摘要:

ISSN 0265-0568 NPRRDF 14(3) 205-308 (1997) Natural Product Reports A journal of current developments in bioorganic chemistry Volume 14 Number 3 CONTENTS ... 111 Hot off the press Robert A. Hill and Andrew R. Pitt Reviewing the recent literature on natural products and bioorganic chemistry 205 Synthesis of amino acids incorporating stable isotopes Nicholas M. Kelly Andrew Sutherland and Christine Willis Reviewing the literature published between 1990 and mid 1996 221 The biosynthesis of the gibberellin plant hormones Jake MacMillan Reviewing the literature published up to September 1996 245 Diterpenoids James R. Hanson Reviewing the literature published in 1995 259 Marine natural products D. John Faulkner Reviewing the literature published in 1995 303 Amaryllidacae alkaloids John R.Lewis Reviewing the literature published in 1995 Cumulative Contents of Volume 14 Number 1 1 Brassinosteroids Shozo Fujioka and Akira Sakurai 11 Quinoline quinazoline and acridone alkaloids (July 1994 to June 1995) Joseph P. Michael 21 Indolizidine and quinolizidine alkaloids (July 1994 to June 1995) Joseph P. Michael 43 Lignans neolignans and related compounds (January 1994 to December 1995) Robert S. Ward 75 Cyclopeptide alkaloids (January 1985 to December 1995) Dimitris C. Gournelis Gregory G. Laskaris and Robert Verpoorte Number 2 83 Recent advances in chemical ecology (July 1992 to December 1995) Jeffrey B. Harborne 99 The role of carbohydrates in biologically active natural products Alexander C.Weymouth-Wilson 111 The biosynthesis of C,-C, terpenoid compounds (1993 to 1995) Paul M. Dewick 145 Natural sesquiterpenoids (1995) Braulio M. Fraga 163 Fatty acids fatty acid analogues and their derivatives (1988 to 1995) Marcel S. F. Lie Ken Jie Mohammed Khysar Pasha and M. S. K. Syed-Rahmatullah 191 Diterpenoid and steroidal alkaloids (mid-1994 to the beginning of 1996) Atta-ur-Rahman and M. Iqbal Choudhary Number 3 205 Synthesis of amino acids incorporating stable isotopes (1990 to mid 1996) Nicholas M. Kelly Andrew Sutherland and Christine Willis 221 The biosynthesis of the gibberellin plant hormones (up to September 1996) Jake MacMillan 245 Diterpenoids (1995) James R. Hanson 259 Marine natural products (1995) D. John Faulkner 303 Amaryllidacae alkaloids (1995) John R.Lewis Articles that will appear in forthcoming issues include Biosynthesis of fatty acids and related metabolites (up to end 1994) Bernard J. Rawlings Biosynthesis of plant alkaloids and nitrogenous microbial metabolites (1995) Richard B. Herbert Phenethylamine and isoquinoline alkaloids (July 1995 to June 1996) Kenneth Bentley Recent progress in chemistry of non-monoterpenoid indole alkaloids (July 1995 to June 1996) Masataka Ihara and Keiichiro Fukumoto Chemistry and biosynthesis of clavulanic acid and other clavams Allan Brown Steroids reactions and partial synthesis (1995) James R. Hanson Monoterpenoids (part 1993 all 1994 part 1995) David H. Grayson Natural products derived from unusual variants of the shikimate pathway H. G. Floss Biosynthesis of polyketides (mid 1993 to end 1994) Bernard J. Rawlings Coumarins (January 1995 to December 1996) A. EstCvez-Braun and A. G. Gonzhlez COPIES OF CITED ARTICLES The Library and Information Centre (LIC) of the RSC offers a first class Document Delivery Service for items in Chemistry and related subjects. Contact the LIC The Royal Society of Chemistry Burlington House Piccadilly London W1V OBN. Tel +44 (0)171 437 8656 -Fax +44(0)171 287 9798 -Email library@rsc.org This service is only available from the LIC in London and not the RSC in Cambridge.

ISSN:0265-0568    

DOI:10.1039/NP99714FP005

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

Natural Product Reports Editorial Board Professor T. J. Simpson (Chairman) University of Bristol Dr J. R. Hanson University of Sussex Dr R. B. Herbert University of Leeds Professor J. Mann University of Reading Professor D. J. Robins U niiversity of G lasgow Dr C. J. Schofield University of Oxford Dr D. A. Whiting University of Notting ham Editorial Staff Editorial Office Dr. Sheila R. Buxton The Royal Society of Chemistry Managing Editor Thomas Graham House Dr Roxane M. Owen Science Park Deputy Editor Milton Road Miss Nicola P. Coward Cambridge Production Editor UK CB4 4WF Dr Carmel M. McNamara Technical Editor Telephone+44 (0) 1223 420066 Mrs Dawn J. Webb Facsimile +44 (0) 1223 420247 Miss Karen L. White E-mail perkin 62rsc.org Editorial Secretaries RSC Server h ttp://chemist ry.rsc. org/rsc/ Natural Product Reports is a bimonthly journal of critical reviews. It aims to foster progress in the study of bioorganic chemistry by providing regular and comprehensive reviews of the relevant literature published during well-defined periods. Topics include the isolation structure biosynthesis biological activity and chemistry of the major groups of natural products-alkaloids terpenoids and steroids aliphatic aromatic and 0-heterocyclic compounds. This is augmented by frequent reviews of the wider context of bioorganic chemistry including developments in enzymology nucleic acids genetics chemical ecology primary and secondary metabolism and isolation and analytical techniques which will be of general interest to all workers in the area.Articles in Natural Product Reports are commissioned by members of the Editorial Board or accepted by the Chairman for consideration at meetings of the Board. Natural Product Reports (ISSN 0265-0568) is published bimonthly by The Royal Society of Chemistry Thomas Graham House Science Park Milton Road Cambridge UK CB4 4WF. 1997 Annual subscription rate f355.00; US$640.00. Customers in Canada will be charged the sterling price plus a surcharge to cover GST. Change of address and orders with payment in advance to The Royal Society of Chemistry The Distribution Centre Blackhorse Road Letchworth Herts. UK SG6 1HN. Air freight and mailing in the USA by Publications Expediting Service Inc. 200 Meacham Avenue Elmont NY 11003.US Postmaster send address changes to Natural Product Reports Publications Expediting Service Inc. 200 Meacham Avenue Elmont NY 11003. Periodicals postage paid at Jamaica NY 11431 -9998. All other despatches outside the UK are by Bulk Airmail within Europe and Accelerated Sutface Post outside Europe. Printed in the UK. Members of the Royal Society of Chemistry should order the journal from The Membership Manager The Royal Society of Chemistry Thomas Graham House Science Park Milton Road Cambridge UK CB4 4WF. 0 The Royal Society of Chemistry 1997 All Rights Reserved No part of this publication may be reproduced stored in a retrieval system or transmitted in any form or by any means electronic mechanical photographic recording or otherwise without the prior permission of the publishers. Printed in Great Britain by Henry Ling Ltd at the Dorset Press Dorchester Dorset.

