment ions under chemical ionization.459 The GCMS analysis of 1-monomycoloyl glycerol fraction of the same bacteria was also studied as per-O-benzoyl derivatives.460 Thermospray mass spectral analyses of corynomycolic acids and their derivatives have been carried out.461 Mass spectral analysis of nicotinates and sulfoquinovosyl monoglycerols from the marine microalga, Heterosigma carterae, confirmed the presence of 16:1(13Z) in the sn-2 position.462 Mixed triacylglycerols of type AAB, ABA and ABC (where A,B,C signify diVerent fatty acyl groups) can be distinguished using atmospheric pressure chemical ionization mass spectrometry.463,464 This technique was applied to the analysis of ·- and „-linolenic acid in berry oils.465,466 Chemical ionization mass spectrometry with nitric oxide as the reactant gas has been used for the determination of the position of the double bond in monoalkenylglycerols isolated from the liver oil of the shark Centrophorus squamosus.467 Mass spectrometric analysis of triacylglycerols using negativeion chemical ionization with ammonia was found to be a rapid method for the semiquantitative determination of triacylglycerol mixtures of various fats and oils.468 A useful method for the identification of fatty acid hydroperoxides by electrospray ionization–tandem mass spectrometry has been described.469 Determination of the structures of aminophospholipids, lysophospholipid regioisomers, cerebroside isomers and tetramethyl diphosphoryl lipid A by a tandem mass spectrometric approach has been reported.470–473 Mass spectral analysis of positional isomers of methyl telluralaurate has been reported.474 8 Biosynthesis and biotechnology 8.1 Biosynthesis The biosynthetic pathways for fatty acid elongation and desaturation in the fungus Neurospora crassa were studied.The results showed the elongation of 16:0 to 18:0 and subsequent stepwise desaturation of 18:0 to 18:1(9Z), 18:2(9Z,12Z) and 18:3(9Z,12Z,15Z).475 ·-Linolenic acid and one of its isomers, 18:3(9Z,12Z,15E), were converted to 18:4(6Z,9Z, 12Z,15Z) and 18:4(6Z,9Z,12Z,15E), respectively, by rat liver microsomes.476 Microsomal ƒ12-desaturase in the yeast Lipomyces starkeyi transformed oleic acid to ·-linoleic acid.477 Incorporation of polyunsaturated fatty acids (PUFAs) into the cellular lipids of the red microalga Porphyridium cruentum showed that only C18 PUFAs were desaturated by ƒ6- desaturase, while C20 PUFAs were desaturated by ƒ5-desaturase.4783-Thia fatty acids, which are strong inhibitors of ƒ6-desaturase in rat liver microsomes, are extensively incorporated into phospholipids and triacylglycerols in rat hepatocytes after 24 h incubation.479 Desaturation of 9- and 10-fluoro fatty acids by yeast Saccharomyces cerevisiae resulted in the production of (E)-flouroalkenoic acids only.480 The biosynthesis of novel .-cycloheptyl fatty acids in Alicyclobacillus cycloheptanicus from phenylacetic acid has been studied.481 8.2 Lipase-catalyzed reactions Novel lipases isolated from the fungus Phoma glomerata,482 Neurospora sp.,483 yeast (Candida, Pichia and Yarrowia sp.) show specific hydrolytic activities.484 Lipase from Penicillium sp.discriminates against diglycerides.485 Surfactant-modified lipases (Rhizopus japonicus) were used for the interesterifi- cation of triglyceride and fatty acid.486 Immobilization of lipases on porous polypropylene caused reduction in esterifi- cation eYciency.487 The role of silica gel in lipase-catalyzed esterification reactions of polar substrates was investigated.488 The transesterification of soybean oil with glycerol, propane- 1,2-diol and methanol by an immobilized lipase in flowing supercritical carbon dioxide for the synthesis of monoglycerides was described.489 A novel compound, 12,13,17- trihydroxy-18:1(9Z) from linoleic acid was obtained by a new microbial isolate Clavibacter sp.490 The enzyme-catalyzed methylation of the fat under supercritical carbon dioxide condition prior to gas chromatographic analysis was described.491 Lipase-catalyzed alcoholysis of cod liver oil was carried out under supercritical carbon dioxide condition.492 EYcient aqueous enzymatic (cellulase, polygalacturonase, protease or ·-amylase) extraction of fresh grated coconut meat gave over 70% yield of oil.493 Aqueous enzymatic extraction of mustard seed and rice bran produced oils which were better with respect to colour and odour than commercial expeller-extracted or Soxhlet-extracted oils.494 Hydroxy fatty acids (75–80% yield) from Lesquerella oil were isolated by a saponification extraction technique involving immobilized Rhizomucor miehei lipase.495 Lesquerolic [14-hydroxy-20:1(11Z)] and auricolic acid [14-hydroxy- 20:2(11Z,17Z)] were also obtained from hydrolyzed lesquerella oil by a low-temperature crystallization procedure.496 The lipase-catalyzed monoesterification of 1-Ohexadecylglycerol in organic solvent was reported using a range of saturated and unsaturated fatty acids.497 (Z)-Hex-3- en-1-yl butyrate, an important flavour and fragrance compound, was synthesized in hexane and solvent-free medium using Mucor miehei and Candida antarctica lipases as biocatalysts. 498 Modification of the fatty acid composition of soy lecithin, principally at its sn-1 position, was achieved by interchange reaction with the methyl ester of individual fatty acids in the presence of lipase from Mucor miehei.499 A low cost selective enzymic production of mono- and di-oleoylglycerols has been developed.500 Lesquerolic acid wax was synthesized via immobilized Rhizomucor miehei lipasecatalyzed esterification of lesquerolic acid [14-hydroxy- 20:1(11Z)] and alcoholysis of lesquerella oil.501 Wax ester production from long-chain alcohol and methyl ester has been investigated with the immobilized thermostable lipase from Mucor miehei.502 Lipase-catalyzed synthesis of regioisomerically pure 1,3-sndiacylglycerols and 1(3)-rac-monoacylglycerols derived from unsaturated fatty acids was accomplished using various types of lipases (including Chromobacterium viscosum, Rhizopus delemar and Rhizomucor miehei).503 Esterification of carotenoic acid with glycerol in the presence of lipase from Candida antartica B gave mixtures of 1-mono- and 1,3-diglycerides.504 618 Natural Product Reports, 1998The acetone extract of germinating rapeseed (Brassica napus L) catalyzes the esterification of specific fatty acids (which do not contain a double bond at the ƒ4 or ƒ6 position) with n-butanol, and also catalyzes the alcoholysis of methyl esters, but not triacylglycerols.505 Interesterification of high-melting palm stearin with liquid oils catalyzed by lipase (Mucor miehei) furnished a low-melting stearin product for use as polyunsaturated fatty acid-rich shortening and margarine fat bases.506 A regioselective synthesis of 6-O-acyl sucrose monoester has been developed through the lipase-catalyzed esterification of sucrose acetals with fatty acids in both organic solvents and under solvent-free conditions.507 Ethyl 6-Odecanoyl glucoside was prepared in microemulsion systems by reacting ethyl glycoside with decanoic acid in the presence of Candida antarctica component B lipase.508 A sugar ester-modified lipase was developed for the esterifi- cation of fatty acids and long-chain alcohols.509 A number of lipases were screened for their ability to transesterify triacylglycerols with short-chain alcohols to alkyl esters for use as biodiesel.510 Purification of „-linolenic acid from borage oil by multi-step enzymatic methods allowed this fatty acid to be enriched to 70–87% after selective esterification.511,512 EPA [20:5(5Z,8Z,11Z,14Z, 17Z)] was readily incorporated into the triacylglycerol of „-linolenic acid-containing Evening Primrose oil by interesterification using immobilized lipase SP435 from Candida antarctica.513 Enzymatic esterification of EPA and DHA [22:6(4Z,7Z,10Z,13Z, 16Z,19Z)] concentrates from seal blubber oil with glycerol was achieved in 94% yield using lipase LP-401-AS from Chromobacterium viscosum.514 Reports on the enrichment of EPA and DHA in fish oils by lipase-catalyzed reactions have been published.515–517 Lipase PS30 (Pseudomonas sp.) was used to incorporate oleic acid into melon seed oil, which increased the level of oleic acid from 13% to 53%, while reducing the level of linoleic acid from 65% to 33%.518 Immobilized lipase-catalyzed production of structured lipids with EPA at specific positions of the glycerol backbone has been described.519 C10 medium-chain mono-, di- and tri-glycerides were produced by lipase-catalyzed reactions from glycerol and capric acid in isooctane.520 Coconut oil has been modified by interesterifi- cation via lipase with methyl 8:0 and 10:0 esters to produce triglycerides rich in medium-chain fatty acids.521 Interesterifi- cation of triolein with ethyl esters of 4:0, 8:0 and 10:0 results in the production of reduced-calorie structured lipids.522–525 The production of structured lipids containing essential fatty acids and 8:0 acid by immobilized Rhizopus delemar lipase was also reported.526 Carica papaya latex was also used in the synthesis of reduced-calorie structured triacylglycerols.527 The eVects of selected substates (10:0 and linoleic acid) on the synthesis of structured lipids by immobilized lipases were studied.528 (+)-Coriolic acid [13S-hydroxy-18:2(9Z,11E)] was obtained by hydroperoxidation of linoleic acid with soybean lipoxygenase-1 to give 13(S)-hydroperoxy-18:2(9Z,11E) acid.The latter compound was chemically reduced to (+)-coriolic acid and isolated by liquid–liquid extraction.529 Solvent polarity has been found to influence product selectivity of lipase-mediated esterification reaction in microaqueous media.530 The lipase from Rhizomucor miehei was shown to recognize molecular aggregation of lipids, which resulted in changes in hydrolysis specificities to polyunsaturated fatty acid ethyl esters in diVerent aggregation states.531 The acyl binding site of Rhizopus delemar pro-lipase and mature lipase was altered through site-directed mutagenesis to improve lipase specificity for short- or medium-chain length fatty acids.532 During the hydrolysis of triolein and its partial glycerides by porcine pancreatic lipase in supercritical carbon dioxide, the regio- and enantio-selectivity of the lipase appears to be controlled by the amount of water present in the system.533 By keeping water activity of the immobilized lipase of Rhizomucor miehei (Lipozyme IM) at an intermediate level, DHA was concentrated by selective partial hydrolysis of egg yolk phospholipids and squid skin phospholipids.534 A mycelial lipase from Rhizopus rhizopodiformis catalyzed the alcoholysis of palm oil mid-fraction in organic solvents eYciently to yield alkyl esters.535 A study of growing cells of Pseudomonas putida at diVerent temperatures lends support to the hypothesis that the isomerization of cis to trans unsaturated fatty acids is an emergency action of cells to adapt membrane fluidity to drastic changes of environmental conditions.536 Heated palm olein, triolein and diolein were used as substrate models to study the behaviour during hydrolysis by porcine pancreatic lipase.537 Biotransformation of oleic acid with Pseudomonas sp.has been found to produce 10-hydroperoxy-18:1(8E), 10-hydroxy- 18:1(8E) and 7,10-dihydroxy-18:1(8E).538 Pseudomonas fluorescens lipase has been found to induce enantioselective acylation of methyl (R,S)-2-hydroxy-2-(hydroxymethyl)hexadecanoate. 539 6-Hydroxy ‰-lactones and 5,6-dihydroxy-20:0/ 22:0 were produced from meadowfoam (Limnanthes alba) fatty acids via a lipase-mediated self-epoxidation reaction.540 Various C18 and C20 polyunsaturated fatty acids fed to the yeast Dipodascopsis uninucleata resulted in the production of 3(R)- hydroxy polyenoic acids.541 In the presence of an immobilized lipase from Candida antarctica unsaturated fatty acid esters and plant oils are epoxidized with hydrogen peroxide.542 12-Hydroxy-18:0 was transformed to 12-hydroxyoctadecanamide by a microbial isolate (Bacillus cereus 50) when grown aerobically in yeast extract.543 The lipase-catalyzed (Candida antarctica) synthesis of a range of amide surfactants by transesterification reactions between various amines and fatty acid methyl esters has been reported.544 The chemoselective enzymatic N-acylation between N-methylglucamine (1-deoxy-1- methylamino-D-glucitol) and oleic acid resulted in the formation of an ion-pair between acid and amine function.545,546 Phospholipase D from Streptomyces sp.has been shown to catalyze the transphosphatidylation reaction of various primary and secondary alcohols with natural phosphatidylcholine. 547,548 The synthesis of six new glycerophospholipids with choline-analogous head groups by phospholipase D has also been described.549 Streptomyces catenulae catalyzed the oxidation of ·-tocopherol to ·-tocopherolquinone.550 8.3 Sources and biological eVects of n-3 polyunsaturated fatty acids Arachidonic acid [AA, 20:4(5Z,8Z,11Z,14Z)] production by the fungus Mortierella alpina was optimized to account for 42% of the total fatty acid.551 ƒ5-Desaturase-defective mutants of Mortierella alpina produced 20:4(8Z,11Z,14Z,17Z) when grown with linseed oil.552 Eicosapentaenoic acid [EPA, 20:5(5Z,8Z,11Z,14Z,17Z)] was isolated from the marine microalga Phaeodactylum tricornutum,553 from a marine bacteria isolated from the intestinal contents of the Pacific mackerel, 554 and from freshwater sponges.555 Docosahexaenoic acid [DHA, 22:6(4Z,7Z,10Z,13Z, 16Z,19Z)] was found in the lipid extract of the fungus Thraustochytrium sp.556 and the marine microorganism Schizochytrium sp.557,558 Methods involving lipase-catalyzed transesterification have been developed to concentrate EPA and DHA from fish oil.559–561 The eVects of curcumin and capsaicin (from red pepper) on arachidonic acid [AA, 20:4(5Z,8Z,11Z,14Z)] metabolism562 and those of dietary AA on human immune response were investigated.563 A study reveals that the AA to EPA ratio in blood correlates positively with clinical symptoms of depression in human beings.564 Controlled feeding of linoleic acid or AA to essential fatty acid deficient rats was used to define the relationship between dietary AA and the inflammatory response evoked during adjuvant-induced arthritis.The results showed that consumption of the average daily amount of AA without concurrent ingestion of linoleic acid did not alter the essential fatty acid deficient state.565 The phospholipid AA level in the blood of human infants born at full term showed a continuous decrease over time when fed formula without AA or DHA as compared to breast-fed infants.566 The eVects of EPA and DHA in the diet (formula) of term infants567–570 and infant rats571 have been studied.A Lie Ken Jie and Pasha: Fatty acids, fatty acid analogues and their derivatives 619comparative study was carried out on breast milk fatty acid composition between Hong Kong and Chongqing (China) Chinese,572 while the total trans fatty acids in Canadian human milk was found to average about 7% of the total fatty acid content.573 The influence of dietary AA on the metabolism in vivo of deuterium-labelled 20:3(8Z,11Z,14Z) in humans was investigated.Based on the concentration of 20:3 in total plasma lipid, the estimated conversion of 20:3 to AA was about 18% and 2% for subjects with a daily intake of 1.7 g and 0.21 g of AA, respectively.574 From the results of the serum phospholipids, no significant diVerence in the retroconversion of dietary DHA as a source of EPA was observed between vegetarians and omnivores.575 A study of the biosynthesis of EPA in the microalga Prophyridium cruentum revealed the incorporation of externally supplied fatty acids into algal lipids which were further metabolized along the n-6 and n-3 pathways. 576 In vitro evidence has been reported for the ƒ8 desaturation of deuterated 20:3(11,14,17) by mouse liver.577 Increased hepatic ‚-oxidation of DHA, elongation of EPA and acylation of lysophosphatidate in rats fed a DHA-rich diet have been observed.578 Dietary deficiency of linoleic acid and other essential fatty acids results in characteristic scaly skin disorder and excessive epidermal water loss.579 EPA and DHA are found to suppress the proliferation of vascular smooth muscle cells through modulation of various steps of growth signals.580 Feeding of post-weanling rats with DHA shows the incorporation of DHA in tissues and enhances bone marrow cellularity.581 The eVect of short-term diets rich in fish, red meat or white meat on thromboxane and prostacyclin synthesis in humans was investigated.582 Polyunsaturated fatty acid regulation of hepatic gene transcription has been reviewed.583 EPA and DHA decrease colonic epithelial cell proliferation in high-risk bowel mucosa.584 The eVect of DHA on mouse mitochondrial membrane properties was studied with results showing a DHA-induced decrease in respiratory control index and membrane potential and an increase in proton movement.585 EPA, but not DHA, was shown to increase mitochondrial fatty acid oxidation.586 Long chain polyunsaturated fatty acids (AA or DHA) were found to influence the fatty acid composition of the visual cell membrane (retina) during development.587–589 Trials of visual attention of preterm infants fed DHA or human milk have been conducted.590–592 Increasing dietary linoleic acid in young rats increases and then decreases DHA in retina but not in brain.593 The eVects of EPA and DHA on the following disorders were reported: disorders of peroxisomal disease,594,595 rheumatic and inflammatory diseases, 596,597 hypoglycemia,598 monocyte phagocytosis599 and schizophrenia.600 The quantity and distribution of DHA in major phospholipids was examined in prenatal rat brain.601 The eVect of dietary DHA on brain composition602 and neural function in term infants603 and the role of essential fatty acids in the function of the developing nervous system604 have been studied.Polyunsaturated fatty acids exert antiarrhythmic actions as free acids rather than in phospholipids.605,606 EPA and DHA prevent ischemia-induced cardiac sudden death in dogs.607 The eVects of EPA and DHA on blood and associated tissues or organs include: reduction of cholesterol,608 platelet and aorta fatty acid levels,609 serum lipid levels,610 plasma lipid levels,611,612 platelet fatty acid levels,613,614 oxidative stress in rat erythrocytes.615 The intestinal lymph absorption of butter, corn oil, cod liver oil, menhaden oil, ethyl esters of EPA and DHA was studied.616 The eVects of triolein or oleic acid on lymphatic recovery of DHA and their intramolecular distribution in lymph triglyceride of rats were investigated.617 Changes in the size and DHA content of adipocytes during chick embryo development have been studied.618 „-Linolenic acid [GLA, 18:3(6Z,9Z,12Z)] containing diet is found to attenuate bleomycin-induced lung fibrosis in hamsters.619 The hypocholesterolemic eYcacies of various polyunsaturated vegetable oils were compared in rats given cholesterol-enriched diets.620,621 The eVects of some vegetable oils containing ·- or „-linolenic acid were studied on the survival time of strokeprone spontaneously hypertensive rats.622 EPA and DHA from diets were readily incorporated in the heart, kidney and lung tissues of weanling rats,623 plasma lipids, erythrocyte membranes and platelets of healthy young men,624 and in the chylomicrons of rats.625 Obese children exhibited significantly higher values of arachidonic acid in the plasma lipids than healthy age-matched controls.626 Low fat-monounsaturated rich diets containing high-oleic peanuts appeared to improve serum lipoprotein profiles in postmenopausal women.627 The eVects of diVerent positional cis and trans isomers of 18:1 on the response of porcine platelets to collagen and thrombin stimulation were determined.628 The ratio of dietary linoleic/ ·-linolenic acid in term infants is an important determinant of the amounts of DHA and AA required to achieve plasma and erythrocyte levels of these fatty acids similar to those of breast-fed infants.629 Hypolipidemic 3-thia fatty acids have been shown to change the fatty acid composition of the liver and heart, as these changes are linked to the activity of hepatic ƒ9-desaturase and the eVect of ‚-oxidation of the fatty acids.630 Dietary sandalwood seed oil, which contains about 30% of 18:2(9A,11E), was found to modify the fatty acid composition of mouse adipose tissue, brain and liver.631 The absorption and transport of cyclic fatty acid monomers derived from heated linseed oil depends on their positioning within the ingested triacylglycerols.632 The presence of small amounts of 21:5(6Z,9Z,12Z,15Z,18Z) in fish oil shows little significance for the biological eVects of these oils, except that it is a strong inhibitor of arachidonic acid synthesis.633 8.4 Lipid peroxidation and autoxidation A short review on lipoxygenase as a versatile biocatalyst has been published.634 The reduction of ferric iron cofactor has been postulated to serve as an electron sink that drives hydrogen tunneling in lipoxygenase catalysis.635 Hydroxy unsaturated fatty acids have been produced by crude lipoxygenase obtained from infected rice plants.636 Rice seed lipoxygenase-2 catalyzed oxygenation of arachidonic acid into a mixture of 5(S)-hydroperoxy-20:4(6,8,11,14) and 15(S)- hydroperoxy-20:4(5,8,11,13).637 Hamberg has studied the stereochemical aspects of fatty acid oxidation involving hydroperoxide isomerases.638 The regiocontrolled oxygenation of arachidonic acid via soybean lipoxygenase gave 15(S)- hydroxy-20:4(5Z,8Z,11Z,13E) on reduction of the corresponding hydroperoxy-20:4 intermediate with triphenylphosphine. 639 Incubation of arachidonic acid with cultured bovine coronary artery endothelial cells resulted in the production of mixtures of 12(S)-, 15(S)- and 11(R)-hydroxy-20:4 and 14,15-, 11,12-, 8,9- and 5,6-epoxy-20:3 isomers.640 Pent-1-ene, which is generated by decomposition of n-6 unsaturated fatty acid hydroperoxides, is further transformed by plant or liver enzymes to 1,2-epoxypentane.641 Oxidation products of unsaturated fatty acid in infarcted porcine heart tissue have been identified as 9,10-epoxy-18:1(12Z) and 12,13-epoxy- 18:1(9Z).642 High levels of hydroperoxides of unsaturated fatty acids are found in injured or damaged animal cells.643 The kinetics of lipid peroxidation in human blood plasma have been investigated.644 A study of the oxidative damage to human skin lipids shows that peroxidation of fatty acids depended on the concentration of cholesterol.645 The rate of lipid peroxidation of n-3 polyunsaturated fatty acids was found not to be suppressed by vitamin E (·-tocopherol) supplementation in the diet.646 The loss of fluorescence of cis-parinaric acid [18:4(9Z,11Z,13Z,15Z)] is a sensitive indicator of lipid peroxidation, which has been applied to the study of peroxidation of unsaturated fatty acids in immune cells.647 Peroxidized natural triacylglycerols were separated by high pressure liquid chromatography and identified by 620 Natural Product Reports, 1998electrospray mass spectrometry.648 Incubation of 13-hydroxy- 12-oxo-18:2(9Z) with a bacterial culture (Ralstonia sp.) exhibited strong monooxygenase activity to give (3Z)-dodecenedioic acid.649 Mannitol was shown to prevent the inactivation of tomato lipoxygenase by hydrogen peroxide.650 Two stable steroidal nitroxyl radicals were found to inhibit lipid peroxidation induced by Fenton’s reagent in both rat liver microsomes and egg phosphatidylcholine liposomes.651 The processes in producing a lag phase in Fe(II)-supported lipid peroxidation in liposomes have been investigated.652 A study of the eVects of oxidative stress on glycerolipid acyl turnover in rat hepatocytes showed that triacylglycerols provided a limited but very dynamic pool of polyunsaturated fatty acids for the resynthesis of phospholipids.653 Lipid peroxidation in a homogenate of porcine heart tissue in the presence of ebselen, 2-phenyl-1,2-benzoisoselenazol-3(2H)-one, caused significant increase of hydroxy fatty acids, while the increase of aldehydic compounds was less.654 Various nonenzymic conditions for conversion of 9(S)-hydroperoxy-18:2(10E,12Z) and 13(S)- hydroxy-18:2(9Z,11E) to the corresponding hydroxy dienoic acids were investigated in vitro.655 A number of dihydroxy and hydroxyoxo fatty acids were obtained after hydrogenation of the hydroperoxy products produced by nonenzymic lipid peroxidation of unsaturated fatty acids.656 The mechanism of linoleic acid hydroperoxide reaction with alkali was investigated. 657 The first direct evidence for lipid–protein conjugation in oxidized human low density lipoprotein was reported.658 Increased levels of lipid oxidation products in low density lipoproteins of patients suVering from rheumatoid arthritis has been observed.659 Factors aVecting the resistance of low density lipoproteins to oxidation have been investigated.660 Feeding experiments have shown that oxidized lipids in the diet are incorporated by the liver into very low density lipoprotein in rats.661 Lipid peroxidation of low density lipoproteins from human plasma and egg yolk is shown to promote accumulation of 1-acyl analogues of platelet-activating factor-like lipids.662 Autoxidation studies have been carried out on sun- flower seed oil663 and on triacylglycerols containing EPA and DHA.664,665 The mechanism of lower oxidizability of EPA than linoleate in aqueous micelles has been studied, which shows that the peroxyl radical derived from EPA is more polar than that from linoleate and thus more likely to diVuse from the core to the micelle surface.666 Oxidation of arachidonic acid by iron ascorbate gives besides other known oxidation products a new aldehydic lipid, 1-hydroxyheptanal.667 9-Epoxy-18:1(12Z) was formed by autoxidation of free linoleic acid or linoleic acid in phospholipids (cardiolipin) by hemoproteins.668 Optical absorption studies of the kinetics of ultraviolet and self-initiated autoxidation of linoleate micelles have been reported.669 The metabolites produced during the peroxisomal ‚-oxidation of linoleate and arachidonate are found to move to microsomes for conversion back to linoleate.670 Highly stereoselective oxidation of arachidonic acid by cytochrome P450BM-3 gave enantiomerically pure 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A. Forse, S. Flickner, D. Pleskow, H. T. Anastopoulos, V. Ritter and G. L. Blackburn, Lipids, 1996, 31, S-313 (supplement). 585 W. Stillwell, L. J. Jenski, F. T. Crump and W. Ehringer, Lipids, 1997, 32, 497. 586 N.Willumsen, H. Vaagenes, O. Lie, A. C. Rustan and R. K. Berge, Lipids, 1996, 31, 579. 587 M. Suh, A. A. Wierzbicki, E. Lien and M. T. Clandinin, Lipids, 1996, 31, 61. 588 H. S. Weisinger, A. J. Vingrys and A. J. Sinclair, Lipids, 1996, 31, 65. 589 M. C. Craig-Schmidt, K. E. Stieh and E. L. Lien, Lipids, 1996, 31, 53. 590 S. E. Carlson and S. H. Werkman, Lipids, 1996, 31, 85. 591 S. H. Werkman and S. E. Carlson, Lipids, 1996, 31, 91. 592 M. H. Jorgensen, O.Hernell, P. Lund, G. Holmer and K. F. Michaelsen, Lipids, 1996, 31, 99. 593 H. M. Su, L. A. Keswick and J. T. Brenna, Lipids, 1996, 31, 1289. 594 M. Martinez, Lipids, 1996, 31, S-145 (supplement). 595 D. De Craemer, M. Pauwels and C. Van den Branden, Lipids, 1996, 31, 1157. 596 J. M. Kremer, Lipids, 1996, 31, S-243 (supplement). 597 D. R. Robinson, M. Urakaze, R. Huang, H. Taki, E. Sugiyama, C. T. Knoell, L. Xu, E. T. H. Yeh and P. E. Auron, Lipids, 1996, 31, S-23 (supplement). 598 G. S. Rambjor, A. I. Walen, S. L. Windsor and W. S. Harris, Lipids, 1996, 31, S-45 (supplement). 599 D. S. Halvorsen, J. B. Hansen, S. Grimsgaard, K. H. Bonaa, P. Kierulf and A. Nordoy, Lipids, 1997, 32, 935. 600 J. D. E. Laugharne, J. E. Mellor and M. Peet, Lipids, 1996, 31, S-163 (supplement). 601 P. Green and E. Yavin, Lipids, 1996, 31, S-235 (supplement). 602 M. M. Christensen and C. E. Hoy, Lipids, 1997, 32, 185. 603 R. A. Gibson, M. A. Neumann and M. Makrides, Lipids, 1996, 31, S-177 (supplement). 604 R. Uauy, P. Peirano, D. HoVman, P. Mena, D. Birch and E. Birch, Lipids, 1996, 31, S-167 (supplement). 605 K. H. Weylandt, J. X. Kang and A. Leaf, Lipids, 1996, 31, 977. 606 J. X. Kang and A. Leaf, Lipids, 1996, 31, S-41 (supplement). 607 G. E. Billman, J. X. Kang and A. Leaf, Lipids, 1997, 32, 1161. 608 L. Froyland, H. Vaagenes, D. K. Asiedu, A. Garras, O. Lie and R. K. Berge, Lipids, 1996, 31, 169. 609 A. J. Sanigorski, A. J. Sinclair and T. Hamazaki, Lipids, 1996, 31, 729. 610 N. M. JeVery, P. Sanderson, E. J. Sherrington, E. A. Newsholme and P. C. Calder, Lipids, 1996, 31, 737. 611 L. G. Cleland, M. A. Neumann, R. A. Gibson, T. Hamazaki, K. Akimoto and M. J. James, Lipids, 1996, 31, 829. 612 G. J. Nelson, P. C. Schmidt, G. L. Bartolini, D. S. Kelley and D. Kyle, Lipids, 1997, 32, 1137. 613 T. Wilkinson, H. M. Aukema, L. M. Thomas and B. J. Holub, Lipids, 1996, 31, S-211 (supplement). 614 G. J. Nelson, P. S. Schmidt, G. L. Bartolini, D. S. Kelley and D. Kyle, Lipids, 1997, 32, 1129. 615 G. Calviello, P. Palozza, P. Franceschelli and G. M. Bartoli, Lipids, 1997, 32, 1075. 616 P. Degrace, C. Caselli, J. M. Rayo and A. Bernard, Lipids, 1996, 31, 405. 617 I. Ikeda, H. Yoshida and K. Imaizumi, Lipids, 1997, 32, 949. 618 K. Farkas, I. A. J. Ratchford, R. C. Noble and B. K. Speake, Lipids, 1996, 31, 313. 619 V. A. Ziboh, M. Yun, D. M. Hyde and S. N. Giri, Lipids, 1997, 32, 759. 620 M. Fukushima, S. Akiba and M. Nakano, Lipids, 1996, 31, 415. 621 M. Fukushima, T. Matsuda, K. Yamagishi and M. Nakano, Lipids, 1997, 32, 1069. 622 M. Z. Huang, S. Watanabe, T. Kobayashi, A. Nagatsu, J. Sakakibara and H. Okuyama, Lipids, 1997, 32, 745. 623 A. Suarez, M. J. Faus and A. Gil, Lipids, 1996, 31, 345. 624 H. M. Vidgren, J. J. Agren, U. Schwab, T. Rissanen, O. Hanninen and M. I. J. Uusitupa, Lipids, 1997, 32, 697. 625 M. S. Christensen and C. E. Hoy, Lipids, 1996, 31, 341. 626 T. Decsi, D. Molnar and B. Koletzko, Lipids, 1996, 31, 305. 627 D. J. O’Byrne, D. A. Knauft and R. B. Shireman, Lipids, 1997, 32, 687. 628 K. W. J. Wahle and L. I. L. Peacock, Biochim. Biophys. Acta, 1996, 1301, 141. 629 C. L. Jensen, H. Chen, J. K. Fraley, R. E. Anderson and W. C. Heird, Lipids, 1996, 31, 107. 630 L. Froyland, L. Madsen, W. Sjursen, A. Garras, O. Lie, J. 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Spiteller and G. Spiteller, Chem. Phys. Lipids, 1997, 89, 131. 656 A. Mlakar and G. Spiteller, Chem. Phys. Lipids, 1996, 82, 25. 657 H. W. Gardner, T. D. Simpson and M. Hamberg, Lipids, 1996, 31, 1023. 658 M. S. Bolgar, C. Y. Yang and S. J. Gaskell, J. Biol. Chem., 1996, 45, 27999. 659 W. Jira, G. Spiteller and A. Richter, Chem. Phys. Lipids, 1997, 87, 81. 628 Natural Product Reports, 1998660 O. Ziouzenkova, S. P. Gieseg, P. Ramos and H. Esterbauer, Lipids, 1996, 31, S-71 (supplement). 661 Staprans, J. H. Rapp, X. M. Pan and K. R. Feingold, J. Lipid Res., 1996, 37, 420. 662 Tokumura, M. Toujima, Y. Yoshioka and K. Fukuzawa, Lipids, 1996, 31, 1251. 663 H. Topallar, Y. Bayrak and M. Iscan, J. Am. Oil Chem. Soc., 1997, 74, 1323. 664 Y. Endo, S. Hoshizaki and K. Fujimoto, J. Am. Oil Chem. Soc., 1997, 74, 543. 665 Y. Endo, S. Hoshizaki and K. Fujimoto, J. Am. Oil Chem. Soc., 1997, 74, 1041. 666 K. Yazu, Y. Yamamoto, K. Ukegawa and E. Niki, Lipids, 1996, 31, 337. 667 A. Mlakar and G. Spiteller, Chem. Phys. Lipids, 1996, 79, 47. 668 H. Iwase, T. Takatori, H. Niijima, M. Nagao, T. Amano, K. Iwadate, Y. Matsuda, M. Nakajima and M. Kobayashi, Biochim. Biophys. Acta, 1997, 1345, 27. 669 K. E. Fygle and T. B. Melo, Chem. Phys. Lipids, 1996, 79, 39. 670 D. L. Luthria, Q. Chen and H. Sprecher, Biochim. Biophys. Res. Commun., 1997, 233, 438. 671 J. H. Capdevila, S. Wei, C. Helvig, J. R. Falck, Y. Belosludtsev, G. Truan, S. E. Graham-Lorence and J. A. Peterson, J. Biol. Chem., 1996, 271, 22 663. Lie Ken Jie and Pasha: Fatty acids, fatty acid analogues and their derivatives 629