ISSN:0265-0568    

DOI:10.1039/NP99714FX013

出版商:RSC    

年代:1997

数据来源: RSC

ISSN:0265-0568    

DOI:10.1039/NP99714BX015

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

Syntheses of amino acids incorporating stable isotopes Nicholas M. Kelly Andrew Sutherland and Christine L. Willis School of Chemistry University of Bristol Cantock’s Close Bristol UK BS8 1TS Covering 1990 to mid 1996 1 Introduction 2 a-Amino acids with hydrocarbon side-chains (alanine isoleucine leucine phenylalanine and valine) 2.1 General methods 2.2 Isotopic labelling of the diastereotopic methyl groups and hydrogens of amino acids 3 a-Amino acids with aliphatic hydroxylated or thiolated side-chains (serine threonine cysteine methionine and tyrosine) 4 a-Amino acids with a carboxylic acid in the side-chain (aspartic and glutamic acids) 5 a-Amino acids with nitrogen in the side-chain (aspar- agine arginine glutamine histidine lysine proline and tryptophan) 6 References 1 Introduction Amino acids incorporating stable isotopes are important for a range of studies within bioorganic chemistry including metabolic studies and to facilitate the elucidation of the 3D-structures of peptides and proteins by NMR spectroscopy.Reflecting the importance of these studies there has been considerable recent effort towards developing methods for the syntheses of amino acids incorporating stable isotopic labels. There have been a number of excellent books’ and review^^,^ on the subject of asymmetric synthesis of unlabelled a-amino acids. However reviews on the synthesis of isotopically labelled amino acids (or isotopomers4) have been rather more limited but do include reports by Yonaha and Soda’ within their article ‘Applications of Stereoselectivity of Enzymes Synthesis of Optically Active Amino Acids and a-Hydroxy Acids and Stereospecific Isotope-Labelling of Amino Acids Amines and Coenzymes’ and by Voges6 in ‘Stereoselective Procedures in the Synthesis of Enantiomerically Pure Isotopi- cally Labelled Compounds’.In 1991 Young gave a valuable summary of methods for the synthesis of isotopomers of a series of pro teinogenic and non-proteinogenic amino acids based on the use of biotransf~rmations,~ and Adriaens and Vanderhaeghe have described methods for the ‘Synthesis of Some Labelled Non-proteinogenic Amino Acids’.8 The most recent review in this area has been compiled by Winkler et al. and describes the major routes for the synthesis of amino acids and their isotopomers based on reaction categories.’ Our review concentrates on methods for the synthesis of I3C-,*H- I8O-and ”N-labelled proteinogenic a-amino acids published in the literature from 1990 to mid 1996.It is structured by class of amino acid. Although there are a variety of ways of grouping amino acids we have used a classification based on four types of side-chain as shown in Table 1. When a particular paper describes a general method for the synthesis of a range of isotopically labelled amino acids then this approach is included in the first section (i.e. a-amino acids with hydrocarbon side-chains) and then referenced back in the appropriate section of the review. 2 a-Amino acids with hydrocarbon side-chains (alanine isoleucine leucine phenylalanine and valine) 2.1 General methods The incorporation of ’’N-labelled amino acids into peptides and proteins is of particular value in the analysis of 3D structure by NMR spectroscopy as it increases the dispersion of signals in the spectra and hence assists assignment of the complex spectra.tert-Butoxycarbonyl- (Boc) protected amino acids are important precursors in peptide synthesis and Ragnarsson and co-workers prepared Boc[”N]glycine via nucleophilic attack of the potassium salt of di-tert-butyl [15N]imidodicarbonate on ethyl bromoacetate followed by removal of one of the Boc groups with a slight excess of trifluoroacetic acid (TFA) and saponification of the ester.’” ’’ Since ethyl [I-”C]- [2-I3C]- and [1,2-’3C2]-bromoacetates are commercially available this approach was simply adapted to give any combination of nitrogen-1 5 and/or carbon- 13 labelled glycine and glycine amides in excellent yield.The use of the lithium salt of Boc,”NH was extended to give a general approach to the enantioselective synthesis of ”N-labelled Boc amino acids from a-hydroxy acids (Scheme l).l2>l3 In situ diazotisation and hydrolysis of an amino acid gives the corre- sponding a-hydroxy acid with retention of configuration at C-2. Protection of the acid as the benzyl ester 1 followed by activation of the alcohol as the triflatet gave 2. Treatment of the triflate with the lithium salt of Boc,I5NH led to formation of the diBoc-protected amino ester 3 with inversion of configu- ration.Selective removal of one Boc group was smoothly achieved with only a slight excess of TFA and finally catalytic hydrogenolysis of the ester 4 furnished the desired ’’N-labelled tTriflate =trifluoromethanesulfonate. Table 1 Classification of amino acids according to side-chain Hydrocarbon Alanine (Aia) Isoleucine (Ile) Leucine (Leu) Phenylalanine (Phe) Valine (Val) Hydroxylated or Nitrogen thiolated Carboxylic acid containing Cysteine (Cys) Aspartic acid (Asp) Asparagine (Asn) Methionine (Met) Glutamic acid (Glu) Arginine (Arg) Serine (Ser) Glutamine (Gln) Threonine (Thr) Histidine (His) Tyrosine (Tyr) Lysine (Lys) Proline (Pro) Tryptophan (Trp) Kelly Sutherland and Willis Syntheses of amino acids incorporating stable isotopes Table 2 Reductive amination of a-keto acids to L-amino acids using dehydrogenases 0 y2 Amino acid dehydrogenase RKC02-Na+ RAC02H NH4+ NADH NAD+ BAA Recycling dehydrogenase For alcohol dehydrogenase A = EtOH and B = MeCHO For formate dehydrogenase A = HC02- and B = CO2 Amino acid Recycling L-Amino acid dehydrogenase dehydrogenase Yield Ref.'NIAla Ala Alcohol 69% 16 ["NIAla Ala Formate 93% 18 [3-I3C]Ala Ala Formate 62% 9 [3-' 3C,1 'NIAla Ala Formate 62% 18 Leu Leu Formate 80% 9 Leu Glu Formate 60% 9 ["NILeu Leu Formate 95% 18 (2S,4R)-[5,5,5-2H3]Leu Leu Formate 85% 19 [''NlIle Leu Formate 74% 67 [''N]Alloile Leu Formate 74% 67 Ile-Alloile mixture Glu Formate 64% 9 [I 'NIPhe Phe Formate 92% 18 Val Glu Formate 57% 9 [ 'NIVal Leu Formate 84% 18 (2S,3R)-[4,4,4-2H3]Val Leu Formate 80% 67 ["NISer Ala Formate 66% 18 Glu Glu Formate 70% 9 ["NJGlu Glu Formate 86% 22 ["N]Glu Glu Alcohol 86% 20 [3- I3C]Glu Glu Alcohol 85% 20 [4-I3C]Glu Glu Alcohol 83% 20 [5-I3C]Glu Glu Alcohol 75% 20 [3,4J3C2]Glu Glu Alcohol 80% 20 [2-2H]Glu Glu Alcohol 70% 21 [3,3-,H,]Glu Glu Alcohol 88% 21 [4,4-,H,]Glu Glu Alcohol 70% 21 RYC02H i RyC02H RYC02CH2Ph based on the use of enzyme catalysed reactions (in particular ~ with dehydrogenases) to establish the stereogenic centre at C-2.NH2 OH OH Cooper and Gelbard" have published the first report for the 1 general enantioselective synthesis of a-[I3N]amino acids using 1 iii immobilised glutamate dehydrogenase to catalyse the reduc- tive amination of a-keto acids to a-amino acids.This method- RvC02CH2Ph -v RvC02CH2Ph iv RyC02CH2Ph ology is valuable for the synthesis of nitrogen-13 labelled I5NHBoc ~~NBoc~ OTf compounds where due to the short half-life of the isotope fast 4 3 2 reaction time is essential. However it is not economic for the vi synthesis of gram quantities of l'N-isotopomers due to 1 the need for stoichiometric amounts of the co-factor NADH. RvC02H Barzu and co-workers overcame this problem by regenerating the NAD' in situ to NADH using an alcohol dehydrogenase- I5NH Boc ethanol recycling system (Table 2). l6 Thus L-[I 'Nlalanine Scheme 1 Reagents i NaNO, H,SO,; ii Cs,C03 DMF PhCH,Br; was prepared in 69% yield from pyruvate using alanine iii Tf20 lutidine CH2C12; iv "NHBoc, BuLi THF; v TFA (1.5 dehydrogenase.equiv.) CH,CI,; vi H,/Pd MeOH Chanatry et al. extended this work using a glutamate dehydrogenase-amino acid transferase-glucose dehydrogenase coupled system (Scheme 2). l7 a-Ketoglutarate was reductively Boc amino acid with approximately 98% ee. This basic aminated to ["N]glutamate which in turn served as an amine approach has been applied to the enantioselective synthesis of donor to other a-keto acids using an amino transferase to give a series of "N-labelled Boc amino acids including (R)-and the corresponding ["Nlamino acids. NADPH used as the (S)-alanine (from ethyl lactate)I2 and (R)-and (5')-leucine as hydride source in the reductive amination step was regenerated In addition using glucose dehydrogenase and [ 'N]ammonium sulfate as well as both enantiomers of ~henylalanine.'~ further isotopically labelled non-proteinogenic a-amino acids the source of isotopic label.In addition the aminotransferase may be prepared from the vast array of both (2R)- and catalysed the exchange of the a-proton so when the reaction 2-*5N]amino acids could be (2S)-hydroxy acids available from the lactate dehydrogenase was conducted in 2H,0 L-[~-~H catalysed reduction of a-keto acids.I4 prepared. Deuteriation was also extended to the P-position A number of general methods for the synthesis of L-amino by pre-exchanging the a-keto acid in 2H,0-base prior to the acids labelled in the a-amino function have been reported enzymatic transformations.This methodology enabled the 206 Natural Product Reports 0 15NH2 Amino transferase -R*C02H + + 15NH2 Glutamate Y 2H 2H de hydrogenase V HO2CAC02Na H#Co2Me HOZC~Co2H *-2H+$C02Me NADP+ NADPH R NHAc R NHAc C6H1206 %Hi207 dehydrogenase Scheme 2 ~-[''NlVal (79%); ~-[''NlLeu (81%); ~-["NlMet (79%); ~-[2,3-~H,, "NIVal (72%); L-[2,3,3-,H3 "N] Leu (65%); ~-[2,3,3-'H,, ''NlPhe (72%)) synthesis of a variety of isotopically labelled a-amino acids including ["NI-Leu -Met and -Val as well as [2,3,3-2H3 "NILeu [2,3-'H2,''N]Val and [2,3,3-2H3,'5N]Phe on a gram scale. Willis and co-workers have used a formate dehydrogenase (FDH) catalysed system to recycle NADH to prepare a series of ~-["N]arnino acids from a-keto acids and amino acid dehydrogenases.l8 Formate dehydrogenase catalyses the oxi- dation of formate ions to carbon dioxide and releases hydride ions which react with NAD' thus driving the reversible reductive amination reaction in the required direction.[''N]Ammonium formate was a convenient source of both isotopic label and formate ions. It was shown that leucine dehydrogenase was a more efficient catalyst than alanine dehydrogenase for the reductive amination of a-keto acids with branched hydrocarbon side-chains giving ["NI-valine -leucine,'* -alloisoleucine and -isole~cine'~ in good yields (Table 2). Alanine dehydrogenase was also used to prepare L-[~-'~C, "Nlalanine from [3-13C]pyruvic acid which in turn was prepared from ['3C]methylmagnesium iodide and diethyl oxalate at low temperature." Winkler et al.have compared various conditions for the reductive amination of a-keto acids to L-amino acids catalysed by glutamate alanine and leucine dehydrogenases.' When FDH was used to recycle NADH they used a mixture of sodium formate and ammonium chloride as the source of the requisite ammonium and formate ions but in general these conditions gave lower yields than with ammonium formate (Table 2). Glutamate dehydrogenase has also proved valuable for the synthesis of a range of iso- topically labelled glutamic acids from 2-oxoglutaric (for further details see section 4). Biotransformations have also been used by Myasoedov et al. to prepare a series of carbon-13 and nitrogen-1 5 labelled amino acids using sodium acetate and ammonium sulfate as the sources of isotopic labels.23 A general method for the conversion of racemic amino acids to homochiral products has been reported by LeMaster and co-workers using an oxidase-aminotransferase coupled sys- The approach has been applied to the synthesis of L-[I-'3C]valine.25 Transformations based upon enzyme catalysis have proved valuable for the preparation of a series of a-deuteriated L-amino acids.26 The approach involves incubation of an unlabelled L-amino acid in D20 with lyophilised E.coli B/It7-A cells abundant in tryptophanase. The relatively broad substrate specificity enables the preparation of a range of a-deuteriated proteinogenic L-amino acids (Val Leu Ile Met Phe His and Arg) as well as a series of tyrosine analogues in reasonable yields (43-76%) and with approximately 95% ee.Homochiral a-and P-deuteriated amino acids have also been prepared by LeMaster and co-workers using cystathionine y-~ynthase.~' Nishiyama and co-workers have used a com- bined chemical-enzymatic approach for the synthesis of L-threo- and L-erythro-[1-' 3C 2,3-2H2]amino acids (including Leu Phe Tyr and further non-proteinogenic amino acids with aromatic side chains) as novel probes for conformational analysis of peptide side chains (Scheme 3).28 Stereoselective vii D-threo7 -D-threo7 + 1 RvNH 2 + 2H 2H L-erythro 7 L-threo 8 Scheme 3 Reagents i AcNHCH,CO,H AcONa Ac,O; ii Na,C03; iii (MeO),POCH(NHAc)CO,Me DBU; iv 'H, catalyst; v acylase 37 T;vi aqu.Na02H Me02H; vii Na02H Ac20 incorporation of deuterium into the a$-positions of protected amino acid 7 was accomplished by catalytic deuteriation of dehydroamino acid derivatives 5 and 6 and was followed by a resolution with acylase giving good yields of the ~-threo-[2,3- 2H2]amino acids 8. For the L-erythro isomers it was then necessary to racemise the remaining D-threo isomer 7 (from the initial resolution) and then to conduct a further resolution. This approach was extended to the preparation of the cor- responding [1-' 3C]amino acids using [1 -'3C]glycine as the starting material. Chen et al. described a general method for the synthesis of a-[2H]amino acids and applied the approach to the preparation of [2-2H]phenylalanine.29 Racemic [2-2H]amino acids were prepared by heating the corresponding amino acid with 0.05 equiv.of benzaldehyde in deuteriated acetic acid. Conversion of the acid to the methyl ester followed by an alcalase catalysed resolution gave the homochiral amino acid with >99% incorporation of deuterium. A further approach to the synthesis of a-deuteriated a-amino acids was reported by Gani and co-~orkers~~.~' and utilises the other major common approach for the synthesis of a-amino acids -namely with chiral auxiliaries and templates. Base catalysed deuteriation of Schollkopfs bis-lactim ethers (3R)- or (3S)-3-isopropyl-2,5-dimethoxy-3,6-dihydropyrazine in refluxing Me02H-2H20 gives the [6-2H2]isotopomer 9 without disturbing the stereogenic centre at C-3 (Scheme 4).91ii C02Me /t-2H H2N R 1 iv + C02Me /.LyRMeMe0 2H t C02H ,+H H2N R Scheme 4 Reagents i Me02H-2H20 KOH (1 equiv.) reflux 3 h; ii BuLi THF then RBr (1.5 equiv.); iii HCl then separate by flash chromatography; iv deprotection Kelly Sutherland and Willis Syntheses of amino acids incorporating stable isotopes Alkylation at C-6 of 9 followed by hydrolysis provided convenient and efficient access to a range of (R)-and (3-a- deuteriated a-amino acids (including Phe Ser and Asp) with approximately 95% ee. A number of chiral auxiliaries and chiral glycine equivalents have been used to prepare a-amino acids including Williams' oxazinones Evans' oxazolidinones 11,33 Schollkopf's favoured the latter method for the preparation of further isotopically labelled amino acids including Boc-[ 1,2-' 3C2, ''Nlalanine Boc-[ 1 ,2-I3C2 ''N]phenylalanine and their 3-deuteriated isotopomers.The basic procedure for the preparation of a-amino acids via the sultam is depicted in Scheme 6 with the synthesis of V MeSy,NY$O2Me * I- 12 SMe Me0aoMe 10 11 \ x r-"do vNH But- NJ S0,l Boc 13 14 1 bis-lactim ethers 12,34Oppolzer's sultams 1335,36 and Seebach's ii + imidazolidinones 1437(for further details see review^'^^). Al-though all have proved extremely valuable in the enantioselec- tive synthesis of a-amino acids it is the latter three which have been used most extensively in the preparation of isotopomers.For example Ragnarsson and co-workers have prepared D-[ l,2-I3C2 "N]leucine a-methyl ester using [1,2-13C2,15N] glycine methyl ester and L-valine methyl ester to assemble the labelled bis-lactim ether 15 (Scheme 5).38 Alkylation of the * 16 1 ii D-Boc-[1,2-l 3C2 15N]leucine Scheme 5 Reagents i BuLi THF then CH,(CH,)CHCH,I; ii HCI then separate; iii Boc,O N-methyl morpholine N-oxide (NMMO) then NaOH lithium salt of the bis-lactim with 1-iodo-2-methylpropane gave 16 which on hydrolysis gave ~-[l ,2-l3C2 "N]leucine a-methyl ester and L-valine methyl ester which were separated by column chromatography. Treatment of the former with Boc,O followed by saponification furnished D-Boc-[ 1,2-I3C2 ''N]leucine in 3 I YOyield calculated from labelled glycine.Although the Schollkopf bis-lactim approach is a versatile and useful procedure for the preparation of a range of isotopi- cally labelled amino acids a major problem is the removal of the valine by-product. A method which overcomes this prob- lem is one devised by Oppol~er~~,~~ where a sultam is used to form the stereogenic centre at C-2. Interestingly Ragnarsson and co-~orkers~~ directly compared the use of the Schollkopf's method with the Oppolzer's approach for the preparation of D-Boc-[~ ,2-I3C2 "N]leucine (the use of the sultam is described in more detail below). They found that although the yields were comparable the enantiomeric excess was 97.2-97.4% via the bis-lactim ether and 99.7% with the sultam.Hence they 208 Natural Product Reports v JlV L-Boc-[1 ,2-13C2,i5N]leucine 19 Scheme 6 Reagents i Me,Al; ii BuLi HMPA CH,(CH,)CHCH,I; iii HCI; iv LiOH THF; v Boc,O L-[ 1,2-'3C2,15N]leucine. Formation of the bis(methylsulfany1) glycinate 17 from ['3C,,15N]glycine proceeds in high yield. Reaction of 17 with the commercially available (2R)-bornane- 10,2-sultam 13 gives 18 which may be alkylated via the lithium salt. For example reaction with 1 -iodo-2-methylpropane gives after crystallisation pure (2R,2'S)-2-{N-bis(rnethylsulfanyl) methylene[1,2-13C2,15N]le~~yl) bornane-l0,2-sultam 19. De-protection of the amine with acid and alkaline hydrolysis of the chiral auxiliary gives homochiral L-Boc-[ 1,2-13C2 "N] leucine in approx 34% yield from labelled glycine.General procedures for the synthesis of ["C]- and [I5N]- amino acids based on the diastereoselective alkylation of [N-(diphenylmethylene)glycinyl]camphorsultam have been For devel~ped.~'?~~ nitrogen-1 5 labelling the { N-(diphenylmethylene)['5N]glycinyl)camphorsultam was synthe- sised from ["N]phthalimide potassium salt prepared in turn from [15N]ammonium chloride. Alkylation with a range of electrophiles has led to the synthesis of carbon-I3 and nitrogen-15 labelled Ala Asn Asp Cys Gln Leu Phe Ser Trp and Val.43 The a-functionalisation of N-acyl sultams 20 with N-electrophiles has proved extremely valuable for the synthesis of a range of I3C-and "N-labelled a-amino acids.6 Oppolzer and co-workers have developed a method which uses l-chloro- I-nitrosocyclohexane (CNC) as the NH2+equivalent (Scheme 7).44-45Reaction of sultam 20 with NaHMDS followed by addition of CNC gives a nitrone which may be hydrolysed to hydroxylamine 21.Reduction of 21 with zinc and acid gave the amine 22 and finally hydrolytic cleavage of the sultam gave a range of pure (R)-amino acids; for example allyl- glycine alanine leucine and valine have been prepared in 5545% yield and >99% ee from 20. Using l-chloro-l-['5N]nitr~~~cyclohexane46 and the enantiomer of sultam 20 \/ v 13 20 22 21 Scheme 7 Rruggents i NaH toluene RCH,COCI; ii Zn aqu. HCl AcOH 0 "C; iii LiOH H,O THF; iv HCl; v Dowex 50 H' Unkefer and Lodwig have used this approach to prepare "N-labelled L-Ala Leu Val Phe and L-[ 1-I3C ''Nlvaline with good yield enantioselectivies (97.2-99.5% ee).47348 Oppolzer has also shown that (E)-a,P-unsaturated enoyl sultams 23 will undergo conjugate addition with either organo- magnesium or organocopper reagents.Trapping of the result- ant enolate with CNC provides a route to a-amino acids with i ii 0 23 1 iii iv y2 +C02H i L-isoleucine Scheme 8 Reugents i EtMgBr; ii CNC H'; iii Zn aqu. HCl AcOH 0 "C; iv LiOH then ion exchange an asymmetric centre at C-3 (Scheme 8). Using this route iso- leucine was prepared in 55% yield from 23 with 90% de at C-3 [and solely the (,!+stereochemistry at C-2].49 This approach has been used extensively by Voges at Sandoz Pharma Basle to prepare a range of isotopically labelled amino acids in good yields and with excellent optical p~rities.~~" A further class of chiral glycine equivalent which has been used widely to prepare isotopically labelled amino acids is imidazolidin-4-one developed by Seebach.Alkylation of Boc-BMI 14 followed by deprotection gives a range of a-amino acids in >99% ee.51p56 Both (R)-and (9-1-(tert-butoxycarbony1)-2-tert-bu t yl- 3 -me th ylimidazolidin-4-ones (Boc-BMI) are commercially available thereby allowing the synthesis of both D-and L-amino acids. In addition 14 is readily prepared from glycine enabling the method to be simply adapted for the incorporation of isotopic labels.57 During the period covered by this review the major use of the Seebach approach has been in the preparation of a-amino acids labelled with carbon-1 1 (e.g.~-[3-"C]alanine and ~-[3-' 'C]phenylalanine5* and DL-[~-''Clglutamic acid and DL-[~-'C] lysine5'). 2.2 Isotopic labelling of the diastereotopic methyl groups and hydrogens of amino acids Leucine and valine residues are important to the three dimen- sional structure of proteins by being involved in hydrophobic interactions. Assignment of the diastereotopic methyl groups of valine and leucine in the NMR spectra would allow protein structures to be more precisely defined. In the light of the importance of these probes several methods for the synthesis of L-leucine and L-valine specifically labelled in either dia- stereotopic methyl group have been reported recently. Young and co-workers described the first totally stereoselective syn- thesis of (2S,4R)-[5,5,5-2H,]leucinein 1992 (Scheme 9).60361 The protected pyroglutamate 24 was used as a template to create the asymmetric centre at C-4.Reaction of 24 with Bredereck's reagent gave the enaminone 25 in 91% yield. Reduction of 25 to the olefin followed by catalytic hydrogen- ation proceeded with complete stereocontrol giving the cis-4-methyl diastereomer 26 as the sole product. Conversion to the acyclic product 27 was achieved with lithium hydroxide in THF with no loss of stereochemical integrity. Reduction of the acid to the dideuteriated alcohol 28 was achieved via a mixed anhydride with sodium borodeuteride and the third deuterium label was introduced via sodium cyanoborodeuteride reduction OGCO2Bu' 0 N I I CO~BU' CO~BU' 24 25 (Bur02C)2N Me xii xiii BurO2CNH -Mh -H& ii iii But02CNH Me jv,V CO~BU'-0 CO~BU' -Bu'O~CNH Me N I BU '02C bC02H Bu'O~C&C2H20H CO~BU' 26 27 28 1 J ix vi Me Bu'O~CNH Me BurO2CNH Me t- Bu'O~C&CH20H 33Ix viii Bu'O~CLCH20TBDMS 32 Bu'O~CLCH20H 30Ix bC2H21Bu'O~C 29Ivii viii H02C KCH2F (2S,4S)-5-fluoroleucine MehY C02Bu' H02C XC2H3 CO~BU' 31 Scheme 9 Reagents i (Me,N),CH; ii H, 10% Pd on C EtOAc; iii LiOH THF; iv ClCO,Bu' Et,N; v NaB2H, ,H,O THF; vi HMPA (PhO),PCH,I; vii NaB(CN),H,; viii 6 M HCl; ix NaBH, H,O THF; x DAST; xi TBDMSCI Et,N DBU; xii Boc,O DMAP Et,N; xiii TBAF AcOH Kelly Sutherland and Willis Syntheses of amino acids incorporating stable isotopes of the iodide 29.Finally deprotection gave (2S,4R)-[5,5,5-2H3]leucine. The use of pyroglutamate intermediates has proved extremely valuable for the synthesis of further derivatives of L-leucine including (2S,4S)-5-fl~oroleucine,~~ (2S,4S)-and (2S,4R)-5,5'-dihydro~y-[5,5-~H,]le~cine,~~ and 4-alkylideneglutamic In the case of the preparation of (2S,4S)-5-fluoroleucine the first step involved reduction of acid 27 to give the primary alcohol 30 which was to be substituted by fluoride (Scheme 9). It was found necessary to use bis-protection of the nitrogen to prevent cyclisation to 31 in the fluorination step. Hence the alcohol was transiently protected as the tert-butyldimethylsilyl (TBDMS) ether 32. Treatment of 32 with Boc anhydride and deprotection of the silyl ether gave alcohol 33 which on reaction with diethyl-aminosulfur trifluoride (DAST) followed by deprotection with hydrochloric acid gave (2S,4S)-5-fluoroleucine.(2S 4RIS)-5,5,5-Trifluoroleucine has been prepared from the correspond-ing a-keto acid by a transamination reaction catalysed by Alcaligenesfaecalis IAM 1015.65 (R)-Pulegone has been used by Hill et al. as the starting material for the synthesis of (2S,4S)-and (2R,4S)-[5,5,5-2H3]leucine(Schemelo).@' (R)-Pulegone 34was converted into 34 35 36 I ii iii AcNH AC02H i s c2Ho 39 A A J ix 38 37 (2S,4S)-[5,5,5-2H3]leucine 40 (2R,4S)-[5,5,5-2H3]1eucine Scheme 10 Reagents i LiAI2H, THF; ii PCC CH,Cl,; iii Na,CO, ,H,O CH 02H;iv (Ph,P),RhCl; v NaIO, K,CO, KMnO,; vi Br, I, PCI,; vii NH,OH; viii Ac,O AcOH; ix hog kidney acylase; x 2 M HCl (R)-citronellic acid 35 and then reduced via the methyl ester with lithium aluminium deuteride to give 36.Oxidation of 36 followed by exchange of the acidic protons for deuterium gave trideuteriated aldehyde 37. Decarbonylation of 37 with Wilkinson's catalyst set up the stereogenic centre in 38 which was to become C-4 in the target molecule. Oxidation of 38 to [2H3]isovalericacid followed by introduction of the a-amino function by the standard methods gave the N-acetyl derivative 39 which was resolved using hog kidney acylase. Hydrolysis of 39 gave the (2S,4S)-diastereomer and acid hydrolysis of the recovered amide 40 gave the (2R,4S)-diastereomer of [5,5,5-2H,]leucine.[The title of this paper has an error and should be (2S,4S)-and (2R,4S)-and not (2S,4S)-and (2S,4R)-as given.] Willis and co-workers have used a chemo-enzymatic approach for the synthesis of L-leucine selectively labelled in either diastereotopic methyl group with deuterium or carbon-13.67 The stereogenic centre at C-4 was established using Evans' chiral auxiliaries (Scheme 11). Thus treatment of the 3-propionyloxazolidin-2-one 41 with sodium hexamethyl-disilazide and C2H31gave the alkylated product 42 with >86% 2 I0 Natural Product Reports 00 00 MedNKO Me\rNKO Ll-C2H3 -7 -7 C2H3 41 42 43 1 1 vii iii 0 MeyBr C2H3 PPh3 C'H3 C2H3 47 46 44 J 1 ix iv Me,$ MenC02Me C02Me C2H3 0 C'H3 48 '[v vi 1.Menc02H y2 C'H3 NH2 MevC02H (2S,4R)-[5,5,5-2H3J1eucine C'H3 (2S,3R)-[4,4,4-2H3]valine Scheme 11 Reugents i NaHMDS C2H31,ii LiAIH,; iii Ph,P Br, PhNO,; iv Mg Et,O THF then (CO,Et),; v NaOH; vi leucine dehydrogenase; vii LiOH H,O,; viii Ph,PCHCN EDCI DMAP; ix 0, MeOH CH,CI,; x lipase leucine dehydrogenase de. Reductive cleavage of the auxiliary followed by reaction of the resultant primary alcohol 43 with Ph3P and Br gave bromide 44.A two-carbon homologation of 44via a Grignard reaction with diethyl oxalate gave the a-keto ester 45. Saponi-fication of the ester followed by reductive amination of the ketone catalysed by leucine dehydrogenase gave (2S,4R)-[5,5,5-2H3]leucinein 85% yield over the two steps. By use of the oxazolidinone prepared from (+)-norephedrine,6g(2S,4S)-[5,5,5-'H3]leucine is readily ~repared.~' This chemo-enzymatic approach to L-leucine is very flexible and facilitates the incor-poration of a combination of isotopes e.g.carbon-13 at C-3 and C-4 using carbon-13 labelled sodium acetates carbon-13 in either diastereotopic methyl group using [13C]methyl iodide and nitrogen-15 by use of ['5N]amm~niumformate in the reductive amination step. The deuteriated derivative 42 has also proved to be a valuable intermediate in the synthesis of (2S,3R)-[4,4,4-2H3]valine(Scheme 1l).I9The chiral auxiliary was cleaved with lithium hydroxide and hydrogen peroxide to give acid 46. A one-carbon homologation of 46 to a-keto ester 48 was achieved via ozonolysis of the P-ketocyanophosphorane 47 with no racemisation of the stereogenic centre.Keto ester 48 was converted directly into (2S,3R)-[4,4,4-2H3]valinein 80% yield in a one-pot procedure involving two enzyme catalysed transformations (Candida cylindracea lipase to hydrolyse the ester and leucine dehydrogenase to catalyse the reductive amination of the ketone). This strategy was extended to the synthesis of further L-amino acids with an asymmetric centre at C-3 including L-[ 15N]isoleucine and [' 5N]alloisoleucine. As with the synthesis of leucine this approach may be simply adapted for the selective labelling at each site of the amino acids. In 1995 the groups of Baldwin in and Chamberlin in Irvine California7' demonstrated the use of aspartic acid as the starting material for the enantioselective synthesis of valine selectively labelled in one of the diastereotopic methyl groups by two completely different strategies.The Oxford group required (2R,3S)-[4,4,4-2H3]valineas an intermediate in the synthesis of (2R)-[2'-2H3]penicillinN which in turn was used to demonstrate the stereospecific ring expansion of penicillin N to deacetoxycephalosporin C by deacetoxy- cephalosporin C synthase from Streptomyces clavuligerus. P-Lactam 49 was prepared from (R)-aspartic acid and trans- alkylated by treatment with LDA and C2H31 giving 50 as the sole product (Scheme 12). Following esterification the 49 50 J ii H3CpN ,CO2 B u - iii 2H3CpN,,C02B~t \ \ 0 Boc 0 TBDMS 52 51 1 iv H p3 BocN..cCH20H COpBu' 53 lv H p 3 C2H3 vi vii BocN..-f%H2SePh -H2N..cMe CO~BU' C02H 54 (2R,3S)-[4,4,4-2H3Jvaline Scheme 12 Reagents i LDA C2H,I; ii Cl,C(C=NH)OBu' BF,-Et,O; iii Boc,O DMAP CH3CN; iv NaBH, MeOH; v N-(phenylseleno)phthalimide Bu,P CH,Cl,; vi Ph3SH reflux; vii TsOH Et,O then ion exchange TBDMS group in 51 was substituted for a Boc group to give 52.Sodium borohydride reduction proceeded smoothly to give alcohol 53 which was deoxygenated via radical reduction of selenide 54. Deprotection using toluene-p-sulfonic acid gave (2R,3S)-[4,4,4-2H3]valine. Chamberlin and co-w~rkers'~ prepared (2S,3S)-[4-'3C] valine from (9-aspartic acid in 54% yield using a very elegant (2S,3S)-[4-'3C]valine Scheme 13 Reagents i LiHMDS I3CH3I; ii DIBALH; iii MsCl Et,N; iv Zn NaI; v H, Pd on C HCl; vi 6 M HCl; vii TsCl py; viii Me,CuLi 13CH3 yC02Me fC02Me i I I BnNAC02Me Bn AC02Me PhFl PhFl 55 56 BnN '%02Me PhFl 59 1 viii v.vi IsCH3r~e L-[3-13CH3]isoleucine 60 _ 13CH3 yCH2OH I Bny AC02Me PhFI 57 BnNAC02Me PhFl 58 I v vi + 13CH3tMe protocol (Scheme 13). Protected aspartate 55 was alkylated with lithium bis(trimethylsily1)amide(LHMDS) and 13CH,I to give 56 as a single diastereomer in 98% yield. Regioselective reduction of the less sterically hindered ester with diisobutyl- aluminium hydride (DIBALH) followed by deoxygenation of the resultant alcohol 57 via Zn-I reduction of the methane- sulfonate gave 58.Finally deprotection gave (2S,3S)-[4-13C] valine. Alcohol 57 also proved to be a valuable intermediate in the synthesis of ~-[3-'~CH,]isoleucine. Activation of the alcohol to the toluene-p-sulfonate 59 followed by displacement with dimethylcuprate gave protected isoleucine 60. Deprotec-tion of 60 by catalytic hydrogenation followed by heating with 6 M hydrochloric acid gave ~-[3-'~C]isoleucine in nine steps and 62% overall yield from aspartic acid. The biosynthesis of the phytotoxin coronatine has been investigated by administering isotopically labelled amino acids to Pseudomonas syringoe pv. gly~inea.~' Isotopomers of a mixture of m-isoleucine and alloisoleucine were simply prepared from 2-bromobutane using [I3C]cyanide and ['5N]ammonium hydroxide as the sources of isotopic labels.Not only have methods been established for the isotopic labelling of either diastereotopic methyl group of L-leucine and L-valine but also the pro-R and pro-S hydrogens of glycine may be selectively labelled for example using the approach described by Kakinuma et al.72 They used a divergent and highly enantioselective method for the synthesis of both enan- tiomers of alanine and for both chirally deuteriated glycines using a diacetone-~-glucos-3-ulosetemplate (Scheme 14). 61 62 n -n +ivn V +I 66 H02C Y2H NH2 (R)-[2-2H]glycine NH2 (S)-[2-2H]glycine 0 64 Scheme 14 Reagents i LiA12H,; ii KHC1,CCN; iii A xylene; iv RuCl, NaIO,; v H'; vi TMSCl py; vii BuLi CH,I; viii K2C03 Reduction of 61 with lithium aluminium deuteride gave the deuteriated allylic alcohol 62 with good regio- and stereo- control.(From the 'H NMR spectrum it was apparent that <5% of the product was labelled at the P-position.) The alcohol was then converted into the imidate 63 and following a [3,3]-sigmatropic rearrangement the trichloroacetamide 64 was formed in 97% yield. Further transformations involving RuC1 oxidation and acid hydrolysis were carried out to afford Kelly Sutherland and Willis Syntheses of amino acids incorporating stable isotopes 21 1 (2S)-[2-2H]glycine in 32% yield. Similar results were obtained starting with isomer 65 giving (2R)-[2-2H]glycine. This approach was extended to the enantioselective synthesis of alanine.Since C-3 of alanine was introduced via reaction of the anion of 61 with methyl iodide to give 66 this method may be simply adapted for the synthesis of isotopically labelled alanine. In 1992 Hegedus et al. reported the synthesis of (9-and (R)-[2-2H]glycine with 82-86% ee via photolysis of optically active chromium carbene complexes (Scheme 1 5).73 Photolysis Ph 2HY C02Me (R)-or (S)67 (R,R)-or (S,S) 68 [2H]glycine (C02Me 2H'.f C02Me v vi iv vii H02CyNH2 ph-fro 2i 69 70 ( R)-[2H]glycine Scheme 15 Reagents i hv Me02H MeCN; ii HCI MeOH; iii H, HCO,H MeOH Pd (OH),; iv 3 M HCI; v Bu'Li THF; vi Me02H; vii Li NH of either (R)-or (S)-carbene complex 67 in Me02H and MeCN produced the deuteriated glycine precursors 68 in excellent chemical yield with almost complete monodeuteriation and high stereoselectivity.Removal of the oxazolidine group fol- lowed by deprotection of the resultant ester gave either mono- deuteriated glycine in 74% overall yield from 67. A second approach to monodeuteriated glycines was achieved in 62% yield via quenching the ester enolate of 69 with Me02H to give 70 with 96% incorporation of deuterium and 84% de (Scheme 15). Hydrolysis of the ester followed by reduc- tive cleavage of the oxazolidinone using lithium and liquid ammonia gave after purification (R)-[2-2H]glycine. Easton and Hutton required the four isomers of [3-2H]phenylalanine to examine the reaction of (R)-phenylalanine with (9-phenylalanine ammonia lya~e.~~ The target compounds were prepared by side-chain bromination of protected phenylalanines followed by deuteriolysis of each of the diastereomeric bromides with deuterium over 5% pal-ladium on carbon.The reduction proceeded with retention of configuration giving access to each of the four isomers of [3-2H]phenylalanine in approximately 98% diastereomeric excess and with 99% incorporation of deuterium. Phenyl- alanines regiospecifically labelled with deuterium in the aromatic ring have been prepared through deuteriolysis of tyrosine tetrazol ethers.75 3 a-Amino acids with aliphatic hydroxylated or thiolated side-chains (serine threonine cysteine methionine and tyrosine) Some of the more general methods for the synthesis of isotopically labelled a-amino acids described in the previous section have been applied to the preparation of a-amino acids with hydroxylated or thiolated side-chains.For example enzyme catalysed procedures have been used to prepare ~-["N]serine'~ ~-["N]methionine'~ ~-[2-~H]methionine~~ and ~-[l-'~C,2,3-~H,]tyrosine,~~ whilst a template based approach has been used to prepare deuteriated ~erines,~',~' carbon- 13 and nitrogen-1 5 labelled cysteine and ~erine.~~ In this section 2 12 Natural Product Reports we have concentrated on literature methods more specifically aimed at the synthesis of isotopomers of a-amino acids with hydroxylated or thiolated side-chains. Serine is an important amino acid not only in its own right but also as a synthetic intermediate to further amino acids e.g L-aspartic acid L-threonine and L-cysteine.Unkefer and co-workers have developed a method for the preparation of isotopomers of L-serine using the serine-type methylotroph Methylobacterium extorquens AM 1.76 In this system C-3 of serine is derived from methanol (via oxidation of methanol to formaldehyde using a methanol dehydrogenase) while C-2 C- 1 and the a-amino group are derived from glycine (catalysed by serine hydroxymethyltransferase). By starting with the appro- priately labelled precursors any of the 2H- I3C- or "N-isotopomers of L-serine may be conveniently prepared on a 40-50 mmol scale. L-Serine prepared in this way has been used in the synthesis of other amino acids. For example pyridoxal phosphate enzymes were used for the conversion of ~-["N]serine into ["N]cysteine tyrosine and trypt~phan.~~ Gorisson et al.have described a method for the resolution of racemic [l-13C]serine as well as the synthesis of [3-13C]cycloserine.77 Resolution of the rac-iC(dinitrobenzoy1)- [1 -13C]serine using [R-(R*,R*)]-2-amino-l-(4-nitrophenyl) propane- 1,3-diol which following crystallisation from methanokthyl acetate and acid hydrolysis gave D-[l -I3C] serine in 26% yield 97% ee. The L-enantiomer was obtained from the mother liquors using a second resolution with quinidine in ethanol followed again by deprotection using acid giving ~-[l-'~C]serine in 29% yield 99% ee. Rapp and co-workers have developed a very versatile approach to the synthesis of I3C- l8O-and 2H-labelled L-serines and L-threonines based on Schollkopf s auxiliary (Scheme 1 6).78 i / cN\,OMe MeOAN' Me0 J...yOCH2PhN ./\J 71 II ii-iv Me0 0 I I + 180H ~-[3-13C]serine ~-[3-W]serine ~-[3-'~O]threonine 0 = 13c Scheme 16 Reagents i BuLi PhCH,'80CH,CI; ii HZ Pd on C AcOH MeOH; iii Ba(OH),; iv H,SO,; v BuLi CITi[NEt,], CH,CH'*O For the synthesis of isotopically labelled L-serine the side chain was introduced on the bis-lactim ether using isotopically labelled benzylchloromethyl ether.In the case of ~-[3-'*0]- serine the benzyl group was removed from 71 by catalytic hydrogenation and then saponification of the methyl ester in 0.05 M solution of aqueous Ba(OH) to prevent I80-exchange with water whereas for ~-[3-'~C]serine the protecting groups were removed under the more usual conditions with 6 M HCl.For the synthesis of isotopically labelled threonine the bis- lactim ether was alkylated with isotopically labelled acetalde- hyde in combination with chlorotitanium tris(diethy1amide). The final ratio of L-threonine to L-allothreonine was found to be 15 I by HPLC analysis. This approach enables L-serine and L-threonine to be labelled at any position or combination of positions in good yields and high enantiomeric excesses. From our search of the literature of the last six years this is the only report of the enantioselective synthesis of L-threonine incorporating stable isotopic labels. However many methods have been reported for the synthesis of unlabelled or radio- labelled threonine and its In theory it should be possible to simply modify some of these approaches for the H2N'112H -FmocNH q+ 0 75 L-threonine Ii incorporation of stable isotopes for example as in the approach described by Blaser and Seebach (Scheme 17).83 72 L-threonine Scheme 17 Reugents i LiHMDS; ii CH,CHO; iii AcOH NH,Cl; iv Pd on C H, then H+ Treatment of Z-BMI with LiHMDS at low temperatures followed by addition of acetaldehyde gave the addition prod- uct 72 with >90% de.Catalytic hydrogenation followed by hydrolysis gave L-threonine in good yield. It has already been shown that the isotopically labelled imidazolidin-4-ones may be readily prepared. An eight step synthesis of L-4-hydroxy-[2,3-'3C2]threonine from [1,2-'3C,]acetylene has been re-ported.84 In addition stereoisomers of 4,4,4-trifluorothreonine were obtained through enzymatic resol~tion.~' Several synthetic routes to isotopomers of allothreonine have been reported.Beaulieu used serine as the starting material for the synthesis of [4,4,4-3H,]allothreonine.86D- or L-serine were converted to L-or D-y,y,y-trichloroallothreonine 74 (via diol 73) which on catalytic hydrogenolysis gave diastereomerically pure L- or D-allothreonine (Scheme 18). z z Z OH 73 I iii OH Z OH D-[4,4,4-3H3]allothreonine 74 Scheme 18 Reagents i Cl,CCO,TMS K,CO, 18-crown-6 90 "C; ii H' MeOH; iii Jones reagent; iv 3H2,20% Pd(OH),/C MeOH Using tritium gas rather than hydrogen L-or ~-[4,4,4-3H3]allothreonine was obtained.Blaskovich and Lajoie reported the stereoselective synthesis of allothreonine and P-[2H]allothreonine from L-threonine (Scheme 19).87 This approach involves protection of the amine with a fluoren-9- ylmethoxycarbonyl group (Fmoc) and the carboxy group with a cyclic ortho ester 75. The ortho ester reduces the acidity of the a-hydrogen preventing racemisation or elimination during elaboration of the side-chain. Swern oxidation of the second- ary alcohol to a ketone 76 followed by reduction with lithium borodeuteride gives the all0 isomer 77. Finally deprotection gives P-[2H]allothreonine in 40% overall yield. A further excel- lent method for the synthesis of L-allothreonine has been reported by Wong and co-w~rkers.