ISSN:0265-0568    

DOI:10.1039/a815607y

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Anthocyanins and other flavonoids J. B. Harborne and C. A. Williams Plant Science Laboratories, University of Reading, Reading, UK, RG6 6AS Covering: January 1995 to December 1997 Previous review: 1995, 12, 639 1 Anthocyanins 2 Lipophilic flavones and flavonols 3 Flavone and flavonol glycosides 4 Chalcones and aurones 5 Flavanones 6 References 1 Anthocyanins Some 85 new anthocyanins have been recorded in plants during the period under review (Table 1).1–41 This is a conservative estimate since several polyacylglycosides reported here may be accompanied by related structures in which one or more of the original substituents are lacking.The majority of new structures have both aliphatic (especially malonyl and also acetyl) as well as aromatic (p-coumaryl, ferulyl, caVeyl, sinapyl) substitution. Most are substituted at the 3- and/or 5-hydroxy positions: but a significant number of 3,7- disubstituted and 3,7,3*-trisubstituted pigments have been described.The three new anthocyanins obtained from Ajuga (Table 1) conform to the normal type found in Labiatae plants, with aromatic acylation on the 3-sugar and malonic acid on the 5-sugar.42 The 3-(3,6-dimalonylglucoside) of cyanidin, reported in Allium victorialis,3 is the first simple disubstituted malonic ester to be characterised. On mild hydrolysis, it is converted to the 3-(3-malonylglucoside), indicating that the malonic acid linked to the 3-hydroxy of glucose is more stable than the more common linkage at the 6-position of glucose.The two acylated cyanidin glycosides, 1 and 2, from Bletilla striata flowers,5 are complex structures, with nine substituents distributed over three hydroxy groups of the parent cyanidin. Substitution at the 3-, 7- and 3*-positions is, however, quite a characteristic feature of anthocyanins from orchid flowers. Other examples can be found in Table 1, under #Laeliocattleya,23 Phalaenopsis31 and Sophronitis.39 Somewhat unexpectedly, the same 3,7,3*-trisubstitution pattern appears in the major pigments of Ceanothus papillosus (Rhamnaceae).6 These are delphinidin 3-rutinoside-7-(6-pcoumarylglucoside)- 3*-glucoside and the related 3*-(6-pcoumarylglucoside) 3.These are present in the bluish flowers of Ceanothus together with kaempferol 3-[xylosyl(1]2)- rhamnoside] as copigment. Copigmentation is quite specific to this kaempferol derivative and the resulting supramolecular complex involving 3 has a UV–VIS spectrum with an extra band in the visible region at 680 nm, as well as bands at 536, 576 and 615 nm.The ratio of flavonol to anthocyanin in the complex is in the order of 8:1.6 Anthocyanins acylated with p-hydroxybenzoic acid appear to occur characteristically in two unrelated plant families, the Campanulaceae and the Ranunculaceae.42,43 Two further sources of such pigments are Consolida armeniaca8 and Delphinium hybridum,9 both in the latter family. Consolida flowers contain the four delphinidin derivatives 4–7, while pink Delphinium flowers have the five pelargonidin glycosides listed in Table 1.O OGlc HO GlcO OH OGlc malonyl p-coumaryl Glc p-coumaryl Glc p-coumaryl O OGlc HO GlcO OH OGlc malonyl caffeyl Glc caffeyl Glc caffeyl O OGlc HO Glc OH OGlc Rha p-coumaryl p-coumaryl OH 6 6 6 6 + 6 6 6 4 1 4 3 6 6 4 6 2 6 + 4 + O OGlc HO GlcO OH OH malonyl p-OH-benzoyl OH Glc p-OH-benzoyl O OGlc HO GlcO OH OH malonyl p-OH-benzoyl OH Glc p-OH-benzoyl Glc O OGlc HO GlcO OH OH malonyl p-OH-benzoyl OH Glc p-OH-benzoyl p-OH-benzoyl O OGlc HO GlcO OH OH malonyl p-OH-benzoyl OH Glc p-OH-benzoyl p-OH-benzoyl Glc 6 6 4 + 4 6 6 6 + 6 4 6 7 + 6 6 6 2 2 2 4 4 6 6 + 5 6 4 Harborne and Williams: Anthocyanins and other flavonoids 631Table 1 New anthocyanins reported in the period 1995–1997 Anthocyanina Sourceb Reference Cy 3-[6-ferulyl-2-(6-ferulylglucosyl)glucoside]-5-(6-malonylglucoside) Ajuga pyrimidalis cell cultures (Labiatae) 1 Cy 3-[2-(6-p-coumarylglucosyl)-6-p-coumarylglucoside]-5-(6-malonylglucoside) Ajuga reptans flower (Labiatae) 2 Dp 3-[2-(6-p-coumarylglucosyl)-6-p-coumarylglucoside]-5-(6-malonylglucoside) Cy 3-(3,6-dimalonylglucoside) Allium victorialis stem (Alliaceae) 3 Cy 3-(3-malonylglucoside) Cy 3-(2-xylosyl-6-caVeylglucoside) Begonia flowers (Begoniaceae) 4 Cy 3-(2-xylosyl-6-p-coumarylglucoside) Cy 3-(2-glucosyl-6-p-coumarylglucoside) Cy derivative 1 Bletilla striata flowers (Orchidaceae) 5 Cy derivative 2 Dp 3-rutinoside-7-(6-p-coumarylglucoside)-3*-glucoside Ceanothus papillosus (Rhamnaceae) 6 Dp 3-rutinoside-7,3*-di(6-p-coumarylglucoside) 3 Dp 3-(6-malonylglucoside)-3*,5*-di[6-(4-glucosyl-p-coumaryl)glucoside](ternatin A3) and four similar Dp glycosides Clitoria ternatea (Leguminosae) 7 Dp polyacylglycosides 4–7 Consolida armeniaca (Ranunculaceae) Pg 3-rutinoside-7-(6-p-hydroxybenzoylglucoside) Delphinium hybridum flowers (Ranunculaceae) 9 Pg 3-rutinoside-7-[4*-glucosyl-(6-p-hydroxybenzoyl)glucoside] Pg 3-(6-malonylglucoside)-7-glucoside Pg 3-glucoside-7-[4*-glucosyl-(6-p-hydroxybenzoyl)glucoside] Pg 3-(6-malonylglucoside)-7-[4*-glucosyl-(6-p-hydroxybenzoyl)glucoside] Cy 3,5-diglucoside with malic acid 8 Dianthus caryophyllus flowers (Caryophyllaceae) 10 Dp 3-rutinoside-5-(6-p-coumarylglucoside) Eustoma grandiflorum flowers, after genetical transformation (Gentianaceae) 11 Dp 3-glucoside-5-(6-p-coumarylglucoside) Cy 3-glucoside-5-(6-caVeylglucoside) Gentiana pink flowers (Gentianaceae) 12 Cy 3-glucoside-5,3*-di(6-caVeylglucoside) Cy 3-glucoside-5-(6-p-coumarylglucoside) Dp 3,3*-diglucoside-5-(6-caVeylglucoside) Gentiana blue flowers (Gentianaceae) 13 Dp 3,3*-diglucoside-5-(6-p-coumarylglucoside) Dp 3-glucoside-5-(6-p-coumarylglucoside) Mv 3-(6-acetylglucoside)-5-glucoside Geranium sylvaticum flowers (Geraniaceae) 14 Mv 3-glucoside-5-(6-acetylglucoside) Geranium pratense flowers (Geraniaceae) 15 Pg 3-glucoside-5-(6-malonylglucoside) Hyacinthus orientalis red flowers (Liliaceae) 16 Pg 3-(6-p-coumarylglucoside)-5-(4-malonylglucoside) Pg 3-(6-p-coumarylglucoside)-5-(6-acetylglucoside) Pg 3-(6-ferulylglucoside)-5-(6-malonylglucoside) Dp 3-(6-p-coumarylglucoside)-5-(6-malonylglucoside) Hyacinthus orientalis blue flowers (Liliaceae) 17 Pt 3-(6-p-coumarylglucoside)-5-(6-malonylglucoside) Cy 3-[2-(6-caVeylglucosyl)glucoside] Ipomoea batatis purple tubers (Convolvulaceae) 18 Pn 3-[2-(6-caVeylglucosyl)glucoside] Cy derivative 9 Ipomoea purpurea brown flowers (Convolvulaceae) 19 Cy derivative 10 I.purpurea violet flowers (Convolvulaceae) 20 Pg analogue of 10 I. purpurea red-purple flowers (Convolvulaceae) 21 Dp-3-(2G-xylosylrutinoside) Linum grandiflorum flowers (Linaceae) 22 Cy derivatives 11–13 #Laeliocattleya cv. mini purple flowers (Orchidaceae) 23 Cy 3-(4-p-coumarylrutinoside)-5-(6-malonylglucoside)-3*-(6-caVeylglucoside) Lobelia erinus flowers (Lobeliaceae) 24 Pg 3-[2-(2-ferulylxylosyl)-6-ferulylglucoside]-5-(6-malonylglucoside) Matthiola incana flowers (Cruciferae) 25 Pg 3-[2-(2-sinapylxylosyl)-6-ferulylglucoside]-5-(6-malonylglucoside) Pg 3-[2-(2-sinapylxylosyl)-6-p-coumarylglucoside]-5-(6-malonylglucoside) Cy 3-malonylsambubioside-7-glucoside Meconopsis grandis, M.horridula and M. betonicifolia flowers (Papaveraceae) 26 Dp 3-(2-galloyl-6-acetylglucoside) Nymphaea#marliacea leaves 27 Dp 3-(6-acetylglucoside) Cy, Pn, Dp and Pt 3-glucoside-5-(6-acetylglucosides) Pelargonium flowers (Geraniaceae) 28 Mv derivatives 14 and 15 Petunia hybrida and P.guarapuavensis flowers (Solanaceae) 29,30 Cy 3-(6-malonylglucoside)-7,3*-di-(6-sinapylglucoside) Phalaenopsis spp. flowers (Orchidaceae) 31 Cy 3-glucoside-7,3*-di-(6-sinapylglucoside) Pn 3-[6-(3-glucosylcaVeyl)glucoside] Pharbitis nil slate flowers (Convolvulaceae) 32 Cy 3-[6-(3-glucosylcaVeyl)glucoside]-5-glucoside Cy 3-(6-succinylglucoside) Phragmites australis inflorescence (Gramineae) 33 Pg 3-[2-(2-caVeylglucosyl)galactoside] Pulsatilla cernua sepals (Ranunculaceae) 34 Cy 3-(6-malonylsambubioside) Ranunculus asiaticus (Ranunculaceae) 35 632 Natural Product Reports, 1998The first macrocyclic anthocyanin 8 has been isolated from flowers of two carnation cultivars by Bloor.10 This is a malic acid ester of cyanidin 3,5-diglucoside, in which the malyl group is linked to both sugars through the 6-hydroxy positions. Not unexpectedly, it is relatively labile and readily undergoes ring opening to furnish the 3-(6-malylglucoside)-5-glucoside.Its discovery raises the question of whether any other acylated anthocyanins, e.g. those with malonic acid attached to the 3-glucose moiety, are capable of occurring in a macrocyclic structure in vivo. The interest in the new anthocyanins based on delphinidin isolated from flowers of Eustoma grandiflorum (see Table 1) lies in the fact that their production has been genetically engineered by introducing a gene for flavonoid 3-glucosyltransferase from Antirrhinum majus into this plant.The original pigments in Eustoma contain galactose as the hexose sugar in the 3-position and this has been partly replaced by glucose during anthocyanin synthesis in the transformed plant.11 Anthocyanins with acetyl substitution are quite rare, although they do occur regularly in the skin of grapes.42 It is interesting that two such derivatives of malvidin 3,5- diglucoside have recently been reported in Geranium flowers.14,15 The acetyl is attached to the 3-glucose in one14 and to the 5-glucose in the other.15 They are apparently diYcult to distinguish since they are identical on HPLC.15 Determination of the position of acetylation depends on 2D 1H NMR, 1H TOCSY and HMBC experiments.15 Acetylated 3,5-diglucosides of other anthocyanins have also been recorded recently in the flowers of Pelargonium28 and Verbena.41 One of the very first polyacylglycosides to be described in the anthocyanin series was the blue peonidin pigment ‘Heavenly Blue’ from Morning Glory flowers (Ipomoea, syn.Pharbitis). Such plants exist in a wide range of colour forms and have continued to yield further examples of complex anthocyanins. Three new structures from I. purpurea include the cyanidin glycosides 9 and 10 and the pelargonidin analogue of 10.19–21 Equally complex are the three polyacylglycosides of the orchid cultivar #Laeliocattleya, pigments 11–13, but here the major complexity is confined to the substitution at the 3*-hydroxy group.22 Blue flower colour in Morning Glory flowers is provided unusually by cyanidin 3*-methyl ether (a peonidin) derivative, since delphinidin is most frequently the anthocyanidin base of blue flower colour.42 Another example of a magenta base providing blue flower colour is the cyanidin glycoside recently described from pure blue flowers of Meconopsis betonicifolia and of two related species.26 The large shift from magenta to blue depends apparently on copigmentation by two kaempferol glycosides, the 3-gentiobioside and the 3-xylosylgentiobioside.The copigment complex is most eVective at a flavonol:anthocyanin ratio of 5.6:1.26 The anthocyanins of Petunia and Solanum have been intensively studied in the past, chiefly in relation to biochemical genetical studies, and would seem to be well known. Nevertheless, new structures have been recorded in both cases.In Petunia hybrida, the first polyacylglycosides 14 and 15 have been discovered. They occur in a mutant cultivar, named ‘Surfinia violet mini’.30 In Solanum, the tuber of a hybrid between S. tuberosum and S. andigena has yielded an acylated pelargonidin 3-rutinoside-5-glucoside, where the usual p-coumaric acid is replaced by caVeic acid.38 The new pigment accompanies the p-coumaryl ester, called pelanin.44 In both pigments, the acyl group is shown to be linked to the 4-hydroxy of the glucose moiety in the 3-position.38 Most emphasis in this review has been given to structural elucidation of new anthocyanins because this is the most active field.Relatively little eVort has been given to other aspects of these pigments, apart from studies of copigmentation which have been mentioned above. In biosynthetic studies, three enzymes involved in acylation have been described and their specificities examined.A succinyl transferase has been obtained from flowers of Centaurea cyanus, a source of cyanidin 3-(6-succinylglucoside)-5-glucoside. As expected, it catalyses the transfer of succinic acid CoA to both cyanidin and pelargonidin 3-glucosides, but not to the corresponding 3,5-diglucosides. It does not appear to be separable from malonyltransferase activity.45 This latter enzyme has been prepared from the leaves of Lactuca sativa, where it again Table 1 Continued Anthocyanina Sourceb Reference Dp 3-(6-malonylsambubioside) Ranunculus asiaticus (Ranunculaceae) 35 Cy 3-(6-p-coumarylsambubioside)-5-glucoside Sambucus canadensis fruits (Caprifoliaceae) 36 Pg 3-(6-malonylglucoside)-7-[6-(4-(6-caVeylglucoside(caVeyl)glucoside] Senecio cruentus pink flowers (Compositae) 37 Pg 3-(6-malonylglucoside)-7-(6-caVeylglucoside) Pg 3-(4-caVeyl-6-rhamnosylglucoside)-5-glucoside Solanum tuberosum#S.andigena tuber (Solanaceae) 38 Cy 3-(6-malonylglucoside)-3*-glucoside-7-(6-caVeylglucoside) Sophronitis coccinea petals (Orchidaceae) 39 Cy 3,3*-diglucoside-7-(6-caVeylglucoside) Cy 3-(6-malonylglucoside)-3*-glucoside-7-(6-ferulylglucoside) Dp 3-(6-p-coumarylglucoside)-5-(6-malonylglucoside) Triteleia bridgensii flowers (Liliaceae) 40 Dp 3-[6-(4-glucosyl-p-coumaryl)glucoside]-5-(6-malonylglucoside) Pg 3-glucoside-5-(6-acetylglucoside) Verbena hybrida flowers (Verbenaceae) 41 Pg 3,5-di(6-acetylglucoside) Pg 3-(6-malonylglucoside)-5-(6-acetylglucoside) Cy 3,5-di(6-acetylglucoside) aAbbreviations: Pg, pelargonidin; Cy, cyanidin; Pn, peonidin; Dp, delphinidin; Pt, petunidin; Mv, malvidin.Sugar linkages are ‚ – for glucose, galactose, xylose and · – for rhamnose. Sugars are all in the pyranose form. Where cinnamic acids are acyl groups, they are assumed to be in the trans-form, although isomerisatoin to the cis-form often occurs during isolation and may be reported as such. bIn alphabetical order according to generic source.O OGlc O OH OH HO COCHOHCH2 O CO 6 6 + 8 Glc O Harborne and Williams: Anthocyanins and other flavonoids 633shows specificity for 3-glucosides, but is inactive on 3,5- diglucosides.46 Cyanidin 3-(6-malonylglucoside) 16 has been incidentally identified for the first time from Lactuca sativa leaves46 and also from dandelion (Taraxacum oYcinale) callus cultures and stems.47 The third acylating enzyme transfers a hydroxycinnamic acid CoA ester to the 5-glucoside of an anthocyanidin 3,5-diglucoside and was obtained from blue petals of Gentiana triflora.It works on both p-coumaric and caVeic acid esters and is relatively non-specific regarding the anthocyanidin 3,5-diglucoside substrate.48 Anthocyanins are significant components of red wine and they undoubtedly undergo chemical changes during the maturing and ageing of such wines. In view of their instability in solution, it is surprising that their colour remains for so long. It has been suggested that they are gradually converted into coloured polymers during ageing.49 Two groups of workers have now shown that one major anthocyanin of wine, malvidin 3-glucoside 17, is capable of undergoing a chemical condensation to produce a stable orange–red pigment absorbing at Îmax 510 nm which is stable to light.One such compound was called vitisin A and characterised as structure 18.50 A similar, presumably identical, pigment was reported from red wine by Fulcrand et al.51 and shown to have structure 19 (see Scheme 1). Such a structure could arise by reaction of 17 with pyruvic acid and this was confirmed by reacting a crude anthocyanin extract of wine with pyruvic acid and isolating the same product.This reaction would also explain the shift from O OGlc HO OH OH HO caffeyl Glc caffeyl Glc caffeyl Glc O O GlcO OH OH HO Glc caffeyl Glc caffeyl Glc + 4 + 13 6 Glc caffeyl 6 6 2 4 O OGlc HO GlcO OH O malonyl p-coumaryl 6 6 3 9 6 6 Glc p-coumaryl 6 Glc caffeyl 6 4 6 6 10 + 4 4 O OGlc HO GlcO OH OGlc malonyl p-coumaryl 12 6 + 6 2 caffeyl 6 Glc 11 caffeyl 6 + 6 4 6 6 O OGlc HO GlcO OH OGlc malonyl caffeyl caffeyl Glc caffeyl 14 15 + 4 4 6 6 + 6 4 4 6 O OGlc GlcO OH OMe HO Rha caffeyl Glc OMe caffeyl O OGlc GlcO OH OMe HO Rha p-coumaryl Glc OMe caffeyl + 6 O OGlc HO OH OH HO malonyl 16 O OGlc OH OH OMe HO O OGlc O OH OMe HO O OGlc O OH OMe HO OMe OMe CO2H O H OH OMe C CH2 HO2C OH + + + 17 19 18 Scheme 1 634 Natural Product Reports, 1998a red to a tawny colour that accrues in wine with ageing.Presumably, the other anthocyanins of red wine are also capable of undergoing the same reaction, so that several similar pyruvic acid adducts may await identification in diVerent wine samples. Several plant surveys for floral anthocyanins have been published. Twenty-five Chilean species of the genus Alstroemeria have variously yielded six pigments: 6-hydroxycyanidin 3-rutinoside 20, 6-hydroxydelphinidin 3-rutinoside, the 3-rutinosides of cyanidin and delphinidin and the 3- malonylglucosides of cyanidin and delphinidin.The Alstroemeria species could be divided into three groups according to the major anthocyanins present.52 In African species of the genus Lobostemon, species with violet blue flowers contain the 3,5- diglucoside, the 3-glucoside and the 3-rutinoside of delphinidin. By contrast, species with red or pink flowers are sources of cyanidin 3,5-diglucoside and the 3-glucoside.53 A survey of ten blue flowered Italian species of the genus Iris showed that the major anthocyanin of the cultivated I.germanica, namely delphanin, was universally present.54 Finally, the distribution of anthocyanins, especially malonylated and succinylated pigments, in the family Compositae has been reviewed.55 2 Lipophilic flavones and flavonols Some 40 new flavone aglycones and 34 new flavonols have been recorded during 1995 to 1997. These are listed in Tables 256–85 and 3, respectively.These discoveries bring the total number of known flavones to 442 and of known flavonols to 537. These lipophilic flavones and flavonols have been reported from a variety of plant tissues, namely from leaves, stems, roots, flowers and heartwood or more generally from underground or aerial parts or even whole plants. Since the flavonoid patterns of the various tissues of a plant usually diVer, it would be scientifically more useful if each tissue or part thereof was separately analysed.This is especially relevant in the case of the more highly O- and C-methylated and isoprenylated flavones and flavonols, which are frequently present in glands on the surface of leaves, stems or flowers. It is therefore unfortunate that, in most recent publications, the authors have not apparently determined whether their new compounds are present on the surface or within the plant tissue. In at least two cases, the authors have extracted with chloroform, which suggests that the flavones they found were probably on the surface of the tissue examined but they make no comment on their location.Amongst the new simple flavones it is very surprising that 7-hydroxyflavone, recently reported from leaves of Clerodendron phlomides (Verbenaceae),56 has not been found before. Table 2 includes a number of reports of methyl ethers of previously known aglycones. These include three tricetin derivatives all reported from diVerent plant families namely the 5,3*-dimethyl ether from Bruguiera gymnorrhiza (Rhizophoraceae),64 the 7,3*,4*-trimethyl ether from leaves of Lethedon tannaensis (Thymelaeaceae)65 and the 7,3*,5*- trimethyl ether from aerial parts of Centaurea incana (Compositae).66 However, the evidence for the identification of the latter compound is not good.The authors seemed to be unaware that they had isolated a new compound and claimed that they compared it with a known marker.Two other newly reported flavones have biogenetically unlikely structures and hence must remain excluded from our table, until better evidence is provided for their unusual patterns of substitution. They are 6,7,4*-trihydroxy-3*,5*- dimethoxyflavone, which strangely lacks a 5-hydroxy (or methoxy) group and 5,5*-dihydroxy-8,3*,4*-trimethoxyflavone, which is extremely unusual in lacking a hydroxy at the 7-position.86 Other new methyl ethers include two rare 5-methoxy compounds: scutellarein 5,4*-dimethyl ether from the whole plant of the parasite Striga passargei (Scrophulariaceae)60 and 6-hydroxyluteolin 5-methyl ether from leaves of Arrabidaea chica f.cuprea (Bignoniaceae).63 A further four 6,8-dihydroxyflavones have been recorded since 1994 including two revised structures for previously isolated compounds: 5,7-dihydroxy-6,8-dimethoxyflavone (revised from 5,8-dihydroxy-6,7-dimethoxyflavone) and 7-hydroxy-5,6,8-trimethoxyflavone (revised from 8-hydroxy- 5,6,7-trimethoxyflavone) from roots of Helichrysum herbaceum (Compositae).59 The other two compounds are both from Scutellaria species (Labiatae): 5,6-dihydroxy-7,8-dimethoxy- flavone from aerial parts of S.ramosissima58 and the unusual 2*-derivative, 5,6,2*-trihydroxy-7,8-dimethoxyflavone from S. baicalensis and S. glabrata.62 Such 2*-substituted flavones are characteristic constituents of the genus Scutellaria and in the period under review two other new examples were found: the simple trisubstituted, 5,7-dihydroxy-2*-methoxyflavone from roots of S.adenostegia57 and 5,7,2*,6*-tetrahydroxyflavone from S. baicalensis and S. glabrata.62 The latter compound was known previously only from synthesis and as an incorrect assignment to a compound from Argemone mexicana seed, which was later found to be luteolin.87 Only four new C-methylflavones have been newly reported including another 2*-hydroxyflavone, 5,2*-dihydroxy-7- methoxy-6,8-di-C-methylflavone from the whole plant of Trianthema portulacastrum (Aizoaceae).71 But a more interesting report is of three C-alkylated flavones, which have a p-hydroxybenzyl moiety attached at the 8-carbon of apigenin, luteolin and diosmetin, respectively which were isolated with the corresponding quercetin and kaempferol derivatives from the aerial parts of Thymus hirtus (Labiatae).72 This is the first report of p-hydroxybenzyl substitution through a carbon–carbon linkage.However, one C-hydroxybenzyl flavonoid has been described previously in the Annonaceae88 but here the aglycone was the flavanone, pinocembrin (5,7-dihydroxyflavanone).During the period 1995–1997 some 15 new isoprenylated flavones have been reported, including a variety of structures from simple prenyls to ring closed furano and pyrano derivatives.73–85 Some of the more exotic structures are illustrated, i.e. 21–31. Two such structures are cyclochampedol 31 from Artocarpus champeden tree bark (Moraceae)85 and Yinganghou A 25 from leaves of Epimedium sagittatum (Berberidaceae).82 The latter co-occurs with the related structures, 3*-prenylapigenin 28, a 3*,5*-diprenylated apigenin 26 and the 3*,4*-pyrano constituents 27 and 29.A reference from 1982 reporting 5-allyloxy-6,7,4*-trimethoxyflavone from heartwood of Tinospora malabarica (Menispermaceae)78 has been included because it has been omitted from previous flavone aglycone check lists.43 The new flavonols reported in the literature between 1995 and 1997 are listed in Table 3.89–112 O-Methylation is usually the final step in the biosynthesis of lipophilic flavones and flavonols, so it is not surprising to find that most of the structures listed are methyl ethers.Since O-methylation can take place at any or all of the free hydroxy groups of flavonols such as quercetin or quercetagetin, the number of possible methyl ethers is considerable. Hence, the first eight compounds in Table 3 are new methyl ethers of well known hydroxy- flavonols.O OGlc HO OH OH HO HO Rha + 6 20 Harborne and Williams: Anthocyanins and other flavonoids 635One is the 5,4*-dimethyl ether of quercetin from leaves of Rhododendron ellipticum (Ericaceae)91 and leaves of Anarthria gracilis (Anarthriaceae).92 There are three new quercetagetin derivatives; the 4*-methyl ether from leaves and stems of Artemisia annua (Compositae),94 the 5,6,7,3*,4*- pentamethyl ether from the leaf exudate of Chromolaena odorata (Compositae)95 and the 3,5,6,3*,4*-pentamethyl ether from the peel oil of Tangerine cv.Dancy (Rutaceae).96 A new pentamethyl ether of myricetin has been recorded as a leaf constituent of another member of the Rutaceae, Murraya Table 2 New flavones reported in the period 1995–1997 Flavone Source Reference Mono-O-substituted flavones 7-Hydroxyflavone Clerodendron phlomoides leaves (Verbenaceae) 56 Tri-O-substituted flavones 5,7-Dihydroxy-2*-methoxyflavone Scutellaria adenostegia roots (Labiatae) 57 Tetra-O-substituted flavones 5,6-Dihydroxy-7,8-dimethoxyflavone Scutellaria ramosissima aerial parts (Labiatae) 58 5,7-Dihydroxy-6,8-dimethoxyflavone Helichrysum herbaceum roots (Compositae) 59 7-Hydroxy-5,6,8-trimethoxyflavone Helichrysum herbaceum roots (Compositae) 59 6,7-Dihydroxy-5,4*-dimethoxyflavone (scutellarein 5,4*-dimethyl ether) Striga passargei whole plant (Scrophulariaceae) 60 5,8-Dihydroxy-7,4*-dimethoxyflavone Helicteres isora leaves (Sterculiaceae) 61 5,7,2*,6*-Tetrahydroxyflavone Scutellaria baicalensis and S.glabrata (Labiatae) 62 Penta-O-substituted flavones 5,6,2*-Trihydroxy-7,8-dimethoxyflavone Scutellaria baicalensis and S. glabrata (Labiatae) 62 6,7,3*,4*-Tetrahydroxy-5-methoxyflavone (6-hydroxyluteolin 5-methyl ether, carajuflavone) Arrabidaea chica f. suprea leaves (Bignoniaceae) 63 7,4*,5*-Trihydroxy-5,3*-dimethoxyflavone) (tricetin 5,3*-dimethyl ether) Bruguiera gymnorrhiza (Rhizophoraceae) 64 5,5*-Dihydroxy-7,3*,4*-trimethoxyflavone (tricetin 7,3*,4*-trimethyl ether) Lethedon tannaensis leaves (Thymelaeaceae) 65 5,4*-Dihydroxy-7,3*,5*-trimethoxyflavone (tricetin 7,3*,5*-trimethyl ether) Centaurea incana aerial parts (Compositae) 66 Hexa-O-substituted flavones 5,2*,6*-Trihydroxy-6,7,8-trimethoxyflavone Scutellaria adenostegia roots (Labiatae) 57 5,3*,5*-Trihydroxy-6,7,4*-trimethoxyflavone (6-hydroxytricetin 6,7,4*-trimethyl ether) Murraya paniculata leaves (Rutaceae) 67 C-Methylflavones 5,7-Dihydroxy-6,8-di-C-methylflavone (Matteuorien) Matteuccia orientalis rhizomes (Aspleniaceae) 68 5-Hydroxy-7-methoxy-6,8-di-C-methylflavone (Desmosflavone) Desmos cochinchinensis (Annonaceae) 70 5,7,3*,4*-Tetrahydroxy-6-C-methylflavone Salvia nemorosa aerial parts (Labiatae) 70 5,2*-Dihydroxy-7-methoxy-6,8-di-C-methylflavone Trianthema portulacastrum whole plant (Aizoaceae) 71 C-Alkylated flavones 5,7,4*-Trihydroxy-8-C-p-hydroxybenzylflavone (8-C-p-hydroxybenzylapigenin) Thymus hirtus aerial parts (Labiatae) 72 5,7,3*,4*-Tetrahydroxy-8-C-p-hydroxybenzylflavone (8-C-p-hydroxybenzylluteolin) 5,7,3*-Trihydroxy-4*-methoxy-8-C-p-hydroxybenzylflavone (8-C-p-hydroxybenzyldiosmetin) Isoprenylated flavones Sanaganone 21 Millettia sanagana root bark (Leguminosae) 73 5,7-Dimethoxy-8-[3+-(2+,5+-dihydro-5+,5+-dimethyl-2+-oxofuryl)flavone (Hookerianum) 22 Tephrosia hookeriana roots (Leguminosae) 74 2+,3+-Dihydro-5-methoxy-3+-(2+*-acetoxy-2+*-methylpropylidene)- 2+-oxofuryl-(4+,5+:7,8)flavone (Tephrorianin) 23 Tephrosia hookeriana aerial parts (Leguminosae) 75 5-Hydroxy-8,3*,4*,5*-tetramethoxy-6,7(2+,2+-dimethyl)pyranoflavone Neoraputia paraensis leaves and stems (Rutaceae) 76 3+-Demethylhaslundin (Hosloppin) 24 Hoslundia opposita leaves (Labiatae) 77 5-Allyloxy-6,7,4*-trimethoxyflavone Tinospora malabarica heartwood (Menispermaceae) 78 7-Hydroxy-5-methoxy-8-(3+-hydroxy)isopent-1-eneflavone Tephrosia emoroides roots (Leguminosae) 79 5,2*,4*-Trihydroxy-7-methoxy-8-prenylflavone (Artocarpetin A) Artocarpus heterophyllus roots (Moraceae) 80 5,4*-Dihydroxy-7,2*-dimethoxy-8-prenylflavone Artocarpus heterophyllus root bark (Moraceae) 81 5,7,2*,5*-Tetrahydroxy-4*-methoxy-3,3*-diprenylflavone Yinyanghou A 25 Epimedium sagittatum leaves (Berberidaceae) 82 Yinyanghou B 26 Yinyanghou C 27 Yinyanghou D 28 Yinyanghou E 29 5,6,7,3*,5*-Pentamethoxy-4*-prenyloxyflavone Ficus maxima leaves (Moraceae) 83 Australone A 30 Morus australis root bark (Moraceae) 84 Cyclochampedol 31 Artocarpus champeden tree bark (Moraceae) 85 636 Natural Product Reports, 1998paniculata97 and a further three new hepta-substituted 6,8- dihydroxyflavonols have been characterised from the peel of Citrus species.96,98,100 C-Methylation occurs more rarely than O-methylation.Nevertheless, six new C-methylated flavonols are recorded in Table 3. Three are quercetin derivatives from leaves of Piliostigma thonningii (Leguminosae):102 the 6-C-methyl-3,7- dimethyl ether, the 6,8-di-C-methyl-3-methyl ether and the O O O O O Ph O Ph MeO MeO O O O O MeO O Ph O HO O Ph O HO O O AcO MeO O O HO Me HO O HO O HO OH O HO O HO OH O O HO O HO R O O O OH O OH OH OH 23 21 22 O HO O 24 26 HO 25 27 R = H 29 R = OH OH OH O 30 28 31 O O OH HO O O OMe O O OH HO O O OH HO O O HO OMe HO MeO O O O MeO OMe OH OMe HO OH OMe OMe HO OH OH O O HO O O OH O O HO HO O OH 32 33 34 35 36 37 38 Harborne and Williams: Anthocyanins and other flavonoids 6373,7-dimethyl ether.However, of special note is the report of two C-methylated flavonols from a fungus since flavonoids are so rarely found in fungi. Thus, 5,4*-dihydroxy-3,6,7- trimethoxy-8-C-methylflavone and its 6-C-isomer were isolated from a culture of Colletotrichum dematium f.sp. epilobii,101 a fungus which is a pathogen of fireweed, Epilobium angustifolium ssp. angustifolium. The latter compound has the same 3,7,8-trimethoxy substitution pattern as the earlier reported chloroflavonin from Aspergillus candidus113 but differs in the presence of a 6-C-methyl group and the absence of a 2*-hydroxy and 3*-chloro group.There are 11 new isoprenylated flavonols, including compounds 32–38, which have nearly all been isolated from root bark tissue. These include three complex structures from the Table 3 New flavonols reported in the period 1995–1997 Flavonol Source Reference Tetra-O-substituted flavonols 7-Hydroxy-3,8,4*-trimethoxyflavone Parkia clappertoniana leaves (Leguminosae) 89 Penta-O-substituted flavonols 5,6-Dihydroxy-3,7,4*-trimethoxyflavone (6-hydroxykaempferol 3,7,4*-trimethyl ether) Tanacetum parthenium leaves, flowers and fruit (Compositae) 90 3,7,3*-Trihydroxy-5,4*-dimethoxyflavone (quercetin 5,4*-dimethyl ether) Rhododendron ellipticum leaves (Ericaceae) 91 Anarthria gracilis leaves (Anarthriaceae) 92 7-Hydroxy-3,6,3*,4*-tetramethoxyflavone (Santoflavone) Achillea santolina aerial parts (Compositae) 93 Hexa-O-substituted flavonols 3,5,6,7,3*-Pentahydroxy-4*-methoxyflavone (quercetagetin 4*-methyl ether) Artemisia annua leaves and stems (Compositae) 94 3-Hydroxy-5,6,7,3*,4*-pentamethoxyflavone (quercetagetin 5,6,7,3*,4*-pentamethyl ether) Chromolaena odorata leaf exudate (Compositae) 95 7-Hydroxy-3,5,6,3*,4*-pentamethoxyflavone (quercetagetin 3,5,6,3*,4*-pentamethyl ether) Citrus sp.