~~,~~ Addition of acetalde- hyde to glycine catalysed by threonine aldolase gave a 93:7 mixture of L-allothreonine to threonine.Although the authors 0 0 77 76 1 iii-v GH I HzN AcozH L-[2-2H]allothreonine Scheme 19 Reagents i (COCI), DMSO CH,Cl, Pr',EtN; ii LiB'H, CH,Cl, MeOH; iii piperidine; iv CF,CO,H H,O; v Cs,CO, MeOH H,O then ion exchange did not report the use of this approach for the preparation of isotopomers it is apparent that since isotopically labelled glycines are readily available (see section 2) then it may be simply modified to prepare isotopically labelled allothreonines. D-Serine and D-cystine specifically labelled at C-3 with deuterium have been prepared from aziridines as shown in Scheme 20.90,91Deuteriated malic acids 7892were converted HA C02H PO \/ H02C HA HB 78 HA HB H t HO HA NCPh3 HO HA NH2 )+ Me02C HA H,3 H02C HA H,3 80 79 J/ Z I -M -+ eHA o 2 c ~ ~ ~ 81 82 83 I I H02C H,3 HA H02C H,3 HA H02C HB HA Scheme 20 into labelled isoserines 79 via a Curtius rearrangement in 30-39% yields.The rearrangement occurred with retention of stereochemistry at the migrating isotopically labelled stereo- genic centre. Methylation of the acid followed by tritylation of the amine gave 80 in excellent yields. Activation of the alcohol as the toluene-p-sulfonate followed by treatment with base afforded the aziridine 81. Reaction of 81 with refluxing 20% aqueous perchloric acid gave nearly quantitative yield of either (2R,3R)-[2,3-2H,]serine or (2R,3,S)-[3-2H]serine depending on the position of isotopic label in the original malic acid.To prepare deuteriated D-cystines it was necessary to convert the N-tritylaziridine 81 to the corresponding N-benzyloxycarbonylaziridine82 prior to reaction with the Kelly Sutherland and Willis Syntheses of amino acids incorporating stable isotopes nucleophile phenylmethanethiol and boron trifluoridediethyl- etherate to give 83. Deprotection was achieved by acid hydrolysis then treatment with sodium in liquid ammonia to give cysteine which was oxidised to give isotopically labelled D-cystine. Aziridines have also proved useful for the synthesis of deuteriated ~-chlor~alanines~~~~~ and ~-prop-2-ynylglycine specifically labelled at C-3.93794 Panigot et al.used an approach based on the Gabriel reaction to prepare isotopically labelled DL-cysteine (Scheme 2l)." Treatment of potassium ["N]phthalimide with ethyl 0 0 84 1I-iii 86 Scheme 21 Reagents i 6 M HCl; ii PhCOCl NaOH; iii EtOH H,SO,; iv LDA PhCH,SCH,Br; v 48% HBr then ion exchange [1,2-'3C2]bromoacetate gave the phthalimide derivative 84 which was hydrolysed to glycine then reacted with benzoyl chloride and esterified to give ester 85. Reaction of 85 with LDA and then benzyl bromomethyl sulfide gave 86 which after deprotection led to ~~-[1,2,3-'~C~ ''Nlcysteine in 16% overall yield. Tryptophan synthase catalyses the nucleophilic displace- ment of the hydroxy group of L-serine. This enzyme can use phenylmethanethiol as the nucleophile in the conversion of serine to (9-benzyl-L-cysteine which is deprotected with sodium in liquid ammonia to yield L-cysteine.A strain of E. coli engineered to overproduce tryptophan synthase was used by Unkefer and co-workers to prepare L-[~-'~C]- and ~-[3-~H,]cysteine.'~ L-methionine may be readily prepared from racemic methio- nine as described by Nakajima et a1.97A very efficient method for the synthesis of L-[' 'C-methyllmethionine has been reported recently which may be simply adapted to prepare the corresponding carbon- 13 labelled material. Treatment of L-homocysteine with potassium fluoride on an alumina support then with ["C]iodomethane gave L-[' 'C-methyl] methionine in 97% yield in 10 min; no purification was required.98 In addition Langstrom and co-workers have described a method for the synthesis of ~-[3-' 'Cltyrosine from DL-[~-''Clalanine using a coupled D-amino acid oxidate-catalase glutamic-pyruvic transaminase and P-tyrosine enzy- matic system which also may be simply adapted for the incorporation of carbon-1 3.993'00 4 a-Amino acids with a carboxylic acid in the side-chain (aspartic and glutamic acids) In the previous section several methods were described for the enantioselective synthesis of isotopically labelled serine an amino acid of interest in its own right and also as a building block to further amino acids.For example in 1992 Lodwig and Unkefer reported the synthesis of L-[~-'~C]- and ~-[3,4- '3C,]aspartic acid from ~-[3-'~C]serine.'~' Protection of [3-"C]serine as the N-Boc derivative and cyclisation under Mitsunobu conditions gave the p-lactone 87 (Scheme 22).Treatment of 87 with potassium [13C]cyanide in DMSO gave nitrile 88 which on acid hydrolysis gave ~-[3,4-'~C,]aspartic 2I4 Natural Product Reports HNBoc H N Boc 1 ii t L-[3,4-13C2]aspartic acid 88 Scheme 22 Reagents i Ph,P DMAD -78°C; ii KCN DMSO; iii 6 M HCl acid in 13.3% overall yield. Similarly ~-[4-' 3C]aspartate was prepared from L-serine and KI3CN. The enantiomeric excess was 96% in each case. LaIuppa and LeMaster also used cyanide as the nucleophile to open a lactone as the key step for the introduction of carbon-13 in the preparation of DL-[~-''N 5-' 3C]glutamic acid.'02 Treatment of 2-bromobutano-4-lactone with potassium ["Nlphthalimide in DMSO gave 89 (Scheme 23) which on reaction with potassium [I3C]cyanide led 00 @;K + BrdO -/ '6 0 89 Ii DL-[5-' 3C 5N]glutamic acid 90 Scheme 23 Reagents i KCN DMSO; ii HCl CH,CO,H to attack on the y-lactone to give 90.Acid catalysed hydrolysis of 90 gave DL-[~-~'N 5-'3C]glutamic acid in 38% overall yield. Isotopically labelled aspartic acid was required by White et al. to examine the biosynthesis of 5-hydroxy-4-oxo-~-n~rvaline."~ They prepared racemic [2-'3C,'5N]- and [4-I3C]- aspartic acid using diethyl [2- '3C]malonate and potassium [''Nlphthalimide as the sources of isotopic labels. Bromination of diethyl malonate followed by nucleophilic displacement of the bromide gave diethyl phthalimidomalonate.Alkylation with ethyl bromoacetate and hydrolysis gave aspartic acid in 65% overall yield. [2-'3C,15N]A~parti~ acid was also used by Baxter et al. to examine the biosynthesis of 3-nitropropanoic acid in the fungus Penicillium atroveneturn. '04 Several enzymatic approaches to the synthesis of isotopically labelled glutamic acid have been reported recently for example the reductive amination of a-ketoglutarate catalysed by glutamate dehydrogenase.' Cappon et al. prepared a series of 2-0xo['~C]glutaric acids on a multigram scale using ethyl bromoacetate and paraformaldehyde as the sources of isotopic labels." Carbon-13 labelled ethyl acrylate was prepared from ethyl bromoacetate and paraformaldehyde via a Wittig reaction (Scheme 24) and then treated with ethyl nitroacetate (also prepared from ethyl bromoacetate) to give diethyl 2-nitroglutarate 91.Ozonolysis of the anion of 91 and subse- quent acid hydrolysis gave 2-oxoglutarate in approximately 50% yield. The enzyme catalysed reductive amination of the a-keto acids was catalysed by glutamate dehydrogenase using alcohol dehydrogenase to recycle the co-factor NADH (Table 2). Using this chemo-enzymatic synthetic route ~-[3-' 'C]- i ii Et02C-Br -EtO2C*PPh3 -Et02Cd H02C-YCo2H NH2 Iiil iii Et02Cd + Et02C-NO2 -Et02C Y--C02Et NO2 91 J iv H02CwC02H ,,i ,,ij EtOzCpC02Et c- NH2 0 L-glutamic acid Scheme 24 Reagents i PPh,; ii NaOH; iii (CH,O),; iv NaOEt; v 0,; vi HCI H,O; vii L-glutamic dehydrogenase -[4-13C]- -[5-l3C]- -[3,4-l3C2]- and -["N]glutamic acids were prepared.This approach was extended to the preparation of ~-[3,3-,H,]- and -[4,4-2H2]glutamic acids (using exchange reac- tions) as well as to the synthesis of a series of L-[,H]- -["N]- and -['3C]glutamines (see section 5 for further details).,' Kragl et al. have used glutamate dehydrogenase to prepare ~-[l~N]glutamic acid on a multigram scale but in this case formate dehydrogenase was used to recycle the NADH and ['5N]amm~ni~m chloride as the souce of isotopic label (Table 2)., Goux et al. have prepared a series of carbon-13 labelled glutamic acids from the correspondingly labelled citrates using commercially available purified enzymes.'05 The citrate is converted to 2-oxoglutarate using an aconitase catalysed iso- merisation to threo-D,-isocitrate followed by an oxidative decarboxylation catalysed by isocitrate dehydrogenase.Finally reductive amination of 2-oxoglutarate is catalysed by L-glutamate dehydrogenase. Starting from '3C-labelled pyruvate acetate and bicarbonate millimolar quantities of L-[ 1-13C]- -[4-13C]- -[5-'3C]- and -[1,3,4-13C3]glutamic acids were prepared. A most valuable method for the synthesis of isotopically labelled L-aspartic acids which we felt must be included even though the papers describing its application pre-date the period of this review is the use of L-aspartase to catalyse the conversion of fumaric acid to aspartic acid. For example Field and Young used L-aspartase in 2H,0 to convert (E)-fumaric acid into (2S,3R)-[3-2H]aspartic acid which in turn was con- verted into (2S,3S)-[3-'H]glutamic acid via a Wolff rearrange- ment of a diazoketone intermediate.'06,'07 Lee et al. lo' used L-aspartase to prepare (2S,3S)-[2,3-'H2]aspartic acid in this case the substrate for the enzyme (E)-[2,3-2H2]fumaric acid was prepared from dimethyl acetylenedicarboxylate with triphenyl- phosphine in 'H,O followed by hydrolysis of the esters. A further example of the use of L-aspartase is by Rohm and Van Etten in the synthesis of [1,3-'3C,]a~partic acid.'" 5 a-Amino acids with nitrogen in the side-chain (asparagine arginine glutamine histidine lysine proline and tryptophan) Isotopically labelled L-glutamic acids have been used to pre- pare both glutamines and prolines incorporating a range of stable isotopes.Four different isotopomers of L-glutamine ([2-,H]- [3,3-'H2]- [4,4-,H,]- and [5-13C]-) were prepared by the conversion of the appropriately isotopically labelled L-glutamic acid using glutamine synthase.,' Using [''Nlammo-nium chloride a nitrogen-15 label was incorporated into the amide group of glutamine. ~-Boc-[4-' 'Nlasparagine and L-Boc-[ 15N]glutamine have been prepared from the corre-sponding Boc protected a-benzyl esters and ["N]ammonia with N,N-carbonyldiimidazole as the coupling agent. lo ~-[3,4-' 3C2]- and ~-['~N]Proline were prepared from the correspondingly labelled L-glutamic acids on a gram scale as shown in Scheme 25."' The approach is based on the ring QC02H +-!- QC02Me H COMe L-proline 94 93 Scheme 25 Reagents i H,O reflux; ii SOCl, MeOH DMF; iii Lawessons' reagent; iv CH,COCl N,N-dimethylaniline; v Bu,SnH AIBN; vi HCI H,O closure of L-glutamic acid to L-5-oxoproline 92 followed by the chemoselective reduction of the amide function without racemisation of the asymmetric centre.The selective reduction was effected by first converting the amide into the thioamide 93 and subsequent reduction with tributyltin hydride to 94. Deprotection under acidic conditions gave isotopically labelled proline with 97% ee after crystallisation. This approach is flexible enabling L-proline incorporating carbon-13 and nitrogen-15 labels at each site or combination of sites to be readily prepared.L-5-Oxoproline methyl ester was also used by Dieterich and Young for the preparation of (2S,3S)-[3-,H]- and (2S,3R)-[2,3-2H2]-proline.'l2 The precursors to the isotopically labelled prolines were (2S,3R)-[3-,H]- and (2S,3S)- [2,3-'H2]-aspartic acids prepared by the well established anti- addition of ammonia to the double bond of fumaric acid discussed in the previous section. Treatment of aspartic acid with trifluoroacetic anhydride gave an anhydride which on opening with methanol gave two monomethyl esters in which the required a-methyl ester 95 predominated (4:l) (Scheme 26). Conversion of 95 into the acid chloride followed by reaction 0 II 0 CF3ANH@~2 Me02C HB HA Me02C HB HA 97 96 C02Me Boc 98 Scheme 26 with diazomethane gave a mixture of diazoketones which were separated by recrystallisation to give the required isomer 96.Photolysis of the diazoketone in dry distilled methanol gave the crude pyroglutamate (via 97) which was purified as the tert-butoxycarbonyl protected derivative 98 with no loss of stereochemical integrity. Conversion of 98 to L-proline then proceeded smoothly following literature precedents. A further synthesis of isotopomers of L-proline was reported by Young and co-workers from ketone 99 (Scheme 27).Il3 The protons at Kelly Sutherland and Willis Syntheses of amino acids incorporating stable isotopes HNZ HNZ H02C4C02Me HoeC02Me 105 106 1 CO~BU' CO~BU' i ii 99 J n iii-v CO~BU' H CO~BU' ~-(3S)-[3-~H]proline 100 Scheme 27 Reagents i 'H,O-silica gel; ii LDA TMSCl 'H,O; iii NaBH, MeOH Et,O; iv TsCl py; v NaBH, DMSO; vi 6 M HCl C-3 of 99 were exchanged using 2H20-washed silica gel in 2H20and THF.Formation of the trimethylsilyl enol ether and quenching with water led to stereoselectiveprotonation at C-3. To complete the synthesis the ketone was reduced with sodium borohydride and the resultant mixture of alcohols converted into the corresponding toluene-p-sulfonates and reduced with sodium borohydride in DMSO giving 100. Using this approach (2S,3S)-[3-2H]-,(2S,3R)-[3-2H]-and (23)-[3,3-2H2]-prolinewere prepared. Baldwin et al. have prepared (2S,4S)-[4-2H]- (2S,4R)-[4-2H]proline to examine the stereochemical course of the hydroxylation of L-proline by proline 4-hydroxylase from Streptomyces griseoviridus P8648.l4 (2S,4R)-Hydroxyproline 101 was converted into its N-Boc 4-tosyl derivative 102 in two steps and deuterium was selectively incorporated by S,2 displacement of the toluene-p-sulfonate using lithium triethyl-borodeuteride in tetrahydrofuran (Scheme 28). For the (4R)-101 102 ~-(4S)-[4-~H]proline Boc Boc H 103 104 ~-(4R)-[4-~H]proline Scheme 28 Reagents i Boc2O NaOH; ii TsCl NaOH; iii LiEt,B2H THF; iv CF,CO,H; v Jones reagent; vi NaBH, MeOH diastereomer Boc-(2S,4R)-hydroxyproline was converted to the (49-tosylderivative 104 via oxidation to the ketone 103 reduction and tosylation. Again reduction with lithium tri-ethylborodeuteride proceeded with good stereocontrol giving after deprotection with trifluoroacetic acid (2S,4R)-[4-2H] proline.Interestingly cyclisation reactions which had proved so valuable in the synthesis of L-proline initially dogged progress in the synthesis of ~-[6-'~C]lysine reported by Sutherland and Willis (Scheme 29).' l5 Chemoselective reduction of commer-cially available N-benzyloxycarbonyl-L-glutamic acid 105 to alcohol 106 was achieved in 60% yield via a mixed anhydride and in 58% yield with diborane. Activation of the alcohol as the toluene-p-sulfonate and displacement with sodium ['3C]cyanide gave 107 (55%) accompanied by Z-L-proline methyl ester 108 (35%). Finally reduction of the nitrile and 107 108 1 iii,iv y2 H2N- C02H L-[6-' 3C]lysine Scheme 29 Reagents i TsCl py; ii NaCN DMF; iii HZ,PtO,; iv 6 M HCl hydrolysis of the methyl ester under standard conditions gave ~-[6-'~C]lysine in quantitative yield from 107.Raap et al. reported a versatile procedure for the preparation of isotopi-cally labelled L-lysine based upon the use of bis-lactim ether of cyclo(D-Val-Gly).' l6 Using K13CN Kl3CI5N I3CH,CN and LiAlD as the sources of isotopic label five isotopomers of L-lysine were prepared incorporating 13C 2H in the 6-and &-positionsand I5N in 3540% yield as illustrated in Scheme 30. In a later paper the utility of this approach was demon-strated by the synthesis of further isotopomers of L-lysine.'" KCN + CI-Br -CI-CN \I . 5TsO-CN >1-CN CH&N OMe I OMe 1 iv OMe OMe vii Jv I H02CwNH2 Ho2CWC02H NH2 NH2 Scheme 30 Reagents i KI; ii BuLi ethylene oxide; iii TsCl; iv BuLi I(CH,),CN; v 4 M HC1; vi LiAIH or LiAI2H,; vii 6 M HCI ~-[2-~H]Histidine have been prepared and ~-[2-~H]arginine from unlabelled L-histidine and L-arginine respectively as described in section 2.26 Cappon et al.described a more general method for the synthesis of isotopically labelled L-histidines which was applied to the synthesis of the 2'-I3C- 1'-l5N-and 3'-15N-isotopomers.11sA 1,5-disubstituted imida-zole ring 109 was constructed in 71% yield via condensation of tosylmethylisocyanide with 3-phenylpropenal and subsequent cycloaddition of benzylamine as shown in Scheme 31. Oxi-dative cleavage of the alkene followed by reduction gave hydroxymethylimidazole 110 which was converted into chloride 111 for coupling with the bis-lactim ether.Reaction of 216 Natural Product Reports Tos L-aspartic acid without the need for a resolution step was also described. In this case ketone 113 was prepared in seven steps ry~QcH2] TosCH~N=C -i ii i,iii ,cH2=N\ from L-aspartic acid. Me3Si Ph As well as alkylation of the sultam as a method to prepare 1iv carbon- 13 and nitrogen-1 5 labelled tryptophan (which is a 111 110 109 OMe OMe OMe I OMe I 112 lviii ix L-histidine Scheme 31 Reagents i BuLi; ii Me,SiCl; iii 3-phenylpropenal; iv BnNH,; v K,OsO,(OH), NaIO,; vi LiAlH,; vii SOCl,; viii 3 M HCl; ix Pd' cyclohexene MeOH 111 with two equivalents of the bis-lactim ether anion gave 112 in 84% yield and finally deprotection gave L-histidine.The synthesis of [' 5N]hydroxymethylimidazole using ["Nlam-monia has been optimised and used in the synthesis of ~~-["N]histidine.I9 Furuta et al. described the synthesis of three types of multilabelled histidines required for pharma- cokinetics and investigations of enzymic reaction mechan- isms.'20 The imidazole ring of 115 was constructed by heating ~~-[3,3,5,5-~H,, ''Nldiarnino acid 114 with NaSCI'N in 2H,0 (Scheme 32). The DL-mercaptohistidine 115 was oxidised with 2H 2H 114 1 ii 2H 2H 116 115 1 iv L-[3,3,5'-2H3,1 ',3'-i5N2]histidine Scheme 32 Reagents i 2H,0 80°C; ii NaSCl'N 2H,0 95 "C; iii Fe,(SO,), H,O 95 T;iv resolution aqueous Fe,(SO,) to give 116 in 86% yield.Resolution was carried out with hog renal acylase on the N-acetyl derivative of 116 to give the corresponding labelled L-histidine. An analo- gous approach for the synthesis of L-histidine (93.8% ee) from general method for the synthesis of amino acids with nitrogen in the side-chain including asparagine glutamine and arginine),42 there have been two further papers published on the synthesis of isotopmers of L-tryptophan within the period of this review. Both used a chemo-enzymatic approach to the target compounds. In the first by van den Berg et al. ~-[3a-'~C]-and -[6-'3C]tryptophan were prepared by reaction of isotopically labelled indoles with L-serine catalysed by tryptophan synthase (Scheme 33).12' The approach used for Ho.-.,f02H + tryptophan mro2H 'N synthetase NH2 H 0 =13c ~-[3a-I 3C]tryptophan Scheme 33 the synthesis of the indole was ideally suited to the incorpor- ation of isotopic labels since the starting materials were cyclohexa- 1,3-dienone methanol ethyl bromopyruvate and ammonia.Unkefer and co-workers have also used a trypto- phan synthase to prepare isotopically labelled tryptophans.'22 In this case they used [3-'3C]-serine [1-15N]- and [2-I3C]- indole as the precursors for the synthesis of L-[P-'~C]- -[1'-I5N]- and -[2'-I3C]tryptophan respectively. The labelled indoles were prepared by the base catalysed cyclisation of appropriately labelled N-formyl-o-toluide. 6 References 1 R. M. Williams Organic Chemistry Series Vol.1 Synthesis of Optically Active a-Amino Acids eds. J. E. 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Pays-Bas 1992 111 517.112 P. Dieterich and D. W. Young Tetrahedron Lett. 1993 34 5455. 113 P. Barraclough P. Dieterich C. A. Spray and D. W. Young in Synthesis and Applications of Isotopically Labelled Compounds 1994 eds. J. Allen and R. Voges Wiley New York 1995 p. 881. 114 J. E. Baldwin R. A. Field C. C. Lawrence K. D. Merritt and C. J. Schofield Tetrahedron Lett. 1993 34 7489. 115 A. Sutherland and C. L. Willis J. Labelled Compds. Radiopharm. 1996 38 95. 116 J. Raap C. M. van der Wielen and J. Lugtenburg Recl. Trav. Chim. Pays-Bas 1990 109 277. 117 J. Raap W. N. E. Wolthuis J. J. J. Hehenkamp and J. Lugtenburg Amino Acids 1995 8 171. 118 J. J. Cappon K. D. Witters J. Baart P. J. E. Verdegem A. C. Hoeck R. J. H. Luiten J. Raap and J. Lugtenburg Red. Trav.Chim. Pays-Bas 1994 113 318. 119 L. A. Silks E. Dunkle C. J. Unkefer J. L. Sudmeier M. Butler and W. W. Bachovchin J. Labelled Compds. Radiopharm. 1995 36 947. 120 T. Furuta M. Katayama H. Shibasaki and Y. Kasuya J. Chem. SOC.,Perkin Trans. 1 1992 1643. 121 E. M. M. van den Berg F. J. H. M. Jansen A. T. J. W. de Goede A. U. Baldew and J. Lugtenburg Recl. Trav. Chim. Pays-Bas 1990 109 287. 122 C. J. Unkefer S. N. Lodwig L. A. Silks 111 J. L. Hanners D. S. Ehler and R. Gibson J. Labelled Compds. Radiopharm. 1991 29 1247. Kelly Sutherland and Willis Syntheses of amino acids incorporating stable isotopes