(Tangerine cv.Dancy) peel oil (Rutaceae) 96 3-Hydroxy-5,7,3*,4*,5*-pentamethoxyflavone (myricetin 5,7,3*,4*,5*-pentamethyl ether) Murraya paniculata leaves (Rutaceae) 97 2*-Hydroxy-3,7,8,4*,5*-pentamethoxyflavone Parkia clappertoniana leaves (Leguminosae) 89 Hepta-O-substituted flavonols 6-Hydroxy-3,5,7,8,3*,4*-hexamethoxyflavone Citrus unshiv fruit peel (Rutaceae) 98 5-Hydroxy-3,7,8,3*,4*,5*-hexamethoxyflavone Murraya paniculata fruit (Rutaceae) 99 7-Hydroxy-3,5,6,8,3*,4*-hexamethoxyflavone Citrus sp.(Tangerine cv. Dancy) peel oil (Rutaceae) 96 8-Hydroxy-3,5,7,3*,4*,5*-hexamethoxyflavone Murraya paniculata fruit (Rutaceae) 99 3,5,6,7,8,3*,4*-Heptamethoxyflavone Citrus spp. (Rutaceae) 100 C-Methylflavonols 5,4*-Dihydroxy-3,6,7-trimethoxy-8-C-methylflavone Colletotrichum dematium f.sp. epilobii fungal culture (Coelomycetes) 101 5,4*-Dihydroxy-3,7,8-trimethoxy-6-C-methylflavone 5,3*,4*-Trihydroxy-3,7-dimethoxy-6-C-methylflavone (3,7,-dimethyl-6-C-methylquercetin) Piliostigma thonningii leaves (Leguminosae) 102 5,7,3*,4*-Tetrahydroxy-3-methoxy-6,8-di-C-methylflavone (3-methyl-6,8-di-C-methylquercetin) 5,3*,4*-Trihydroxy-3,7-dimethoxy-6,8-di-C-methylflavone (3,7-dimethyl-6,8-di-C-methylquercetin) 5-Hydroxy-3,7,3*,4*-tetramethoxy-6-C-methylflavone (6-C-methyl-3,7,3*,4*-tetramethylquercetin) Leptospermum laevigatum leaf wax (Myrtaceae) 103 C-Alkylated flavonols 3,5,7,4*-Tetrahydroxy-8-C-p-hydroxybenzylflavone (8-C-p-hydroxybenzylkaempferol) Thymus hirtus aerial parts (Labiatae) 72 3,5,7,3*,4*-Pentahydroxy-8-C-p-hydroxybenzylflavone (8-C-p-hydroxybenzylquercetin) Isoprenylated flavonols 3,5,7-Trihydroxy-4*-methoxy-8-(3+-methoxyisopentyl)flavone (brevicornin) 32 Epimedium brevicornum aerial parts (Berberidaceae) 104 4*-Hydroxy-3,6,3*,5*-tetramethoxy-7,8-pyranoflavone 33 Diospyros peregrina fruit (Ebenaceae) 105 Petalopurpurenol 34 Petalostemon purpureus root (Leguminosae) 106 Velloquercetin 3*,4*-dimethyl ether 35 Vellozia graminifolia whole plant (Velloziaceae) 107 8-Geranylkaempferol (tomentosanol C) Sophora tomentosa roots and stems (Leguminosae) 108 5,7,2*,5*-Tetrahydroxy-4*-methoxy-3,3*-diprenylflavone Artocarpus heterophyllus root bark (Moraceae) 109 3,5,7,4*-Tetrahydroxy-8,3*-diprenylflavone (broussoflavonol F) Broussonetia papyrifera root bark (Moraceae) 110 3,5,7,3*,4*-Pentahydroxy-8,5*,6*-triprenylflavone (broussoflavonol G) Broussonetia papyrifera root bark (Moraceae) 111 Artelasticin 36 Artocarpus elasticus wood (Moraceae) 112 Artelastochromene 37 Artelasticin 38 638 Natural Product Reports, 1998Table 4 New flavone glycosides reported in the period 1995–1997 Flavone Source Reference 5,6-Dihydroxy-7-methoxyflavone (Negletein) 6-glucoside Colebrookea oppositifolia bark (Labiatae) 115 6-Rhamnosyl(1]2)fucoside Origanum vulgare (Labiatae) 116 5,7-Dihydroxy-6-methoxyflavone (Oroxylin A) 7-glucosyl(1]3)rhamnoside Eupatorium africanum whole plant (Compositae) 117 5,7,2*-Trihydroxyflavone 7-glucoside Scutellaria ramosissima aerial parts (Labiatae) 118 2*-glucoside Apigenin 7-Apiosyl(1]6)glucoside Gonocaryum calleryanum leaves 119 7-(2G-Rhamnosyl)gentiobioside Lonicera gracilepes var.glandulosa leaves (Caprifoliaceae) 120 7-[6+-(3-Hydroxy-3-methylglutaryl)glucoside] Chamaemelum nobile flowers (Compositae) 121 7-(2+-E-p-Coumarylglucoside) Echinops echinatus flowers (Compositae) 122 7-(3+-p-Coumarylglucoside) Stachys aegyptica aerial parts (Labiatae) 123 7-(3+-Acetyl-6+-E-p-coumarylglucoside) Blepharis ciliaris aerial parts (Acanthaceae) 124 Apigenin 4*-methyl ether (Acacetin) 7-Rhamnoside Peganum harmala aerial parts (Zygophyllaceae) 125 7-(2G-Rhamnosyl)rutinoside Buddleia oYcinalis flowers (Loganiaceae) 126 7-Rhamnosyl(1]2)glucosyl(1]2)glucoside Peganum harmala aerial parts (Zygophyllaceae) 125 7-Rhamnosyl(1]2)glucosyl(1]2)glucosyl(1]2)glucoside Peganum harmala whole plant (Zygophyllaceae) 127 7-[6+-Glucosyl-2+-(3+-acetylrhamnosyl)]glucoside Peganum harmala whole plant (Zygophyllaceae) 125 Scutellarein 6-methyl ether (Hispidulin) 7-Rhamnoside Picnomon acarna aerial parts (Compositae) 128 Scutellarein 5,4*-dimethyl ether 7-Glucoside Striga passargei whole plant (Scrophulariaceae) 129 7-(4Rha-Acetylrutinoside) 5,7,8,4*-Tetrahydroxyflavone (Isoscutellarein) 7-Glucosyl(1]2)xyloside Sideritis spp.aerial parts (Labiatae) 130 5,7,2*,6*-Tetrahydroxyflavone 2*-Glucoside Scutellaria baicalensis hairy root cultures (Labiatae) 131 5,2*-Dihydroxy-7,8-dimethoxyflavone 2*-Glucoside Andrographis paniculata roots (Acanthaceae) 132 Luteolin 5-Glucuronide-6+-methyl ester Dumortiera hirsuta gametophytes (Hepaticae) 133 5-Rutinoside Salvia lavandulifolia spp.oxyodon aerial parts (Labiatae) 134 7-Glucosyl(1]4)-·-L-arabinopyranoside Cassia glauca seed (Leguminosae) 135 7-Xylosyl(1]6)glucoside (7-primeveroside; cesioside) Dacrydium spp.(Podocarpaceae) 136 Halenia corniculata whole plant (Gentianaceae) 137 7-Robinobioside Pteris cretica fronds (Adiantaceae) 138 7-Sophoroside Pteris cretica aerial parts (Adiantaceae) 139 7-(6+-p-Benzoylglucoside) Vitex agnus-castus root bark (Verbenaceae) 140 7-Glucosyl(1]6)[(4+*-caVeyl)glucoside] Lonicera implexa leaves (Caprifoliaceae) 141 7-(6++-Acetylallosyl)(1]3)glucosyl(1]2)glucoside (Veronicoside A) Veronica didyma (Scrophulariaceae) 142 Luteolin 3*-methyl ether (Chrysoeriol) 7-·-L-Arabinofuranosyl(1]6)glucoside Tagetes patula whole plant (Compositae) 143 7-Neohesperidoside Morinda morindoides leaves (Rubiaceae) 144 Luteolin 4*-methyl ether (Diosmetin) 7-Arabinosyl(1]6)glucoside Galium palustre (Rubiaceae) 145 7-Xylosyl(1]6)glucoside Luteolin 5,3*-dimethyl ether 7-Glucoside Pyrus serotina leaves (Rosaceae) 146 4*-Glucoside 6-Hydroxyluteolin 6-Rhamnoside Erythroxylum leal-costae leaves (Erythroxylaceae) 147 6-Glucoside-7-[6+*-(3-hydroxy-3-methylglutaryl)glucoside] Frullania teneriVae (Hepaticae) 148 7-[6+-(3-Hydroxy-3-methylglutaryl)glucoside]-3*-glucuronide Frullania cesatiana (Hepaticae) 148 Harborne and Williams: Anthocyanins and other flavonoids 639Table 4 Continued Flavone Source Reference 6-Hydroxyluteolin 6,3*-dimethyl ether 7-Rutinoside Kichxia elatine aerial parts (Scrophulariaceae) 149 8-Hydroxyluteolin (Hypolaetin) 8-Rhamnoside Erythroxylum leal-costae leaves (Erythroxylaceae) 147 8-Hydroxyluteolin 4*-methyl ether 7-(6+*-Acetylsophoroside) Sideritis syriaca aerial parts (Labiatae) 150 5,7-Dihydroxy-6,8,4*-trimethoxyflavone (Nevadensin) 5-Glucoside Lysionotus pauciflorus aerial parts (Gesneriaceae) 151 5-Gentiobioside 5,2*,6*-Trihydroxy-6,7-dimethoxyflavone 2*-Glucoside Scutellaria baicalensis roots (Labiatae) 152 5,7,2*,4*,5*-Pentahydroxyflavone (Isoetin) 4*-Glucuronide Adonis aleppica whole plant (Ranunculaceae) 153 5,7,4*-Trihydroxy-3*,5*-dimethoxyflavone (Tricin) 7-‚-L-Arabinopyranoside (Setaricin) Setaria italica leaves (Gramineae) 154 5,2*,6*-Trihydroxy-6,7,8-trimethoxyflavone 2*-Glucoside Scutellaria baicalensis roots (Labiatae) 152 5,7-Dihydroxy-6-C-methylflavone 7-Xylosyl(1]3)xyloside Mosla chinensis (Labiatae) 155 5,7-Dihydroxy-6,8-di-C-methylflavone (Matteuorien) 7-[6+-(3+*-Hydroxy-3+*-methylglutaryl)glucoside Matteuccia orientalis rhizomes (Aspleniaceae) 68 Stachysetin 39 Stachys aegyptiaca aerial parts (Labiatae) 156 Table 5 New flavonol glycosides reported in the period 1995–1997 Flavonol Source Reference 3,6,7-Trihydroxy-4*-methoxyflavone 7-rhamnoside Setaria italica leaves (Gramineae) 157 Kaempferol 3-Rhamnosyl(1]2)-·-L-arabinofuranoside (Arapetaloside B) Artabotrys hexapetalus leaves (Annonaceae) 158 7-Glucosyl(1]3)rhamnoside Rhodiola crenulata roots (Crassulaceae) 159 7-Sophoroside Crocus sativus stamens (saVron) (Iridaceae) 160 7,4*-Diglucoside Cassia javanica flowers (Caesalpinaceae) 161 3-Glucosyl(1]2)galactosyl(1]2)glucoside Nigella sativa seeds (Ranunculaceae) 162 3-Rhamnosyl(1]2)[glucosyl(1]4)]glucoside Allium neapolitanum whole plant (Liliaceae) 163 3-Rhamnosyl(1]2)galactoside-7-·-L-arabinofuranoside Indigo hebepetala flowers (Leguminosae) 164 3-Robinobioside-7-·-L-arabinofuranoside 3-Neohesperidoside-7-rhamnoside Sedum telephium subsp.maximum leaves (Crassulaceae) 165 3-Neohesperidoside-4*-glucoside Pseuderucaria clavata aerial parts minus flowers (Cruciferae) 166 3-Neohesperidoside-7,4*-diglucoside 3-(2G-Rhamnosylrutinoside)-7-glucoside (Mauritianin 7-glucoside) Alangium premnifolium leaves (Alangiaceae) 167 3-[2+-(Z)-p-Coumarylglucoside] Eryngium campestre aerial parts (Umbelliferae) 168 3-(6+-CaVeylglucoside) Pteridium aquilinum aerial parts (Dennstaedtiaceae) 169 3-(5+-Ferulylapioside) Pteridium aquilinum aerial parts (Dennstaedtiaceae) 170 3-(6+-Acetylglucoside) Picea abies needles (Pinaceae) 171 3-(3+,4+-Diacetylglucoside) Minthostachys spicata aerial parts (Labiatae) 172 3-(6+-CaVeylglucoside)(1]4)rhamnoside Rorippa indica whole plant (Cruciferae) 173 3-(6+*-Sinapylglucoside)(1]2)galactoside Thevetia peruviana leaves (Apocynaceae) 174 3-[2+,4+-Di-(E)-p-coumarylrhamnoside] Pentachondra pumila leaves and stems (Epacridaceae) 175 3-(2+-p-Coumarylglucoside)-7-glucoside Ononis vaginalis and O.sicula aerial parts (Leguminosae) 176 3-[6+*-Acetylglucosyl(1]3)galactoside] Ricinus communis roots (Euphorbiaceae) 177 3-(6+-Acetylglucoside)-7-rhamnoside Ligustrum walkeri flowers (Oleaceae) 178 3-Xyloside(1]2)rhamnoside-7-(4++-acetylrhamnoside) (4++-acetylsagittatin A) Kalanchoe streptantha leaves (Crassulaceae) 179 3-Neohesperidose-7-[2+-(E)-p-coumarylglucoside] Allium ursinum whole plant (Liliaceae) 180 3-Neohesperidoside-7-[2+-(E)-ferulylglucoside] 640 Natural Product Reports, 1998Table 5 Continued Flavonol Source Reference 3-Glucosyl(1]4)(6+*-sinapylglucosyl)(1]2)galactoside Thevetia peruviana leaves (Apocynaceae) 174 3-(2++-Sinapylglucosyl)(1]4)(6+*-sinapylglucosyl(1]2)galactoside 3-Neohesperidoside-7-[2+-(E)-p-coumaryllaminaribioside] Allium ursinum whole plant (Liliaceae) 180 Kaempferol 7-methyl ether (Rhamnocitrin) 4*-Glucoside Cotoneaster simonsii leaves (Rosaceae) 181 3-Apiosyl(1]5)apioside-4*-glucoside Mosla chinensis (Labiatae) 182 Kaempferol 4*-methyl ether (kaempferide) 3-(4Rha-Rhamnosylrutinoside) Sageretia filiformia leaves (Rhamnaceae) 183 3-Acetate Afromomum hanburyi (Zingiberaceae) 184 6-Hydroxykaempferol 7-Alloside Tagetes erecta flowers (Compositae) 185 8-Hydroxykaempferol (Herbacetin) 3-‚-D-Glucofuranoside Jungia paniculata whole plant (Compositae) 186 3-Rhamnoside-8-glucoside Ephedra aphylla aerial parts (Ephedraceae) 187 Herbacetin 7-methyl ether 3-Sophoroside Ranunculus sardous pollen (Ranunculaceae) 188 3-[2+-(E)-Ferulylglucoside] Ranunculus sardous pollen (Ranunculaceae) 189 Quercetin 3-Rhamnosyl(1]2)-·-L-arabinofuranoside (Arapetaloside A) Artabotrys hexapetalus leaves (Annonaceae) 158 3-Rhamnosyl(1]3)galactoside Myrsine africana leaves (Myrsinaceae) 190 3-Galactosyl(1]2)rhamnoside Embelia schimperi leaves (Myrsinaceae) 191 3-Laminaribioside Pteridium aquilinum aerial parts (Dennstaedtiaceae) 192 3-Glucosyl(1]4)galactoside Rumex chalepensis leaves (Polygonaceae) 193 3-Rhamnoside-3*-glucoside Myrsine seguinii leaves (Myrsinaceae) 194 3-Glucosyl(1]2)galactosyl(1]2)glucoside Nigella sativa seeds (Ranunculaceae) 162 3-Neohesperidoside-7-rhamnoside Sedum telephium subsp.maximum leaves (Crassulaceae) 165 3,7,4*-Triglucoside Allium cepa scales (Alliaceae) 195 3-Glucosyl(1]4)rhamnoside-7-rutinoside Myrsine africana leaves (Myrsinaceae) 196 3-(6+-n-Butylglucuronide) (parthenosin) Parthenocissus tricuspidata leaves (Vitaceae) 197 3-(2+-Galloylrutinoside) Euphorbia ebractedata aerial parts (Euphorbiaceae) 198 3-(6+*-CaVeylgentiobioside) Lonicera implexa leaves (Caprifoliaceae) 141 3-(6+*-Sinapylglucosyl)(1]2)galactoside Thevetia peruviana leaves (Apocynaceae) 174 3-(6+-Malonylglucoside)-7-glucoside Ranunculus fluitans leaves (Ranunculaceae) 199 3-(6++-CaVeylsophorotrioside) Pisum sativum shoots (Leguminosae) 200 3-(6++-p-Coumarylsophorotrioside) 3-(6++-Ferulylsophorotrioside) 3-(6++-Sinapylsophorotrioside) 3-(6++-Ferulylglucosyl)(1]2)galactosyl(1]2)glucoside Nigella sativa seeds (Ranunculaceae) 162 3-CaVeylsophoroside-7-caVeylglucoside Ranunculus fluitans leaves (Ranunculaceae) 199 3-CaVeylsophoroside-7-ferulylglucoside Quercetin 3-methyl ether 5-Glucoside Asplenium trichomanes-ramosum fronds (Aspleniaceae) 201 7-·-L-Arabinofuranosyl(1]6)glucoside Lepisorus ussuriensis whole plant (Polypodiaceae) 202 7-Rutinoside Bidens leucantha leaves (Compositae) 203 7-Gentiobioside Lonicera implexa leaves (Caprifoliaceae) 141 Quercetin 7-methyl ether (Rhamnetin) 3-Laminaribioside Pteridium aquilinum aerial parts (Dennstaedtiaceae) 204 3-Gentiobioside Cassia fistula roots (Leguminosae) 205 Quercetin 3*-methyl ether (Isorhamnetin) 3-Rhamnoside Oxytropis lanata aerial parts (Leguminosae) 206 3-Apiosyl(1]2)galactoside Vernonia galamensis spp. nairobiensis leaves (Compositae) 207 3-Laminaribioside Pteridium aquilinum aerial parts (Dennstaedtiaceae) 192 3-Xylosylrobinobioside Nitraria retusa leaves and young stems (Nitrariaceae) 208 3-(4Rha-Galactosylrobinobioside) 3-Rhamnosyl(1]2)[glucosyl(1]6)]glucoside Allium neopolitanum whole plant (Liliaceae) 163 3-Rhamnosyl(1]2)[glucosyl(1]6)]glucoside 7-glucoside 3-Rhamnosyl(1]2)[gentiobiosyl(1]6)]glucoside) 3-(3+*-Ferulylrhamnosyl)(1]6)galactoside Herniaria fonanesii aerial parts (Caryophyllaceae) 209 3-(4+-Sulfatorutinoside) Zygophyllum dumosum parts (Zygophyllaceae) 210 Harborne and Williams: Anthocyanins and other flavonoids 641wood of Artocarpus elasticus (Moraceae):112 two triprenylated, artelasticin 36 and artelastochromene 37 and a dipyranodiprenyl constituent, artelastin 38.The structure of another triprenylated flavonol from root bark of Broussonetia papyrifera (Moraceae),111 3,5,7,3*,4*-pentahydroxy-8,5*,6*- triprenylflavone has been revised from 3,5,7,3*,4*- pentahydroxy-8,2*,6*-triprenylflavone previously named broussoflavonol E.110 Flavones, flavonols and their glycosides (see next section) continue to attract the attention of biologists, because of their Table 5 Continued Flavonol Source Reference Quercetin 4*-methyl ether (Tamarixetin) 3,7-Diglucoside Zanthoxylum bungeanum pericarps (Rutaceae) 211 Quercetin 3,4*-dimethyl ether 7-Glucoside Zanthoxylum bungeanum pericarps (Rutaceae) 211 7-Rutinoside Bidens leucantha leaves (Compositae) 203 Bidens pilosa var.radiata aerial parts (Compositae) 213 7-2G-Rhamnosylrutinoside Bidens leucantha leaves (Compositae) 203 7-2G-Glucosylrutinoside Quercetin 7,4*-dimethyl ether (Ombuin) 3-Arabinofuranoside Coccinia indica roots (Cucurbitaceae) 212 Quercetin 5,7,3*,4*-tetramethyl ether 3-Galactoside Sesbania aculeata (Leguminosae) 214 6-Hydroxyquercetin (Quercetagetin) 6-Glucoside Tagetes mandonii aerial parts (Compositae) 215 7-[6+-(E)-CaVeylglucoside] Eupatorium glandulosum leaves (Compositae) 216 7-(6+-Acetylglucoside) Quercetagetin 6-methyl ether (Patuletin) 3-(2+-Ferulylglucosyl)(1]6)[apiosyl(1]2)]glucoside Spinacia oleracea leaves (Chenopodiaceae) 217 Quercetagetin 6,3*-dimethyl ether (Spinacetin) 3-(2+-Apiosylgentiobioside) Spinacia oleracea leaves (Chenopodiaceae) 217 3-(2+*-Ferulylgentiobioside) 3-(2+-p-Coumarylglucoside(1]6)[apiosyl(1]2)]glucoside 3-(2+-Ferulylglucoside(1]6)[apiosyl(1]2)]glucoside Quercetagetin 6,7,3*-trimethyl ether (Veronicafolin) 3-Glucosyl(1]3)galactoside Eupatorium africanum (Compositae) 218 8-Hydroxyquercetin 7,8-dimethyl ether (Gossypetin 7,8-dimethyl ether) 3,3*-Disulfate Erica cinerea flowers (Ericaceae) 219 3,5,7,3*,4*,5*-Hexahydroxyflavone (Myricetin) 3*-Rhamnoside Davilla flexuosa leaves (Dilleniaceae) 220 3-Rhamnoside-3*-glucoside Myrsine seguinii leaves (Myrsinaceae) 194 3,4*-Dirhamnoside 3-(Acetylrhamnoside) Betula pubescens leaves (Betulaceae) 221 3-(3+,4+-Diacetylrhamnoside) Myrsine africana leaves (Myrsinaceae) 222 8-Hydroxymyricetin 8-methyl ether 3-Rhamnoside Erica verticillata aerial parts (Ericaceae) 223 8-Hydroxymyricetin 8,5*-dimethyl ether 3-Rhamnoside Erica verticillata aerial parts (Ericaceae) 223 8-Hydroxymyricetin 8,3*,5*-trimethyl ether 3-Rhamnoside Erica verticillata aerial parts (Ericaceae) 223 5,4*-Dihydroxy-6,7,8,3*-tetramethoxyflavone (Africanutin) 4*-Galactoside Eupatorium africanum whole plant (Compositae) 218 3,6,7,8,3*,4*-Hexahydroxy-5*-methoxyflavone 7-Neohesperidoside Hibiscus vitifolius (Malvaceae) 224 8-Prenylkaempferol[Noranhydroicaritin-3,5,7,4*-tetrahydroxy-8- (3+,3+-dimethylallylflavone)] 8-Prenylkaempferol 4*-methyl ether (Anhydroicaritin) 3-[2+*,6+*-Diacetylglucosyl(1]3)-4+-acetylrhamnoside]-7-glucoside (Epimedin K) Epimedium koreanum aerial parts (Berberidaceae) 225 3-[4+*,6+*-Diacetylglucosyl(1]3)-4+-acetylrhamnoside]-7-glucoside Epimedium koreanum aerial parts (Berberidaceae) 226 8-(3+-Hydroxy-3+-methylbutyl)kaempferol 4*-methyl ether (Icaritin) 3-Rhamnosyl(1]2)rhamnoside (Wanepimedoside A) Epimedium wanshanense whole plant (Berberidaceae) 227 8-(„-Methoxy-„,„-dimethyl)propyl kaempferol 4*-methyl ether 7-Glucoside (Caohuoside D) Epimedium koreanum aerial parts (Berberidaceae) 228 8-Prenylquercetin 4*-methyl ether 3-Rhamnoside (Caohuoside C) Epimedium koreanum aerial parts (Berberidaceae) 229 642 Natural Product Reports, 1998presence in many food plants, as well as in commonly available medicinal plants.Currently, most attention is being given to their anti-inflammatory and antioxidative activities. This is apparent in the proceedings of a recent conference entitled ‘Flavonoids and Bioflavonoids 1995’ which was published in 1996 and contains a variety of papers emphasising the biological properties of particular structures.114 3 Flavone and flavonol glycosides Some 52 new flavone glycosides115–156 and 110 new flavonol glycosides157–236 have been reported during the period 1995–1997. These are listed in Tables 4 and 5, respectively and include acylated and sulfated derivatives as well as those with only sugar substituents.This brings the total of known glycosides to 569 flavones and 1141 flavonols. Among the new flavonoid monoglycosides are an unusual 5-deoxy derivative, 3,6,7-trihydroxy-4*-methoxyflavone 7-rhamnoside from Setaria italica,157 the 8-rhamnoside of hypolaetin (8-hydroxyluteolin) and the 6-rhamnoside of 6- hydroxyluteolin from Erythroxylum leal-costae.147 Two other new monorhamnosides are acacetin 7-rhamnoside125 and hispidulin (scutellarein 6-methyl ether) 7-rhamnoside128 from Peganum and Picnomon respectively (Table 4).A 1989 report of tricin 7-‚-L-arabinopyranoside from leaves of Setaria italica154 is included in Table 4, since it was accidentally omitted from previous flavonoid lists. In all known flavonoid glycosides, glucose is present in the ‚-D-pyranose form, so that a report of herbacetin 3-glucofuranoside in Jungia paniculata (Compositae)186 needs to be very secure. Unfortunately, the NMR data supporting the furanose form for the sugar are not completely convincing. 237 Furthermore, the authors did not apparently bother to hydrolyse their glycoside to confirm that the sugar was, indeed, glucose. More data are really needed before we can be sure that a flavonol glucofuranoside is present in this plant. An unusual finding is of the 7-alloside of 6-hydroxykaempferol from Tagetes erecta.185 Both allose and apiose are uncommon in glycosidic combination with flavonoids. In the present lists allose is otherwise seen only acetylated at the 6+*-position in combination with luteolin in a new trisaccharide, allosyl(1]3)glucosyl(1]2)glucose, from Veronica didyma.142 There are five reports of flavonoid apiosides, including one flavone glycoside, apigenin 7-apiosyl(1]6)glucoside from Gonocaryum calleryanum,119 which is an isomer of the well known apiin, apigenin 7-apiosyl(1]2)glucoside from parsley.The flavonol apiosides include a new disaccharide, apiosyl(1]5)apiose in combination with kaempferol 7-methyl ether as the 3-apiosyl(1]5)apioside-4-glucoside in Mosla chinensis (Labiatae).182 Also from the Labiatae are four further reports of 2*-hydroxyflavone 2*-glycosides from Scutellaria species.118,131,152 This is a rare substitution pattern but very characteristic of this labiate genus. 5-Glycosylation is also comparatively rare and there are only two new records, the 5-glucoside and 5-gentiobioside of 5,7-dihydroxy- 6,8,4*-trimethoxyflavone (nevadensin), both from Lysionotus pauciflorus.151 A new monosaccharide has been discovered in combination with a flavonoid during the review period, fucose, a sugar which is a common constituent of algal and plant polysaccharides.It was found as a disaccharide in combination with the simple flavone negletein, 5,6-dihydroxy-7-methoxyflavone as the 6-rhamnosyl(1]2)fucoside in Origanum vulgare.116 Two further new disaccharides were found in combination with flavones: glucosyl (1]4)-·-L-arabinopyranose in seed of Cassia glauca135 attached at the 7-hydroxy of luteolin and xylosyl(1]3)xylose at the 7-hydroxy of 5,7-dihydroxy-6-Cmethylflavone in Mosla chinensis.155 Three new disaccharides have been found linked to the 3-hydroxy of quercetin.These are all new linkages of previously known sugar combinations i.e. rhamnosyl(1]3)galactose from Myrsine africana,190 galactosyl(1]2)rhamnose from Embelia schimperi191 and glucosyl(1]4)galactose from Rumex chalepensis.193 Another glucosylgalactose with a (1]3) linkage was found attached at the 3-hydroxy of quercetagetin 6,7,3*-trimethyl ether in Eupatorium africanum.218 There are three new linear trisaccharides: glucosyl(1]2)- galactosyl(1]2)glucose found in combination with the 3-hydroxy of quercetin and kaempferol in seeds of Nigella sativa, allosyl(1]3)glucosyl(1]2)glucose already mentioned above142 and rhamnosyl(1]2)glucosyl(1]2)glucose, which occurs attached to the 7-hydroxy of acacetin in Peganum harmala125 together with the corresponding new linear tetrasaccharide, rhamnosyl(1]2)glucosyl(1]2)glucosyl(1]2) glucose.127 Two new branched trisaccharides have been characterised: rhamnosyl(1]2)[glucosyl(1]4)]glucose from Allium neapolitanum163 at the 3-position of kaempferol and glucosyl (1]4)[glucosyl(1]2)]galactose from Thevetia peruviana (Apocynaceae), where it occurs in mono- and di-acylated form with sinapic acid in combination with kaempferol at the 3-hydroxy group.174 Two new flavonoid sulfates have been reported recently.One is gossypetin 7,8-dimethyl ether 3,3*-disulfate from flowers of Erica cinerea.219 The other is isorhamnetin 3-(4+-sulfatorutinoside) from Zygophyllum dumosum.210 There are 50 new acylated derivatives, 13 flavones and 37 flavonols. These include one new acylating acid, n-butanoic acid, which was determined at the 6+-position of quercetin 3-glucuronide in leaves of Parthenocissus triscuspidata.197 Four further papers describe another unusual acylating acid, 3-hydroxy-3-methylglutaric acid.One of the four reports is of a C-methylflavone derivative: 5,7-dihydroxy-6,8-di-C-methyl- flavone 7-[6+-(3+*-hydroxy-3+*-methylglutaryl)]glucoside from rhizomes of Matteuccia orientalis.68 There are five new isoprenylated flavonol glycosides two of which are complex with three sugars and three acyl groups: anhydroicaritin 3-[2+*,6+*-diacetylglucosyl(1]3)-4+-acetylrhamnoside]- 7-glucoside225 and the corresponding 4+*,6+*- diacetylglucosyl isomer from Epimedium koreanum.225 Recently, three glycosides of 8-prenylkaempferol (noranhydroicaritin) thought to contain a (1]4) sugar linkage have been reassigned as having a (1]2) linkage.230 Thus, baohuoside III, the 3-rhamnosyl(1]4)rhamnoside from Epimedium davidii231 has been corrected to the 3-rhamnosyl(1]2)rhamnoside, which is identical to 2+- rhamnosylikarisoside A from E.koreanum.232 Similarly, baohuoside V [the 3-rhamnoside(1]4)rhamnoside-7-glucoside] from E.davidii231 has been reassigned as the corresponding (1]2) linked glycoside, diphylloside B already known from E. diphyllum233 and baohuoside VI [anhydroicaritin 3- rhamnosyl(1]4)rhamnoside] from E. davidii231 and E. pubescens234 as epimedin C previously identified in E. sagittatum235 and E. koreanum.236 The most complex flavone glycoside reported to date is stachysetin 39 from Stachys aegyptiaca.156 It is composed of O O HO O OH O CH 2 HO HO HO O C O C O O CH2 O O HO O OH O HO HO HO OH OH 39 Harborne and Williams: Anthocyanins and other flavonoids 643two molecules of apigenin 7-glucoside linked at the 6+ of the two glucose molecules to truxillic acid, a dimeric phenylpropanoid made up of two molecules of p-coumaric acid.This compound is of biosynthetic interest in that there are two possibilities for its formation: i.e. whether the truxillic acid is formed first and then combines with two molecules of apigenin 7-glucoside or whether two preformed molecules of apigenin 7-(6+-p-coumarylglucoside) condense together.Both acylation and isoprenylation can convert what are usually water-soluble flavonoid glycosides into lipophilic substances. A number of such structures listed here in Tables 4 and 5 may well have some lipophilic properties. This is apparently true of a 7-methylherbacetin 3-(2-ferulylglucoside) isolated from the pollen of Ranunculus sardons. Preliminary screening by HPLC retention time suggested that it was a flavone aglycone.189 One way of converting lipophilic aglycones into water soluble glycosides is by feeding them into plant or microbial cultures.For example, when quercetin is fed to cell cultures of a Vitis hybrid, it was converted to the 3,7,4*-triglucoside.238 More recently, the methylated flavone, 5,3*-dihydroxy- 7,2*,4*,5*-tetramethoxyflavone, was converted by microbial cultures of Cunninghamella elegans into the expected 3*-glucoside.239 The raison d’être for the proliferation of flavone and flavonol glycosides in nature (see e.g.Tables 4 and 5) continues to intrigue plant scientists. Some progress has been made recently in explaining the presence of these compounds or related phenolics in the epidermis of leaves and in plant pollens. The ability of UV-B radiation to damage DNA, RNA and proteins as well as to impair processes like photosynthesis is well established. The leaf epidermis plays a major role in attenuating this radiation and the presence of flavonoids and other phenolics in the vacuole of such epidermal cells provides a means of absorbing the damaging radiation.A clear example of this has now been demonstrated in the case of the needles of Scots pine, Pinus sylvestris. Two main compounds, isoquercitrin 3+,6+-di-p-coumarate and the same kaempferol 3-glucoside derivative, have been found to be induced in seedlings under simulated global radiation. The concentration of the acylated kaempferol 3-glucoside reaches 2.4 µmol g"1 fresh wt.240 Interestingly, two other compounds, catechin and kaempferol 3-glucoside, are also present in needle extracts but neither of these substances increases in concentration under UV treatment. The possibility that increases in flavonoid concentrations on the leaf surface can also contribute to UV-B protection has been put forward for two Gnaphalium species, members of the Compositae.In G. vira-vira, two O-methylated flavones, araneol and 7-methylaraneol, increase in concentration under UV-B radiation,241 while in G.luteo-album two flavonols, calycopterin and 3*-methoxycalycopterin, showed significant increases in concentration. By contrast, a third surface flavonol in G. luteo-album actually decreased in amount.242 In the case of plant pollens, it has long been recognised that flavonol glycosides are widely present and apparently contribute to the yellow colour in the pollen. What is striking is that the two glycosides most frequently encountered are the 3-sophorosides of kaempferol and quercetin and furthermore, if these are not present, closely related 2+-O-glycosides of a flavonol 3-glucoside are normally encountered.Further examples are two methylated herbacetin 3-sophorosides reported in Ranunculus, Raphanus and Klea pollens.188 The function of flavonoids in pollen is still uncertain in most plants, but in the case of Petunia hybrida, there is rather good evidence that it Table 6 New chalcones reported from 1995–1997 Chalcone Source Reference HYDROXYCHALCONES Leridal chalcone 40 Petiveria alliacea aerial parts (Phytolaccaceae) 244 2*,4*,4-Trihydroxy-3-prenylchalcone Glycyrrhiza glabra roots (Leguminosae) 245 2*,4*,2,4-Tetrahydroxy-3*-prenylchalcone Maclura pomifera cultures (Moraceae) 246 2*,4*,6*,4-Tetrahydroxy-3*-C-geranylchalcone Humulus lupulus resin (Cannabinaceae) 247 5*-Prenylxanthohumol 41 2*,3*,4*,4-Tetrahydroxychalcone 4*-(6+-p-coumarylglucoside) Bidens leucantha leaves (Compositae) 203 2*,3*,4*,3,4-Pentahydroxychalcone 4*-(4+,6+-diacetylglucoside) Bidens pilosa aerial parts (Compositae) 213 Gemichalcone A 42 Hypericum geminiflorum heartwood and root (Guttiferae) 248 Gemichalcone B 43 2*,4*,4-Trihydroxy-3*-methoxychalcone Helianthus annuus leaves (Compositae) 249 PYRANO- AND FURANOCHALCONES Glyinflanin G 44 Glycyrrhiza inflata roots (Leguminosae) 250 Glychalcone A 45 Glycosmis citrifolia leaves (Rutaceae) 251 Glychalcone B 46 Pyranochalcone 47 Neoraputia magnifica stems (Rutaceae) 252 Pyranochalcone 48 Humulus lupulus resin (Cannabinaceae) 247 Pyranochalcone 49 Lonchocarpus subglaucescens roots (Leguminosae) 253 Furanochalcone 50 Anthyllisone 51 Anthyllis hermanniae aerial parts (Leguminosae) 254 Anthyllin 52 OH OH HO O HO OHC MeO Me OH O Ph OH OH HO O MeO RO 40 41 42 R = ferulyl 43 R = p-coumaryl 644 Natural Product Reports, 1998has a critical role in subsequent pollen tube growth, once pollination has occurred.Structure–activity relationships have been explored in the case of Petunia, and a kaempferol diglycoside does appear to be the most active constituent.243 4 Chalcones and aurones Nineteen miscellaneous chalcones have been isolated as new structures in the period under review (Table 6).244–254 Half the compounds were obtained from either the Compositae or the Leguminosae, two families well known to accumulate these flavonoid types.Chalcones have been less frequently encountered in the other five families listed under sources in Table 6. Leridal chalcone 40, the first compound listed, is a rare example of a chalcone with an aldehyde function. The prenylated chalcone 41 and the pyranochalcone 48 were expectably found in hop resin, a known rich source of isoprenylated polyphenols. A mixture of nine related flavonoid structures, including the three listed here, occur together in all nine hop varieties tested.They are all present in similar amounts, so that they cannot be utilised as chemotaxonomic markers.247 Isoprenylated chalcones 42 and 43 from Hypericum geminiflorum are distinctively novel in having the aromatic acyl groups, ferulic or p-coumaric acid, attached through the O-prenyl substituents. The p-coumarate ester 43 occurs in both the (Z)- and (E)-configurations, whereas 42 is only present as the (Z)-form.248 Nine pyrano or furano substituted chalcones, compounds 44–52 have been encountered recently in various plant tissues (Table 6).The chalcones 49 and 50 are only two of 23 related flavonoid structures obtained from roots of Lonchocarpus subglaucescens; other classes present are flavones, flavonones and isoflavonoids. Notable features of the two chalcones and the other structures are pyrano- or furano-rings, as well as methylenedioxy substituents.253 Aurones are biogenetically related to chalcones and are conveniently listed with them.There appear to be only two reports describing five new structures. Thus, the rhizomes of Cyperus capitatus (Cyperaceae) have yielded 4,6,3*,4*- tetramethoxyaurone, together with the 5-C-methyl derivatives of 6,3*,4*-trihydroxy-4-methoxy- and 6,3*-dihydroxy-4,4*- dimethoxyaurone 53.255 The other report refers to aerial parts of Bidens pilosa, which have given a new chalcone (Table 6) as well as two acetylated glucosides of 6,7,3*,4*- tetrahydroxyaurone, the 6-(2+,4+,6+-triacetylglucoside) and the 6-(3+,4+,6+-triacetyl