ISSN:0265-0568    

DOI:10.1039/NP9971400205

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

Biosynthesis of the gibberellin plant hormones Jake MacMillan IACR-Long Ashton Research Station Department of Agricultural Sciences University of Bristol Long Ashton Bristol UK BS18 9AF Covering Up to September 1996 Previous review J. E. Graebe Ann. Rev. Plant Physiol. 1987 38,419 1 Introduction 2 Overview 3 Stage A mevalonic acid to ent-kaur-16-ene 3.1 Metabolic steps 3.2 Stereochemistry 3.3 Enzymology 4 Stage B ent-kaur-16-ene to GA,,-aldehyde 4.1 Metabolic steps 4.2 Stereoc hemis try 4.3 Enzymology 5 Stage C GA,,-aldehyde to C20- and C,,-GAs 5.1 Metabolic steps 5.1.1 Higher plants in vitro 5.1.2 Higher plants in vivo 5.1.3 Higher plants other pathways 5.1.3.1 Early 2P-hydroxylation 5.1.3.2 12a- and 12P-Hydroxylation 5.1.3.3 15P-Hydroxylation 5.1.3.4 9,ll-Didehydro- and 9,15-cyclo-GAs 5.1.4 Fungal cultures 5.1.4.1 Gibberella fujikuroi 5.1.4.2 Other fungi 5.2 Enzymology 5.2.1 General 5.2.2 Purification and cloning 5.2.3 Multifunctionality 5 2.4 Substrate specificity 5.3 Stereochemistry 5.3.1 Gibberella fujikuroi 5.3.2 Higher plants 5.4 Mechanistic considerations 6 Concluding remarks 7 References 1 Introduction The discovery of the gibberellins (GAS) as natural plant hormones and their commercial exploitation provides an excellent example of the way in which curiosity-driven basic research leads to wealth creation.This serendipitous story has been described in many reviews (e.g. ref. 1 and references cited therein). It progressed from a Japanese farmer's interest in the bakanae disease of rice via the identification of the causative fungus as Gibberella fujikuroi (Saw.) Wr [the sexual form of Fusarium moniliforme (Sheldon)]. There followed the isolation of GAS from cultures of this fungus and the discovery that GAS occur in higher plants,2 in which they control many aspects of plant growth and de~elopment.~,~ The two basic structures and nomenclature of these plant hor- mones are shown in Fig. 1. The trivial nomenclature' whereby individual GAS are allocated a number in the series GA to GA, is used in this report. At the time of writing n=108.These GAS are oxidised variants of the two basic C20- and C,,-GAS shown in Fig. 1 and include the 9,15-cycloC,,-GAS (see Section 5.1.3.4). The structures and occurrence of GA to Mac Millan Biosynthesis of the gibberellin plant hormones 12 20 '1-C20-Gibberellins,e.g. GA12 ent-gibberell-l6-ene-7,19-dioic acid C19-Gibberellins,e.g. GAg ent-20-norgibberelI-16-ene-7,19-dioicacid 19,lO-lactone Fig. 1 Gibberellin structures and nomenclature GA, are provided in an overview6 of the chemistry of the GAS.The structures of the more recent GAS are shown where appropriate. Because of the biological importance of the GAS there has been great interest in their biosynthesis and there are many reviews for example refs. 3 7 and 8.Since the most recent review8 on the biosynthetic pathways many of the remaining details of the pathways have been established. There has also been progress in elucidating the enzymology of the pathways. Many of the enzymes have been characterised and their genes cloned; this aspect is discussed in a recent review.' A compre- hensive review of the current status of GA biosynthesis is therefore timely. The GA biosynthetic pathways were first investigated in the fungus G. fujikuroi in which the GAS are produced in relatively large concentrations. For example the end-product GA, accumulates to many grams per litre of culture medium in commercial fermentations. Up to the late 1960s these early fungal studies relied on radiotracer methods. In higher plants the amounts of GAS are much lower and therefore progress depended on the development of appropriate technologies.In particular the development of GC-MS" and the realisation that radiochemical labels as well as stable isotope labels could be detected by mass spectrometry' were important factors. Another facilitating discovery was the development of cell-free homogenates'2 derived from seed tissue in which GAS occur in relatively high concentrations (pg g -') and numbers (e.g. over 30 different types in apple seeds). In non-reproductive tissues of growing plants where the GA levels are low (ng g ') rapid ~ progress was only made possible with the advent of capillary GC-MS' together with the use of GA biosynthesis mutants. Such mutants of the fungus G.fujikuroi and of higher plants in which specific steps in the GA biosynthetic pathway are blocked have been critical in identifying genes of the pathway and the function of their products. Some of these mutants have also been used to determiqe which GAS are bioactive without further metabolism. A detailed discussion of these mutants is beyond the scope of this review. However the use of particular mutants will be discussed where relevant to the biosynthesis 22 1 Table 1Key to names of plants Abbreviated name Common name Full name A. thaliana Arabidopsis Arabidopsis thaliana B. rapa Turnip Brassica rapa C. maxima Pumpkin Cucurbita maxima C. pep0 Marrow Cucurbita pep0 C. sativus Cucumber Cucumis sativus D.dolichopetala 'Jacaranda do cerrado" Dalbergia dolichopetala H. vulgare Barley Hordeum vulgare L. esculentum Tomato Lycopersicon esculentum M. domestica M. macrocarpus Wild cucumber Apple Malus domestica Marah macrocarpus 0. sativa Rice Oryza sativa P. abies Spruce Picea abies P. coccineus Runner bean Phaseolus coccineus P. sativum Pea Pisum sativum P. vulgaris French bean Phaseolus vulgaris R. communis Castor bean Ricinus communis R. sativus Garden radish Raphunus sativus S. bicolor Sorghum Sorghum bicolor S. edule Chocho Sechium edule S. pentandra Bay willow Salix pendantra S. oleracea Spinach Spinacia oleracea T. aestivum Wheat Triticum aestivum Z. mays Maize Zea mays at C-1 C-7 C-12 and C-18 in GA from [2-I3C]MVA was determined15 by I3C NMR spectroscopy.The regio- and stereo-specificity of the incorporation of the H-atoms from MVA into GA in cultures of G. fujikuroi are also shown in Scheme 1. The significance of these labelling patterns is discussed in later Sections. Two major discoveries concerning the intermediates between MVA and GA, were made by Cross and co-workers using cultures of G. fujikuroi. First they i~olated'~"~ ent-kaur-16-ene and showed" that it was metabolised to GA,. It is of interest to note that this conversion together with the knOwn16,19,20 absolute stereochemistry of ent-kaur-l6-ene established the absolute stereochemistry of the GAS at the same time as did crystallographic Secondly Cross et al.' found that GA,,-aldehyde was a very efficient pre- cursor of fungal GAS.It is convenient therefore to discuss GA biosynthesis in the three stages A B and C specified in Scheme 1. 3 Stage A mevalonic acid to ent-kaur-16-ene Information on the intermediates between MVA and ent-kaur- 16-ene (Scheme 2) comes from the use of cell-free preparations initiated by the seminal studies of Graebe et a1.I2 These authors showed that homogenates from the endosperm-nucellus of immature seeds of M. macrocarpus (formerly Echinocystis macrocarpus) catalysed the formation of ent- kaur- 16-ene from MVA. Subsequently the formation of ent-kaur-16-ene from MVA was also observed for cell-free preparations from other sources including the young ~eeds,~~.~~ young fruit26 and shoot tips27 of P.sativum; imma- ture seeds2' and seedlings29 of R.communis; endosperm3' of C. maxima; young seedlings of 2. mays3' and L. es~ulentum;~~ and rny~elia~~.~~ germinating grain of H. v~1gai-e;~~ of G. fujikuroi. The highest rates of conversion of MVA were obtained in the endosperm homogenates from Marah and Cucurbita and ent-kaur-16-ene was the major product (ca. 40% yield). In other cases the formation of squalene and phytoene predominated.26,31 3.1 Metabolic steps The intermediacy of geranylgeranyl pyrophosphate (GGPP) between MVA and ent-kaur-16-ene was established by its formation from MVA and its conversion to ent-kaur- 16-ene using the M. macrocarpus system;36 the free alcohol geranyl- geranyl (GGOH) is not however an intermediate since it is not incorporated into GAS by resuspension cultures of G.fujikuroi.l2 The bicyclic intermediate between GGPP and ent-kaur- 16-ene was shown to be ent-copalyl pyrophosphate (ent-CPP) by Schechter and West34 using G. fujikuroi. (ent- CPP is used throughout for ( -)-copalyl pyrophosphate to distinguish it from (+)-copalyl pyrophosphate in analogous fashion to the use of ent-kaur-16-ene for ( -)-kaur-16-ene. It is recommended that this nomenclature be universally adopted.) Using a cell-free extract from G. fujikuroi Schechter and West34 identified GGPP and eat-CPP as products of MVA and demonstrated the metabolism of GGPP to ent-CPP and ent- CPP to ent-kaur-16-ene. The metabolism of ent-CPP to ent- kaur- 16-ene also occurred34 in soluble enzyme preparations from M.macrocarpus and R.communis. Thus the formation of ent-kaur-16-ene from CPP occurs in two steps further defined in Sections 3.2 and 3.3. 3.2 Stereochemistry The stereochemical details of the formation of ent-kaur- 16-ene are shown in Scheme 3. The depicted all-trans cyclisation of GGPP to ent-GPP has been established by (i) the exclusive labellingI4 of C-18 and not C-19 in GA from [2-I4C]MVA; (ii) the retention37 of the 4-pro-R hydrogen from MVA at C-3 and biological activity of the GAS. For higher plants the abbreviated Latin names are used; a key to these names is provided in Table 1. 2 Overview The diterpenoid nature of the GAS was initially e~tablished'~ for cultures of G. fujikuroi by the incorporation of four I4C atoms from [2-'4C]mevalonic acid (MVA) into GA and by degradative location of two of the labels at C-7 and C-18 (see asterisks in Scheme 1).Later the position of all four I3C-labels ent-kaur-16-ene GAI2-aldehyde j Stage C 1 C~S-GAS Scheme 1 Outline pathway from (3R)-mevalonic acid 222 Natural Product Reports I IPP DMAPP GPP c2 + c3 ent-kaur-16-ene ent-CPP GGPP FPP I I c phytoene squalene Scheme 2 MVA to ent-kaur-16-ene and (2-9; and the dem~nstration,~ that the 4-pro-R hydrogen from MVA at C-3 in ent-kaur-16-ene is lost by hydroxylation with retention of configuration. This is one of the few docu- mented cases of the generally accepted mechanism for the all-trans cyclisation of GGPP. Details (Scheme 3) of the cyclisation of GGPP to ent-CPP MVA have been elucidated as follows.First ent-CPP labelled with tritium at C-17 in the E-configuration (Ha) was incubated with a lyophilised homogenate from the endosperm of M. macrocarpus to give ent-kaur- 16-ene containing tritium at the 15-endo-po~ition.~~ Secondly the 14P-H in GA (and therefore in ent-kaur- 16-ene) is derived4' from the 5-pro-S hydrogen of MVA in cultures of G. fujikuroi and this has also been confirmed39 by the incorporation of the (1 S)-3H of GGPP into the 14P-position in ent-kaur-16-ene by the M. macrocar-pus system. These results define the stereochemistry of the GGPP H5R formation of the bicyclo[3.2. lloctane in ent-kaur-16-ene as illustrated in Scheme 3. Supplementary ~onfirmation~~ of the C-12-C-16 migration comes from the the retention of 13C-13C coupling between carbons 16 and 17 in ent-kaur-16-ene formed from [3,3'-13C2]MVA in a cell-free extract from G.fujikuro i. 3.3 Enzymology ent-C P P 1 Plant enzymes involved in the mevalonoid pathway to GGPP anti-!&' C-17 on reface of C-13 have been reviewed by Cha~pell.~~ The enzymes that catalyse the cyclisation of GGPP to ent-CPP and ent-CPP to ent-kaur- 16-ene were originally named43 ent-kaur- 16-ene synthetase (activity A and activity B) and this unsatisfactory nomencla- ture has been continued as ent-kaurene synthetase A (KSA) and ent-kaurene synthetase B (KSB). It is recommended that the name ent-CPP synthase be used for the enzyme that catalyses the step from GGPP to ent-CPP and that ent-kaur- I 16-ene synthase be used for the enzyme that catalyses the step from ent-CPP to ent-kaur-16-ene.Early attempts to purify both enzymes from cell-free extracts from G. fujikuroi pro- ~ided~~ a partially purified protein complex M ca. 430 kDa containing both ent-CPP synthase and ent-kaurene synthase activity. Partial separation of both activities was achieved44 from endosperm of M. macrocarpus. Each protein had an estimated M of ca. 82 kDa. The mixed protein preparations from both sources converted ent-CPP derived from GGPP more efficiently to ent-kaurene than added ent-CPP implying a cooperative channelling of the ent-CPP synthase product to the ent-kaurene synthase. More detailed information on ent-CPP synthase comes from the cloning45 of the GAI locus in A.thaliana using the GA-responding dwarf mutant gal and a genomic subtraction technique. This gene has been to encode an ent-CPP synthase with a molecular mass of 86 kDa which is imported into pea chloroplasts and processed to a ent-kaur-16-ene 76 kDa protein (see later). An ent-kaur-16-ene synthase has Scheme 3 Formation of ent-kaur-16-ene from MVA stereochemistry been purified (291-fold) from immature seeds of C. maxima.47 and mechanism MacMillan Biosynthesis of the gibberellin plant hormones It has M 81 kD and catalyses the conversion of ent-CPP to ent-kaur-16-ene with K 0.35 p~ for ent-CPP an optimal pH of 6.8-7.5 and a requirement for Mg2+ Mn" or Co2+. The gene coding for this enzyme has been cloned and expressed in Escherichia coli; the recombinant protein has been shown to catalyse the conversion of ent-CPP to ent-kaur- 16-ene.48 The amino acid sequences of these ent-CPP and ent-kaur- 16-ene synthases are similar to those of other plant cyclases (for a review see ref.49). Both contain N-terminal amino acid sequence^,^^,^^ characteristic of a transit peptide for targetting to plastids and the in vitro translated protein of the ent-CPP synthase is imported into and processed by the pea chloro- plast~.~~ These observations agree with evidence" for the localisation of the synthesis of ent-kaur-16-ene in the plastids of three plant species. It will be of interest to determine if there is also a plastidic route to ent-kaur-16-ene via the recently discovered non-mevalonoid derived isopentenyl pyrophos- phate (IPP).5' The ent-kaur-16-ene synthase contains the DDxxD motif that may be involved49 in the metal-complexed pyrophosphate group and in assisting ionisation of the pyro- phospate.Interestingly formation of ent-CPP from GGPP does not involve the pyrophospate and the ent-CPP synthase sequence does not contain this motif. 4 Stage B ent-kaur-16-ene to GA,,-aldehyde The pathway from ent-kaur-16-ene to GA,,-aldehyde is shown in Scheme 4. The branch pathways to the kaurenolides and seco-ring B ent-kaurenoids are included because of their enzymological interest. The evidence for these pathways and their stereochemical details are discussed in the following Sections.4.1 Metabolic steps The discovery that ent-kaur- 16-ene" and GA,,-aldeh~de~~ were efficiently metabolised to GA in cultures of G. fujikuroi prompted the search for intermediates between these two precursors. Chronologically evidence for the steps 1 to 5 ent-kaur-1 6-ene enf-kaur-16-en-19-01 GAl 2-aldehyde ent-7a-hydroxykaur-16-en-19-oic acid Is (Scheme 4) comes firstly from metabolic studies using fungal cultures then cell-free systems from immature seeds of plants and finally vegetative plant tissue. Initially 14C-labelled substrates were used and the products were identified by combinations of radio-TLC GC-radio counting and isotope dilution. Thus in fungal cultures ent-kaur- 16-en- 19-01 12,52s3 ent-kaur- 16-en- 19-a1,54 ent-kaur- 16-en-19-oic and ent-7a-hydroxykaur- 16-en- 19-oic a~id~~,~~ to be were shown metabolised to GA,.The same kind of evidence was obtained for the following individual steps; steps 1 2 and 3 using homogenates from seeds of M. ma~rocarpus;~~ steps 1 4 and 5 using homogenates from seeds of C. maxima5* and P. sati-v~rn;~~ and step 5 in G. fujikuroi More definitive evidence for steps 4 and 5 was obtained59 in the cell-free system from P. sativum using 14C-labelled substrates and GC-MS identification of products. More recently each of the steps 1 to 5 have been established6' for young shoots of 2. mays by applying the (1 7-13C 1 7-,H)-labelled substrate for each step and identifying the product of each step by GC-MS and Kovats retention indices.Importantly in the latter study each of the six intermediates were also shown to occur naturally in the shoots of 2. mays by determining its isotopic dilution following the addition of each of the (1 7-I3C 17-3H)-labelled intermediates to shoot extracts. In other systems that have been used to study the pathway to GA,,-aldehyde there is fragmentary evidence that the intermediates are endogenous e.g. ent-kaur-1 6-ene,17,61 ent-kaur-16-en-19-01,'~,~' ent-kaur-16-en- 1 9-a16' and ent-kaur-16-en- 19-oic acid62 in the mycelia of G. fujikuroi; and ent-kaur- 1 6-ene,63,64 ent-kaur-16-en- 19- 01~~ and ent-kaur- 16-en- 19-oic in the microsomal prep- arations of immature seeds of C. maxima. Curiously the only direct identification (by GC-MS) of GA,,-aldehyde has been made65 in extracts of mature seeds of P.sativum although immature seeds of this and other species provide the most efficient cell-free systems for the in vitro formation of GA,,- aldehyde from its precursors. However the identification of GA,,-aldehyde in developing seeds of P. sativum has been established66 by the isotopic dilution of [14C]GA aldehyde that had been added to and recovered from a homogenate of the seeds. ent-kaur-16-en-19-oic acid ent-kaura-6,16-dien-l9-oicacid l9 fujenal ent-6a,7a-dihydroxykaur-l6-en-l9-oic acid kaurenolides ent-6a,7a-epoxykaur-16-en-l9-oic acid Scheme 4 ent-Kaur-lBene to GA,,-aldehyde (bold arrows) kaurenolides and fujenal pathways and stereochemistry 224 Natural Product Reports In addition to the metabolic evidence for each of the steps from ent-kaur- 16-ene to GA,,-aldehyde the overall conver- sion of MVA to GA,,-aldehyde has been realised using cell-free systems from C.maxima end~sperm,~~ P. coccineus seeds6' and S. edule endosperm and embryos.69 The C. maxima system in particular has been very useful for the prepara- tion of GA,,-aldehyde (and other GA intermediates) for the metabolic studies described in Section 5. The branch pathway from ent-7a-hydroxykaur- 16-en- 19-oic acid to ent-6a,7a-dihydroxykaur-16-en-l9-oic acid (step 6 Scheme 4) has been shown7' to occur in the C. maxima system by labelling studies and GC-MS identification of the product. Earlier,7' using 14C-labelled material and cultures of G.fujikuroi ent-6a,7a-dihydroxykaur-16-en-19-oic acid was shown to be present by isotopic dilution and converted to fujenal (step 7). The kaurenolides (R'=R2=H and R'=OH R2=H; Scheme 4) in cultures of G. fujikuroi and the kaurenolide (R'=R2=OH) occurs in C. pe~o~~ and C. maxim^.