glucoside).213 Both chalcone and aurone structures are known to exhibit significant biological activity and some further examples have been reported since the last review.The known pedicin (2*,5*-dihydroxy-3*,4*,6*-trimethoxychalcone) inhibits tubulin assembly into microtubules and thus has anticancer activity with an IC30 value of 300 µM.256 A number of known chalcones isolated from Myrica serrata (Myricaceae), were tested for antifungal and antibacterial activity.A Cmethylchalcone, aurentiacin A, was inhibitory, whereas related dihydrochalcones were inactive.257 Interestingly, the Cmethylaurone, 6,3*-dihydroxy-4*-methylaurone, a synthetic compound, proved to show strong antiviral activity in experiments using tomato ring spot virus. Quercetin and twelve other flavonols showed similar antiviral activity.258 Finally, inhibition of platelet aggregation is a characteristic of natural phenolics, and isobavachalcone (2*,4*,4-trihydroxy-3*- prenyl) has been found to have this property in rabbit blood.259 5 Flavanones New flavanones are listed in Table 7.260–287 They comprise 15 simple hydroxy (or methoxy) derivatives, 25 prenylated OH O O O OH OH OMe O O R OH OMe MeO O OMe OH OH O O OH OMe O O MeO OMe O MeO OMe OH O OH O OH O O O O HO OH OH HO O OMe MeO 44 45 R = H 46 R = OMe 47 48 49 50 51 52 O HO Me MeO O CH OH OMe 53 Harborne and Williams: Anthocyanins and other flavonoids 645Table 7 New flavanones reported between 1995–1997 Flavanonea Source Reference HYDROXYFLAVANONES 6,7,8-Trihydroxy-5-methoxyflavanone Isodon oresbius whole plant (Labiatae) 260 5,7-Dihydroxy-8,4*-dimethoxyflavanone Chromolaena subscandens leaves (Compositae) 261 7,8-Dihydroxy-6,4*-dimethoxyflavanone Tecoma stans flowers (Bignoniaceae) 262 5,2*,3*-Trihydroxy-7-methoxyflavanone Iris tenuifolia underground parts (Iridaceae) 263 5,3*-Dihydroxy-7,2*-dimethoxyflavanone 5,2*,5*-Trihydroxy-6,7-dimethoxyflavanone Dioclea grandiflora roots (Leguminosae) 264 5,6,7,3*,4*,5*-Hexamethoxyflavanone Neoraputia magnifica stems (Rutaceae) 252 5,2*-Dihydroxy-6,7,6*-trimethoxyflavanone Scutellaria comosa roots (Labiatae) 265 5,7-Dihydroxy-6,8-di-C-methyl-3*-methoxyflavanone Matteuccia orientalis whole plant (Aspleniaceae) 266 5,7-Dihydroxy-8-C-methyl-4*-methoxyflavanone Amaranthus caudatus flowers (Amaranthaceae) 267 5,2*,3*-Trihydroxy-6,7-methylenedioxyflavanone Iris tenuifolia underground parts (Iridaceae) 263 5,2*-Dihydroxy-6,7-methylenedioxyflavanone 2,5-Dihydroxy-7-methoxy-8-C-methylflavanone Friesodielsia enghiana stem bark (Annonaceae) 268 2,5-Dihydroxy-7-methoxy-6-C-methylflavanone 5,7-Dihydroxy-8-C-methylflavanone 6-aldehyde Petiveria alliacea aerial parts (Phytolaccaceae) 244 ISOPRENYLATED FLAVANONES 8-Geranyl-7,4*-dihydroxyflavanone Sophora prostrata roots (Leguminosae) 269 7,4*-Dihydroxy-6,8,3*-triprenylflavanone 4*-Hydroxy-7-methoxy-8-prenylflavanone Mundulea suberosa stem bark (Leguminosae) 270 5,4*-Dihydroxy-2-methoxy-8-prenylflavanone 3*-Geranyl-5,7,4*-trihydroxyflavanone Macaranga pleiostemona leaves (Euphorbiaceae) 271 7,4*-Dihydroxy-8-lavandulyl-2*-methoxyflavanone Sophora alopecuroides roots (Leguminosae) 272 8-(2-Hydroxy-3-methylbut-3-enyl)naringenin Sophora tomentosa roots (Leguminosae) 108 6-(1,1-dimethylallyl)naringenin Monotes engleri leaves (Dipterocarpaceae) 273 6-(1,1-dimethylallyl)eriodictyol 6-(1,1-dimethylallyl)eriodictyol 3*-methyl ether 5,7,3*,4*-Tetrahydroxy-6-(‚-hydroxyethyl)-8-prenylflavanone Derris laxiflora leaves and twigs (Leguminosae) 274 6-Geranyl-5,7,2*,4*,6*-pentahydroxy-8-prenylflavanone Sophora tomentosa roots and stems (Leguminosae) 108 5,4*-Dihydroxy-2+,2+-dimethylpyrano[5+,6+:7,8]flavanone Maclura pomifera cell cultures (Moraceae) 275 5,7-Dihydroxy-8-prenyl[6+*,6+*-dimethylpyrano[2+*,3+*:4*,3*]flavanone Euchresta formosana roots (Leguminosae) 276 5,7-Dihydroxy-6-C-methyl-8-prenylflavanone Dalea caerulea (Leguminosae) 277 Glyflavanone A 55 Glycosmis citrifolia leaves (Rutaceae) 251 Glyflavanone B 56 Abyssinin I 54 Erythrina abyssinica stem bark (Leguminosae) 278 5,7,4*-Trihydroxy-5*-methoxy-3*-prenylflavanone Erythrina abyssinica stem bark (Leguminosae) 278 5,7,4*,5*-Tetrahydroxy-2*,3*-diprenylflavanone 2+*,3+*-Epoxylupinifolin 57 Derris reticulata stems (Leguminosae) 279 Dereticulatin 58 4+,5+-Dihydro-5-methoxy-5+-isoprenylfurano [2+,3+:7,8]flavanone Tephrosia emoroides roots (Leguminosae) 280 6-Methoxy-6+,6+-dimethylpyrano[2+,3+:7,8]flavanone Lonchocarpus subglaucescens roots (Leguminosae) 253 5,6-Dimethoxy-[2+,3+:7,8]furanoflavanone GLYCOSIDES 7-Hydroxyflavanone 7-glucoside Clerodendron phlomides leaves (Verbenaceae) 281 5,7,2*-Trihydroxyflavanone 7-glucoside Scutellaria ramosissima aerial parts (Labiatae) 282 Naringenin 4*-rhamnoside Crotalaria striata stems (Leguminosae) 283 Naringenin 7-[3+-acetyl-6+-(E)-p-coumarylglucoside] Blepharis ciliaris aerial parts (Acanthaceae) 284 5,7,8,4*-Tetrahydroxyflavanone 8-glucoside Calluna vulgaris flowers (Ericaceae) 285 7,8,3*,4*-Tetrahydroxyflavanone 7-(2+,4+,6+-triacetylglucoside) Bidens pilosa aerial parts (Compositae) 213 5,7-Dihydroxy-6-C-methylflavanone 7-xylosyl(1]3)-xyloside Mosla chinensis (Labiatae) 286 5,7-Dihydroxy-6,8-di-C-methylflavanone 7-[6+-(3+*-hydroxy-3+*-methylglutaryl)glucoside] Matteuccia orientalis rhizomes (Aspleniaceae) 287 5,7-Dihydroxy-6,8-di-C-methyl-4*-methoxyflavanone-7-[6+-(3+*-hydroxy-3+*- methylglutaryl)glucoside] aNaringenin=5,7,4*-trihydroxyflavanone; eriodictyol=5,7,3*,4*-tetrahydroxyflavanone. 646 Natural Product Reports, 1998compounds and nine glycosides. Flavanones have an asymmetric carbon at C-2 and can be obtained in optically active form. In most cases where the optical rotation has been determined, they have the (2S)-configuration. In describing the prenylated derivatives, the abbreviation prenyl is used for 3,3-dimethylallyl. Among the 15 simple flavanones (Table 7), it is surprising that the 5-methoxy-6,7,8-trihydroxy structure has not been encountered before.This occurs in Isodon oresbius, a member of the Labiatae family, and has been named oresbiusin after the species name.260 Of the other structures, the most notable are the four flavanones from Iris tenuifolia, which have the rare 2*,3*-dihydroxylation pattern in the B-ring.263 Another rarity in the flavanone series is a 2-hydroxy group and yet two such structures have been encountered in Friesodielsia enghiana.They readily undergo dehydration at the 2,3 position and expectedly, they co-occur in the above plant with the related flavones.268 In the previous report43 for the years 1992–1994, no less than 23 new prenylated flavanones were listed. This number has dropped oV slightly, since there are about 15 prenylated structures and 10 flavanones with pyrano- or furanosubstituents in Table 7.The latter class are illustrated here with the structures 54–58. Most of the prenyl derivatives have been found in the Leguminosae, a family represented here by the genera Dalea, Derris, Erythrina, Euchresta, Lonchocarpus, Sophora and Tephrosia. This family continues to be a rich source of such flavonoids. The Dipterocarpaceae, a large family of Malaysian trees, is very diVerent botanically from the Leguminosae. It is interesting that the three prenylated flavanones now reported in Monotes (Dipterocarpaceae) have a distinctive isoprenyl substitution, where the most common 3,3-dimethylallyl (prenyl) side chain is replaced by 1,1-dimethylallyl.273 Laxiflavin, a flavanone with the rare ‚-hydroxyethyl substituent at C-6, occurs in Derris laxiflora.274 It inhibits proteintyrosine kinase activity and hence is a potential new anticancer drug.Cytotoxicity is exhibited by many prenylated flavanones 54 57 58 55 R = H 56 R = OMe O O HO HO O OMe O R OMe O MeO O O OH O O O OH O O OH O Table 8 New flavanonols reported in the period 1995–1997 Flavanonol Source Reference 3,7,4*-Trihydroxyflavanone Tecoma stans flowers (Bignoniaceae) 289 3-Acetoxy-5,7-dihydroxy-4*-methoxyflavanone Aframomum hanburyi (Zingiberaceae) 290 3,5,2*,3*-Tetrahydroxy-7-methoxyflavanone Iris tenuifolia underground parts (Iridaceae) 263 3,5,3*-Trihydroxy-7,2*-dimethoxyflavanone 3,5,7,4*-Tetrahydroxy-3*,5*-dimethoxyflavanone Tecoma stans flowers (Bignoniaceae) 289 3,5,7-Trihydroxy-6-C-methyl-4*-methoxyflavanone Amaranthus caudatus flowers (Amaranthaceae) 291 3,5,3*,4*-Tetrahydroxy-7-(2,3-en-3-methylbutyloxy)flavanone Pterocaulon alopecuroides aerial parts (Compositae) 292 3,5,7-Trihydroxy-6-prenyl-8-methoxyflavanone Dioclea grandiflora root bark (Leguminosae) 293 (2R,3R)-3,5,7,2*,4*-Pentahydroxy-6-(3-hydroxy-3-methylbutyl)-8-lavandulylflavanone (Kosamol A) 59 Sophora flavescens roots (Leguminosae) 294 3,5,7,4*-Tetrahydroxyflavanone (Aromadendrin) 4*-glucosyl(1]6)xyloside Dalbergia latifolia arterial parts (Leguminosae) 295 3,5,7,8,4*-Pentahydroxyflavanone (Callunin) 8-(2+-acetylglucoside) Calluna vulgaris flowers (Ericaceae) 285 3,5,7,4*-Tetrahydroxy-6-p-hydroxybenzylflavanone Cudrania tricuspidata stem bark (Moraceae) 296 3,5,7,4*-Tetrahydroxy-6,8-di-p-hydroxybenzylflavanone (2R,3R)-3,5,7,3*,4*-Pentahydroxy-6*-hydroxymethylflavanone Trifolium alexandrium seeds (Leguminosae) 297 Dipyranoflavanone 60 Mundulea suberosa leaves (Leguminosae) 298 Sanggenol F 61 Morus cathayana root bark (Moraceae) 299 Sanggenol G 62 Sanggenol H 63 Sanggenol I 64 Sanggenol K 65 2-Prenylflavanone 66 Sorocea ilicifolia roots (Moraceae) 300 2-Prenylflavanone 67 2-Prenylflavanone 68 Harborne and Williams: Anthocyanins and other flavonoids 647and has been demonstrated for the flavanones of Monotes273 and Derris reticulata.279 Trypanocidal activity has been found in the simple 5,4*-dihydroxy-7-methoxyflavanone. This flavanone at a concentration of 500 µg ml"1 gives a 100% kill rate on the protozoan parasite responsible for Chagas’ disease.288 This compares with gentian violet, a drug in present use, which produces a 50% kill at 31 µg ml"1.The number of new flavanonols (or dihydroflavonols) discovered during 1995 to 1997 is 23, with fewer structures than in the flavanone series. These are gathered together in Table 8.289–300 Most of the substitution patterns are as expected, with O-methylation, C-methylation, prenylation and glycosylation all being represented.As with the flavanones, a major source of new structures is the family Leguminosae. Sophora flavescens, for example, elaborates kosanol A 59, a compound with a C10 lavandulyl substituent at C-8 and a hydrated C5 prenyl unit at C-6.294 Again, Mundulea suberosa produces a novel flavanonol 60 with two pyran units fused to the A-ring.298 The most structurally exciting new flavanonols are from the family Moraceae,299,300 where a series of complex structures 61–68 have been uncovered.Most of them have an ether linkage between the 2*- and 3-positions and seven of the eight have an isoprenoid group at the 2-position. Sanggenol H 63 must be unique among the known flavonoids in having a C15 farnesyl sidechain attached at this position. A noticeable feature of the new structures reported recently for flavanones as well as for chalcones (Table 6) is the relatively small number of new O-glycosides described.This may reflect some emphasis among phytochemists for examining the lipophilic rather than the water soluble fractions of plant extracts. 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ISSN:0265-0568    

DOI:10.1039/a815631y

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

The biosynthesis of steroids and triterpenoids GeoVrey D. Brown Chemistry Department, The University of Hong Kong, Pokfulam Rd, Hong Kong Covering: January 1990 to December 1996 Previous review: 1990, 7, 459 1. 2. Introduction Eukaryotes 2.1 Biosynthesis of isopentenyl pyrophosphate from acetyl-Coenzyme A 2.2 Biosynthesis of squalene epoxide from isopentenyl pyrophosphate 2.3 Conversion of squalene epoxide into lanosterol in animals and fungi 2.3.1 Conversion of lanosterol into cholesterol in animals 2.4 2.3.2 Biosynthesis of steroidal hormones in vertebrates 2.3.3 Biosynthesis of vitamin D and bile acids 2.3.4 Conversion of lanosterol into ergosterol in fungi Conversion of squalene epoxide into cycloartenol or ·- and ‚-amyrins in higher plants 2.4.1 Biosynthesis of sitosterol and campesterol 2.4.2 Biosynthesis of plant steroids and triterpenes as secondary metabolites Biosynthesis of steroids in insects Biosynthesis of steroids in marine organisms Prokaryotes Biosynthesis of isopentenyl pyrophosphate in eubacteria Biosynthesis of hopanoid triterpenes from isopentenyl pyrophosphate in eubacteria Biosynthesis of core isoprenoid lipids in archaebacteria Evolutionary considerations References 2.5 2.6 3.3.1 3.2 3.3 4. 5. 1 Introduction The papers discussed in this review were selected mainly from perusal of Biological Abstracts on CD-ROM and of the major organic and bio-organic chemistry journals for the appropriate period.The sheer volume of publications in what could be loosely construed as the field constituting ‘biosynthesis of steroids’ has dictated that several reports have, of necessity, been excluded from this review; in particular, many publications from the extensive ‘medical’ literature are not cited. Those references which are cited are intended to be of interest to the biosynthetic chemist and deal, in the main, with delineation of metabolic pathways and the mechanistic chemistry of the transformations involved in steroid and triterpenoid biosynthesis (as revealed by isotopic labelling studies) as well as studies of the enzymes involved in eVecting these transformations (including enzyme inhibition).References dealing exclusively with the regulation or expression of enzymes involved in steroid biosynthesis and/or their localisation to a particular tissue or sub-cellular compartment are generally not included; similarly, clinical studies related exclusively to disease conditions or hereditary abnormalities resulting from specific deficiencies in steroid metabolism are generally not covered.The field of steroid and triterpenoid biosynthesis, as interpreted above, has undergone a revolution since the previous review, owing to the wide availability of new chemical, biochemical and biological tools. The application of modern techniques in purification and structure elucidation (e.g. 2D NMR spectroscopy) by chemists has allowed new insights into steroid transformations at the molecular level, whilst the now Brown: The biosythesis of steroids and triterpenoids routine ability of molecular biologists to clone and sequence DNA which codes for the enzymes involved in steroid biosynthesis has already been of great value in providing large quantities of pure enzyme for study, and will surely become even more important in the future (such references are also included for this reason).Traditionally, most studies of steroid metabolism have been made with mammals and other eukaryotes. However, recent evidence has emerged that these biosynthetic pathways to steroids are not universal to all organisms: in particular, there is strong reason now to believe that an alternative pathway to isopentenyl pyrophosphate exists in some prokaryotes (and perhaps in the chloroplasts, of presumed prokaryotic origin, found in higher plants).This important new finding is reflected in the structure of this review, which diVers from that of its predecessors in making a major demarcation between prokaryotes and eukaryotes. In addition, the author has chosen (perhaps controversially) to extend this phylogenetic theme to include the fifth kingdom of archaebacteria. Although no steroids or triterpenes as such are reported from these organisms, archaebacterial membranes are composed of core lipids which are apparently of isoprenoid origin.The broad picture of biosynthesis in the five kingdoms is summarised in Scheme 1. EUBACTERIA Glyceraldehyde 3-phosphate OPP OPP n IPP n = 2,3ARCHAEBACTERIA Acetyl-CoA n = 1 Archaebacterial core lipids EUKARYOTES, ARCHAEBACTERIA Hopanoids Squalene EUBACTERIA Cholesterol Squalene epoxide Sitosterol EUKARYOTES ANIMALS 2 Eukaryotes Ergosterol FUNGI Scheme 1 Comparative biosynthesis of isoprenoid structural components of membranes in the five kingdoms 2.1 Biosynthesis of isopentenyl pyrophosphate from acetyl-Coenzyme A In mammals and yeast, the first step in the synthesis of mevalonic acid (MVA; 4) from acetyl-Coenyzme A HIGHER PLANTS653(acetyl-CoA; 1) is the Claisen-type condensation of two molecules of 1 to form acetoacetyl-Coenzyme A (acac-CoA; 2) mediated by the enzyme acetoacetyl-CoA thiolase (AACT; EC 2.3.1.9).This is followed by incorporation of a second molecule of 1 in an aldol-type process to produce (S)-3- hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA; 3), under the control of HMG-CoA synthase (HMGS; EC 4.1.3.5) (Scheme 2). Cytosolic AACT associated with sterol biosynthesis has been studied from yeast1 and the human cytostolic thiolase has been assigned to chromosome 6q25.3-q26.2 OH O O O ii i SCoA SCoA O HO2C OH HO2C HO2C HO2C OP OH 4 6 5 OH vi vii OPP 7 8 OH viii 9 ix 10x OPP 3 11 + O SCoA 1 2 3iii v iv 22 OPP 23 OPP22 O 3O 12 Scheme 2 Biosynthesis of squalene epoxide in eukaryotes; Enzymes: i, AACT; ii, HMGS; iii, HMGR; iv, MVAK; v, MVAPK; vi, MPD; vii, IPP isomerase; viii, FPS; ix, SQS; x, SE. P=phosphate Investigations of the higher plant Catharanthus roseus showed that both thiolase and synthase activities co-purified3,4 and it was also proposed that in radish seedlings5 a single monomeric protein, referred to as AACT/HMGS, was responsible for both catalytic activities.6,7 The AACT gene from radish was later cloned8 and the HMGS protein obtained from cDNA of Arabi- 654 Natural Product Reports, 1998 dopsis thaliana was shown to be devoid of AACT activity.9 Analogues of acetyl-CoA have been synthesised for studies of a variety of biosynthetic pathways, including the terpenoid pathway, which utilise this precursor.10,11 Syntheses and activities of a number of HMGS inhibitors have been described, including the known natural product ‚-lactone 1233A (13)12 (an irreversible inhibitor also referred to as L-659,699),13,14 its structural analogues15–21 and the new hypolipidemic compound lifibrol (K12.148).22,23 A fungalderived „-lactone incorporating a long alkyl chain is also CO2H HO O O 13 HO HO CO2Na OH O O H O H R2 R1 O 17 OO 14 R1 = H; R2 = O O 15 R1 = Me; R2 = O O 16 R1 = Me; R2 = PO3H2 n R1 OH HO 1 186 R2 2C O OH HOHO2C O 19 R1 = ; R O 2 = O CO2HOAc O R2 = PO3H2 OAc 20 R1 = ; O OH O O 21 R1 = ; R2 = OAc O 22 R1 = ; R O 2 = N O S S 23 Inhibitors of the early steps of steroid biosynthesisreported as an inhibitor of HMGS.24 Labelling experiments have proven that the inhibitory eVect of 1233A is specific to HMGS25 and it is proposed that Cys-129 of the recombinant human enzyme is covalently modified by the ‚-lactone group of this inhibitor.13 Avian HMGS cDNA has been sequenced26,27 and it has been shown that His-264 is important in the active site of this enzyme28 as well as cysteine residues previously implicated in the mechanism (in particular Cys-129).29 The cytoplasmic and mitochondrial HMGS genes in mice (Hmgcs1 and Hmgcs2) have been mapped to chromosomes 13 and 3 respectively30 and both human cytostolic HMGS31 and mitochondrial HMGS genes32 have been sequenced for the first time (the mitochondrial HMGS gene from rats has also been isolated).33 Cytosolic HMGS is believed to be associated with cholesterol biosynthesis in mammals, whereas mitochondrial HMGS is concerned with ketogenesis.The sequence of the HMGS gene (hcs) from the fission yeast Schizosaccharomyces pombe has been shown to have a high degree of homology with that from mammalian sources.34 It has been suggested that HMGS from Hevea brasiliensis regulates the synthesis of rubber in latex35 and two HMGS genes from Blatella germanica (which is also not involved in sterol biosynthesis) have also been cloned.36,37 The next step in the biosynthesis of isopentenyl pyrophosphate involves reduction of HMG-CoA by (S)-3-hydroxy-3- methylglutaryl-CoA reductase (HMGR; EC 1.1.1.34).Unlike the preceding transformations, this reaction is irreversible under physiological conditions and thereby constitutes a key committed step in the biosynthesis of steroids in eukaryotes.In consequence, a very large number of syntheses continue to be reported of potential novel inhibitors of HMGR (and of building blocks employed in their synthesis)38–83 with a view to obtaining new drugs capable of lowering plasma and low density lipoprotein cholesterol levels in man via regulation of its biosynthesis. Most of these compounds are structurally closely related to the established fungal-derived inhibitors mevastatin (compactin, ML-236B; 14), lovastatin (mevinolin, monacolin K; 15) and simvastatin (16),84–87 which incorporate a ‚-hydroxy-‰-lactone functionality, or to pravastatin (17),88,89 which contains the ring-opened equivalent (development of these inhibitors has been reviewed by Endo).90 Molecular modelling approaches to the rational design of such HMGR inhibitors have been described.91,92 Enantiomerically pure (R)- HMG-CoA has been obtained by biotransformation and shown to be a competitive inhibitor of the (S)-enantiomer (which is the natural substrate).93 An alternative strategy to that of enzyme inhibition, is the design of seco-oxysterol inhibitors which operate at the level of transcription of HMGR94,95 (25-hydroxyvitamin D3 analogues have also been shown to be eVective suppressors of HMGR).This strategy is based on the observation that several naturally occurring oxysterols (i.e. sterols bearing a second oxygen function in addition to that at C-3) are reported to regulate HMGR production in mammals by a feedback mechanism involving an oxysterol binding protein96–99 (this topic has been reviewed).100 25-Hydroxycholesterol is established as one such potent inhibitor but there is conflicting evidence as to the eYcacy of 27-hydroxysterols.101,102 A report that the most important suppressor of HMGR in vivo is cholesterol itself (in mice at least) also contradicts current wisdom: the authors claim that the ƒ5-bond is the important structural feature for such activity whilst oxygenation at sites other than the 3-position is unimportant.103 Several attempts have been made to identify or synthesize analogues of cholesterol, lanosterol and bile acids which may function as inhibitors of HMGR:104–113 various oxylanosterols appear to regulate enzyme expression posttranscriptionally,114–117 a mechanism of action previously ascribed only to non-steroidal molecules.The 4,4-dimethyl steroids are stated to be more potent inhibitors of HMGR than their 4,4-bis-nor-methyl counterparts.118 It has been proposed that HMGR is also involved in the regulation of biosynthesis of phytosterols in higher plants,119 which normally contain a Brown: The biosythesis of steroids and triterpenoids small family of isogenes for this enzyme, diVerentially expressed in regard to location and time and probably specific to diVerent sub-cellular isoprenoid pathways120–122 (in contrast to vertebrates which contain only one HMGR gene).The situation is complex and opinions remain divided as to the importance of HMGR as the regulatory step in steroid biosynthesis in higher plants;123–125 the topic has been reviewed.126 HMGR from the prokaryote Pseudomanas mevalonii (EC 1.1.1.88) has been shown to be a catalytic analogue of the mammalian enzyme although it diVers in being a soluble protein lacking a membrane anchor domain, and normally functions in a catabolic sense [hence a requirement for NAD(H) co-factor rather than NADP(H) as utilised by the eukaryotic enzyme]. However, the similarities to mammalian HMGR, together with the availability of large quantities of this enzyme have made it an attractive subject for crystallographic studies.The crystal structure of HMGR from P. mevalonii127,128 shows a dimer with an unusual binding-fold for HMG-CoA at the interface of the two sub-units.129 His-381 has been demonstrated to perform an important function as a general base in the catalytic domain130 and Glu-83 is judged to be the acidic residue functional in catalytic formation of the mevaldate thiohemiacetal intermediate 24.131 HMGR from Syrian hamster was also found to be a homodimer with its active site located at the sub-unit interface.132 Initial results confirmed that His-865 (which corresponds to His-381 of P.mevalonii) was catalytically important in this species;133 later studies with mutant enzyme demonstrated that both Glu-558, and Asp-766 were also involved in the catalytic activity and a revised mechanism for catalysis by mammalian HMGR has been proposed based on these findings, (Scheme 3) which diVers slightly to that proposed previously for P.mevalonii.134 O O O H H Asp-766 O Asp-766 O– Asp-766 O H O O O HO HO HO SCoA H SCoA CO2H CO2H CO2H 24 3 NADP(H) NADP(H) HN OH HO N O His-865 H H O Glu-558 CO2H H 655 4 Scheme 3 Proposed catalytic mechanism for mammalian HMGR Phosphorylation of Ser-871135 impairs the catalytic function of nearby His-865,136,137 perhaps as a result of electrostatic interaction with the introduced phosphate group.138 This finding is of some interest because in vivo regulation of mammalian HMGR activity is known to occur via reversible phosphorylation of this enzyme which is catalysed by an AMP-activated kinase (AMPK).139 In higher plants HMGR activity has also been shown to be regulated by a protein kinase cascade140–142 in which plant HMG-CoA reductase kinase A recognises similar structural motifs as other members of the SNF1 protein kinase family.143,144 Inactivation of rat liver HMGR upon treatment with hydrogen peroxide is interpreted as evidence for an active thiol group in the catalytic domain of this enzyme.145 HMGR from pea plastids has been partially purified;146 the purified enzyme from the human parasite Schistosoma mansoni has been characterised as a single protein of molecular weight 66 000.147 cDNA for HMGR from radish148 encodes a polypeptide of 583 amino acids containing two membrane-spanning domains, which is apparently a common feature for plant HMGRs7 (by contrast, mammalian HMGRs contain eight transmembrane helices).149–151 A gene which encodes one of the three HMGRs from Hevea brasiliensis152,153 has been expressed in tobacco; data for this species supported the suggestion that HMGR is a key limiting enzyme in phytosterol biosynthesis in higher plants.154 Three HMGR genes (hmg1, hmg2 and hmg3) isolated from a potato tuber library155 were diVerentially expressed when challenged by fungal pathogens; it was suggested that hmg1 is involved in steroid biosynthesis, whilst hmg2/hmg3 are concerned with sesquiterpenoid– phytoalexin biosynthesis. Two HMGR genes are reported from Arabidopsis thaliana156 and four from tomato:157 single copies are known from Catharanthus roseus,158 Nicotiana sylvestris,159 rice160 and wheat.161 The gene encoding HMGR from the fungal pathogen Ustilago maydis has also been isolated and identified162 and HMGR from Syrian hamster has been expressed in Escherichia coli.163 The fission yeast Schizosaccharomyces pombe is the only known unicellular eukaryote to contain a single copy of HMGR (as observed for mammals).164 Sequences for several zygomycetes fungi have been compared.165 Several studies of HMGR genes not associated with steroid biosynthesis have been reported from species such as Blatella germanica,166 Phycomyces blakesleeanus and Gibberella fujikuroi.167 Although mevalonic acid (MVA; 4) is always biosynthesised as the (R)-isomer; a chemical synthesis of the unnatural (S)- isomer has been reported which may be of future interest for biosynthetic studies.168 The conversion of (R)-mevalonic acid into isopentenyl pyrophosphate (IPP; 7), the C5 building block required for biosynthesis of squalene, requires three consecutive phosphorylation reactions proceeding via (3R)- mevalonate-5-phosphate (5-phosphomevalonate; MVAP; 5) and (3R)-mevalonate-5-pyrophosphate (5-pyrophosphatemevalonate; MVAPP; 6), which are catalysed by the enzymes mevalonate kinase (mevalonate-5-phosphotransferase; MVAK; EC 2.7.1.36), mevalonate-5-phosphate kinase (phosphomevalonate kinase; MVAPK; EC 2.7.4.2) and mevalonate- 5-pyrophosphate decarboxylase (mevalonate pyrophosphate anhydrodecarboxylase; MPD; EC 4.1.1.33).Studies of all these enzymes have been reported from sea bass169 and an enzyme assay has been developed for all three.170 These enzymes (and those involved in subsequent formation of farnesyl pyrophosphate) are associated with the peroxisomes.171–175 Several substrate analogue inhibitors of MVAK have been designed and their eYciency as competitive inhibitors has been correlated with their ability to fit into the active site of the enzyme.176 The ERG12 gene from yeast which codes for MVAK has been sequenced and found to be identical with the yeast gene RAR1; the corresponding enzyme is predicted to contain 443 amino acids and have a molecular weight of 48 500 Da.177 Human MVAK has been mapped to chromosome 530 and both the human enzyme178 and that from Arabidopsis thaliana179 have been cloned. There is a consensus that mevalonate kinases from fungal, mammalian and plant sources all show strong homologies in sequence.The ERG8 gene from yeast has been cloned180 and a 424 amino acid sequence deduced for yeast MVAPK; the human cDNA which encodes MVAPK171 is predicted to be a 191 amino acid protein.MPD catalyses phosphorylation of MVAPP at the tertiary hydroxy group and the subsequent anti-elimination of carbon dioxide and inorganic phosphate which results in IPP. From studies of various inhibitors the transition state for the elimination step is suggested to have considerable carbocationic character and to be stabilised by the 3-methyl group in the substrate.181 In support of this proposal, a proline-containing transition state analogue has been shown to be a very potent inhibitor of this enzyme.182 Phenylalanine and some of its metabolites are inhibitors of MPD but not of the two preceding phosphorylating enzymes,183,184 as is the case for phenylacetate and phenylpyruvate.184,185 HMGR is also known to be inhib- 656 Natural Product Reports, 1998 ited by these compounds and a secondary regulatory role for MPD in cholesterol biosynthesis has been proposed.Rat liver MPD has been purified and shown to be a homodimer of 45 kDa,186 which agrees well with reports of enzyme structure from chicken and pig liver, but contradicts previous reports that the rat enzyme is a tetramer. The recombinant human enzyme is also a homodimer of 43 kDa subunits.187 Yeast strains deficient in the MPD gene (ERG19) have been isolated.188 2.2 Biosynthesis of squalene epoxide from isopentenyl pyrophosphate Isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IPP isomerase; EC 5.3.3.2) catalyses the reversible antarafacial 1,3-allylic rearrangement of the homoallyllic substrate IPP (7) to its allylic isomer, dimethylallyl pyrophosphate (DMAPP; 8).Electrophilic DMAPP then acts as a primer for subsequent prenyl transfer reactions in the steroid biosynthesis pathway. The catalytic mechanism for this enzyme is thought to involve protonation–deprotonation steps catalysed by active site acidic and basic residues located on either side of the substrate double bond.Cys-139 in yeast isopentenyl isomerase has been shown to be essential for catalytic activity189,190 and has also been demonstrated to form a thioether linkage with the inhibitor 3-methyl-3,4-epoxybutyl diphosphate.191 Glu-207 was subsequently identified as a second catalytically important group.192 Cloned IPP isomerase from Schizosaccharomyces pombe contains the same essential Cys and Glu catalytic residues193 previously identified from Saccharomyces cerevisiae192 (the IDI1 gene from S.cerevisiae has been mapped to chromosome XVI).194 The human gene has been sequenced and the enzyme product characterised;195 IPP isomerase from rubber latex has also been partially purified and characterised.196 The enzyme farnesyl pyrophosphate synthase (FPS; EC 2.5.1.10) catalyses the biosynthesis of farnesyl pyrophosphate (FPP; 9) by a ‘head-to-tail’ 1’,4-condensation of IPP (7) and DMAPP (8) to form the C10 compound geranyl pyrophosphate (GPP) which then serves as the allylic substrate for a second condensation with IPP to produce the C15 compound FPP (Scheme 4).FPS is localised in the peroxisomes172,173 as is the case for the preceding phosphorylating enzymes. Christensen and Poulter have performed enzymatic syntheses, using recombinant organisms, to generate FPP which is selectively labelled either in the first or third isoprene unit, or in all three, for future use in biosynthetic studies.197 Enzymic studies with bisubstrate analogues of IPP and DMAPP have provided the first direct evidence for a sequential condensation of IPP followed by elimination of hydrogen in the prenyl chain elongation process.198 Conformational analysis of such FPS inhibitors suggested that the hydrocarbon moieties of the two substrates may occupy a central volume in the enzyme active site flanked on top and bottom by the diphosphate residues.199 Diphosphate modified analogues of FPP have been studied as potential substrates of FPS,200 and results support a mechanism involving initial ionisation of the pyrophosphate group, prior to the condensation and elimination steps discussed above; the importance of the pyrophosphate group is suggested to lie in its ability to complex with divalent cations such as magnesium, thereby providing a good leaving group. Evidence from 13C NMR spectroscopy, supported by molecular orbital calculations, has provided the first direct evidence that a delocalised C10-allylic cation (cf.GPP) is generated during the biosynthesis of FPP.201 Various GPP analogues incorporating oxygen atoms in their alkyl chains serve as substrates for FPS.202 Formation of (E,E)-farnesol by FPS from Pisum sativum follows an anomalous reaction course in which the allylic residue adds to the re-re face rather than the si-si face of IPP.203 The X-ray structure of avian FPS has revealed a large cavity formed from ten ·-helices with two aspartate-rich sequencesAsp Asp Asp Asp Asp Asp Mg Mg Mg 4 PPO PPO 7 1¢ + OPP– O PP Mg Mg Asp Asp Asp Asp 8 Asp Asp Asp Asp Mg Mg PP+ PPO PPO 9 H OPP– OPP– Mg Mg Asp Asp Scheme 4 Proposed model for conversion of IPP (7) and DMAPP (8) into FPP (9) by FPS Asp Asp (SQS, farnesyl diphosphate: farnesyl diphosphate farnesyl transferase; EC 2.5.1.21); it has been suggested from studies Asp Asp 15) synthase into a catalyst for formation of geranylgeranyl 20), farnesylgeranyl (C25) and longer chain prenyl pyrophoson either wall of the cavity.204 In total, five highly conserved regions from the large isoprenyl diphosphate synthase family – which include these two aspartate-rich domains205 – were located in the vicinity of this putative substrate binding pocket.The aspartate-rich domains have previously been implicated in binding the pyrophosphate groups of both substrates via a magnesium bridge and in promoting catalysis.206–209 Further X-ray work, coupled with analysis of sequence alignments for this family and site-directed mutagenesis has lead to the hypothesis that the bulky aromatic residues Phe-112 and Phe-113 on the floor of the active site play a critical role in determining the length of chain-elongation by confining the size of the binding pocket available to the growing prenyl chain: reduction in the size of these residues can convert FPP (C (C phates.207 Blanchard and Karst have shown that a single nucleotide change in the yeast ERG20 gene, resulting in substitution of Lys-197 by Glu results in preferential formation of geraniol (C10)210 (yeasts deficient in both ERG20 and ERG19 excreted neither geraniol nor farnesol).188,211 The role of conserved arginine near the C-terminus of the enzyme is not clear.212 FPS from Capiscum annuum has been purified and characterised.213 Rat FPS has been cloned;214 a purification of a recombinant human FPS expressed in Escherichia coli215 has been described and the nucleotide sequence of the ispA gene from E.coli itself has been determined.216 The human gene has been sequenced217 and mapped to chromosome 3.30 The FPS gene has been isolated and cloned from the yeast Kluyveromyces lactis218 and two ascomycete fungi.219 FPS has been cloned from the higher plants Lupinus albus,220,221 Artemisia annua,222 Arabidopsis thaliana223 and Hevea brasiliensis.224 A.thaliana contains two diVerentially expressed isoforms of this enzyme;225 FPS from the guayale rubber plant also contains two enzymes.226 The occurrence of FPS isozymes in higher plants is reminiscent of the situation for HMGR and may be similarly explained by the need for regulation of multibranched isoprenoid pathways emanating from FPP in higher plants. Synthesis of the ‘head-to-head’227 triterpene squalene (SQ; 10) from two molecules of FPP occurs in two stages; initial condensation of two molecules of FPP to form presqualene diphosphate (PSPP; 25) followed by subsequent reductive rearrangement of PSPP, requiring NAD(P)H, to SQ (Scheme 5).Both steps occur under the direction of squalene synthase Brown: The biosythesis of steroids and triterpenoids Asp Asp Mg + PPO –OPP + IPP (7) O 7 H PPO OPP– Mg Mg Asp Asp Asp Asp Asp Asp Mg 7 PPO+ PPO– Mg 1 3 H + PPO R R R GPP –OPP –OPP 9 H H 1¢ 3¢ PPO R R R 9 25 R R + R H PPO H 1¢ 1 10 R NADP(H) 657 R = Scheme 5 Conversion of FPP (9) to SQ (10) via PSPP (25) with yeast that a single catalytic site promotes both reactions.228 Poulter has conducted a study of initial velocities for synthesis of PSPP and squalene over a wide range of FPP and NADPH concentrations using soluble recombinant enzyme, which indicated a sequential mechanism for addition of the two FPP residues.229 Studies with radio-labelled phosphorylated bifarnesol have shown it to be a bona fide intermediate in the biosynthesis of PSPP, thereby providing further evidence against any type of concerted farnesyl/presqualene diphosphate change.230 Poulter has presented evidence for cyclopropylcarbinyl cationic intermediates in the rearrangement of PSPP to SQ, such as those depicted in Scheme 5,231 from the observation that, in the absence of NADPH, the substrate was rapidly converted first to PSPP and subsequently to cisdehydrosqualene and hydroxysqualene as well as the triterpenoid hydroxybotryococcene, which incorporates a 1*–3 rather than a 1*–1 linkage between the two C15 precursors.232 Biller has proposed a model for the SQS active site which led to the rational design of a potent SQS inhibitor.233 Enantiomerically pure (+)-PSPP, which may be a useful tool for future mechanistic studies, is now available by synthesis.234SQS is thought to occur at a critical branch-point for isoprenoid synthesis in eukaryotes.235 This, together with the role of SQS as the first committed step in the biosynthesis of cholesterol, has prompted a large number of synthetic studies describing a wide diversity of inhibitors of this enzyme, in the continuing search for new drugs for the treatment of hypercholesterolemia in man236–253 and the subject has been reviewed.254 A novel radiometric assay for SQS activity, intended for development of new inhibitors, has been described255 and an improved method for analysis and purification of the FPP substrate has been reported.256 The most important new SQS inhibitors to be discovered are the bisphosphonates (18 and other isomeric long chain alkyl phosphates),257–263 which are thought to mimic the FPP substrate, and the zaragozic acids264 (ZGAs, squalestatins265–268), polyketide269,270 fungal metabolites which are active at nano- to pico-molar concentrations271,272 and may mimic the reaction intermediate PSPP.ZGAs A (squalestatin 1; 19),273,274 B (20), C (21)275 and D (22)276 share a polar 2,8-dioxabicyclo[3.2.1]octane-4,6,7-trihydroxy-3,4,5- tricarboxylic acid core and vary in the composition of the lipophillic 6-O-acyl and 1-alkyl side-chains.Absolute stereochemistries of ZGA-A277 and ZGA-C278 have been determined by X-ray crystallography. Investigators have probed the eVects of chemically modifying both the core279–290 and the C-6/C-1 side-chains291–294 on inhibitory activities of ZGAs (directed biosynthesis has also been employed to obtain side-chain modified analogues).295–297 Monocyclic analogues prepared by cleavage of the ZGA bicyclic core298,299 and by total synthesis300 are also described (acyclic mimics of ZGA301 and alkyl citrates incorporating ZGA-A and -B side-chains302 have moderate biological activity).Synthesis of a zaragozic acid– PSPP hybrid has been described.303 Syntheses of ZGA-A/ ZGA-C C-1 side-chains304 and the core unit305 have culminated in a total synthesis of (+)-ZGA-C.305–308 Finally, although ZGAs are competitive inhibitors of SQS, ZGA-A has been shown to be also capable of bringing about a mechanismbased irreversible inactivation and it is suggested that ZGA-A will prove useful in further evaluating the unique catalytic mechanism of this enzyme.309 The fungal metabolite schizostatin described in 1995 represents another new structural class of natural product potent SQS inhibitors.310–312 SQS activity has been purified from dandelion,313 tobacco314 and daVodil microsomes315 and the SQS genes from Glycyrrhiza glabra,316 Nicotiana benthamiana317 and Arabidopsis thaliana318 have been cloned.The ERG9 gene from yeast has also been cloned319–321 and a soluble yeast SQS truncated at the membrane-spanning C-terminus has been overexpressed in Escherichia coli to provide milligram quantities of enzyme for use in studies of enzyme kinetics.322,323 The secondary structure for cloned rat SQS expressed in E. coli has been predicted:324 a 315 amino acid domain at the centre of the protein contains the catalytic site, which can be released in a soluble form by partial proteolysis.325 Mouse (Fdft1)326 and human (FDFT1)327,328 SQS genes have been mapped to chromosomes 1430 and 8p22-p23.1329 respectively.Analysis of amino acid sequences predicted from human and yeast cDNAs suggests a high degree of structural and functional conservation between these two enzymes.330 Squalene epoxidase (squalene monooxygenase, SE; EC 1.14.99.7) catalyses the first oxygenation step in sterol biosynthesis which is suggested to be another one of the rate-limiting steps in this pathway.The enzyme requires molecular oxygen, FAD, NADPH and NADPH-cytochrome P-450-reductase, and is the only known flavoprotein to catalyse the epoxidation of an olefin. No exchange of tritium was detected in the conversion of squalene (SQ; 10) labelled at the 3-position into (3S)-2,3-oxidosqualene (2,3-epoxysqualene; SO; 11), apparently ruling out any oxametallacyclic species in the mechanism.331 SE from pig liver and SE from rat332 have been shown to further convert SO to (3S,22S)-2,3;22,23-squalene dioxide (SDO; 12) with about half the eYciency of the first oxi- 658 Natural Product Reports, 1998 dation333 (6,7- and 10,11-oxidosqualenes were also found to serve as substrates).334 The ability of SE to produce SDO from SQ is of relevance in the production of oxysteroids (see next section), which are implicated as regulators of steroidogenesis (see HMGR).Specific aYnity labelling of the squalene binding site of SE, which would be of considerable utility in probing the mechanism of this little understood enzyme, remains an elusive goal;335 a series of squalene analogues bearing photolabile functionalities have been prepared, but were found to be poor inhibitors.336 NB-598 (23) was the first mammalian SE inhibitor reported to decrease serum cholesterol levels in vivo;337–340 NB-598 was derived by structure–activity modifications based on the allylamine trebinafine,341,342 which is a potent inhibitor of fungal SE343,344 (and also of ƒ7- and ƒ14-reductases).345 Halazy has since reported syntheses of analogues of this mammalian inhibitor;346,347 syntheses of several other SE inhibitors,348,349 many of which are analogues of squalene, have also been described.350–355 SE inhibitors254,356 and their structure–activity relationships357 have been reviewed and a convenient synthesis of 3H-labelled SQ for use in future biosynthetic studies has been reported.358 Porcine SE has been purified and its properties described;359 rat SE was purified by aYnity chromatography360 and the recombinant enzyme characterised.332 Studies of SE from Saccharomyces cerevisiae with both NB-598 and trebinafine inhibitors demonstrated that the properties of this enzyme diVered considerably from those previously established for either rats or Candida albicans, including a suggested very tight binding of FAD.361 The ERG1 gene from S.cerevisiae which encodes SE was cloned362 and later mapped to the right arm of chromosome VII.363 The cloned rat gene was shown to exhibit 30% homology in a deduced protein sequence with that from S. cerevisiae364 but 93% homology with the SE sequence from mouse:365 putative FAD-binding and transmembrane domains were identified in both species. 2) 2.3 Conversion of squalene epoxide into lanosterol in animals and fungi The cyclization of (3S)-2,3-oxidosqualene (SO; 11) to lanosterol (27) by oxidosqualene-lanosterol cyclase (OSC; EC 5.4.99.7) in animals and fungi has fascinated organic chemists for the past thirty years and there has recently been a renaissance in the study of the mechanistic details of this process.366 Animal and fungal OSCs have been reviewed.367 Three chemical syntheses of the SO substrate required for biosynthetic studies have been reported358,368,369 and a synthesis of a protostane steroid, closely related to the protosterol cation intermediate produced by the cyclization (26), has been described.370 The biosynthesis of ganoderic acids in the fungus Ganoderma lucidum, has been studied using (1,2-13C acetate:371 labelling patterns have proven for the first time that SO is a precursor of 3·-hydroxylanostanes in fungi, as in other eukaryotes.Several attempts towards chemical mimicry of the enzymatic cyclization of SO continue to be described. The main diYculties which chemists must overcome are, firstly, formation of an A/B-trans, 9,10-syn, B/C-trans ring junction stereochemistry, which requires that SO adopts a thermodynamically less favoured prechair–preboat–prechair conformation372 and secondly formation of a six-membered C-ring by an energetically unfavourable anti-Markovnikov process (see Scheme 6).Model reaction studies have indicated that neither of these diYculties are insuperable, however, given careful substrate design.372–374 A number of radical-initiated biomimetic cyclizations of polyene SO analogues have been described375–377 in one of which the steroid nucleus (rings A–D) is formed cleanly, albeit as a mixture of isomers at the 17-position (steroid numbering), and with 9,10-anti stereochemistry (see above).378,379 Investigation of cationic biomimetic cyclizations of SO analogues has resulted in some successes in forming23 11 H Enz�CH+ 6 O 19 14 15 H 2 10 7 3 18 H H H HO H H H HO H+ H Hvia 28, 29, 30 ?+ 10 H 9 HO 11 (SO numbering) 20+ 17 H H H Scheme 6 Postulated cyclization of SO (11) to lanosterol (27) by OSC 27 Several acyclic and alicyclic compounds, designed to mimic postulated high energy intermediates (e.g. 26, 28�C30) formed in the cyclization process, which contain a nitrogen atom at the presumed cationic site, have been found to be potent inhibitors of OSC355,392�C409 thereby providing further evidence for the operation of a stepwise mechanism throughout the cyclization process. (Replacement of the methyl groups at C-10 and C-15 of SO by ethyl groups resulted in a monocyclic triterpene, providing evidence for participation of cationic intermediate 29,410 however replacement of the C-6 and C-19 methyls by hydroxymethyl groups had no eVect on the cyclization reaction).411 Substitution of sulfur atoms at the putative cationic centres [C-6, C-10, C-14 and C-19 (SO numbering)] of SO also resulted in competitive inhibition.412�C414 Several miscellaneous inhibitors of OSC, designed as hypercholesterolemic and antifungal agents have been described415�C419 and the topic of OSC inhibition has been reviewed.254,356 MDL 28,815, previously believed to be an inhibitor of OSC, has now been shown to exert its inhibition of cholesterol biosynthesis by inhibition of enzymes later in the pathway.420 Two adjacent aspartate residues in the most highly conserved region of the vertebrate OSC were equally labelled by the irreversible mechanism-based inhibitor 29-[3H]methylidene-2,3-oxidosqualene421�C423 and are possibly involved in stabilising the protosterol cation424 (cf.stabilisation of allylic cations by the aspartate-rich motif in FPS). Although yeast lanosterol-cyclase and plant cycloartenolcyclase were not so labelled,425 studies with this inhibitor have provided the first information regarding the structural details of the active/binding site of OSC in vertebrates426 which may incorporate a Gln-, Trp-containing motif.427 Irreversible inhibition by maleimide squalene derivatives428 and 3-carboxy- 4-nitrophenyl-dithio-1,1*,2-trisnorsqualene429 suggests that thiol groups are essential to the activity of OSC in S.cerevisiae and these residues may be located at the enzyme active site. 26 (sterol numbering) pentacyclic triterpene skeletons (all with a 9,10-anti ring junction), but not the steroid nucleus.380�C383 OSC must be able to force the unfavourable prechair�Cpreboat�Cprechair conformation on the SO substrate, a proposal which is supported by the observation that an SO analogue lacking the 10-methyl group is converted to a product with unnatural 9,10-anti stereochemistry by the enzyme.384 The question of whether cyclization in such model reactions proceeds by a concerted or a stepwise mechanism is not easily resolved.However, Borc¡¦ic¡ä has used kinetic isotope eVects to demonstrate participation of the e-system in the solvolysis of 2-chloro-2,3-dihydrosqualene, which provides circumstantial evidence for involvement of at least two double bonds in a concerted mechanism.385 In similar fashion, it has sometimes been assumed that the four enzymatically-induced cyclizations of SO occur in a concerted process to form the protosterol cation (26), followed by a series of four concerted 1,2-shifts to generate the lanosterol skeleton (27) (Scheme 6).However, Corey has now provided strong direct evidence that at least two discrete carbocations are involved in the cyclisation process.The first tertiary carbocation is proposed to arise from Markovnikov-type formation of a five-membered C-ring366,386 (see comments in preceding paragraph) which then undergoes ring expansion by a 1,2-shift to generate a secondary carbocation.384,387 The ensuing series of 1,2-migrations involved in transformation of the protosterol cation to lanosterol may be induced by weak binding of this protosterol cation with OSC.388 The folding of SO depicted in Scheme 6 diVers from that previously proposed389,390 in that the 18,19-double bond (SO numbering) adopts a conformation leading to a 17-side-chain substituent in the protosterol cation (sterol numbering); such a conformation more naturally accounts for the observed (20R)-configuration (steroid numbering) of the steroid side-chain attained following hydride migration.391 Brown: The biosythesis of steroids and triterpenoids H HO + 2 H 28 H6 H + HO H H H HO 10 29+ H H 24 25 30D C A B HO H HHH 659OSC from Candida albicans requires a histidyl residue in addition to the thiol groups previously identified.430 Rather surprisingly, an SO analogue in which sulfur replaced carbon at C-18 has been identified as an irreversible mechanism-based inhibitor of rat OSC431 (sulfur analogues are normally considered to be competitive inhibitors). Attempts to design SO analogues incorporating photoreactive groups, in order to photolabel the enzyme, have been unyme characterisation has been undertaken with purified OSC from rat liver432,433 and a partially purified cyclase from yeast434 (a highly purified yeast enzyme was also obtained by aYnity chromatography).435 The ERG7 gene from Saccharomyces cerevisiae, coding for OSC, has been cloned436,437 and the product of the ERG7 gene from Candida albicans438,439 was shown to display considerable homology with squalene-hopene cyclase (see section 3.2).440 The enzyme from a cDNA of Schizosaccharomyces pombe is over 50% identical to other lanosterol synthases;441 mutation studies of conserved tryptophan residues suggest that these are not important for catalytic activity (apparently refuting suggestions that tryptophan residues control carbocation formation in the cyclization process).The human lanosterol synthase gene has been mapped to chromosome 21q22.3442 and has been cloned.443,444 Predicted amino acid sequences from cloned rat OSC445 showed ca. 40% homologies with those from fungal and plant sources.446 Finally, OSC is able to eVect not only transformation of (3S)-2,3-monoepoxysqualene (SO) to lanosterol but can also convert (3S,22S)-2,3;22,23-diepoxysqualene (SDO; see previous section) into 24(S),25-epoxylanosterol (31) thereby, ultimately leading to 24(S),25-epoxycholesterol (32) and other oxysterols, which have been implicated as repressors of HMGR (see previous section).The so-called ‘squalene dioxide pathway’ of steroid biosynthesis (Scheme 7) initiated by this enzyme has been extensively reviewed.447–450 Kinetic studies with mammalian OSC have in fact revealed a preference O O 12i O 24 25 HO H 31ii O HO 32 Scheme 7 The squalene dioxide pathway in mammals: i, OSC; ii, several steps – see section 2.3.1 660 Natural Product Reports, 1998 for the diepoxide as substrate451 (however, 2,3:18,19- dioxidosqualene is a non-competitive inhibitor of rat OSC452,453 and other dioxidosqualenes also show a less pronounced inhibitory eVect).454 The novel compound BIBX 79 has been described as a selective inhibitor of OSC455 which exerts maximum eVect in impeding steroidogenesis at intermediate concentrations: the rationale for this unusual observation is that, at intermediate concentrations, BIBX 79 eVectively suppresses cyclization of SO, but not of the preferred SDO substrate.Consequent channelling of substrate into the squalene dioxide pathway rather than into formation of the primary metabolite cholesterol (via lanosterol) results in formation of oxysterols, which then further reduce cholesterol biosynthesis by inhibiting steroidogenesis at the level of HMGR (however, higher concentrations of SDO inhibit cyclization of both SO and SDO, precluding such regulation of HMGR). 14DM in Scheme 9.) 14DM 2.3.1 Conversion of lanosterol into cholesterol in animals Bloch has given a historical review of cholesterol biosynthesis covering the period from the 1930s to the 1960s.456 In contrast to the elegant simplicity by which several ring-forming and rearrangement reactions are catalysed by a single enzyme in the formation of lanosterol from SO, the subsequent conversion of lanosterol to cholesterol, involving removal of three angular methyl groups and introduction of a double bond at the 5-position, involves a sequence of a further sixteen additional steps, all of which are catalysed by little-studied membrane-bound enzymes, several of which are located in the peroxisomes457 (as well as microsomes).First amongst these steps is removal of the 14·-methyl group catalysed by lanosterol 14·-demethylase (P-45014DM). The 14-methyl group is first converted to an aldehyde by two successive oxidations, followed by a third oxidation which is proposed to form a 14-formyloxy intermediate458 (proceeding by a Baeyer–Villiger type rearrangement), from which formic acid is eliminated to form a conjugated 8,14-diene.(However, see mechanism proposed for fungal P-450 Particular interest has been shown in developing lanosterol derivatives modified at the 14-methyl group and the 15- position, which can have a dual action in preventing cholesterol biosynthesis by serving both as inhibitors of P-450 and providing feedback suppression of HMGR (by virtue of their structural relationships to oxylanosterols).118,459–465 Bossard has also described steroidal acetylenes substituted at the 14-methyl group as mechanism-based inhibitors of this enzyme.466 RS-21607, a non-steroidal azole inhibitor, preferentially inhibits the third oxidation step,467 as has been observed for the fungal P-45014DM azole inhibitor ketoconazole, which also inhibits rat P-45014DM.468 The fungal P-45014DM inhibitor SSF-109 also inhibits rat P-45014DM.417 P-45014DM has been purified from the Syrian hamster,469 pig liver470 and humans.471 cDNAs for the human472,473 and rat474 CYP51 genes have been cloned and expressed and shown to have lanosterol 14·-demethylase activity.DNA sequences for several species and phyla are highly conserved475 and this is suggested to be an ancient gene.476 Sterol-ƒ14-reductase (14-reductase) catalyses the NADPHdependent reduction of the 14-double bond of ƒ8,14-diene steroids created in the previous step (ƒ7,14-dienes are also reduced): a purified reductase from rat appears to be a 70 000 Da protein composed of two subunits.477 MDL 28,815, previously believed to be an inhibitor of OSC, has been shown to inhibit sterol-ƒ14-reductase in rat.420 Sterol-8-isomerase (ƒ8]ƒ7-isomerase) from rat which catalyses the reversible conversion of ƒ8-steroids to their corresponding ƒ7- isomers has been purified and found to have a weight of 21 000 Da.478 The deduced amino acid sequence from a murine ƒ8]ƒ7-isomerase encoding cDNA has been shown to be highly similar to human emopamil-binding protein (of unknownfunction).479 Both 14-reductase and 8-isomerase activities undergo negative feedback regulation by cholesterol.477,478 In the final steps of cholesterol biosynthesis, the molecular oxygen-requiring NADPH-dependent enzyme lathosterol-5- desaturase (lathosterol-5,6-dehydrogenase) catalyses the introduction of a new ƒ5-double bond into conjugation with the existing ƒ7-double bond to form 7-dehydrocholesterol (7-DHC; 54). The properties and kinetics of this enzyme have been studied480 and a human cDNA (SC5DL), homologous to the fungal ERG3 gene, which encodes sterol C-5 desaturase, has been isolated.481 The final step in the modification of the steroid nucleus is mediated by 7-dehydrocholesterol-ƒ7-reductase (3‚- hydroxysteroid ƒ7-reductase; ƒ5,7-sterol-ƒ7-reductase) which converts 7-DHC to cholesterol.482 Assays for evaluation of activity of this enzyme has been described483,484 and inhibition studies with AY 9944, BM 15.766 and YM 9249 have been reported.477,484,485 The clinical disorder Smith–Lemli–Opitz syndrome may be caused by a deficiency in this enzyme.486–489 Reduction of the ƒ24,25-double bond in the side-chain is achieved by sterol-ƒ24-reductase: the stereochemistry of hydrogen addition to C-25 for the mammalian enzyme has now been determined directly by 13C NMR spectroscopy and is the same as that previously demonstrated in insects.490.scc 2.3.2 Biosynthesis of steroidal hormones in vertebrates Mammalian steroidal hormone biosynthesis is a huge field of study and the direction of research is heavily influenced by applications to medicine. General topics which have been reviewed include neurosteroids,491–497 biosynthesis of androgens and estrogens,498–500 in particular testosterone biosynthesis in Leydig cells,501,502 corticosteroid biosynthesis503,504 and biosynthesis of placental steroid hormones.505 Reviews of particular classes of enzymic activity involved in steroid biosynthesis have covered P-450-dependent enzymes,506–509 oxidative bondcleavage reactions510 and hydroxysteroid dehydrogenases.511 The inhibition of steroid biosynthesis has been reviewed512–517 and the application of such inhibitors in the treatment of cancer has been discussed.518,519 Several reviews concerning various aspects of regulation and expression of enzymes involved in steroidogenesis – a topic not covered in this review – have appeared.520–525 The biosynthetic route to all vertebrate steroidal hormones (Scheme 8) is generally assumed to begin with the side-chain cleavage of cholesterol (33) at C-20/C-22 initiated by cytochrome P-450scc (Cyp11A1, cholesterol side-chain cleavage cytochrome P-450) which is the rate-limiting step in steroidal hormone biosynthesis.(However, evidence for an alternative ‘sesterterpene pathway’ of steroid biosynthesis has been provided by Tait.526) Studies with a reconstituted P-450 system have provided direct evidence that cholesterol is converted to pregnenolone (PREG, 34) sequentially via (22R)-22-hydroxycholesterol and (20R,22R)-20,22-dihydroxycholesterol.527,528 (20S)-22-Thiacholesterol [although not the (20R) diastereoisomer] is a mechanism-based inhibitor of this enzyme.529 A steroidal mechanism-based fluorogenic probe has been synthesised and used to confirm the location of P-450scc on the inner surface of mitochondrial membranes.530 The interactions between this membrane-bound enzyme and ferredoxin, which mediates electron transfer to the enzyme, have been shown to be electrostatic in nature531 and to depend on the presence of specific charged amino acids.532,533 Isocaproaldehyde (4-methylpentanal), the other C6 product of side-chain cleavage, is further metabolised by aldose reductase, an enzyme concerned with detoxification of aldehydes derived from lipid peroxidation.534 The bovine P-450scc gene has been expressed in Escherichia coli535 and the rat gene has been cloned.536 The human CYP11A1 gene has been localised to chromosome 15q23-q24.537 21 steroid Following conversion of cholesterol to the C PREG, there are two metabolic pathways available for further Brown: The biosythesis of steroids and triterpenoids elaboration of steroidal hormones: the 4-ene pathway involving progesterone (PROG; 35) and 17·-hydroxyprogesterone (17·-OH-PROG; 37) leading to androstenedione (4-androst- 3,17-dione; A-dione; 39); and the 5-ene pathway in which PREG is converted to dehydroepiandrosterone (DHEA; 38) via 17·-hydroxypregnenolone (17·-OH-PREG 36).538,539 Lombardo has shown that the 4-ene pathway dominates in the production of testosterone in human testes,540 whilst estrogen synthesis in the bovine placenta541 and Trichogaster trichopterus542 follows the 5-ene pathway.ƒ5-Steroids can be converted to the corresponding ƒ4-steroids at two sites in the 5-ene pathway in the sea star Asteria rubens.543 In Bufo arenarum, the 4-ene route to aldosterone is microsomal whilst the 5-ene route is mitochondrial.544 The enzyme 3‚-hydroxysteroid dehydrogenase/ƒ5–ƒ4- isomerase (3‚-HSD; EC 1.1.1.51/1.1.1.145; EC 5.3.3.1) connects the two pathways by catalysing an oxidation/ isomerisation reaction which converts a variety of 3‚-hydroxy- 5-ene steroids into their 3-keto-4-ene equivalents.545–549 (N.B.Kumar has proposed an alternative conversion, involving superoxide dismutase and peroxidase.550) The most important substrates are PREG, which is converted into PROG (a synthesis of [19-2H3]PROG and [18-2H3]PROG for use in biosynthetic studies has been described)551 and DHEA, which is converted to A-dione. 