^'.^^ Evidence for the pathway to the kaurenolides shown in Scheme 4 has been obtained from C. maxima endosperm homogenate and from cultures of G. fujikuroi. Using the C. maxima system it was e~tablished~~ that ent- [14C]kaura-6 16-dienoic acid was formed from ent-['4C]kaur-16-en-19-oic acid (step 8) and its precursors although not from ent-7a-hydro~y-['~C]kaur-16-ene-19-oic acid. Also the [14C]-6 16-dienoic acid was converted into 7P-hydroxy- ['4C]kaurenolide (R' =R2=H) and 7P,12a-dihydroxy-[14C]kaurenolide (R' =H R2=OH); the intermediacy of the 6P,7P-epoxide was inferred.In fungal cultures a detailed confirmed that ent-kaura-6,16-dienoic acid was a pre- cursor of 7p-hydroxykaurenolide and it was shown that a synthetic sample of the presumed intermediate the 6P,7P- epoxide was rapidly converted into 7P-hydroxykaurenolide in aqueous buffer. This also eliminated the intermediacy of the parent kaurenolide and ent-6P,7a-dihydroxykaur-16-en- 19-oic acid. The metabolism of 7P-hydroxykaurenolide to 7P 1 8-dihydroxykaurenolide has been dem~nstrated~~ in fungal cultures by l4C-1abelling. 4.2 Stereochemistry The results of stereospecific labelling with tritium and deuter- ium are summarised in Scheme 4 where the relevant hydrogen atoms are appropriately labelled.Step 2 from ent-kaur-16-en-19-01 to ent-kaur-16-en-19-al has been to involve the loss of the 19-pro-R hydrogen in a microsomal enzyme preparation from M. macrocarpus seeds. The evidence comprised the preparation of the two [19-3H,]- diastereoisomers of ent-kaur- 16-en-1 9-01 distinguished by 'H NMR spectroscopy and determining the 3H:'4C ratios in the products from doubly labelled substrates. The stereochemistry of the steps subsequent to ent-kaur- 16- en- 19-oic acid were determined using eat-kaur- 16-en- 19-oic acid labelled with deuterium at the ent-6a- 6P- 7a-or 7P-po~itions~~ (Had respectively in Scheme 4). The metab- olism of these labelled substrates were examined76 in a micro- soma1 enzyme preparation from the endosperm of C.maxima seeds and in cultures of G. fujikuroi. The deuterium content of the metabolites from each substrate was determined by GC-MS. The results from both systems showed that (i) in steps 4 and 6 ent-6a- and ent-7a-hydroxylation occurs with retention of configuration; (ii) in step 8 the formation of ent-kaur-6,16-dien- 19-oic acid (and the kaurenolides) occurs by stereoselective loss of the ent-7a-hydrogen and non-selective loss of the ent-6a- and ent-6P-hydrogens; and (iii) in Step 5 the formation of GA,,-aldehyde occurs with the loss of the ent-6a- and 7a-hydrogens and the retention of the ent-6P-hydrogen. These results complement a previous study7' showing that GA ,-aldehyde was formed from ent-7a-hydroxy-[6,6-3HH,,'4C]kaur-16-en-19-oic acid with the loss of one tritium.Also in a conference proceedings Graebe77 reported that ent- 7a-hydro~y[6a-~H, ''C]kaur-l 6-en-19-oic acid was metabolised MacMillan Biosynthesis of' the gibberellin plant hormones by the C. maxima system to GA,,-aldehyde and ent-6a,7a- dihydroxykaur- 16-en- 19-oic acid with loss of the tritium label and with an isotope effect of 10-12 while ent-[6P-3H,'4C]-7a- hydroxykaur-16-en-19-0icacid was metabolised to the same products with retention of tritium. All of these results con- flict with an earlier conclusion by Hanson et that the formation of GA,,-aldehyde involved the retention of both hydrogens at the C-6 position in its kaurenoid precursor. This conclusion was based on the low incorporation (0.005%) of [1-3H,,l-'4C]geranyl pyrophosphate (GPP) into GA,,-aldehyde containing the same 3H,:'4C ratio as the substrate in cultures of G.fujikuroi. However the bulk of direct labelling sudies are consistent with the stereochemistry shown in Schemes 4 and 5 for the ring contraction of ent-7a-Enz-Fe4+=0 Enz-Fe3+:OH Ll ent-7a-hydroxykaur-16-en-19-oic acid ,,,,' 1 ent-6a,7a-dihydroxykaur-l6-en-l9-oic acid GAI2-aldehyde Scheme 5 Formation of GA,,-aldehyde and ent-6a,7a-dihydroxykaur-16-en-19-oic acid from ent-7a-hydroxykaur- 16-en- 19-oic acid via a common intermediate hydroxykaur- 16-en- 19-oic to GA,,-aldehyde. The enzymology and the accompanying formation of ent-6a,7a-dihydroxykaur-16-en-19-oic acid are discussed in the Section 4.3.4.3 Enzymology The steps 1 2 3 and 4 (Scheme 4) were shown54 to be catalysed by the microsomal fraction of the M. macrocarpus system requiring 0 and a reduced pyridine nucleotide (pref- erably NADPH). Steps 1 and 3 are inhibited by CO and the inhibition is reversed by light with maximum effectiveness at 450 nm; reduced microsomes from M. macrocarpus end~sperm~~ and from cotyledons25 from P. sativum gave CO-difference spectra with maxima at 420 and 450 nm. These properties studied in some detail79 for the M. macrocarpus system accord with those of cytochrome P450 mixed-function oxidases." Step 4 in G. fujikuroi shows'' the same character- istics. Each of the steps 14 may be catalysed by different catalytic sites" and explain the existence6' of a mutant B1-41a of G.fujikuroi blocked for step 3. Steps 5 and 6 (Scheme 4) at the branch from ent-7a- hydroxykaur- 16-en- 19-oic to the first ent-gibberellane in the pathway and to ent-6a,7a-dihydroxykaur-16-en- 19-oic acid appear to be catalysed by the same enzyme in the C. maxima system.82 Both activities are catalysed by the microsomal fraction with the same optimal cofactors (0 and NADPH) pH (7.0-7.4) and temperature (30 "C) and similar Michaelis- Menton kinetics. Thus both reactions may have a common intermediate perhaps the C-6 free radical in Scheme 5 from which GA,,-aldehyde may be formed by a 1,2-radical shift driven by discharge of the radical. None of the enzymes have been purified. Several GA-responding dwarf mutants have been characterised that may have lesions at some of the steps 1-5 (Scheme 4).For example step 5 may be blocked in the nu mutant of P.sati~um,'~ step 4 or 5 may be blocked in the gib-2 mutant of L. es~ulentum,'~ and step 1 or 2 may be blocked in the ga2 mutant of A. th~liana.~~ However none of the genes for these functions have been cloned. The only cloned gene that may function in this part of the pathway is the 03 locus in 2.mays.86The predicted protein sequence has high similarity to the cytochrome P450 superfamily including the highly conserved signature FxxGxxCxG. However the gene product has not been characterised or its function determined. 5 Stage C GA,,-aldehyde to C2,,- and C,,-GAS 5.1 Metabolic steps While the biosynthetic pathway from MVA to GA,,-aldehyde is the same in the fungus and all higher plant systems that have been studied in contrast the pathway from GA, aldehyde differs in detail depending on the organism.However the conversion of the C,,-GAS to the C,,-GAS has a common basis (see Scheme 6 and carbon numbering). This basic sequence comprises the progressive hydroxylation at C-20 12 r? J isolated as I"' & R' I C02H C02H Scheme 6 General pathway from GA,,-aldehyde to C,,-GAS non- early 3,13-hydroxylation pathway when R' =R2=H; early 3-hydroxylation pathway when R'=OH R2=H; early 13-hydroxylation pathway when R'=H R2=OH 226 Natural Product Reports from the methyl group to the hydroxymethyl group (always isolated and identified as the &lactone) then to the aldehyde and finally to the y-lactone with loss of C-20 as C0,.87 This sequential oxidation at C-20 has been shown to occur before or after hydroxylation of GA,,-aldehyde or GA, at the 3- and 13-positions.Thus three pathways to the C,,-GAS have been established (a) non-early 3 P 13-hydroxylation; (b) early 13-hydroxylation; and (c) early 3P-hydroxylation. An early 3P,13-hydroxylation pathway to C,,-GAS may also exist but the evidence is incomplete (see later). 12a-Hydro~ylation~~~~~ of GA,,-aldehyde and 2P-hydroxylation of GA,,90,91 and of GA,,, have been demonstrated but conversion of 2P- and 12a-hydroxy-C2,-GAs to 2P- and 12a-hydroxy-C1,-GAs has not been established.Pathways to 11-hydroxy- 1 5P-hydroxy- 9,ll -didehydro- and 9 15-cyclo-GAS have been little studied (see Section 5.1.3). Studies on the pathways post-GA,,-aldehyde began by using fungal cultures. However for clarity the evidence for the pathways is presented in three sub-sections in the order in vitro systems from higher plants; in vivo systems using intact plants and fungal cultures. The enzymology stereochemistry and mechanisms of the steps are discussed separately. 5.1.1 Higher plants in vitro Since developing seeds accumulate a plethora of GAS in relatively high concentrations extracts from seeds have been used extensively for in vitro studies of the pathways beyond GA,,-aldehyde. Thus homogenates of seed tissue have been used from C. maxima,58~82~88.89~93-97 P.vul-P. garis,99~100 P. coccineus,lo' ,Io2 and M. macrocarpus. lo3-'05 Cell-free homogenates have also been used from leaves of S. ~Zeracea,~~ from germinating grain of H. vulgare,'06 and from anthers of 0.sativa. lo7 Fractionation of these homogen- ates has provided partially purified enzyme preparations from endosperm of C. maxima,'08~'09 cotyledons of P. VUZ-garis' and P. sativum,117'"8and leaves of S. oleracea.' l9 More recently molecular cloning has provided recombinant enzymes for some of the biosynthetic steps from C. M. macrocarpus,'05 S. oZeracea'21and A. thaliana,'22-'25and these proteins have been used to establish specific steps in the pathways The properties of the known biosynthetic enzymes are discussed in Section 5.2.The pathways defined by the in vitro2tudies with homogenates purified native enzymes and recombinant protein are presented schematically. Scheme 7 shows the steps from GA,,-aldehyde to the initially formed C,,-GAS GA, GA, GA, and GA, and Scheme 8 shows the subsequent steps from these C,,-GAS. In these schemes the full arrows show each of the steps that were established by GC-MS identification of the labelled compound (shown after the arrow) as a metabolite of its labelled precursor (shown before the arrow). The multiple full arrows show multiple steps and the dashed arrows show individual steps that are inferred by analogy or from indirect evidence. For the non-3p 13-hydroxylation pathway to GA (Scheme 7) each step from GA,,-aldehyde has been established for in vitro systems from P.vulgaris,"' and H. vulgare,'06 while each step from GA, has been shown in homogenates from P. ~ativum.~* Some of the details require comment. Oxidation of GA, occurs at C-20 to give the alcohol isolated as the &lactone GA,,. Further oxidation at C-20 to the aldehyde GA,, occurs either on the opened lactone in systems from non-vegetative tiss~e~*~'~~ or on the &lactone in the system from vegetative tissue.106 This distinction is also shown for the step from GA to GA19 in the early 13-hydroxylation path- way to GA, and is discussed later. The aldehyde GA24 is then converted to GA with the loss of C-20 as CO and not as H,C =0 or HC0,H in the P. sativum system.87 The individual steps to GA that have been confirmed in other systems are noted in Scheme 7.For the early 3P-hydroxylation pathway from GA,,-aldehyde to GA, all the steps except one have been HO* CHO C02H G A ,-aidehyde GA12-aldehyde GAS3-aldehyde 56.82.68.93.97.108 c maxima 62 93 P. vulgaris loo H. vulgare lob 1 1 1 HO@ 8 CO2H COpH GA14 c a9.9?.io9,1zo p. sativum 98 P. sativum g8,118 P vulgaris P. vulgaris C maxima 82 93 M. macrocarpus lo5 S oleracea OQ lZ1 H. vulgare lo6 P. coccineus '02 A thaliana lZ2 H. vulgare lo6 0 sativa107 i.A thaliana 122,123 i i t HO* 0-CHp *C maxima g3 96 lo8 P vulgaris '00 co --> P vulgaris l1 (both opened lactone) HO HO C02H G A38 P. sativum 96 (opened lactone) P.sativum 9a (opened lactone) P. vulgaris'OO(opened lactone) S.oleracea (lactone) H. vulgare '06 (lactone) H. vulgare '06 (lactone) I COpH C02H GA36 GA23 C. maxima lz0 C maximag3 S.oieracea t* P sativum 96 C maximag5 a1 1 P sativum 96 p. vulgaris100 @ C maxima'20 * C maximaag 93 P vulgaris 'O0 __-* P. coccineus lo2 __.> C maxima 120 /-/ vulgare lo6 A. thaliana 123 HO C maxima'20 s o/eracea90.'2' HO C02H ; COpH I COpH C02H COpH GA17 GA28 GA13 7 7 HO HO GA4 G A20 GAl I I Scheme 7 Biosynthetic steps established in vitro from GA,,-aldehyde to C,,-GAS;dashed arrows indicate steps not direct determined (see text); horizontal rows contain GAS at the same C, oxidation level; vertical rows show non- early 30- and early 13-hydroxylation pathways and intermediates in a possible early 3p 13-hydroxylation pathway demonstrated using enzyme preparations from C.maxima Individual steps from GA, to GA, in other systems are seeds.82,93.95 The step from GA,, or its open lactone to GA, recorded in Scheme 7. As in the case of the step from GA, to has not been directly confirmed; the lactone was not metabo- GA24 the C-20 alcohol of GA, appears to be the the true and the opened lactone has not been examined because precursor of GA, in cell-free systems from non-vegetative li~ed,~ curiously the opened 6-lactone of GA, of the anticipated (mistaken?) 3-epimerisation during its tissue of P. ~ativurn;'~ preparation by alkaline hydrolysis of the lactone. is 3P-hydroxylated to GA, by a cell-free preparation from For the early 13-hydroxylation pathway to GA,, P.vuIgaris embryos."' However GA, is metabolised to GAl9 13-hydroxylation of GA ,-aldehyde to GAS,-aldehyde has as the &lactone in cell-free preparations from leaves of S. been noted in embryo homogenates from P. vulgaris"' and oleraceago and germinating grain of H. vu/gare.lo6 As noted The step from GA,,-aldehyde to GA, has in Section 5.1.2 GA, was also converted" to GA, in shoots P. cocczneus.101~102 not been reported but the formation of GA, from GA,,- of Z. mays without the opening of the lactone. Thus the aldehyde via GA, has been observed in several in vitro 6-lactone serves as a precursor of the C,,-GAS in vegetative systems noted in Scheme 7. All the steps to GA19 from tissues while the corresponding hydroxy acid may be the GA,,-aldehyde via GA, and GA, have been established intermediate in non-vegetative tissues (see Section 5.3.2).in vitro for germinating grain of H. vu/gare;lo6 the step from An early 3P 13-dihydroxylation pathway from GA,,-GA, to GA, could not be established but can be assumed aldehyde to GA has been considereds9 but there is incom-since GA, was converted to GA, (9%) as well as GAl9 (53%). plete evidence for its presence in vitro. Only the last step from MacMillan Biosynthesis of the gibberellin plant hormones 227 GA23 (Scheme 7) has been established12' using the fusion protein expressed in E. coli from the cloned C-20 oxidase from C. maxima endosperm (see Section 5.2). Also shown in Scheme 7 is the formation of GA23 in a cross-over conversion from GA19 in the early 13-hydroxylation pathway using a partially purified 3P-hydroxylase from endosperm108 of C.maxima. A logical precursor of GA23 is GA, but its stepwise formation and the conversion of GA, to GA23 has not been established. The only evidence for this pathway is indirect and comes from the observations that GA, is metabolised'OO to GA, in a cell-free system from P. vulgaris embryos and that incubation of GA, with increasing concentrations of a soluble enzyme preparation from C. maxima endosperm gave,89 progressively GA,, GA23 GA28 and GA . The C-20 aldehyde intermediates are the immediate precur- sors of the C,,-GAs but they are also oxidised to the C,,-GA tricarboxylic acids.Thus GA24 gives9 GA25 GA, gives9 GA13 GA, gives'20 GA17 and there is indirect evidence" for the conversion of GA, to GA28 (see Scheme 7). However these tricarboxylic acids and their C- 19,C-2O-anhydrides do not appear to be precursors of the C,,-GAS because GA, is not metab~lised,~ to GA while neither GA,, nor its C-19,C- 20-anhydride is metab~lised,~ to GA,. The C,,-GAs that are initially formed from the C,,-GAS are further oxidised in vitro (see Scheme 8 and refs. therein). 3P-Hydroxylation of GA to GA and of GA, to GA has been observed in most of the enzyme preparations studied. 3P-Hydroxylation of GA, to GA occurs1oo in the P.vulgaris system but 13-hydroxylation of GA to GA, has only been observed as the opened lactone on the P.sativum system., 13-Hydroxylation of GA to GA has not been recorded. 2P-Hydroxylation is a common feature both in vitro and in vivo studies (see Section 5.1.2) leading to biologically inactive GAS and all of the steps GA,/GA,, GA,,/GA, and GA,/GA, have been observed in one or more of the commonly used in vitro systems as documented in Scheme 8. The direct formation of GA, from GA has not been reported. ID-Hydroxylation has only been reported in one system namely a homogenate of M. macrocarpus ernbryo~.'~~~'~~ 2,3-Dehydrogenation of GA to 2,3-dehydro-GA9 occurs in the M. macrocarpus and of GA, to GA in the P. ~ulg~ris"'~~~''~~''~ and H. vulgare'06 systems. The conversion of 2,3-dehydro-GA9 to GA, and of GA to GA and traces of the related GA,, occurs in the M.macrocarpus system.103,105The conversion of GA to GA is also catalysed by enzyme preparations from germinating grain of H. vulgare106and anthers of 0.sativa.114 The conversion of GA to GA also occurs in vivo (Section 5.1.2). The steps dehydro-GA,/GA and GAJGA, are unique enzymatic reactions. In G. fujikuroi,the origins of GA and GA are different (see Section 5.1.4.1). The stereochemis- try and mechanism of the formation of GA, GA and GA, are discussed in detail in Sections 5.3 and 5.4. 