3‚-HSD has been found in both mitochondria and microsomes552,553 and this family of enzymes has been reviewed.554 A single protein is responsible for both oxidation and double bond isomerisation activities: thus, bovine 3‚-HSD was obtained as a homogeneous 81 kDa protein which exhibited both properties (in contradiction of an earlier report).555 The physiologically more important 3‚-hydroxy-5-ene-substrates bind to 3‚-HSD without the requirement for prior binding of the NAD+ co-factor (although 3‚-hydroxy-5·-reduced steroids do require prior binding).556 A model for enzyme action has been proposed in which NADH, produced during the initial oxidation step, induces a conformational change in this bifunctional protein,557 thereby triggering the isomerase reaction at the same active site.558 (However, Luu-The has also proposed the presence of distinct dehydrogenase and isomerase active sites.559) AYnity labelling of human placental 3‚-HSD indicated that His-262 and Cys-183 were in close proximity in the 3-dimensional structure of the enzyme.560,561 At least two cysteines562 were indicated at or in the vicinity of the single563 NAD+ binding site: from comparative amino acid sequence analysis a structural model for the bovine 3‚-HSD protein has been presented in which the NAD+ binding site and the membrane-anchoring segment were localised564 (membrane association has been shown to be important in stabilising the enzyme).565,566 Six distinct mouse 3‚-HSD cDNAs have been isolated and characterized545,567–573 and all these genes are closely linked together on chromosome 3.574 Mouse 3‚-HSDs IV and V have 3-ketosteroid reductase activity (and employ NADPH rather than NAD+) but are devoid of ƒ4,5-isomerase activity:570 they may be involved in inactivation of steroid hormones [e.g.conversion of 5·-dihydrotestosterone (46) to 5·-androstane- 3‚,17‚-diol],575 rather than in the 5-ene and 4-ene pathways of steroidogenesis. Four isoforms of the rat enzyme have been cloned:576–579 type III is an NADPH-utilising 3-ketosteroid reductase, whilst types I and IV also display 17‚-hydroxysteroid dehydrogenase activity (see later).580 This secondary activity is presumed to be the result of active site-binding of the steroid substrate in an inverted orientation to that normally found for the primary 3‚-HSD activity.In addition, the type I rat enzyme has much higher specificity for PREG and DHEA than type II.581 Several studies of human 3‚-HSD have been described582–590 and the genes for human type I and II enzymes have been localised to chromosome 1p13.1.591,592 cDNA clones encoding macaque,593 chicken,594 hamster (three isozymes)595 and trout 3‚-HSD596 have been isolated. Some mutations in the human gene have been 661ii HO O Dienone 41 iii Andien-b 40 iii 22 i 3 5 PHEROMONES 20 O 17 16 ii O HO HO 33 PREG 34 iii PROG 35 iii 21 4-ene/5-ene O O HO 17a-OH-PREG 36 iii O 17a-OH-PROG 37 iii 19 HO O A-dione 39 iv DHEA 38 iv OHOHii ii ii O A-diol 42 T 43v HDHT 46 O vii, aromatase; viii, P-450c21; ix, P-45011‚; x, P-450aldo; xi, 11‚-HSD characterised in relation to the clinical disorder congenital adrenal hyperplasia.597–600 Enzyme function is almost completely abolished by mutation in Asn-100, which is conserved in all 3‚-HSDs, and is suggested to be of crucial importance to enzyme function.601 Studies with the classical 3‚-HSD inhibitors trilostane (2·- cyano-4·,5·-epoxy-17‚-hydroxyandrostan-3-one) and cyanoketone (2·-cyano-4,14,17·-trimethyl-17‚-hydroxy-5-androstan- 3-one)602 have helped confirm603 the hypothesis that an enzyme distinct from that involved in the sex hormone-producing pathway is involved in the conversion of androsta-5,16-dien-3‚-ol (andien-‚; 40) to androsta-4,16-dien-3-one (dienone; 41) in the pheromone-producing 16-androstene pathway in pigs.549 The Scheme 8 Overview of steroidal hormone biosynthesis in mammals.i, P-450SCC; ii, 3‚-HSD; iii, P-45017·; iv, 17‚-HSD; v, 5AR; vi, 3·-HSOR; Cleavage of the C-17–C-20 bond in the C azasteroid 4-MA, previously described as a 5·-reductase inhibitor, has been shown to also inhibit 3‚-HSD;604 the anti-fertility agent gossypol also inhibits this enzyme.605 21 steroids PROG and PREG to yield the corresponding C19 steroids A-dione and DHEA (4-ene and 5-ene pathways, respectively) is mediated by P-45017· (P450c17; 17·-hydroxylase-17,20-lyase; 17·-hydroxylase-17,20-desmolase; P450XVII; CYP17; EC 1.14.99.9).This enzyme may also produce 17·-OH-PROG or 17·-OH-PREG as products, which then serve as substrates for biosynthesis of C21 glucocorticoids (see later). The enzyme requires NADPH-cytochrome P-450-reductase (or its equivalents);606,607 cytochrome b SEX HORMONES HO 662 Natural Product Reports, 1998 O viii O O DOC 48 11 viii O 11-Deoxycortisol 49 O vii HO OH vii HO OHOHvi HO H 3a-Adiol 47 OH O O H HO O ALDO 52 x OH O OH 18 HO Oix or x O Cortcosterone 51 OH OOH OHix MINERALCORTICOIDS OH O HO O Cortisol 50 xi OH OOH OCortisone 53 OH GLUCOCORTICOIDS O O E1 44 iv E2 45 OH5 is also utilised and may14DM and Some amino acid residues in rat P-450 play an important role in regulating the balance between 17·- hydroxylation and 17,20-lyase activities,608 thereby determining the extent of sex hormone synthesis versus glucocorticoid biosynthesis.609,610 Other activities demonstrated for P-45017· include formation of 17·-hydroxyandrogens, 16·- hydroxylation611 and lysis of PREG to andien-‚612–614 (the substrate for the 16-androstene pathway).615 An attempt to select for these varied P-45017· activities by construction of chimeric proteins was unsuccessful, perhaps because enzyme activity is controlled by subtle and unpredictable aspects of tertiary structure.616 Phe-343 in rat P-45017· has been shown to enhance lyase activity for steroids in the 4-ene pathway.617 Although a single enzyme is now generally held to be responsible for both hydroxylase and lyase activities, Zachmann has pointed out that there are discrepancies between this finding and some clinical results.618–620 Isotopiclabelling studies have provided evidence that the mechanism of acyl-carbon cleavage involves FeIII-OOH reacting with the carbonyl group to form a peroxy adduct (Scheme 9).621–626 If the enzyme has previously eVected 17·-hydroxylation (which is believed to occur by oxene rather than peroxide chemistry),627 then subsequent ionic rearrangement of the peroxide adduct leads to elimination of acetic acid and formation of a 17-ketosteroid; if not, then decomposition of the peroxide leads to a carbon radical which may undergo either disproportionation628,629 or oxygen-rebound630 processes to yield 16-androstadienes or 17·-hydroxyandrogens respectively.(N.B. similar mechanisms have been proposed for acyl-carbon bond cleavage processes catalysed by fungal P-450 aromatase.631) C17–C20 cleavage of PROG by a Pseudomonas species may involve a Baeyer–Villiger type oxidation, rather than those mechanisms discussed above.632 17· are associated with 17· lyase activity (e.g. Arg-346, Arg-357) and others with hydroxylase activity (e.g.Arg-363), suggesting that the steroid substrate-protein interaction must change during the course of the two consecutive reactions.633 Ser-106634 in the human enzyme is crucial for both activities and may play a role in the active site,635 whilst His-373 is important in heme binding.636 Using a variety of techniques, such as sequence alignment, secondary structure prediction and molecular mechanics, a tentative model for the active site of P-45017· has been proposed in which there are two binding modes for the substrate, reflecting the dual function of the enzyme.637 Homology modelling studies with related bacterial cytochromes 3·/20‚-hydroxysteroid dehydrogenase638 and P-450cam,639 which were validated by site-directed mutagenesis, have also been used to produce models of P-450 enzyme structure. Ahmed has described a molecular model of azole inhibitors bound to a substrate–heme complex640 and Kuhn-Velten has made detailed experimental investigations of enzyme kinetics and inhibition.641,642 Several investigators have studied the importance of the hydrophobic N-terminal sequence, which is involved in membrane-insertion, for folding of this protein into its correct structural form.616,643–646 A number of steroidal inhibitors incorporating a variety of functional groups at the 17- and 20-positions have been designed:647–653 the most potent such inhibitor so far synthesised contains a 3-pyridyl group at the 17-position654,655 and was developed from the observation that various 3- and 4-pyridylacetic acid esters are inhibitors of this enzyme.656–659 This compound may find application in treatment of prostatic cancer519 since clinical use of the imidazole inhibitor, ketoconazole,660,661 is limited by its non-selective inhibition of other P-450 enzymes.Other inhibitors include the synthetic 4-amino-steroid MDL 19687662 and various imidazolyl- and pyridyl-substituted compounds,663–666 as well as naturallyoccurring tetrahydroquinoline667 and ‚-carboline668 alkaloids.7·-Thiospironolactone, derived from the mineralocorticoid agonist spironolactone, has been suggested to be a mechanismbased suicide inhibitor.669–671 Transforming growth factor- Brown: The biosythesis of steroids and triterpenoids P-45017aOHO FeIII–OOH 2017 16 O FeIV–O• OOH P-450 Aromatase O H 19 10 FeIII–OOH 23O O HO 14DM14 8 H FeIII–OOH Scheme 9 Mechanism proposed for the acyl-carbon cleavage reaction performed by P-45017·, aromatase and fungal P-45014DM ‚-1 is a non-competitive inhibitor of 17·-hydroxylase activity,672 and testosterone has been shown to regulate its own biosynthesis by specific inhibition of 17·-hydroxylase activity.673 Purification of both pig674 and calf675 testicular P-450 17· activity has have been described.The gene coding for P-450 been located to chromosome 19 in mouse676 and chromosome 10q24.3 in humans.677 P-450c17 cDNAs from dogfish shark,678 trout,679 pig ,680,681 rat,682 guinea pig683,684 and mouse676 have been sequenced; the genes from dogfish shark,685 cattle686 and humans687 have been expressed in + FeIII + H2O OH + FeIII FeIII–O• H+ OH O O• FeIII • + FeIII–O• + CH3COOH FeIII–O• H+ + FeIII + H2O OHO O FeIII O H O FeIII–OOH + FeIII + CH3COOH O OH FeIII O + HCOOH O OH OH O• O O + FeIII–O• FeIII FeIII–O• H+ • + HCOOH17·s 663Escherichia coli (the bovine enzyme was also expressed in insect cells using baculovirus688).Several mutant human CYP17 genes have been studied in relation to clinical disorders.689–693 17‚-Hydroxysteroid dehydrogenase (17‚-HSD; 17‚- hydroxysteroid oxidoreductase; EC 1.1.1.62) is the key enzyme for subsequent conversion of 17-ketosteroids to 17‚- hydroxysteroids.Two particularly important reactions catalysed are the conversion of A-dione to the active male sex hormone testosterone (T; 43) and transformation of the female sex hormone estrone (E1; 44) to the more potent estradiol (E2; 45). 17‚-HSD, which belongs to the short-chain alcohol dehydrogenase family, has been reviewed.694 Four distinct human isozymes of 17‚-HSD have been characterised so far:695–697 type 1 is active in forming E2,520 type 3 converts A-dione to T and type 2 has both activities as well as a 20·-HSD activity.698 The type 1 enzyme was studied by X-ray crystallography and shown to be a dimer of identical sub-units699 containing a Tyr-X-X-X-Lys sequence (where X is any amino acid) and a serine residue at the active site (these are also conserved features for other enzymes in the short-chain dehydrogenase family).700,701 The complex of 17‚-HSD1 with E2 was also crystallised and interactions between Ser-142, Tyr-155, His-221 and Glu-282 of the enzyme and the substrate were demonstrated.702 Tyr-155 was suggested to be involved in acid/base catalysis, while Lys-159 may participate in binding the NADP(H) co-factor. Site-directed mutagenesis of Tyr-155 and His-221 resulted in significant loss of catalytic activity.703 2Several estradiol derivatives containing substituents at C-16 have been synthesised and/or evaluated as inhibitors of E biosynthesis704–707 in a drive to find complementary approaches for the treatment of breast cancer; the contraceptive agent nomegesterol acetate has also been shown to inhibit 17‚-HSD catalysed E2 biosynthesis.708 Both retinoic acids709 and the HMG-CoA reductase inhibitor, simivastatin,710 are inhibitors of the A-dione 17-keto reductase activity which produces T.An FPLC purification of placental 17‚-HSD has been reported.711 cDNAs for rat type 1712 and type 2713 17‚-HSDs have been characterised and 17‚-estradiol dehydrogenase cDNAs from pig714 and mouse715 have been obtained.The human type 1 gene has been mapped to chromosome 17q12-q21716 and type 2 was mapped to 16q24.717 The enzyme steroid 5·-reductase (3-oxo-5·-steroid-ƒ4- dehydrogenase, 5AR, EC 1.3.99.5) is involved in the reduction of the ƒ4,5-double bond which converts T to 5·- dihydrotestosterone (DHT; 46), the more potent androgen involved in male sexual diVerentiation.Both rats718–720 and humans718, 721–725 express two isozymes of 5AR. Amino acids possibly involved in substrate and co-factor binding in the human type 2 enzyme have been identified from a study of naturally occurring mutants.726 The NADP(H) co-factor binding site of rat 5AR has been identified by photo-labelling experiments727 and enzyme kinetics have been studied.728 Following the success of the 4-azasteroid inhibitor finasteride,729,730 which is currently approved for the treatment of benign prostatic hyperplasia, further 4,17- and 17-substituteddiazasteroids731,732 and 6-azasteroids733–738 continue to be described as inhibitors of this enzyme.Many other steroidal inhibitors, such as the 3-androstene-3-carboxylic acid epristeride,739,740 have also been designed to mimic the A-ring enol that is a presumed intermediate formed during the conjugate reduction of T.649,741–746 A few non-steroidal synthetic inhibitors have also been described.747–750 Finasteride has been shown to act as a mechanism-based inhibitor: a tetrapeptide sequence was identified in the rat enzyme which confers sensitivity to finasteride,751 and a mechanism for finasteride inhibition, involving participation of the proposed enolic intermediate in eVecting covalent modification of the co-factor has been suggested.729 The major degradation route for DHT in male accessory sexual tissues is to 5·-androstane-3·,17‚-diol (3·-Adiol; 47), catalysed by 3·-hydroxysteroid oxidoreductase (3·-HSOR; 664 Natural Product Reports, 1998 The biosynthesis of C EC 1.1.1.50).752 Another 3·-HSOR also converts 5·- dihydroprogesterone to 3·,5·-tetrahydroprogesterone in the female pituitary.753,754 A synthesis of potential mechanismbased inhibitors of 3·- and 3‚-hydroxysteroid dehydrogenases has been reported.755 18 estrogens from C19 androgens involves two sequential hydroxylations at the 19-position to produce an aldehyde, which then undergoes oxidative acylcarbon cleavage, resulting in aromatisation of the steroid A-ring and elimination of formic acid.All three reactions are catalysed by a complex consisting of the aromatase cytochrome P-450 and NADPH-cytochrome P-450 reductase (P-450aromatase; aromatase; estrogen synthase; EC 1.14.14.1) and several mechanisms have been postulated at each step.499 Both T and A-dione serve as substrates, resulting in formation of E2 and E1 respectively. Theoretical studies of various proposed mechanisms of carbon–carbon cleavage have been reported;756,757 however, model reaction studies with 19-oxoandrostenedione analogues758,759 have supported the proposition that a 19-hydroxy-19-ferric peroxide intermediate is involved in the enzymatic acyl-carbon bond cleavage step (cf.mechanisms proposed for acyl-carbon cleavage by P-45017· and fungal P-45014DM in Scheme 9).The precise mechanism by which this peroxy intermediate is converted into the aromatic product remains unclear: results from model studies suggest that 2,3-enolization may be an important prior requirement for carbon–carbon bond cleavage760,761 (which is thereby assisted by immediate attainment of aromaticity in the A-ring), whilst experiments involving aromatase itself with various 19-demethyl substrate analogues have supported a cleavage mechanism involving formation of a radical at the 10-position.762 (Alternatively, somewhat inconclusive enzymatic experiments by the same investigators may implicate a 2-hydroxy species in the third oxidation step.763) Site-directed mutagenesis has established that the region comprising Gln-298 to Val-313 is important for the active site of the human enzyme.764–766 Predictions from molecular modelling, confirmed by site-directed mutagenesis, were consistent with the conclusion that Pro-306 causes a bend in the I-helix, which is required in forming the substrate-binding pocket, and that the carboxylic acid of Glu-302 is important for catalytic activity.767 Studies of the kinetic properties of several aromatase mutants in the presence of inhibitors also suggested that Pro-308 and Asp-309 play critical roles in determining substrate specificity and catalytic capability.768 Inhibitors of aromatase are very important with regard to treatment of estrogen-dependent cancers, in particular breast cancer: the current status, design and mechanism of action of such inhibitors has been extensively reviewed.769–776 Clinical trials have been reported for arimidex,777,778 letrozole (CGS 20267),779,780 exemestane (FCE 24304)781 and fadrozole hydrochloride (CGS 16949A).782,783 Inhibition of aromatase by R76713 has been shown to be due exclusively to the (+)-S-isomer784 (vorozole) which is now under clinical evaluation.Structure–activity relationships of some aromatase inhibitors have been described785,786 and several syntheses of potential new787–795 and known796 steroidal inhibitors substituted at the 4-, 6-, 7- and 19-positions have been reported; most non-steroidal inhibitors incorporate 3- and 4-pyridyl groups656,659,664,797,798 (cf. P-45017·). 14·-Hydroxyandrost-4- ene-3,6,17-trione, produced by microbial transformation of A-dione, is a mechanism-based irreversible inhibitor.799 5·-Dihydronorethindrone, a metabolite of norethindrone (which is a component of the contraceptive pill), inhibits aromatase.800 The antifungal agent ketoconazole is claimed to be an in vitro inhibitor of aromatase660 and tobacco alkaloid derivatives are also described as inhibitors.801,802 The gene encoding human aromatase803 (CYP19) has been expressed in insect cells using a baculovirus vector.804 Aromatase cDNAs have been obtained from mice805 and cattle.806 Entry into the glucorticoid and mineralocorticoid classes of steroid hormones is controlled by the NADPH-dependent5 may regulate the activity of 11‚ enzyme steroid 21-hydroxylase (P450c21; CYP21; EC 1.14.99.10) which catalyses the conversion of both PROG to 11-deoxycorticosterone (DOC; 48) and 17·-OH-PROG to 11-deoxycortisol (49)807 (leading to mineralocorticoids and glucorticoids respectively).There is suggested to be a single CYP21 gene encoding P450c21 in pigs:808 the situation in humans is more complex and both a functional CYP21A1 (21B) gene and a presumed non-functional CYP21A1P (21A) pseudogene have been described809–812 Several mutant forms of human CYP21 exist, many of which may result from the close proximity of these two genes.813–821 The clinical condition congenital adrenal hyperplasia is over 90% associated with deficiency of this enzyme822,823 and a rapid PCR protocol for determination of mutations in 21-hydroxylase has been described.824,825 From mutation studies, Cys-428 has been proposed to be the heme ligand and the presence of a methyl group at the ‚-carbon of Val-281 has been shown to be required for heme-incorporation and consequent enzymatic activity.826 Mutations in Ile-172 (believed to be involved in the membrane-anchoring domain) and Arg-356 in the substratebinding site resulted in loss of activity.827 21-Methyl substituted steroids have been described as mechanism-based inhibitors.828 Cytochrome b P450c21 (cf.P-45017·).829 Steroid 11‚-hydroxylase [cytochrome P-450(11‚)] catalyses the final steps of biosynthesis of glucocorticoids and mineralocorticoids, eVecting the conversion of 11-deoxycortisol (49) to cortisol (50) and DOC to aldosterone (ALDO; 52). The role of this enzyme830–834 in aldosterone biosynthesis835,836 has been reviewed (an alternative pathway to aldosterone via 21-deoxyaldosterone is also known).837 A single enzyme is responsible for the final steps of aldosterone synthesis in pigs838 and bullfrogs,839 there are two distinct 11‚- hydroxylases known from mice,840 cattle841 and man,842 whilst four are now described from rat842–848 (of which one is a pseudogene).Isolation of cDNAs for both human enzymes has demonstrated that the product of CYP11B1 (11‚- hydroxylase; P-45011‚; P450c11‚) can 11‚-hydroxylate both 11-deoxycortisol and DOC to cortisol (50) and corticosterone (51) respectively, whereas that of CYP11B2 (aldosterone synthase; P-450aldo; P450c11AS; P450c18) can eVect synthesis of ALDO from DOC849–852 by performing 11‚- hydroxylation as well as two successive 18-hydroxylation reactions853 (18-hydroxylase and oxidase activities were previously ascribed to the ‘hypothetical enzymes’ corticosterone methyl oxidases I and II).854 Phe-66 and Ser-126 are important for maintaining both activities in the bovine enzyme.855 Cortisol is also metabolised to 18-hydroxycortisol and 18-oxocortisol by this enzyme.856,857 Consequently, P-450 is involved in the synthesis of glucocorticoids, whereas P-450aldo is involved in the synthesis of mineralocorticoids.The enzymes share 93% homology and are encoded by structurally similar genes located in tandem on chromosome 8q22.858 (An 11‚-hydroxylase from the fungus Curvularia lunata has also been shown to catalyse 11‚-hydroxylation of a variety of steroids including 11-deoxycortisol and DOC.)859,860 The putative steroid- and heme-binding regions of P-450(11‚) have been identified by amino acid sequence alignment of these enzymes from various animal species.833 18-Vinylprogesterone and 18-ethynylprogesterone have both been found to be potent mechanism-based inhibitors of aldosterone biosynthesis861 and 18-vinylprogesterone preferentially inhibits the 18-hydroxylation step.862 A hypothetical model for aldosterone synthesis, involving a single active site which undergoes a conformational change in order to bring about 18-hydroxylation as opposed to 11‚-hydroxylation has been proposed on the basis of studies of enzyme kinetics and a comparison of inhibition by these two compounds.863 18-Ethynyl-deoxycorticosterone is also a mechanism-based inhibitor864,865 of the last steps of aldosterone biosynthesis (although 18-nitro-oxyandrostenedione apparently is not).866 Brown: The biosythesis of steroids and triterpenoids Other inhibitors include mespirenone,867,868 etomidate,661 fadrozole hydrochloride,782 norharman,668 gossypol869 and acetophenone natural products.870 The antiparasitic drug suramin probably exerts activity by inhibiting replenishment of reducing equivalents in mitochondria which are required by this mitochondrial enzyme, rather than by directly inhibiting the hydroxylase itself.871 The ability of this enzyme to aVect hydroxylation at more than one site in the steroid nucleus is reminiscent of that of P-450scc, and EPR studies have confirmed similarities in the active sites of the two enzymes.872 Human P-450(11‚) genes have been expressed in hamster873 and yeast874 and found to be functional in both cases. Mutations in both human CYP11B1875–877 and CYP11B2878–882 genes have been studied and the subject has been reviewed.858 The enzyme 11‚-hydroxysteroid dehydrogenase (11‚-HSD), which catalyses the interconversion of biologically active cortisol (50) to inactive cortisone (53) in man, and of corticosterone to 11-dehydrocorticosterone in rodents, has been reviewed.830,883 Two forms of this enzyme are known from both man and rodents: low aYnity NADP(H)-dependent 11‚-HSD1 acts predominantly as an oxoreductase, whilst 11‚-HSD2 is a high-aYnity NAD-dependent uni-directional dehydrogenase.884 Human 11‚-HSD has been cloned.885 Licorice is a well known inhibitor of 11‚-HSD886 and other enzymes.887 The metabolism of cortisol also involves cortisol ƒ4-5‚-reductase, dihydrocortisol-3·-oxidoreductase888 and possibly cortisol 6‚-hydroxylase.889 3; 3 3 2.3.3 Biosynthesis of vitamin D and bile acids Conversion of 7-dehydrocholesterol (7-DHC; provitamin D 54) to vitamin D3 (cholecalciferol, calciol, 56) proceeds via two pericyclic reactions: the first a photochemical conrotatory electrocyclic opening of the B-ring to produce the (Z)- hexatriene previtamin D3 (55) and the second a thermal [1,7]-sigmatropic hydrogen shift (presumed to be antarafacial) which yields cis-vitamin D3 (Scheme 10).It has long been recognised that casual exposure to sunlight is suYcient to provide most humans with their vitamin D requirement via conversion of cutaneous stores of 7-DHC to previtamin D3.However, only low concentrations of vitamin D3 and 7-DHC were found in cats and dogs, and it has been suggested that, unlike herbivores and omnivores, these animals are unable to synthesise vitamin D in the skin, and are mainly dependent on dietary intake.890 Bernasconi has presented evidence that a photoreaction from 7-DHC may also be operative in the biosynthesis of vitamin D3 in plants, as described for animals.891 Feeding experiments with labelled cholesterol in fish have demonstrated the absence of a nonphotochemical pathway of vitamin D synthesis.892 An exceptionally clean and eYcient photoconversion of 7-DHC to previtamin D3 in aqueous solution has been eVected in the presence of a naphthalene co-polymer893 acting as both photosensitizer and screen against other possible photoreaction products, such as lumisterol and tachysterol.894 The quantum yield for photolytic opening of the diene ring for various 1·-trialkylsilyloxy-7-DHC analogues was found to decrease with increasing size of the alkyl group,895 whilst 1·-hydroxy-7-DHC itself underwent novel photoisomerisation reactions, involving cleavage of the 1,10-bond, to an appreciable extent.896 Primary deuterium kinetic isotope eVects of 11.4 and 5.5 have been reported for the thermal sigmatropic rearrangement reaction of pentadeuteriated previtamin D 897 and pentadeuterio 1·,25-dihydroxyprevitamin D 898 (deuteriated at the 9- and 19-positions) which seem to be more reasonable for the proposed pericyclic mechanism – involving linear and symmetrical H-transfer from H-19 to H-9 – than previously reported values (kH/kD~45). At 37 )C, previtamin D3 and vitamin D3 exist in an 14:86 equilibrium ratio, which is attained after ca. 1 week in hexane. However, kinetic and 66519 1 10 7 3 HO 54 22 hn 23 iii OH HO HO 59 A3 HO 243 CO2H 3 OH913 3. OH23 Scheme 10 Biosynthesis and degradation of 1,25-(OH)2-D3 (58).i, CYP27; ii, 1·-OH-ase; iii, 24-hydroxylase 56 25-OH-D3 catabolism (which is suggested to proceed in the sequence: 24-hydroxylation, oxidation of the 24-OH group to a ketone, 23-hydroxylation and cleavage of the C-23/C-24 bond).922 Human 1,25-dihydroxyvitamin D3 24-hydroxylase has also been cloned.923 Two major pathways924,925 (Scheme 11) are now known for the catabolism of cholesterol to the bile acids chenodeoxycholic acid (CDCA; 75) and cholic acid (CA; 74) and this topic has been reviewed.925,926 Regulation of hydroxylases involved in bile acid biosynthesis has also been reviewed.927 In the liver only, the initial rate-limiting step involves conversion of cholesterol to 7·-hydroxycholesterol (60) by microsomal cholesterol 7·-hydroxylase (P-450c7; CYP7; EC 1.14.13.17) (the ‘neutral’ pathway).In the liver and elsewhere in the body,928,929 the mitochondrial enzyme sterol 27-hydroxylase930–932 eVects initial hydroxylation at C-27 of the sidechain, with subsequent 7-hydroxylation by a diVerent933–935 mitochondrial936,937 or microsomal937 7·-hydroxylase which is active towards both 27-hydroxycholesterol (61) and 25-hydroxycholesterol935,938 (the ‘acidic’ pathway).The acidic route leads predominantly to CDCA via either 5-cholestene- 3‚,7·,27-triol (62)936,939 or 3‚-hydroxy-5-cholestenoic acid (63)937 and a single 7·-hydroxylase is probably involved in both branches.940 The neutral pathway may be connected to the acidic pathway by side-chain oxidation of C27 steroids with the 3-oxo-ƒ4-7·-hydroxy nucleus.941 Cyclosporin A may be an inhibitor of the acidic pathway.942 Synthesis of several 2H-labelled potential intermediates in both bile acid biosynthetic pathways have been described.943 The following description relates to the major neutral pathway.944 Studies with cloned mouse cholesterol 7·-hydroxylase have shown that Leu-209 is located at a critical site in the heme–substrate binding pocket.945 Synthetic sterol probes were used to demonstrate that the presence of a 3‚-hydroxy group was essential for interaction between substrate and enzyme.946 The regulation of 7·-hydroxylase has been reviewed.927 In vivo, cholesterol is required to maintain 7·-hydroxylase activity947 (operating at the level of transcription); surprisingly, oxysterols also stimulate this enzyme in vivo,948 while progesterone is an inhibitor.949 Deoxycholic acid also regulates 7·-hydroxylases.950 Purification of both rat951,952 and human951 enzymes have been described.The CYP7 gene has been cloned from Syrian hamster,953,954 mouse,955 rat956–961 and humans962–966 and a cytosolic catalytically active enzyme, truncated at the membrane-binding N-terminus, has been obtained from both human967 and rat968 58 thermodynamic studies of the previtamin D3 to vitamin D3 conversion in human skin have revealed a much higher rate than for the same reaction in hexane, and it was suggested that interaction with intracellular lipids and/or proteins was responsible.899 In vitro studies have shown that the presence of ‚-cyclodextrins in aqueous solution can also cause a 40-fold rate enhancement relative to the reaction in hexane.900 Incorporation of an 11-hydroxy group into the previtamin D nucleus and expansion of the A-ring to a heptacycle resulted in a rate acceleration and a shifting of the equilibrium towards the vitamin side which was explained as a result of increased A-ring strain,901 whilst contraction of the A-ring to a pentacycle also led to a rate acceleration, for reasons which are not immediately obvious.902 (Similarly, an enhanced rate of conversion for 1·-hydroxyprevitamin D3 diacetate in benzene as compared with other solvents is not easily explained.903) Substitution of the 3-hydroxy group in previtamin D2 by electron-withdrawing groups shifts the equilibrium to the previtamin side, which has been explained in terms of Û*-� interactions.904 Thermal rearrangement of an unnatural (7Z) form of vitamin D has also been studied.905 3 is biologically inert and requires two successive Vitamin D hydroxylations to form 1,25-dihydroxyvitamin D [1,25- (OH)2-D3; 58] which is highly conformationally labile906 and has the important physiological function of maintaining blood calcium in the normal range.Cloned sterol-27-hydroxylase, previously identified as eVecting 27-hydroxylation of C-triol in the bile acid pathway (see later), was shown also to function as a vitamin D3 25-hydroxylase.907–911 Further hydroxylation of (25-OH-D 25-hydroxyvitamin D 3; 57) is achieved by a 25-hydroxyvitamin D3-1·-hydroxylase (1·-OHase);912 studies with cloned CYP27 have shown that this enzyme also possesses 1·-OHase activity for 25-OH-D Okamura has described several 25-hydroxyvitamin D tors of 1·-OHase.914–916 3 analogues as inhibi- The steady state concentration of active 1,25-(OH)2-D3 is determined by the relative rates of biosynthesis from 25-OH-D3 by 1·-OHase and catabolism by pathways involving 23- and 24-oxidations:917 the 24-oxidation pathway ultimately leads to side-chain degradation and formation of C calcitrioic acid (59), which can be excreted.918 Evidence has been presented that 1·-OHase and 24-hydroxylase activities are due to distinct polypeptides encoded by diVerent genes.919 Human 25-hydroxyvitamin D3-24-hydroxylase has been cloned920, 921 and shown to be a multicatalytic enzyme catalysing most, if not all, of the reactions in the C-24/C-23 pathways of 666 Natural Product Reports, 1998 11 HO 55 Cis-Vitamin D3 25 OH i ii 71 HO HO 57O 27 OH OH ii Acidic Pathway ii HO HO HO 63iii 61iii O OH OH ii 3 5 OH OH HO HO HO 65 62 OH60 iv iv iv O OH OH ii ii OH OH OH O O O 64vi, vii v CDCA 75 HO OH HO O H OH66vi, vii O C-diol 69 ii 22 HO SCoA OH b-Oxidation of side-chain OH O 25 THCA 68O ix O OH HO SCoA H OH HO H DHCA 70 viii O C-triol 67 ii 22 D24-THCA 71 x Neutral Pathway 33i 12 O HO OH OH O OH SCoA OH HO TeHCA 72 x H OH HO H CDCA 75 O O THCA 68 viii O SCoA HO OH 24-Keto-THC-CoA 73 O OH HO H OH CA 74 CA 74 Scheme 11 Acid and neutral pathways of bile acid biosynthesis.i, 7·-hydroxylase; ii, sterol 27-hydroxylase; iii, 7·-hydroxylase (diVerent to i); iv, 3‚-hydroxy-ƒ5- steroid oxidoreductase; v, sterol 12-hydoxylase; vi, 5‚-reductase; vii, 3·-HSD; viii, ‚-oxidation – see inset; ix, THCA-CoA oxidase; x, 2-enoyl-CoA hydratase/3-hydroxy-CoA dehydrogenasecDNAs expressed in Escherichia coli.The human gene has been localised to 8q11-q12.969 Prough has described 7-oxocholesterol obtained from 7·-hydroxycholesterol by the newly discovered enzyme 7·-hydroxycholesterol dehydrogenase (7·-HCD) in hamster.970 The second step in the neutral pathway is conversion of 7·-hydroxycholesterol to 7·-hydroxycholest-4-en-3-one (64) (now available by synthesis)971 by NAD+-dependent 3‚-hydroxy-ƒ5-C27-steroid oxidoreductase, catalysing both C-3 oxidation and concomitant double bond isomerisation from ƒ5 to ƒ4.The same enzyme accepts both 7·-hydroxycholesterol (60) and 3‚,7·-dihydroxycholest-4- enoic acids (e.g. 65) as substrate and is therefore common to both neutral and acidic pathways972 (however, no activity was found toward C19/C21 steroids and this enzyme is therefore distinct from the 3‚-HSD oxidoreductase involved in steroidal hormone biosynthesis). A quantitative HPLC method for measuring the activity of this enzyme has been developed973 and a clinical condition associated with enzyme deficiency has been reported.974 Sterol 12·-hydroxylase, catalysing the 12·-hydroxylation of 64, occurs at a branch point in the neutral pathway and is responsible for the balance between formation of CA and CDCA in bile acid biosynthesis. The enzyme has been purified to homogeneity from rabbit; the amino terminus was sequenced and found to be quite diVerent from that of other known P-450s.975 Cloning and sequencing of the rabbit CYP12 gene has confirmed that this cytochrome P-450 should be assigned a family of its own.976 ƒ4-3-Ketosteroid 5‚-reductase (5‚-reductase) catalyses the reduction of the the ƒ4-double bond to give a steroid with A/B rings in the cis-configuration.A cDNA has been cloned from rat977,978 and the cloned and expressed human enzyme utilises both 7·,12·-dihydroxycholest-4-en-3-one (66) and 7·-hydroxycholest-4-en-3-one (64) as substrates979 and is therefore involved in both branches, but shows no activity towards PROG (C21) or A-dione (C19) (there was detectable activity towards cortisol, however).A 3·-hydroxysteroid dehydrogenase (3·-HSD), which completes the reduction of the conjugated 3-keto system, has been cloned.980 24 27-Hydroxylase catalyses the first step in side-chain degradation leading to the C bile acids. Cloned sterol 27-hydroxylase (CYP27; EC 1.14.13.15) from man and rabbit is capable of performing three monooxygenation reactions at the 27-methyl group, converting 5‚-cholestane-3·,7·,12·-triol (C-triol; 67) into 3·,7·,12·-trihydroxy-5‚-cholestanoic acid (THCA; 68) by way of the 3·,7·,12·, 27-tetrol (27-OH-C-triol) and the corresponding 2aldehyde;981–983 the same enzyme also converts 5‚-cholestane-3·,7·-diol (C-diol; 69) into 3·,7·-dihydroxy-5‚-cholestanoic acid (DHCA; 70) via 5‚-cholestane-3·,7·,27-triol, albeit less eYciently.984 Mutations of the sterol 27-hydroxylase gene associated with the clinical condition cerebrotendinous xanthomatosis985,986 have been studied.Sterol 27-hydroxylase also has 25-hydroxylase activity907,913,987 (see vitamin D section). The final steps in the synthesis of bile acid involve ‚-oxidation of the sterol side-chain in a manner analogous to that of the peroxisomal ‚-oxidative cleavage of fatty acids.This topic has been reviewed.988 Biosyntheses of CDCA from DHCA and CA from THCA have been shown to proceed by the same pathway989 involving esterification with acetyl-CoA, introduction of a ƒ24-double bond, hydration of the double bond to a C-24 alcohol, oxidation to a 24-ketone990 (24-keto- THC-CoA is now available by synthesis)991 and cleavage of the C-24–C-25 bond to generate a 24-acid with accompanying elimination of propanoic acid (the conversion of THCA to CA only is depicted in Scheme 11).Both (25S)- and (25R)-THCAs were utilised by crude peroxisome preparations992 and all four possible 24,25-diastereoisomeric products of the intermediate 3·,7·,12·,24-tetrahydroxy-5‚-cholestanoyl-CoA (TeHCA; 72) – identified by comparison with synthetic tetrols993 (pentols are also available)994 – could be detected using either (25R)- or 668 Natural Product Reports, 1998 (25S)-THCA as substrates with mitochondrial preparations.995 However, in peroxisome preparations, both (25R)- and (25S)- THCA were converted only to 24R,25S-TeHCA via the intermediate (24E)-3·,7·,12·-trihydroxy-5‚-cholest-24-enoyl-CoA (ƒ24-THCA; 71).996 It may be possible to reconcile these somewhat conflicting results with the expectation that enzymes involved in the ‚-oxidation pathway should be stereospecific by noting the outcome of experiments with purified enzyme preparations.Thus, introduction of the double bond has been found to be catalysed by a distinct peroxisomal THCA-CoA oxidase, which is specific for the 25S diastereoisomer of THCA,997,998 even though this compound is expected to be present as the 25R diastereoisomer (as a result of the stereospecificity of the preceding steroid 27-hydroxylase reaction).Consequently, a THCA-CoA racemase999 (epimerase) must also be present in order for the THCA-CoA oxidase catalysed conversion of (25R)-THCA to ƒ24-THCA to occur.1000 ƒ24-THCA is then formed stereospecifically as the (E)-isomer.1001 Similarly, although the peroxisomal enzyme 2-enoyl-CoA hydratase/3- hydroxy-CoA dehydrogenase1002 was able to eYciently hydrate the double bond in ƒ24-THCA to (24S,25S)-TeHCA, the dehydrogenase component of the enzyme was then virtually inactive towards this product,1003 although the 24S,25R isomer was eYciently transformed: possibly, as noted in the preceding step, an additional racemase is required to interconvert the (25R) and (25S) forms, so that this enzyme may then convert ƒ24-THCA into 24-keto-THC-CoA (73) (alternatively more than one dehydrogenase may be present).Une has shown that purified mitochondria can further metabolise DHCA and THCA to the ƒ22-unsaturated analogues of CDCA (3·,7·-dihydroxy-5‚-chol-22-enoic acid; ƒ22- CDCA) and CA (3·,7·,12·-trihydroxy-5‚-chol-22-enoic acid; ƒ22-CA); whereas only CDCA and CA were obtained in peroxisomes.1004 3·,7‚-Dihydroxy-5‚-chol-22-en-24-oic acid (ƒ22-UDCA) has also been described as a metabolite of ursodeoxycholic acid (UDCA); it is speculated that this product arises from incomplete ‚-oxidation of UDCA.1005 2.3.4 Conversion of lanosterol into ergosterol in fungi The biosynthetic pathway leading from lanosterol (27) to the C28 steroid ergosterol (80) in yeast is shown in Scheme 12 and will form the basis for the discussion of ergosterol biosynthesis in this section (however, variations in the order of these reactions are encountered in other ergosterol-producing fungi).1006 The function,1007 structural requirements1008 and biosynthesis1009 of ergosterol as a bulk constituent of fungal membranes have been reviewed. All eight genes controlling the transformation of lanosterol to ergosterol in yeast have now been characterised;320,1010,1011 mutations associated with some of these genes have been reviewed.1012 Other reviews have appeared covering topics such as pharmaceutical and agrochemical inhibitors1013–1020 and the role of heme-containing proteins in the biosynthetic pathway.1021 One pressing reason for investigation of the ergosterol biosynthetic pathway is the increasing resistance of human fungal pathogens to known azole anti-fungal drugs, which is a particularly serious problem for patients undergoing prolonged AIDS therapy.1022 Lanosterol 14·-demethylase (sterol 14·-demethylase; P-45014DM; CYP51) is the first enzyme encountered in the transformation of lanosterol by yeast.(However, isolation of 24-methylene-24,25-dihydrolanosterol (eburicol) from Schizosaccharomyces pombe and S. octosporus has lead to the suggestion that an alternative ergosterol biosynthetic pathway also exists in yeast, similar to that found in the filamentous fungi, which involves a prior transmethylation at C-24 of lanosterol;1023 in addition P-45014DM from S.cerevisiae, for which lanosterol is the natural substrate, is also able to utilise eburicol.1024) In accordance with the proven 14,4,4- demethylation sequence for steroid biosynthesis in fungi shown in Scheme 12, it has been demonstrated that the activity24 8 4 HO 27 8 HO 7 1033 79v imazalil,1031,1044,1045 HO zole,1031,1033,1041–1043 1058 of fungal P-45014DM is unaVected by the presence of a 4‚-methyl group in the substrate: by contrast, in higher plants, for which the demethylation sequence 4,14,4- is followed (Section 2.4.1), P-450OBT·14DM was found to be unable to catalyse demethylation of 4‚-methylsteroids.1025 From experiments with isotopically labelled substrates it is suggested that the acyl-carbon bond cleavage reaction catalysed by recombinant P-45014DM from Candida albicans1026 follows the same mechanism as that proposed for P-45017· and aromatase in mammals (see Section 2.3.2, Scheme 9).The structure– function relationships of P-45014DM from Saccharomyces cerevisiae have been reviewed.1027 Azole antifungal1028 pesticides and drugs are known to act primarily as inhibitors of P-450 binding to the heme iron of the prosthetic group through their azole nitrogen. Several biological studies consistent with this role have been reported for the inhibitors itraconazole,1029– fluconazole,1032–1037 tetraconazole,1038–1040 ketocona- SSF-109,1046,1047 diniconazole,1048 SDZ 89-485,1049 flusilazole,1050 tebuconazole,1046,1050 fenarimol,1051 triadimenol,1052 prochloraz,1053,1054 cyproconazole1046 and various imidazole-1-carboxylates1055 in a wide variety of species.(Inhibition of P-45014DM was normally determined either by detecting a decrease in 4-desmethyl steroids and/or an increase in 14·-methyl steroids following administration, or from an investigation of UV diVerence spectra on binding of CO to the P-450).Resistance to azole fungicides has been studied using P-45014DM mutants from Ustilago maydis1056– and S. cerevisiae.1059 Novel azole inhibitors continue to be developed1060–1064 and synthetic methods have been reviewed.1065 Non-azole,1066 ster- Brown: The biosythesis of steroids and triterpenoids HO Scheme 12 Biosynthesis of ergosterol (80) from lanosterol (27) in yeast (genes encoding enzymes shown in parentheses).i, P-45014DM (ERG11); ii, ƒ14-reductase (ERG24); iii, C-4 sterol methyl oxidase (ERG25); iv, SMT (ERG6); v, ƒ7]ƒ8 SI (ERG2); vi, 5-desaturase (ERG3); vii, ƒ22-desaturase (ERG5); viii, ƒ24(28)-sterol reductase (ERG4) oidal1067 and the first natural product1068–1070 inhibitor (which coordinates the heme iron by a basic glycine substituent) have also been described. The ERG11 gene encoding P-450 been cloned from Ustilago maydis,1071 Erysiphe graminis hordei1072 and Candida glabrata.1073 The CYP51A1 gene from Saccharomyces cerevisiae has been expressed in tobacco1074 and the CYP51 gene encoding eburicol 14·-demethylase from Penicillium italicum has also been cloned.1075 The morpholine fungicide fenpropimorph has been con- firmed to inhibit at least three enzymes in the ergosterol pathway, including ƒ14-reductase,1076–1078 the enzyme which removes the ƒ14 double bond created by 14-demethylation; the piperidine fungicide piperalin is a relatively poor inhibitor of this enzyme.1079 A25822 B, a 15-azasteroid natural product which is thought to inhibit ƒ14-reductase by mimicking the putative C-14 cation generated during the course of the reduction (morpholine inhibitors are also believed to act in this manner), has now been obtained by chemical synthesis1080 and a synthesis of an A25822 B side-chain analogue has been reported.1081 New inhibitors with potential as fungicides include morpholine and piperidine derivatives,1063 guanidinium and amidinium derivatives1082 and 7-aminocholesterol.1083 The erg3 gene in Neurospora crassa, which may encode ƒ14-reductase,1084 has been sequenced;1085 the ERG24 gene has been cloned from Saccharomyces cerevisiae1086,1087 and Schizosaccharomyces pombe.1088 Strains defective in ERG24 accumulate ignosterol (ergosta-8,14-dienol);1089 which is a suitable sterol for aerobic growth.1090 The yeast ERG25 gene which encodes C-4 sterol methyl oxidase, one of three enzymes required to remove the two 80 HO iv HO vii ii 5 14DM by 24 22 7 8 14DM has 14 78 4 HO 76iii iii HO vii HO 77viii HO 6694-methyl groups in 4,4-dimethylzymosterol (76) has been cloned1010 (the other enzymes are a sterol 4-decarboxylase and a 3-ketosteroid reductase). 6-Amino-2-n-pentylthiobenzothiazole1091 is an inhibitor of sterol demethylation at C-4:1092 accumulation of 4-methylzymosterol (77) and 4-methylfecosterol in the presence of this inhibitor is interpreted as evidence that the two 4-demethylations are non-equivalent,1093 and that diVerent enzyme systems may be involved in 4-demethylation of 4,4-dimethylsterols and 4-methylsterols, as has been observed for higher plants.Following removal of the three methyl groups from the steroid nucleus, a new methyl group is now introduced into the side-chain by (S)-adenosyl-L-methionine:ƒ24(25)-sterol methyltransferase (SMT, EC 2.1.1.41).The structural features of sterols necessary for binding to yeast SMT have been determined from studies of inhibitors and structurally modified sterols, leading to a hypothetical model of the sterol–enzyme complex.1094 SMT catalyses transfer of a methyl group from (S)-adenosyl-L-methionine (AdoMet; SAM) to the si-face of the 24,25-double bond to yield a C-25 cation intermediate, followed by a 1,2-hydride shift of H-24 to C-25 (now confirmed to occur via what was the opposite re face of the double bond in yeast)1095 to produce a C-24 cation, which is subsequently converted to the ƒ24(28) product (see Scheme 14).Several steroids incorporating either S or N heteroatoms at the 24- and 25-positions, which were designed to mimic these proposed high energy cationic intermediates, have been found to be potent inhibitors of SMT.1096 The polyene antimycotic amphotericin B also inhibits this enzyme at high concentrations.1097 The yeast ERG6 (SED6)1098 gene which codes for SMT has been expressed in Escherichia coli and shown to convert zymosterol (78) to fecosterol (79).1099 Isomerisation of the ƒ8-double bond in fecosterol is mediated by sterol ƒ8]ƒ7 isomerase (ƒ8]ƒ7 SI; C-8 sterol isomerase).The mechanism of isomerisation is proposed to involve a C-8 cation intermediate: in consequence, many of the morpholine inhibitors of ƒ14-reductase (which is presumed to generate the adjacent C-14 cation as an intermediate) are also eVective inhibitors of this enzyme.1076,1077,1079,1082,1100–1102 Molecular modelling studies of fenpropimorph have indicated that the totally extended conformation is probably responsible for inhibitory activity of ƒ8]ƒ7 SI.1078 Novel morpholine inhibitors continue to be developed1063,1103 and other inhibitors also designed to mimic the putative cation intermediate have been described.1082,1104 7-Aminocholesterol1083 and the immunosuppresant SR 317471105 both inhibit this enzyme.The sigmaligand haloperidol specifically photoaYnity-labelled ƒ8]ƒ7 SI and may be of value in future studies to map the active site.1106 The ERG2 gene which codes for ƒ8]ƒ7 SI has been cloned from Saccharomyces cerevisiae,1107,1108 Magnaporthe grisea1109 and Ustilago maydis.1110 Yeast strains doubly mutant in ERG6 and ERG2 accumulate zymosterol as the major sterol component.1111 The ƒ7-double bond is next converted to a ƒ5,7-diene system in the B-ring by ƒ7-sterol 5-desaturase (C-5 sterol desaturase; 5-desaturase).The first inhibitors of 5-desaturase have been reported, incorporating ·-face heteroatoms in the vicinity of C-5.1112 The ERG3 gene which encodes 5-desaturase has been cloned from Candida glabrata1073 and Saccharomyces cerevisiae1113 and the yeast gene has been transferred to an Arabidopsis thaliana mutant deficient in this enzyme.1114 Mutations in the ERG3 gene have been used to probe the physiological role of membrane ergosterol in yeast1115 and defects in 5-desaturase in Ustilago maydis have been shown to be associated with resistance to azole antifungals (see P-45014DM).1116 The last two steps in ergosterol synthesis are mediated by ƒ22-desaturase (C-22 sterol desaturase) and ƒ24(28)-sterol reductase, which between them first introduce a new double bond in the side-chain (resulting in a steroid with two conjugated diene functionalities) and then remove the existing ƒ24(28) double bond.The ƒ22-desaturase, which is a P-450 cytochrome (P-450 61), has been purified from yeast1117 and the ERG5 gene 670 Natural Product Reports, 1998 which encodes this enzyme has been cloned.1011 Piperalin and fenpropidin both inhibit ƒ24(28)-sterol reductase.1079 2.4 Conversion of squalene epoxide into cycloartenol or ·- and ‚-amyrins in higher plants Several reviews concerning all aspects of plant steroid biosynthesis have appeared in the first half of this decade.1118–1123 Steroid biosynthesis in higher plants diVers from that in animals and fungi in that the cyclization product from (3S)- 2,3-oxidosqualene (SO) is either the 9,19-cyclopropylsteroid cycloartenol, or pentacyclic triterpenes, such as ·- and ‚-amyrins.Cycloartenol (81), the precursor of phytosterol primary metabolites such as stigmasterol and sitosterol (85), is formed by the action of 2,3-oxidosqualene-cycloartenol cyclase (OSCC, EC 5.4.99.8) whereas the huge variety of pentacyclic triterpenoid secondary metabolites encountered in plants, are generated by the action of 2,3-oxidosqualene-‚-amyrin cyclase (OS‚AC; EC 5.4.99.-).Both enzymes have been reviewed.367 SO substrate analogues in which C-10, C-19 and C-23 of SO (11) were replaced by N, were potent inhibitors of both enzymes, and are believed to mimic the corresponding carbocation intermediates formed during the cyclization process1124 (cf.oxidosqualene-lanosterol cyclase; Scheme 6); however, a monocyclic hydroxypiperidine analogue designed to mimic a high energy C-6 intermediate showed marked diVerences with respect to inhibition of OSCC and OS‚AC.396 U18666A, a known inhibitor of OSCC,1125 may also aVect the later stages of phytosterol biosynthesis.1126 The properties of both OSCC and OS‚AC purified from pea seedlings have been reported1127 and the cycloartenol synthase gene (CAS1) has been cloned from Arabidopsis thaliana and expressed in yeast.1128 Various elicitors have been reported to control metabolic flux between the steroid and the triterpene pathways, probably by acting at the level of these cyclases.1129,1130 It has been established that all of the three hydrogen rearrangements which follow the three backbone rearrangements in the biosynthesis of the pentacyclic triterpene ursolic acid are 1,2- hydride shifts, as expected for the accepted cyclization– rearrangement mechanism postulated in the formation of ·-amyrins.1131 A biomimetic synthesis of ‚-amyrin, employing a polyene which incorporated fluorine as a cation-stabilising auxiliary, has been reported.1132 2.4.1 Biosynthesis of sitosterol and campesterol Whereas animal cell membranes consist predominantly of a single steroid (cholesterol), plant cell membranes incorporate a complicated mixture of phytosterols, including sitosterol [(24R)-24-ethylcholesterol; 85], stigmasterol [(24S)-24-ethylcholesta-5,22-dienol], campesterol [(24R)-24-methylcholesterol; 88] and 22,23-dihydrobrassicasterol [(24S)-24- methylcholesterol; 24-epicampesterol; 87].Rather surprisingly, given the similarity in structure, sitosterol has been shown to be a membrane reinforcer, whereas stigmasterol is not.1133–1135 A detailed study of sterol biosynthesis in Zea mays has demonstrated that the composition of these and other steroids varies as a function of both plant organ and stage of development.123 An overview of the early stages of phytosterol biosynthesis, which are common to most plants, is provided in Scheme 13.Subsequent transformations of the steroid nucleus parallel those already discussed for animals and fungi, but higher plants are also able to further methylate the side-chain (Scheme 14), although the order in which such modifications to the nucleus and the side-chain occurs is variable.Biosynthesis of sitosterol from cycloartenol commences with transfer of a methyl group from (S)-adenosyl-L-methionine (AdoMet; SAM) to C-24 of cycloartenol, catalysed by the enzyme (S)-adenosyl-L-methionine: ƒ24(25)-sterol methyl transferase (SMT) to yield ƒ24(28)-methylene cycloartanol (82), which has been suggested to be the rate-limiting step in phytosterol biosynthesis.123,1136 An alternative methylation reaction yielding a ƒ23(24)-steroid (86) has been shown to24 19 9 ii i 11 HO HO 82 81 19 8 9 iv 3 HO O v 83 14 7 HO HO 3-Me)methionine1148 and (1,2- 13 Scheme 13 The early stages of phytosterol biosynthesis from SO (11) in higher plants.i, OSCC; ii, SMT; iii, 4-methyloxidase; iv, sterone reductase; v, COI the sequence 4,14,4 (in contrast to animals and fungi where the sequence is 14,4,4 – see sections 2.3.1 and 2.3.4).The 24-methylenecycloartanol substrate is suggested to adopt a bent conformation and it is the 4·-methyl group which is removed first.1150,1151 A reaction pathway proposed for removal of this 4-methyl group to yield cycloeucalenol (83), which is based on the isolation of reaction intermediates, is similar to that described previously for animals.1151 The initial oxidation step requires molecular oxygen and NAD(P)H, but does not involve cytochrome P-450.1152 The product of decarboxylation is suggested to be a 4·-methyl 3-ketosteroid (the remaining methyl group – which was ‚ in the 4,4-dimethyl substrate – has converted to the ·-configuration): Rahier has isolated an NADPH-dependent sterone reductase, with a preference for 4·-methylsteroids, which is capable of converting such ketones to 3‚-hydroxysteroids and has suggested that this is a constitutive component of the 4-demethylation complex.1153 The methyl oxidase and sterone reductase enzymes involved in oxidative 4-demethylation have been reviewed.1154 Removal of the second 4-methyl group at a later stage in phytosterol biosynthesis probably occurs by the same pathway, although a diVerent oxidative system seems to be involved.1151 Steryl esters,1155 formed under conditions of excess MVA supply, may regulate the rate of C-4 demethylation.1156 Following removal of the 4·-methyl substituent, the 9‚,19- cyclopropyl group in cycloeucalenol is opened to a ƒ8(9)- double bond by the enzyme cycloeucalenol-obtusifoliol isomerase (COI); this enzyme is inhibited by the systemic fungicide fenpropimorph.1157–1160 The subsequent removal of the 14·-methyl group of obtusifoliol (84) is performed by the cytochrome P-450-dependent obtusifoliol-14-·-demethylase (P-450OBT·14DM).This enzyme has been characterised from Zea mays and shown to require the presence of a ƒ8-double bond, but to reject substrates incorporating a 4‚-methyl substituent,1161 in accordance with the pathway shown in Scheme 13.Several azoles have been identified as inhibitors of 85 84 require a diVerent SMT.1137 SMT from sunflower uses cycloartenol preferentially over other substrates including zymosterol (78) (in contrast to the yeast enzyme which is specific for zymosterol – see preceding section): only a nucleophilic centre at C-3 and the ƒ24-double bond appear to be obligatory structural features for substrate binding and methylation; in addition, the cycloartenol nucleus is suggested to assume a flat (rather than a bent) conformation in the enzyme–substrate complex.1136 Nes has proposed that the methyl group adds to C-24 of the double bond from the si-face in Zea mays,1138 as found for fungi and marine organisms (although previous investigators have identified addition to the re-face for this species), and this is supported by results from Ajuga reptans.1139 Subsequent hydride migration of H-24 to C-25, converting the C-25 cation to a C-24 cation, would then be expected to occur via what was the re-face of the double bond1140,1141 and Seo has obtained experimental evidence for re-face hydrogen migration in Physalis peruvina.1142 A variety of inhibitors designed to mimic these presumed cationic intermediates have been investigated;1143 of these (24R,S),25- epiminolanosterol, incorporating an aziridine group at C-24(25), was found to be the most potent.1144 The cDNA for soybean SMT has been cloned1145,1146 and an SMT-encoding cDNA from Arabidopsis thaliana expressed in a yeast vector was found to be capable of performing two sequential methylations at the C-24 position of the steroid side-chain, leading to 24-ethylidene steroids (see later).1147 Results of feeding experiments with L-(2H C2)acetate/(2-13C,2-2H3)acetate1149 with cultures of Amsonia elliptica suggested that the side-chain saturated 24-epimeric phytosterols dihydrobrassicasterol (24‚-; 87) and campesterol (24·-; 88) are produced by initial formation of a ƒ24(28)-sterol which is isomerised to a ƒ24,(25)-sterol that may then undergo regiofacially specific reduction of the ƒ24(25)-double bond to yield either product.1149 It has now been directly established1150 that in higher plants the three methyl groups of the steroid nucleus are removed in Brown: The biosythesis of steroids and triterpenoids 28 23 24 iii HO OH iii 4 HO OH O 671P-450OBT·14DM1162–1168 (cf.inhibition of fungal P-45014DM) and new azole inhibitors have been described.1169,1170 7-Oxoobtusifoliol analogues are the first inhibitors reported to interact with the substrate-binding domain.1171 Reviews of inhibition1172 and substrate specificity1173 of this enzyme have appeared.P-450OBT·14DM from Sorghum bicolor has been partially sequenced and may belong to the CYP51 family found in animals and fungi: if so, this will be the only P-450 family so far known to be conserved across these phyla.1174 The ƒ14-double bond introduced during the 14- demethylation step is removed by ƒ14-reductase and it has now been shown directly that the resulting ƒ8-steroid is transformed to a ƒ5-steroid in three stages: initial isomerisation of the double bond to the 7-position, introduction of a new conjugated double bond at the 5-position and finally reduction of the ƒ7-double bond.1175 ƒ8]ƒ7-Isomerase (ƒ8]ƒ7 SI), catalysing the first step in this sequence, is inhibited by morpholine fungicides,1157,1159 which also inhibit ƒ14- reductase1159 (cf. inhibition of both these enzymes in fungi).Introduction of the ƒ5-double bond, to yield ƒ5,7-sterols, is catalysed by ƒ7-sterol C-5-desaturase (EC 1.3.3.2; lathosterol oxidase, 5-desaturase) which has been characterised for the first time from Zea mays and shown to require O2 and NADH; this enzyme is stated to be similar to other eukaryotic desaturases.1175 An Arabidopsis thaliana mutant which is deficient in C-5 desaturase has been identified1114 and a cDNA encoding this activity was later isolated.1176 The conjugated ƒ7 -double bond is reduced by the NADPH-dependent enzyme ƒ5,7- sterol-ƒ7-reductase (ƒ7-reductase) which has also been cloned from A.thaliana.1177 ƒ7-Reductase is inhibited by morpholine fungicides1178 and 6-aza-B-homosteroids:1179 both observations are consistent with a previously proposed mechanism, analogous to that described for ƒ14-reductase and ƒ8]ƒ7 SI, which would involve initial protonation at C-8 to generate a C-5/7 delocalised cation. The squalene epoxidase inhibitor terbinafine may also inhibit this enzyme.345 3- Formation of the C29 steroid sitosterol (85) requires the transfer of a second methyl group to the side-chain 24(28)- double bond by the enzyme (S)-adenosyl-L-methionine sterol methyltransferase (SMT).When supplied with L-(2H Me)methionine, tissue cultures of Physalis peruviana incorporated a maximum of four deuterium atoms into the 24·-ethylsterol sitosterol, indicating that biosynthesis proceeds through a 24-ethylidene intermediate [branch (a); Scheme 14],1180 whilst up to five deuterium atoms appeared into the 24‚-ethylsterols (89) of Trichosanthes kirilowii, indicating that a 24-ethylidene intermediate is not involved in this transformation [branch (b); Scheme 14].1181 Results from Ajuga reptans have confirmed this pathway to 24‚- ethylsteroids.1139 Triazole fungicides may interfere with the second transmethylation step.1168 The unique quaternary carbon containing side-chain of 24-methylene-25-methylcholesterol in Phaseolus vulgaris was shown to arise from methylation of 24-methyldesmosterol,1182 demonstrating for the first time that higher plants are able to synthesise steroids with highly alkylated quaternary carboncontaining side-chains – a biosynthetic capability previously only attributed to sponges (see Section 2.6).This compound has also been reported for the first time from a Chlorophyte freshwater alga;1183 a Chlorophyte alga has been shown to be able to de-alkylate steroid side-chains,1184 which again is a capability previously only ascribed to insects and marine sponges. 