213,3P-Epoxidation of GA to GA has been observed in a partially purified preparation of a 3P-hydroxylase from P. vulgaris embryos114 and in a homogenate from seeds of M. macro-carpus;103the latter system also catalyses the conversion of 2,3-dehydro-GA9 to 2f3,3f3-epoxy-GA9.In summary the results from the in vitro studies provide a wealth of detailed information on the pathways from GA, aldehyde. However they may not reflect the true situation in intact plants. An interesting feature is cross-over between the pathways at each level of oxidation at C-20 up to and including the initially formed C,,-GAS. Yet there is no cross-over thereafter for example between the 2P-hydroxy- C,,-GAS. The observed metabolic grid may be a consequence of a loss of compartmentation in the preparation of the homogenates. This point is returned to in Section 5.1.2. 5.1.2 Higher plants in vivo The in vivo metabolism of GAS was initially conducted with developing seeds. These early studies using ,H-labelled sub- strates have been thoroughly reviewed by Sponsel'26 and are not discussed here.Attention is focussed on later work in which individual steps from GA,,-aldehyde have been estab- lished by the criteria described in Section 5.1.1. Scheme 9 shows the established steps from GA,,-aldehyde to the C19- GAS that may be bioactive without further metabolism (see P. sativum 58 C.maxima 57 I 0 sativa107 P. sativum 98 P. vulgaris I16 P. vulgaris 12 j3 P. vulgaris"24. 116 M. macrocarpus1°3 H. vu/gare 106 C. maxima 88 P. vulgaris P. sativum 58 HO A. fhaliana lZ5 (opened lactone) M. macrocarpuslo5 I HO I co -' C02H @C02H M. macrocarpus 0.sativa114 H. vulgare I co -' @C02H Io5 + -' HO GA7 2,3-de hydro-GA9 M.macrocarpus'03J '03 GA3 M.macrocarpus P. vu/garislll,114 PP,BP-epoxy-GAg GAG Scheme 8 C,,-GAS in vitro conversions 228 Natural Product Reports later) and Scheme 10 shows the further metabolism of these Vegetative tissues of Z. mays contain GA ,-aldehyde6' and they contain GAS characteristic C,,-GAS. The most complete information comes from studies GA,,. 152,153 In additi~n,'~~?'~~ using shoots of 2. mays and the results of these studies are of the three pathways indicated by the in vitro studies (Scheme discussed in detail. For clarity the sequential steps that have been established from GA,,-aldehyde for shoots of Z. mays are highlighted by bold arrows and bold references in Schemes 9 and 10; discontinuous steps that have been determined in other plants are documented in normal print and they are discussed separately where appropriate.7). Most of these endogenous GAS namely GA,, GA,, GA,, GA19 GA,, GA29 GA, GA, GA and GA, belong to the early 13-hydroxylation pathway; the others are GA,, GA24 GA and GA (non-early 3,13-hydroxylation pathway) and GA and GA, (early 3-hydroxylation pathway). All steps in the early 13-hydroxylation pathway from GA,,-aldehyde to * p-p __-J 4 P. sativum --J 6 CHO CHO C02H GAB3-aldehyde GAI2-aldehyde P.sativum 137 ~ z. mays91 gp P. sativum 137,738 I I C02H C02H C02H C02H GA53 (312 i?. mays 91 S.oleracea 140 ow\ I C02H GA44 Z. mays 91 1 Z. mays 91 S. pe ndanta 45 C. sativus 149 IR.safivus 47 1 o n,OH on Z. mays 126,130 6.rapa j42 S.pendanta 143 R.sativus 147 z.mays 127,128,130 A. thaliana I3l 0.sativa I31 6.rapa 14* P. sativum 135.136 R. sativus 47 S. oleracea I39 P. abies 148 H. vulgare 146 C. sativus 149 R. sativus 147 S. bicolor 50 1D.dolichopetala 51 Z.mays l29 A. thaliana 29 0.sativa 129 6.rapa 142 S. pendanta 44 R. safivus147 0.dolichopetalaI5l I GA3 I I I J Scheme 9 Steps established in vivo from GA,,-aldehyde to C,,-GAS that are bioactive per se (see text); dashed arrows indicates steps not directly determined MacMillan Biosynthesis of the gibberellin plant hormones GA, have been demonstrated in vivo in young plants of 2. mays.91As shown in Scheme 9 GA,,-aldehyde is converted to GA, which is 13-hydroxylated to GA, and then converted stepwise to GA, via GA, and GA,,.It should be noted that GA, is formed from GA, without prior opening of the &-lactone providing another example of the apparent difference between vegetative and non-vegetative tissues in this respect (see Section 5.1.1). Gibberellin A, has been shown to be at a branch point. It is 3P-hydro~ylated'~~~'~~ to GA and 2,3- de~aturated'~~,'~~ to GA (Scheme 9). Gibberellin A is then con~erted'~~''~~ to GA by the unique ene-hydroxylation- rearrangement reaction noted in Section 5.1.1 and discussed later in Section 5.4.Thus the in vitro transformations of GA, and GAS noted in Section 5.1.1 also occur in vivo in shoots of 2.mays. Later steps in the early 13-hydroxylation pathway in 2. mays plants are continued in Scheme 10 where it is shown that GA, is also metab~lised'~~"~~ to GA29 and thence GA,,-catabolite and that GA is 2P-hydro~ylated'~~ to GA8. The apparent metabolism'28 of GA, to 3-epi-GA1 has subse- quently been to be artefactual at least in the case of 2.mays. The only step that has not been determined is that to GA17 from presumably GA, (Scheme 9). In summary the in vitro studies with Z. mays plants have established each step in the early 13-hydroxylation pathway form GA,,-aldehyde to all the endogenous 13-hydroxy-GAs except GA,,. Since all the steps from ent-kaur-16-ene to GA,,-aldehyde have also been documented for this plant (see Section 4.l) the complete path- way from ent-kaur-16-ene to the endogenous 1 3-hydroxy-GAS in the vegetative shoots of 2.mays has been elucidated.Indi- vidual steps in the early 13-hydroxylation pathway have been documented in vivo in other plants as detailed in Schemes 9 and 10. Particular attention has been paid to the conversion of B. rapa 142 1 P.abies 148 GAS See Scheme9 GA, to GA because of the biological importance of GA (see later). For the non-early 3,13-hydroxylation pathway to GA, the only in vivo evidence is the formation'49 of GA from GA, in shoots of C. sativus and there is no evidence for an early 3p-hydroxylation pathway to GA,. Evidence for these two pathways could be obtained from detailed metabolic studies using A.thaliana seedlings which are known'55 to contain most of the GAS of these two pathways. However only incomplete and preliminary data are a~ailable.'~ 3p-Hydroxylation of GA to GA has been shown to occur in vivo in the four plant species noted in Scheme 9. 13-Hydroxylation of GA to GA, and GA to GA has also been demonstrated in the plants listed in Scheme 9. However in contrast to the in vitro studies (Scheme 7) there have been no reported cases of cross-over from possible C-20 alcohol or aldehyde intermediates to other pathways. This absence of a complex metabolic grid may be because few of these inter- mediates have been examined in vivo in the same detail as in vitro. Nevertheless cross-over has not been observed for GA,, GA, or GA19.The C,,-GAS shown in Scheme 9 are further hydroxylated then oxidised or conjugated as shown in Scheme 10. The 2p-hydroxylation of GA, to GA29 and of GA to GA8 in 2. mays plants have been discussed previously. These and the other known examples of hydroxylation at the lp- 2a- 2p- 12a- and 12P-positions are recorded in Scheme 10 with references to the species in which specific steps have been determined. To complete the metabolic picture conjugation of GAS has been shown to occur in many plants. The most definitive evidence has been shown for germinating seeds of D. dolichopelata' s1 in which the following transformations GAPS-catabolite GA60 ~ i'. mays 129 A. thaliana 129 0. sativa 129 B. rapa 142 H.vulgare 146 OH GA7j.12P-OH 230 Natural Product Reports GA81 ___) HO HO@ C02H G A4 HO-' 2.mays 128 S. pedandra 145 H. vulgare 146 R. sativus 147 .._______________ HO HO GA6 GAB-catabolite Scheme 10 In vivo metabolism of C,,-GAS I ,n n,OH GAS8 GA99 Fig. 2 Natural 2P-hydro~y-C~~GAs have been established GA, to GA1-3~-0-glucuronic acid and GA1-3~-0-glucoside; GA to GA,-3P-O-glucoside; GA to GA -3P-0-glucoside; and GA to GA3-3P-O-glucoside. Also the hydrolysis of [ 17-13C]GA,,[6-2H]glucosyl ester and recon- jugation of [17-'3C]GA, and its metabolites have also been observed. 56 The biological significance of the results from the in vivo metabolic studies comes from metabolic studies using dwarf mutants that are blocked for specific steps in the GA bio- synthetic pathway.The application of GAS to these mutants before the blocked step has no effect on growth but appli- cation of GAS after the blocked step restores normal growth. Thus a knowledge of the the endogenous GAS and of the biosythentic pathway provides a means of identifying the GA(s) that promote stem elongation without further metab- olism. Of particular importance is the step from GA, to GA that has been shown to be blocked in the dl mutant of Z. mays,127 the le mutant of P. sati~um'~'(Mendel's original dwarf pea) the dy mutant of 0.~ativa'~~ and the ga4 mutant of A. thaliana13' Bioassays using such mutants have led to the conclusion that GA is the main GA of the early 13-hydroxylation pathway that promotes stem elongation without the need for further metabolism (for reviews see refs.157 and 158). The biological importance of the step from the biologically inactive GA, to the bioactive GA explains the attention that this metabolic step has received (see refs. in Scheme 9) in terms of both genetics and environmental influ- ences. Recently it has been shown'30 that the dl mutant of Z. mays is also blocked for the metabolism of GA, to GA and GA to GA and that GA and GA are as active as GA in promoting the growth of the dl mutant. Thus at least for Z. mays GA and GA are also active without further metabolism. The hormonal status of GA and GA is less clear. Both are bioactive but GA is metabolised to GA, and GA is metabolised to GA in a range of plants as detailed in Scheme 9.It remains to be determined if GA and GA are bioactive without metabolism to GA,. 5.1.3 Higher plants other pathways 5.1.3.1 Early 2P-hydroxylation 2P-Hydroxylation of C,,-GAS is well documented in vivo and results in the loss of biological activity (e.g. GA to GA, Scheme 10). However there are indications that that there may also be an early 2P-hydroxylation pathway in vivo from GA,,. Of the 2P-hydroxylated C,,-GAS shown in Fig. 2 the occurrence of GA,, GA and GA, in higher plants has recently been e~tablished;'~' confirmation of the identification of 2P-hydroxy-GAI awaits its rational synthesis. By analogy with the established pathways (e.g. Scheme 9) these GAS can be placed in the sequence 2P-hydroxy-GA,,/GA,,/GA9,/ GA,,.The first step has been shown" for a cell-free extract from leaves of S. oleracea and the sequence is indicated by the finding" for shoots of 2. mays that GA, is metabolised to MacMillan Biosynthesis of the gibberellin plant hormones 2P-hydroxy-GA,, GA, and GA,, yet GA, and GA, were not formed from GA, and GA,, respectively. On the other hand GA, is reported, to be metabolised to GA, in S. oleracea plants. Direct evidence for a linear 2P-hydroxylation pathway must await further metabolic studies. It would be of particular interest to determine if GA, were converted to the corresponding C,,-GA and provide an alternative pathway to GA51 that has been establised via the non-early hydroxylation pathway to GA (see Schemes 9 and lo).5.1.3.2 12a- and 12fLHydroxylation Five 1 l-hydroxy- and twenty 12-hydroxy-GAS have been identified from higher plants. As shown in Scheme ll(a) 12a-hydroxylation of C2,- and C,,-GAS has been estab-in homogenates from C. maxima endosperm and embryos; in these in vitro systems pathways have been docu- mented to 1 2a-hydroxy-C2,-GAs with oxidation levels at C-20 of the alcohol (12a-hydroxy-CAl , 12a-hydroxy-GA3,) and the acid (GA,, 12a-hydroxy-GA2 and -GA,,). 12a- and 12P-Hydroxylation of C2,-and C,,-GAS have also been observed in vivo. As shown in Scheme 10 GA is 12a-hydroxylated to GA, in Z. mays,1290. ~ativa,'~~ and A. thali~na,'~~ and it is 12P-hydroxylated to GA7 in H. vulga~e'~~ and A. thaliana.', However direct evidence is lacking for steps from 1 2a-hydroxy-C2,-GAs to C ,,-GAS.Interestingly the methyl esters of GA,, GA,, GA and GA are 12a- and 12p-hydroxylated in cultures of spores of the fern Lygodium japonicum. These metabolic studies,160p162 shown in Scheme ll(b) were undertaken following the identifi~ation'~~ of GA methyl ester as a native inducer of antheridia in prothallia of this fern (see also Section 5.1.3.4). 5.1.3.3 15P-Hydroxylation The known 15P-hydroxy-GAs shown in Fig. 3 can be arranged in putative biosynthetic sequences from GA, and GA, via the (as yet) unnatural 15P-hydroxy-GA, and the natural 1 SP-hydroxy-GA, (GA,,,) by analogy with the path- ways shown in Schemes 7-10. However no relevant metabolic studies have been reported.Thus elucidation of their bio- synthetic origin and that of the highly oxygenated GA32 GA, and GA, remains a challenge. 5.1.3.4 9,ll-Didehydro- and 9,15-cyclo-GAs An equally challenging problem is the biosynthetic origin of the 9,ll-didehydro-GAS (GA,, GA8 and GA,,) and the more recently characterised 9,l 5-cyclo-GAS (~~,03~,08). Both structural types shown in Fig. 4,cooccur with 15P-hydroxy- GAS in seeds of M. domestic^'^^-'^^ and all three types may have common biosynthetic precursors e.g. a C-15 free radical. The structures are drawn in Figs. 3 and 4 in a way that emphasises this possible biosynthetic relationship but no per- tinent metabolic studies have been reported. One point of biological and biosynthetic interest is the relationship of the 9,ll -dehydro-GAS and the 9,l 5-cyclo-GAS to the antheridio- gens which induce antheridia formation in fern prothallia.Thus GA, occurs166 as the methyl ester in Lygodium japonicum; GA104 (1 P-OH-9,l 5-cyclo-GA9) occurs167 in Ane-mia mexicana; and GA,, (3a-OH-9,1 5-cyclo-GA9) occurs168 in Anemia phyllitidis with the related antheridic acid'69,'70 (Fig. 4). All three compounds induce antheridium formation. Metabolic studies with cultures of A. phyllitidis have shown'68 that antheridic acid is formed from GA,, via GA107 and that GA, is formed from GA103. 5.1.4 Fungal cultures 5.1.4.1 Gibberella fujikuroi In cultures of G. fujikuroi the major pathway from GA,,- aldehyde begins by 3P-hydroxylation to GA,,-aldedyde171 (Scheme 12).This early 3P-hydroxylation pathway to GA is accompanied by the minor non-early hydroxylation pathway 231 a OH -Ref. 88 Ref. 88 GA12 aldehyde (Scheme 7) :C02H C02H 12a-OH-GA12 Ref. 97 II (374 Ref. 88/ 12a-OH-GA12-aldehyde JRef. 88 PH GAl2 (Scheme 7) HO Ref. %'\ PH 1& Ref. 97 \ 12a-OH-GA15 PH __-J -Ref,97 12a-OH-GA37 ,?H __-J -Ref. 89 H O --J S HO HO HO I C02H C02H 1 C02H C02H 4 C02HC02H :C02H C02H GA39 12a-OH-GA43 GA43 OH PH Ref. 89 941 GAI3 (Scheme7) GAg -Ref. 88 (Scheme 7) HO b PH OH I Ref. 162 P + GA12 Me ester R = H 12a-OH-GA12 Me ester R = H 12P-OH-GAI2 Me ester R = H GAI4 Me ester R = OH GA74 Me ester R = OH 1 2P-OH-GAI4 Me ester R = OH _____) R GAg Me ester R = H GA70 Me ester R = H GA69 Me ester R = H GA4 Me ester R = OH GA58 Me ester R = OH GAT Me ester R = OH Scheme 11 12-Hydroxylation.(a) Fragmentary evidence for a 1212-OH pathway in cell-free systems from C. maxima endosperm and embryos. (b) 12a- and 12P-Hydroxylation in cultures of Lygodium japonicum spores via GA, to GA,. There is a cross-over branch from GA, appr~ach,'~','~~ using a single set of conditions are shown in to GA14. The essentials of these pathways were originally Scheme 12 where the bold arrows show the major steps the postulated by Cross et al.23Evidence for the steps is derived normal arrows show the minor steps and the dashed arrows from studies with different wild-type strains and from single show multiple steps; all the GAS in the scheme occur in gene mutants blocked for GA biosynthesis.This evidence has cultures of the fungus. been thoroughly reviewed by Bearder.172 The most complete For the major pathway the steps from GA,,-aldehyde to results come from studies using a mutant B1-41a in which GA via GA,,-aldeyde GA14 GA and GA were established. GA biosynthesis is blocked6' at the step between ent-kaur-16- It was also shown that GA was not converted to GA and en-19-a1 and ent-kaur-16-en-19-oic acid (see Section 4.1). In that GA16 was neither converted to GA nor formed from the near-absence of endogenous GAS unlabelled substrates GA,. The studies confirmed earlier work on separate steps were used together with GC-MS to identify the metabolites using various wild type strains e.g. GA to GA and GA and ,H-labelled metabolites were used with GC-RC to to GA3,I7 and the formation of GA1,175 and GA, from quantify the metabolites.The results of this integrated GA,. Scheme 12 also accommodates the observations that 232 Natural Product Reports R2 // unnatural R1 = R2 = H GAloo R' = H; R2=OH HOgC R' dR2 JI OH CO2H CO2H C02H v"' // GA45 R' = R2 = H GA76 R' = H; R2 = OH GA67 R' = H; R2 = OH GA75 R' = R2 = OH G&3 R' =OH; R2 = H GA72 R' = R2 = OH GA86 Fig. 3 15P-Hydroxy-GAS GA ,-aldehyde,23 GA,,-aldehyde GA,,23 and GA,', serve as precursors of GA,. In a time-course study of the formation of GAS from ent-[ 17- 14C]kaur- 16-en- 19-01'~~ and ent-[17-1 ,C]kaur- 16-en- 19-oic acid55 it was concluded that GA was formed from GA, as well as from GA and GA,.Pitel et al.', also recorded a 0.6% conversion of GA into GA when G. fujikuroi strain ACC917 was cultured on a non-synthetic medium containing corn steep liquor conditions that also led to substantial accumulation of GA,. A low radiochemical conversion (2.7%) of GA to GA was also noted',* in cultures of the mutant R-9 lacking the 13-hydroxylation activity for GA,. Thus 1,2-dehydrogenation of GA to GA can occur to a limited extent in some strains of the fungus. The 1,2-dehydrogenation of GA to GA is accompanied by la-hydroxylation of GA to GA16,173'174 and 2a-hydroxylation of GA to GA,,.'76 This la-and 2a-hydroxylation in fungal cultures contrasts with the lp- and 2P-hydroxylation found in higher plants (Section 5.1.2) an observation that led to the elucidation of the stereochemistry of 1,2-dehydrogenation (see Section 5.3.1).The minor pathway from GAl,-aldehyde via GA, to GA mirrors the major pathway. The further metab~lism'~~ of GA in cultures of the mutant B1-41a parallels that of GA, namely 1,2-dehydrogenation to A'-GA, 2a-hydroxylation to GA40 and 13-hydroxylation to GA,,. A further metabolite GA 1 and its possible precursor shown in Scheme 12 were also identified. There is no late 3P-hydroxylation of GA to GA,. MacMillan Biosynthesis of the gibberellin plant hormones antheridic acid Fig. 4 12,15-Cyclo-GAs 9( 1 1)-didehydro-GAs and antheridiogens None of the metabolic studies have identified the steps from GA14 to GA or from GA, to GA,.Although the possible intermediates shown in Scheme 12 have been identified in cultures of G. fujikuroi GA,, GA,, and GA, were neither formed from GA14 nor metabolised to GA in cultures of the mutant B1-41a.17 Parallel results'73 were obtained for GA,, GA, and GA25. Also in cultures of the wild-type strain ACC 917 GA13 was not c~nverted~~~'*~ to C,,-GAS. A sugges-tion'*' that GA1,-19,20-anhydride was converted to GA in 0.015% yield could not be c~nfirmed.'