2.4.2 Biosynthesis of plant steroids and triterpenes as secondary metabolites In addition to the primary metabolites discussed in the previous section which have a function in membrane stabilisation, plant steroids can also serve as secondary metabolites with a wide variety of roles.Phytoecdysteroids such as ecdysone (92) and 20-hydroxyecdysone (93) are plant compounds which 672 Natural Product Reports, 1998 biosynthesis.1190–1193,1195–1197 scc in section 2.3.2).1213,1214 mimic the eVect of insect moulting hormones. They diVer from conventional plant steroids in possessing a 6-oxo-ƒ7-nucleus with a cis-A/B ring junction, no alkyl substitution of the side-chain, and hydroxylation at the 2-, 14-, 20, 22-, and 25-positions.Unlike insects, plants are capable of the de novo biosynthesis of phytoecdysteroids, which is thought to proceed from acetate and mevalonate via cholesterol and lathosterol and has been studied in Spinacia oleracea,1185–1188 Zea mays,1189 Serratula tinctoria,1190 Ajuga reptans,1191,1192 and Polypodium vulgare.1193 The biosynthesis, regulation and distribution of phytoecdysteroids has been reviewed1194 and plant tissue culture seems to be a particularly promising vehicle for studying phytoecdysteroid Ecdysone 20-monooxygenase from spinach, which catalyses formation of 20-hydroxyecdysone from ecdysone, has been shown to be a cytochrome P-450.1198 The brassinosteroids are a new and important group of plant growth hormones.Feeding experiments with Catharanthus roseus, Triticum aestivum, Nicotiana tabacum and Oryza sativa involving labelled precursors and GC-MS analysis have determined that the most biologically active and widely occurring brassinosteroid, brassinolide (BL; 105), is derived from campesterol (88) by the following sequence: 881199] campestanol (94)1199]6·-hydroxycampestanol (95)1199]6- oxocampestanol (96)1199/cathasterone (97)1200]teasterone (TE; 98)1201,1202]3-dehydroteasterone(99)1203–1205]typhasterol (TY; 100)1201,1202]castasterone (CS; 101)1201,1206]105; and have also indicated the presence of an alternative pathway, in which the 6-oxo-group is introduced at the last step proceeding as follows: 3-dehydro-6-deoxoteasterone (102)1207]6- deoxotyphasterol (103)1207]6-deoxocastasterone (104)1208] 101]105.Brassinolide can be metabolised by 23-Oglycosylation in mung bean,1209 while 24-epi-brassinolide and 24-epi-castasterone have been shown to undergo 25-hydroxylation/1210glycosylation,1211 conjugation to fatty acids1212 and side-chain cleavage to 20-ketopregnanes, following introduction of a 20-hydroxy group (cf. mechanism of P-450 Biosynthesis of 5‚-cardenolides in Digitalis species1215 (Scheme 15) is believed to occur in three stages: (i) conversion of cholesterol to 3‚,14‚,21-trihydroxy-5‚-pregnan-20-one (115) via pregnenolone (34) and progesterone (35), (ii) construction of a butenolide ring at C-20/C-21 and (iii) glucosidation at C-3.Several of the enzymes involved in forming the 3‚-hydroxy-5‚-pregnane skeleton have been isolated and characterised, including ƒ5-3‚-hydroxysteroid dehydrogenase/ƒ5- ƒ4-ketosteroid isomerase (3‚-HSD) which converts pregnenolone to progesterone,1216 progesterone-5‚-reductase1217–1219 forming 5‚-pregnane-3,20-dione (114) (suggested to be the first committed step in cardenolide biosynthesis),1220 and 3‚-hydroxysteroid 5‚-oxidoreductase.1218,1221,1222 (N.B. progesterone 5·-reductase1220 and 3·-hydroxysteroid 5‚-oxidoreductase1223 are also normally present and may serve to remove precursors such as 35 and 114 from the cardenolide pathway, thereby contributing to the overall regulation of cardenolide biosynthesis).Formation of the butenolide ring (117) has been suggested to involve transfer of a malonyl moiety to the 21-hydroxy group to form a malonyl hemi-ester (116) (catalysed by malonyl-coenzyme A: 21-hydroxypregnane 21-hydroxy-malonyltransferase) and subsequent Claisen-type condensation of the malonyl moiety with the C-20 ketone, accompanied by decarboxylation1224 (rather than Claisen-type condensation as the initial step, as previously assumed).Enzymes involved in the final glycosylation stage have been described from Digitalis species1225,1226 and Nerium oleander.1227 The biosynthesis of 5·-cardenolides in Asclepias curassavica has also been studied1228–1230 and the topic of cardenolide biosynthesis has been reviewed.1231 Steroidal sapogenins such as diosgenin (106)1232 and steroidal alkaloids such as solanidine (107),1233 compounds probably involved in defence against fungal pathogens, are believed to be biosynthesised from cholesterol (Nohara hasiv 24 i H Me ii 33 25 + FUNGIH 91 23 86 Brown: The biosythesis of steroids and triterpenoids 87 (24b) 88 (24a) Scheme 14 Modifications to the steroid side-chain in fungi, plants and insects (numbers refer to characteristic side-chains of certain sterols – nucleus not specified).i, si-face methylation by SMT; ii, re-face hydride migration by SMT; iii, SMT; iv, ƒ24-reductase isolated a 26-aminocholestanol derivative which may be an intermediate in the formation of the steroidal alkaloids).1234 Sequential addition of single monosaccharides to the free 3-OH group of these steroid aglycones, resulting in steroidal saponins and steroidal glycoalkaloids respectively, is catalysed by UDP-glucose:sterol ‚-D-glucosyltransferase (UDPGSGTase).Several cytosolic UDPG-SGTases with specificities for either steroidal sapogenins1235,1236 or steroidal alkaloids1237–1241 have been reported: by contrast, ‘general purpose’ UDPG-SGTases appear to be membraneassociated.1239–1243 The biosynthesis of steroidal glycosides in Dioscorea species1244 and steroidal glycoalkaloids in potato1245 has been reviewed. The withanolides are a large group of biologically active steroids from the Solanaceae family which are characterised by a lactone-containing side-chain and oxidised A/B rings.The putative precursor, 24-methylenecholesterol, is suggested to undergo cleavage between C-24 and C-25 during withanolide side-chain modification in Acnistus breviflorus,1246 while oxidation at C-22 and C-26 was shown to be suYcient to account for lactol formation in the side-chain of Nic-1 (108) in Nicandra physaloides.1247 Formation of the unusual six-membered aromatic D-ring in Nic-1 occurs by ring expansion with oxidative inclusion of the C/D angular methyl substituent.1248 Limonoids are a group of potent insect anti-feedants of which possibly the best known is azadirachtin from the Indian Neem tree (Azadirachta indica).However, limonin (109)1249 and nomilin (110),1250,1251 highly degraded seco-A, seco-D ring steroids which are responsible for the bitter taste of citrus juice, have been the subject of most metabolic studies. The enzyme which interconverts the D-ring lactone dilactone, limonin, with the non-bitter monolactone, limonoate A-ring lactone, has been purified.1252 Other transformations such as the glycosylation of nomilin1253 or its conversion to nominilic acid1251 and the oxidation of limonoate A-ring lactone by limonoate dehydrogenase1254 have also been described.lathyris,1262–1264 Biosynthetic studies have been made of the pentacyclic friedooleane triterpene bryonolic acid1255–1258 and the monocyclic iridal triterpenes.1259–1261 More general studies of triterpene biosynthesis are reported from Euphorbia oYcinale,1265 Dryopteris Taraxacum crassirhizoma1266 and Glycyrrhiza glabra.1267,1268 2.5 Biosynthesis of steroids in insects As for other animals, insects require cholesterol both as a component of membranes and as a substrate for production of hormones; however, they are unable to manufacture their own steroids.Consequently, many insects must rely on dietary sources of cholesterol or convert the major C28 and C29 phytosterols to cholesterol (e.g.sawflies,1269 Locusta migratoria1270 and Manduca sexta1271). The dealkylation of phytosterols to cholesterol1272,1273 (Scheme 14) and alternative strategies adopted by phytophagous insects which are unable to eVect side-chain dealkylation,1273 have been reviewed. Inhibition of insect steroid metabolism has also been reviewed.1274 Side-chain cleavage of the 24,28-epoxides of fucosterol (90) and isofucosterol, which creates a ƒ24-double bond and eliminates acetaldehyde, is suggested to involve migration of H-25 anti to C-24 of the cleaved epoxide group.1275 Subsequent conversion of the ƒ24-steroid desmosterol (91) to cholesterol (33) requires a sterol ƒ24-reductase: studies with Manduca sexta have shown that a variety of sterols can be reduced1276 and the microsomal enzyme has been characterised.1277 The C-25 hydrogen is introduced stereospecifically from the si-face of the double bond.1272 The most intensively studied area of insect steroid biosynthesis is the modification of dietary steroids to ecdysteroids, the hormones which control insect moulting behaviour.(N.B. biosynthesis of vertebrate-type hormones has also been reported from a few insect species – the significance of which has been reviewed.1278,1279) Ecdysteroid biosynthesis,1271,1280,1281 and specific topics such as suicide INSECTS 28 O 24 H 90 O–+ 28 24 iii 82 HIGHER PLANTS H(a) (b) + iii(a) H (b) H iii(b) H24 89 85 673R6 R1 24 22 25 20 14 2 R7 R2 A 7 B6 R3 R4 H O O HO 106O 92 R1 = R4 = H; R2 = R3 = R5 = R6 = R7 = OH 93 R1 = R2 = R3 = R5 = R6 = R7 = OH; R4 = H 111 R1 = R5 = R6 = R7 = H; R2 = OH; R3R4 = O 112 R1 = R2 = R5 = R6 = R7 = H; R3R4 = O 113 R1 = H; R2 = R5 = R6 = R7 = OH; R3R4 = O 3 B OOA O A¢ 674 Natural Product Reports, 1998 H inhibition1282 and P-450 monooxygenases involved in biosynthesis have been reviewed.1283 The dietary or endogenous origin of ecdysteroids in gastropods remains an open question.1284 The universally observed first step in ecdysteroid biosynthesis is conversion of cholesterol to 7-dehydrocholesterol (7-DHC; 54)1285 mediated by a microsomal cytochrome P-450 monooxygenase;1286 cholesterol analogues modified with a 5·,6·-epoxy or imino group are mechanism-based suicide inhibitors of this enzyme1287 and the conversion is suppressed by the steroid 5·-reductase inhibitor L-645390.1288 Luu has described a synthesis of (1,2-3H2)-7-DHC of high specific activity for use in biosynthetic studies.1289 The precise details by which the steroid nucleus of 7-DHC acquires 6-keto and 14·-hydroxy groups are still not resolved, although several possible intermediates have been described.1290–1292 The cis-A/B ring junction appears to arise from a 3-oxo-ƒ4-intermediate1293 which is acted upon by a 5‚-reductase: one possible reduction product of this enzyme, 14·-hydroxy-5‚-cholest-7-ene-3,6-dione (5‚-diketol; 111), was found to be eYciently converted to ecdysone (92)1294 whereas 5‚-cholest-7-ene-3,6-dione (5‚-diketone; 112) was not,1295 emphasising that 14·-hydroxylation is probably an early step in the biosynthetic sequence.Sequential 25-, 22R- and 2‚-hydroxylation reactions constitute the later stages of ecdysteroid biosynthesis and suicide-substrate type acetylenic or allenic inhibitors have been designed to inhibit hydroxylations at each of C-25,1296–1298 C-221297–1303 and C-2.1304 A variety of P-450 inhibitors were also observed to aVect the 109 R5 R7 23 R1 R2 3 O C O HO D O HR3 R4 O OO R5 23 22 6 N R6 26 107 AcO 17 O22-hydroxylase, suggesting that this is the most sensitive of the terminal hydroxylases towards inhibition.1305 3-Dehydroecdysone (113) is converted to ecdysone (92) by 3‚-dehydroecdysone-3‚-reductase: this NAD(P)H-dependent enzyme has been purified and characterised from Spodoptera littoralis.1306 Ecdysone-20-monooxygenase (EC 1.14.19.22) which then converts ecdysone to 20-hydroxyecdysone (93) has also been characterised from cricket1307 and EPR studies of this cytochrome P-450 have been reported.1308 RH 5849 is an inhibitor of this enzyme;1309 azole-derivatives of metyrapone show a greater inhibitory aYnity toward insect ecdysone 20-monooxygenase than to mammalian microsomal monooxygenases, opening up the possibility of designing selective inhibitors of 20-hydroxyecdysone biosynthesis.1310 Somewhat unexpectedly, ecdysone does not appear to be a precursor of 20-hydroxyecdysone in the terrestial amphipod Orchestia cavimana.1311 2.6 Biosynthesis of steroids in marine organisms Marine organisms are characterised by steroids with unusual structural features such as an A-nor or 19-nor nucleus and multiply alkylated side-chains, which sometimes contain cyclopropane or cyclopropene rings.Steroid structure and biosynthesis have been reviewed.1312–1315 Radiolabelling experiments have defined two steroid biosynthetic pathways in the sea cucumber Eupentacta fraudatrix: the first involves transformation of squalene to parkeol, and subsequent demethylation to 4·,14·-dimethylcholest-9(11)-en-3‚-ol and 14·-methylcholest- 9(11)-en-3‚-ol; the second proceeds through squalene to 110 23 20 25 24 OH HO HO H O OH 24 OD 105 O 26 22 OH 2 5 O O OH 108 OOH CO2H OO20 O ii i 35 34 3 5 O H 114iii OH 21 O 14 OH HO H H O O HO CoAS 115iv OH O O O O O O OH O O OH O – CO2 – H2O OH OH OH 117 116 Scheme 15 Biosynthesis of cardenolides in higher plants.i, 3‚-HSD; ii, progesterone 5‚-reductase; iii, 3‚-hydroxysteroid-5‚-oxidoreductase; iv, malonyl-coenzyme A:21-hydroxypregnane-21-hydroxy-malonyl transferase lanosterol and then to 5·-cholest-7-en-3‚-ol and its xyloside.1316 Most sponges are also now believed to be capable of de novo sterol biosynthesis.1317 An investigation of eleven sponges revealed that most were able to produce between 40–80% of their own sterol requirement with the rest coming from dietary sources1318 (although all species studied contained ƒ22-sterols, none were biosynthetically able to introduce the ƒ22-double bond).The sea cucumber Stichopus californicus is able to reversibly isomerise ƒ5-double bonds in steroids of dietary origin to ƒ7-double bonds probably via a ƒ5,7- intermediate;1319 the sponge Dysidea fragilis is also able to transform dietary ƒ5-sterols to ƒ5,7-sterols.1320 Biosynthesis of 19-norsterols in the sponge Axinella polypoides was suggested to involve conversion of a dietary ƒ5-3‚-hydroxy sterol to a ƒ4-3-keto-intermediate: this functional group was then well placed to assist 19-decarboxylation once the 19-methyl group has been oxidised to a carboxylic acid.1321 Marine sponges are able to dealkylate the side-chain of dietary steroids in a manner analogous to the oxidative pathway used by insects1322,1323 (see Scheme 14); however, unlike insects, sponges are also capable of the reverse process, S-adenosyl-L-methionine-mediated (SAM) alkylation.Cellfree extracts have been used to study both dealkylation1322 and alkylation1324 reactions and, rather surprisingly, both processes may be operative at the same time.1323 Djerassi has demonstrated that H+ loss from the tertiary cation generated during the conversion of 24-methylenecholesterol to isofucosterol/fucosterol [Scheme 14; arrow iii(a)] occurs antito the original methylation (Scheme 14; step iii).1325 The three methylations involved in the biosynthesis of the quaternarycarbon containing side-chain of mutasterol (118) have been shown to occur in the order C-24, C-26, C-25.1326 Biosynthesis of various 23-methylated side-chain steroids such as gorgosterol (119), dinisterol (120) and peridinosterol (121) is thought to involve more than one SAM-sterol methyl transferase.1327 Brown: The biosythesis of steroids and triterpenoids Djerassi has previously proposed that the disparate ensemble of structurally diverse C29 and C28 sterols with unique cyclopropane-containing and acyclic olefinic side-chains from Haplosclerid sponges could be accounted for by a single unified biosynthetic scheme, involving rearrangements of a nonclassical cation (Scheme 16; C29 only shown).The formation of this ion is now suggested to occur by removal of H-23 which is catalysed by an aberrant ƒ22-desaturase.1328,1329 Ab initio calculations for the rearrangement of protonated ethylcyclopropane cations support some of the unusual cation rearrangements which are postulated to occur in this biosynthetic scheme.1330 The acyclic side-chain of 24- propylidenecholesterol may be formed from a protonated cyclopropyl intermediate.1331 3 Prokaryotes 3.1 Biosynthesis of isopentenyl pyrophosphate in eubacteria The early steps of isoprenoid biosynthesis in several eubacteria are clearly diVerent from those in eukaryotes.1332 Rohmer originally proposed that eubacterial IPP was derived from pyruvate and a triose phosphate, rather than acetyl-CoA, in a pathway reminiscent of the biosynthesis of L-valine.1333 It was later shown that the C5 isoprene units of eubacteria arise from condensation of thiamine-activated acetaldehyde (C2; 123), derived from pyruvate (122) decarboxylation, with the carbonyl group of glyceraldehyde 3-phosphate (GAP; 124) (C3 ), derived from dihydroxyacetone phosphate, which is followed by a transposition step (Scheme 17).1334 It is now generally accepted that chloroplasts, the almost ubiquitous sub-cellular organelles found in plants, have a prokaryotic origin, and may be viewed as prokaryotic symbionts which co-exist inside the cells of their eukaryotic partner.In this connection, Rohmer has provided evidence from the 13C-labelling pattern of plastid-derived steroids in a green alga fed with 13C-labelled glucose and acetate that the early steps of isoprenoid biosynthesis in plastids also proceed by the GAP/ pyruvate (Rohmer) pathway1335 and not via the classical acetate/mevalonate pathway previously described for animals and fungi.Bach has suggested that the operation of two independent routes to IPP in higher plants (i.e. cytosolic mevalonate biosynthesis and a compartmentalised Rohmer pathway)121 might go a long way to explaining several ambiguous findings concerning isoprenoid biosynthesis123 in this kingdom.However, other workers have failed to detect this pathway in chloroplasts.1336 3.2 Biosynthesis of hopanoid triterpenes from isopentenyl pyrophosphate in eubacteria The C35 bacteriohopanepolyols are amphiphilic molecules, unique1337 to eubacteria, which are postulated to perform a role in membrane-stabilisation1338 and are believed to be analogous in function to cholesterol, sitosterol and ergosterol in eukaryotes.They are formed from a pentacyclic triterpene (C30) nucleus, derived from squalene, and a C5 n-alkyl polyfunctionalized side-chain, derived from a D-pentose (C3 sidechains are also known), which are linked together by a carbon–carbon bond. Sometimes additional methyl groups are introduced at the 2- and 3-positions of the triterpene nucleus and sugar residues may be attached to the side-chain via glycosidic or ether linkages. Several reviews have appeared concerning the structure, function and biosynthesis of hopanoids.1339–1343 Although biosynthesis of IPP in eubacteria diVers from that in eukaryotes (see previous section), the conversion of IPP to squalene is believed to be broadly comparable between the two.Thus, Zymomonas mobilis was found to accumulate farnesyl pyrophosphate (FPP; 9), presqualene diphosphate (PSPP; 25), squalene (SQ; 10), hop-22(29)-ene (diploptene; 67526 25 24 118 H 2425 23H23 25 24 H B–: Scheme 16 Highly alkylated steroid side-chains from sponges (118–121) and a unified biosynthetic scheme for biosynthesis of sterol side-chains in Haplosclerid sponges H HO O ON+ N+ H+O – HO O S 122 H+ O H PO OH 124 HO N+ N: S H O S HO S 123 OH OP H+ HO O H OP O H O OH OH OP Scheme 17 GAP/pyruvate (Rohmer) pathway to IPP (7) in eubacteria 125) and hopan-22-ol (diplopterol; 126).1344 A eubacterial IPP isomerase from Rhodobacter capsulatus is the smallest such enzyme yet identified and may consist mostly of the fundamental catalytic core for the enzyme.1345 An FPS has been isolated from Escherichia coli216 and FPS from the prokaryote Bacillus stearothermophilus has been studied by X-ray crystallography.1346 Neither of the cysteine residues were found to be essential for catalytic function;1347 other results parallel those for the eukaryotic enzyme: in particular Asp-244 and Asp-255 have an essential catalytic function whilst Lys-47 and Lys-183 are important in binding.1348 In addition, a conserved domain of hydrophobic Phe-Gln residues may be important for interaction with the alkyl 676 Natural Product Reports, 1998 2824 22 23 119 25 H24 Me 23 + H 25 H24 Me 23 OPP 7 moieties of the substrates.1349 Mutagenesis of the amino acid at position 81 (five residues before the aspartate-rich motif) alters the chain length of the final product (which is found to be inversely proportional to the accessible surface area of the substituted amino acid);1350 thus, FPS can be converted into a geranylgeranyl phosphate synthase (GGPS).1351 The FPS from B.stearothermophilus has less tolerance for oxygen-containing artificial substrates when compared with the eukaryotic enzyme.1352 FPS from B.stearothermophilus1353,1354 has been cloned. The mechanism by which prokaryotic squalene synthase converts FPP to SQ in Zymomonas mobilis may be diVerent from that described for eukaryotes since in the absence of NADH an unidentified conversion product, not corresponding to presqualene alcohol, was isolated in good yield.1355 128 23 23 121 120 22 OH 29 126 125 30 35 33 NH2 OH 34 OH 32 31 OH 127 OH 3The cyclization step, in which squalene-hopene cyclase (SHC; EC 5.4.99.-) forms the triterpene component of bacteriohopanepolyols, diVers from that for eukaryotic sterol biosynthesis in that SQ itself, rather than 2,3-oxidosqualene (SO), is the natural substrate and that cyclization proceeds from an all-prechair transition state rather than a prechairpreboat-prechair-chair conformation.Bacterial squalenecyclases have been reviewed.367 Eubacterial SHCs generally have quite low substrate specificities and will also accept both (3R)- and (3S)-SO, resulting in formation of 3·- and 3‚-hydroxyhopanes.As found for the eukaryotic oxidosqualene cyclases, conserved aspartate residues (Asp-376 and Asp-377) are important for the catalytic activity of SHC and it has been suggested that these amino acids are present in order to stabilise the charge of intermediate cations generated during the cyclization.1356 2-Aza-2,3-dihydrosqualene has been found to inhibit SHC,1357 which would certainly be consistent with such a mechanism.SHC has been purified and characterised from Alicyclobacillus acidocaldarius (Bacillus acidocaldarius)1358 and Rhodopseudomonas palutsris.1359 The SHC gene has been cloned from Zymomonas mobilis,1360 Bradyrhizobium japonicum1361 and A. acidocaldarius1362 and expressed in Escherichia coli in each case. A C-ribosylhopane (aminobacteriohopanetriol; 127), which is a postulated intermediate in the biosynthesis of the bacteriohopanepolyols from the C30 triterpenes, has been obtained by synthesis.1363 Methionine has been identified as the source of the transferred methyl group in a novel series of C-31 methylated hopanoids from Acetobacter europaeus.1364 Gammacerane triterpenes, such as tetrahymanol (128), which are isomeric with hopanes, are also encountered in eubacteria, although they are not formed by SHC.1359 However, a squalene-tetrahymanol cyclase has been reported from the lower eukaryote protozoon Tetrahymena thermophila which is similar to SHC in several respects, including low specificity and use of SQ as a substrate1365 [(3S)- and (3R)-SO were also converted to gammacerane-3‚,21·-diol and gammacerane-3·,21·-diol by the enzyme from T.pyriformis].1366 3.3 Biosynthesis of core isoprenoid lipids in archaebacteria Eukaryotes and eubacteria are believed to have diverged from the archaebacteria at an early stage in evolution. Archaebacterial cell membranes incorporate no structural steroids or triterpenes such as those found for eukaryotes and eubacteria, but instead consist predominantly of saturated C20 (sometimes C25 or C40) linear isoprenoid ether derivatives of glycerol (or more complex polyols) such as 130, rather than the glycerol fatty acid esters found in other organisms. These core lipids are formed from IPP by the action of geranylgeranyl diphosphate synthase (GGPP synthase; GGPS) and 3-Ogeranylgeranylglyceryl phosphate synthase (GGGP synthase) (Scheme 18). Archaebacterial core lipids are included in this review because of shared aspects of function and biosynthesis (via the isoprenoid pathway) with steroids and triterpenes found in the other four kingdoms. The isoprenoid pathway in archaebacteria leading to the core lipids is thought to resemble that in eukaryotes. Thus, archaebacteria contain a 3-hydroxy-3-methylglutaryl Coenzyme A reductase (HMGR) involved in the synthesis of mevalonate and this enzyme is inhibited by lovastatin (mevinolin) just like its eukaryotic counterpart.1367 The HMGR gene from Haloferax volcanii has been expressed in Escherichia coli and is suggested to employ a similar catalytic mechanism to that proposed for eukaryotes.1368 An IPP isomerase has also been described from Methanobacterium thermoautotrophicum and Halobacterium halobium.1369 The GGPP synthase from M. thermoautotrophicum catalyses formation of both FPP and GGPP from IPP and DMAPP, in contrast to eubacteria and eukaryotes where distinct FPP synthases and GGPP synthases are assigned specific func- Brown: The biosythesis of steroids and triterpenoids OH PO 23 OH + i 7 PPO OH PO + OPP O 129ii ii + PPO O PO O + OPP 25) 677 130 Scheme 18 Biosynthesis of arachaebacterial core lipids: i, GGPS; ii, GGGP synthase tions.1370,1371 Random chemical mutagenesis of GGPS (EC 2.5.1.29) from Sulfolobus acidocaldarius has shown that the enzyme activity can be channelled into synthesis of longer prenyl chains [e.g. farnesylgeraniol diphosphate (FGPP) (C and hexaprenyl diphosphate (C30)] and led to the suggestion that a single amino acid (Phe-77), located five residues upstream from the aspartate-rich motif involved in binding the pyrophosphate moiety of the substrates, regulates chain elongation1372 (cf. results obtained with eukaryotic FPS in section 2.2 and eubacterial FPS in section 3.2). [Interestingly, a ‘true’ FGPP synthase (EC 2.5.1.X), which synthesises the C25-prenyl diphosphate required for the C25 moiety of C20,C25 diether lipids of haloalkaliphilic archaebacteria, has now also been described.1373] GGPS has been purified and characterised from Methanobacterium thermoformicicum;1374,1375 genes encoding GGPS have been cloned from M. thermoautotrophicum (idsA)1371 and Sulfolobus acidocaldarius.1376 Although it is often assumed that geranylgeranyl diphosphate (GGPP; 129) is always synthesised de novo by GGPS, Ohnuma has demonstrated an alternative possibility (perhaps a salvage pathway) involving phosphorylation of geranylgeraniol by geranylgeraniol kinase and geranylgeranyl phosphate kinase.1377 Poulter has studied the subsequent prenyl transfer reaction of GGPP to glycerol catalysed by GGGP synthase and concluded that two distinct prenyltransferase activities are responsible for sequential formation of the two ether bonds at the 3- and 2-positions of (S)-glyceryl phosphate.1378 He has also found that the substrate specificity is similar for prenyltransferases from M. thermoautotrophicum and H. halobium.1369 4 Evolutionary considerations In this review, the distinctions in biosynthesis of isoprenoidderived membrane components between mammals (cholesterol), fungi (ergosterol), plants (sitosterol), eubacteria (bacteriohopanepolyols) and archaebacteria (archaebacterial core lipids) have been stressed. These are not hard and fast divisions: for example, Giner has reported an isolated example of biosynthesis of cycloartenol, lanosterol and hopanes in the higher plant Euphorbia lathyris.1379 However, the apparent generality of such observations has lead some authors to speculate on the evolution of steroid and triterpene metabolism. Ourisson, for example, has interpreted the unusualcomposition of Euphorbia latex as evidence that cycloartenol, which would have functioned as a reinforcing constituent in early plant membranes, is a primitive state and that the shift towards cyclization to lanosterol is a more advanced state.1380 Similarly, it has been shown from X-ray crystallography that prokaryote hopanoids occupy a smaller volume than cholesterol (for example) and it was suggested that hopanoids are primitive membrane components which have been replaced by the more eVective steroids in eukaryotes during the course of evolution.1338 This might then account for the observation that amongst the eukaryotes only plants retain a significant capacity to biosynthesise triterpenes – and only then as secondary metabolites. One scenario envisaged for this shift in metabolic capability is that cyclization of SQ to hopanes evolved at a time when there was no oxygen in the atmosphere, and that utilisation of SO to form steroids by eukaryotes evolved later in response to the appearance of atmospheric oxygen. Only further research will be able to shed more light on these fascinating hypotheses. 5 References

ISSN:0265-0568    

DOI:10.1039/a815653y

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Book review Carbohydrate chemistry Ed. G.-J. Boons Blackie 1998 508 pp. ISBN 0751403962 Over recent years the carbohydrate field has witnessed a series of significant advances that have contributed greatly to our increased awareness of the potential and importance of glycosides and related glycoconjugates. It is also apparent that this is an area characterised by the breadth of disciplines involved and we now have new and eYcient methods for synthesis that provide a host of diVerent carbohydrate structures. Many of these molecules have therapeutic potential and the study and characterisation of these structures also demands access to sensitive and sophisticated analytical and computational methods. The aim of this book is to bring together the contributions associated with these diVerent disciplines and to highlight to the reader both the connectivities involved and the varied directions that research is following.The book’s contributors eleven in all are very experienced in their fields and their eleven chapters bring together a series of topics with the chemistry of complex oligosaccharides and glycoconjugates as a focus. The first four chapters are of an introductory nature and are similar to those found in more standard textbooks. These include discussions of the structural and conformational properties of simple monosaccharides the nature and use of protecting groups the manipulation and functionalisation of monosaccharides and methods available for the synthesis of O-glycosides.One might argue that this information can be found elsewhere but these chapters are very clearly presented well referenced and oVer to the less experienced reader an up-to-date introduction to the field and more importantly set the scene for the remainder of the book. The bulk of this book deals with the construction properties and study of complex carbohydrates. The strategic and tactical issues associated with oligosaccharide synthesis are presented and this is followed by a discussion of the chemistry of Oand N-linked glycopeptides neoglycoconjugates cyclic oligosaccharides (cyclodextrins) and modified carbohydrates (aza- carba- thio- and phosphasugars C-glycosides and other carbohydrate mimics). Rene Roy’s contribution on the rapidly developing topic of neoglycoconjugates was the highlight for me in the way that he set about illustrating the diversity complexity and possible utility of structures now available.Chapter 10 deals with the therapeutic potential of complex sugars and the final chapter focuses on the physical methods available for characterising carbohydrate structures. The book does reach its goal of bringing together the contributions and interplay associated with those disciplines involved with the development of the chemistry of complex carbohydrates. The individual chapters are of a high standard and are generally well balanced. The editor has also ensured that this text is both manageable but also highly informative and certainly stimulating. The breadth of the subject is there for all to see and I would strongly recommend this book to those either already active or contemplating becoming involved in carbohydrate chemistry. Timothy C. Gallagher University of Bristol UK 697

ISSN:0265-0568    

DOI:10.1039/a815697y

出版商:RSC    

年代:1998

数据来源: RSC