~~ The failure to identify intermediates led to the suggestion that GA14 may not be on the direct pathway to C,,-GAS. This question was posed from the observations that GA14 was metabolised less efficiently than GA,,-aldehyde to GA3,171 and that GA14 accumulated following incubations with ent-kaur- 16-en- 19-oic acid ent-7a- hydroxykaur- 16-en- 19-oic acid GA ,-aldehyde and GA 14-aldehyde', in cultures of the mutant B1-41a.Indeed there is one report'" that GA14 was not metabolised by the GA,- producing strain ACC917. Therefore it has been suggested that the intermediates between GA,,-aldehyde and GA are at the C-7-aldehyde oxidation level. '*' A tentative that GA1,-7-aldehyde was metabolised to GA in 12.9% yield could not be confirmed;1s3 authentic GAl,-7-aldehyde and its 19,20- anhydride were largely unmetabolised and partially hydro- lysed to GA13 in cultures of the mutant B1-41a and the wild-type strain CMI 58289. As shown in Section 5.1.1 the intermediacy of the GA, opened lactone and of GA,, have been demonstrated using cell-free systems from seeds of higher plants.The failure to demonstrate metabolism of GA, and GA,, and of their non-3-hydroxylated counterparts GA 5 and GA,, is probably explained by their poor transport from the medium to the mycelium of the fungus. No cell-free fungal systems have been reported that support the steps from GA,,-aldehyde to GA,. The relative amounts of the GAS produced in fungal cul- tures is influenced by the pH levels. It has been reported'* that cultures of the wild-type strain ACC 917 buffered at pH 7 favour the production of GA and GA over GA,. With non-early 3-hydroxylation early 3-hydroxylation gp-J;a,cj- HO I CHO CHO 8 C02H C02H GA12-aldehyde GA14-aldehyde t/ 1,2-dehydro-GAg + f-GAll artefact ? GA3 Scheme 12 Early 3p- and non-early 3P,13-hydroxylation pathways in G.fujiikuroi mutant B1-41a;'71,'73,'79 *major; -minor; -+ more than one step cultures of the mutant B1-41 under these condition^,'^^ GA and GA7 were not metabolised and the metabolism of GA, was suppressed. Thus GA, aldehyde was metabolised mainly to GA14. Another effect of the higher pH was to suppress the partitioning of GA, to the 3P-hydroxylation pathway in the mutant B1-41a; at pH7 two-thirds of added GA, was metabolised to mainly GA with traces of GA24 and GA,, while GA, and its metabolites were not detected. The effect of the higher pH may be the result of increased transport of the substrates rather than altered enzyme activity.5.1.4.2 Other fungi Two other fungal species are known to produce GAS. Cultures of Sphaceloma species produce'85 GA and no GA, GA or GA,. Thus unlike G. fujikuroi this fungus appears to be 234 Natural Product Reports unable to synthesize 2,3-dehydro- or 1 3-hydroxy-GAS. How- ever no biosynthetic studies have been reported. Cultures of a Phaeosphaeria species L487 contained'86,' 87 GA,, GA,, GA,, GA,, GA, GA, GA,, GA, and GA,. All of these GAS were shown to be produced from GA,,-aldehyde. In addition it was shown that GA was metabolised to GA via both GA and GA, (Scheme 13). This pathway is unlike that in G. fujikuroi and more like that found in higher plants. 5.2 Enzymology 5.2.1 General No fungal extracts that catalyse the conversion of C,,-GAS to C,,-GAS have been described.All information on the enzymes involved in stage C (Scheme 1) comes from studies on higher plants. on HO GA20 Scheme 13 Phaeosphaeria species L487 steps's6,1s7from GA to GA The precursors of GA,,-aldehyde are lipophilic and the formation of GA,,-aldehyde from ent-kaur-16-ene is catalysed by microsomal P4.50 enzymes (Section 4.3). The metabolic steps from GA,,-aldehyde lead to progressively more hydrophilic GAS and they are catalysed by soluble oxidases discussed later. GA,,-aldehyde occurs at this transition and its immediate metabolism is catalysed by both types of enzyme. Thus the 7-oxidation to GA, (Scheme 7) occurs in micro- soma1 and soluble enzyme preparations from endosperm and embryos of C.and in enzyme preparations from H. vulgare.Io6 The 12a-hydroxylase activity for GA,,- aldehyde in C. maxima seeds is also both microsomal" and ~oluble.'~ The 30-hydroxylation of GA,,-aldehyde to GA14- aldehyde and subsequent 7-oxidation to GA, are catalysed by a soluble enzyme preparation from the endosperm of C. maxima.82The 13-hydroxylase activities that convert GA,,- aldehyde to GAS,-aldehyde in P. vulgaris'O' and GA, to GAS in C. maxim^,"^^^ P. vulgaris,'OOP. sativum9' and H. vulgare,Io6 are all microsomal. In S. oleraceago however GA, is converted to GAS by a soluble enzyme fraction and this is the only example of a soluble 13-hydroxylase. Indeed the cross-over steps in vitro from the non-early 3P,13-hydroxylation pathway to the early 13-hydroxylation pathway namely GA, to GA,, GA24 to GA19 (Scheme 7),and GA to GA, (Scheme 8) also appear to be microsomal reactions; they may therefore be artefactual since the substrates are the products of soluble oxidases.*C02H Yo + CH2 + O2 + Fe2+ y2 'C02H The identification of the soluble enzymes as 2-oxoglutarate- dependent oxidases was first made by Hedden and Graebe.96 Using an S,, preparation from C. maxima endosperm that had been filtered through Sephadex G-25 they showed that each of the steps from GA,,-aldehyde (Scheme 7) to GA, (Scheme 11) were dependent on 2-oxoglutarate and Fe2'. Confirmation"' of this conclusion was subsequently obtained using partially purified GA,,-aldehyde 7-oxidase7 GA, 20-oxidase and GA 5 3 P-oxidase from C.maxima endosperm. In particular it was shown that 2-oxoglutarate was a co-substrate for the oxidation of GA, to GA, and GA, by the 20-oxidase (Scheme 14). Thus 2-0~0[.5-'~C]glutarate was oxidised stoichiometrically to [''C]succinate and GA 5/24 and approximately equimolar amounts of I4CO were released from 2-0xo[l-~~C]glutarate. It was further established that 0 was the source of oxygen by showing that five "0-atoms were incorporated into GA, when GA ,-aldehyde was incubated with "02and a mixture of the 7- 20- 3P- and 2P-oxidases (Scheme 14). To date this is the only study'" showing that 2-oxogluarate is a cosubstrate for the soluble GA oxidases. Indeed there is one report,'I3 using a five-fold purified 3P-hydroxylase from seeds of P.vulgaris of the failure to show the cooxidation of 2-oxogluarate to succinate but the observed oxidation of ascorbic acid to threonic acid. Never- theless it is generally accepted that soluble oxidases requiring Fe2+ 2-oxoglutarate and ascorbate as cofactors catalyse the steps shown in Scheme 7 from GA, to GA (early non-Sp 13-hydroxylation pathway) GA, to GA (early 30-hydroxylation pathway) and GAS to GA (early 13-hydroxylation pathway). The conversions of the C ,-GAS shown in Scheme 8 are also catalysed by such soluble oxi- dases. It has become general practice to describe these enzymes as GA-dioxygenases. However in a recent and general review"' on dioxygenases from animals plants and micro- organisms attention is drawn to the problems of nomencla- ture.To avoid such problems GA oxidase is used here to describe the soluble 2-oxoglutarate-dependent enzymes of GA biosynthesis. 5.2.2 Purification and cloning Native proteins that have been partially purified from non- vegetative tissues and characterised as 2-oxoglutarate-dependent GA oxidases are 7- 3P- and 20-oxidases from C. maxima,'08 a 20-oxidase from P.sativum,' I' 2P-oxidases from P. vulgaris' and P. sativum and 3 P-hydroxylases from P. vulgaris. 'Opl There is only one report on the partial purification of a GA oxidase from vegetative tissue namely 20-oxidases from leaves of S. oleracea."' The methods used for the purification of the GA oxidases have been re~iewed."~ In general the enzymes are present in very low amounts in OW\ + C02H crude enzyme preparation * I8O2+ 2-oxoglutarate + Fez+ GA1 2-aldehyde GA43 Scheme 14 Cooxidation of GA, and 2-oxoglutaric acid and 180-labelling by enzyme preparations from C.maxima endo~perrn~~~'~~ MacMiIlan Biosynthesis of the gibberellin plant hormones 235 plant tissues; they are unstable and lose most of their activity on purification. However a 20-oxidase from the endosperm of C. maxima has been purified"' to near-homogeneity as the native protein by ammonium sulfate precipitation gel filtra- tion anion-exchange and hydrophobic-interaction chromatog- raphy. This protein M 45 kDa is multifunctional catalysing successive steps in the oxidation of GA, to GA,, GA24 and GA,, and of GA, to GA,, GA19 GA, and (putatively) GA2 (see Scheme 7).The amino acid sequences of the N-terminus and of two peptides from a tryptic digest were found to contain an identical sequence of nine amino acids that did not include conserved sequences of other plant dioxygen- ases. A polyclonal antiserum to a synthetic peptide (VF- GGSDESK) based on this sequence was subsequently used'20 to screen a cDNA expression library prepared from the mRNA of C. maxima cotyledons and isolate cDNA clones encoding a GA 20-oxidase. Heterologous expression in E. coli gave a recombinant protein that catalysed the progessive oxidation of GA, to GA15 GA, and GA25 and (less efficiently) GA, to GA,, GA19 and GA,,. These activities were completely depen- dent on 2-oxoglutarate and were reduced when Fe2' and ascor- bate were omitted.These properties and the M of 43.3 kDa predicted from the cDNA clone mirror those of the purified native protein from the endosperm. This seminal work"' on the purification and cloningI2' of the GA 20-oxidase from immature seeds of C. maxima led rapidly to the cloning and heterologous expression of a series of GA 20-oxidases. In one case,', a genomic DNA library was screened using cDNA of the GA 20-oxidase from C. maxima as a heterologous probe. In the other cases detailed as follows increasingly refined degenerate oligonucleotides were used to amplify genomic DNA or cDNA by the polymerase chain reaction. Thus one genomic clone has been obtained from S. oleracea,'2' three genomic clones from A.thaliana'22 and a cDNA clone from M. macrocarpus.lo5 All have been expressed in E. coli and their recombinant proteins display multifunction- ality (see Section 5.2.3). One of these genomic clones'23 was essentially identical to one isolated'22 independently from A. thaliana and shown to be tightly linked to the ga5 semi-dwarf locus which is impaired in GA 20-oxidase activity. Further- more it was shown that the ga5 mutant contains a G+A point mutation that inserts a translational stop codon in the protein coding sequence. Since the ga5 mutant is only a semi-dwarf requiring application of a GA after the block for normal growth the mutant must contain other C-20 oxidases. The deduced sequences of these GA 20-oxidases have 52-8 1% iden-tity and contain highly conserved regions.Two histidine motifs (e.g. H,,,XD and H298R in the C. maxima enzyme) may be associated with the metal binding site and correspond to the metal binding site in isopenicillin synthase identified by X-ray crystallographic studies ' and site-directed mutagenasis. 91 There is also a NYYPXCXXP motif that may identify the 2-oxoglutarate binding site and a LPWKET motif that may be associated with binding of the GA substrate. Other GA 20- oxidases have recently been cloned (see ref. 9). In many plant species there are multiple enzymes indicating tissue specific regulation of GA biosynthesis. Three 30-hydroxylases have been cloned although only the details of one has been published. The GA4-locus identified in A.thaliana from the GA-responding dwarf mutant ga4 has been cloned'24 using T-DNA tagged progeny from transgenic plants generated by Agrobacterium root transformation. The deduced amino acid sequence has 22% identity with the C. maxima 20-oxidase and contains similar metal and 2-oxoglutarate binding motifs. This clone has been expressed in E. coli and the recombinant protein has been to convert GA efficiently to GA and less efficiently GA, to GA,. 5.2.3 Multifunctionality All the GA 20-oxidases are multifunctional in that they catalyse some or all of the steps in the sequential oxidation of 236 Natural Product Reports GA, andlor GAS at C-20 to the alcohol (GA,,/GA,,) the aldehyde (GA,,/GAI9) the acid (GA,,/GA,,) and the CI9-GA (GA,/GA,,) (see Scheme 7).They do however differ in the ratio of the acid and y-lactone that is formed. This difference appears to be subtle in terms of protein sequences. For example the 20-oxidase from C. maxima produce^'^' the (2-20 acid and little of the y-lactone while that from M. macrocarpus converts GA, to GA in high yield;"' yet the two proteins show 81% identity at the amino acid level."' The mechanism of these successive oxidation steps by a single enzyme is discussed in Section 5.4. There is also metabolic and biogenetic evidence that some 3P-oxidases are multifunctional. Thus the partially purified 3p-hydroxylase from P. vulgaris embryos catalyses 3p-hydroxylation"1~"3 of GA, to GA, 2P-hydroxylation' l3 of GA, to GA29 2,3-desaturation1' of GA, to GA, and '9' 2,3-epoxidation1 l4 of GAS to GA6 (see Scheme 8).This multifunctionality is supported by kinetic data' l3 for the formation of GA, GAS and GA29 from [17-'3C,17-3H]GA2 and [2P-,H 17-I3C,17-3H]GA20 and by the fact that double reciprocal plots of the rate of formation of GA, GAS and GA29 from [17-I3C,17-,H]GA2 versus 2-oxoglutarate con-centrations are parallel.Il3 A 3P-oxidase present in shoots of Z. mays also appears to be multifunctional. As described in Section 5.1.2 (Scheme 9) the steps GA,,,/GA and GA,,,/GA,I GA, occur in shoots of normal 2. mays. However in the dwarf1 single gene mutant all of these steps are blocked,13' suggesting that the product of the DZ-gene catalyses the 3P-hydroxylation of GA, to GA, the 2,3-desaturation of GA, to GA, and the 3P-hydroxylation of GAS with 2,3/1,2- rearrangement of the double bond to give GA,.However definitive proof of the multifunctionality of the 3p-hydroxylases from P. vulgaris embryos and from shoots of Z. mays must await the isolation of the purified enzymes or fusion proteins from cDNA clones. 5.2.4 Substrate specificity The 2-oxoglutarate-dependentGA oxidases from higher plants display less than strict substrate specificity. For example most of the known 20-oxidases accept both GA, and GAS to a greater or less degree and the recombinant proteins from the cDNA clones (see e.g. ref. 120) convert each of the C-20 aldehydes GA24 GA, and GA23 to the corresponding y-lactones GA, GA, and GA (see Scheme 7). The partially purified 3 P-hydroxylase from P.vulgaris embryos metabolises GA to GA4 as well as GA, to GA,;'" GA, and GA, may also be substrates since they competitively inhibit the formation of GA from GA2,.'10 As noted earlier there is no direct evidence on the properties of the fungal enzymes that convert C2,-GAS to C,,-GAS. However there is indirect evidence that these enzymes also have lax substrate specificity. A wide range of 'unnatural substrates' are metabolised to 'unnatural' fungal GAS by cultures of G. fujikuroi mutant B1-41a blocked6' for GA biosynthesis between ent-kaur- 16-en-19-a1 and ent-kaur- 16-en- 19-oic acid (Section 4.3) and by wild-type cultures blocked for GA biosynthesis by the addition of synthetic plant growth retardants. The subject has been comprehensively reviewed by Bearder' 72 and only three illustrative examples are provided.First the semi-synthetic GA1,-2-en-7,19-diol is convertedIg2 in 20% yield to the non-fungal GA (Scheme 15). Second trachylobanic acid is metabolised to a series of 12,16-cyclo analogues of fungal GAS including 12,16-~yclo-GA,'~~ (Scheme 15). Third the unnatural substrate ent-1 5a-hydroxykaur- 16-en- 19-oic acid is metaboli~ed'~~ to the 15p-hydroxy analogues of the fungal GAS GA,, GA, and GA, (Scheme 12) and the higher plant GAS GA,, GA,, GA,, GA6 and GA,, shown in Fig. 3. However there are limits and attempts to produce enantiomeric GAS from kaur- 16-ene were un-successfu1.19',196 GAI2-2-ene-7,1 9-diol GAS Ref. 193 w-MVA C02H H0TC02H Trachylobanic acid Scheme 15 Two examples of the metabolism of ‘unnatural’ substrates by cultures of G.fujikuroi 5.3 Ster eochemistr y Scheme 16 Incorporation of specifically tritiated MVA into GA and 5.3.1 Gibberella fujikuroi GA in cultures of G. fujik~roi~~,’~~ The stereochemical course of the incorporation of each of the pro-R and pro-S hydrogens of MVA into the metabolites of G. fujikuroi has been determined.37,’97 Two results of relevance to respectively. Thus in cultures of G. fujikuroi the formation of the formation of the C,,-GAs from the C,,-GAS are summar- GA via GA from GA [see Scheme 17(a)] occurs by cis-ised in Scheme 16. The labelling is based on I4C:,H ratios 1a,2a-dehydrogenation. This conclusion has been directly con- chemical conversions and the established all-trans cyclisation firmedIo3 using cultures of the mutant Bl-41a of G.fujikuroi in of GGPP into ent-kaur-16-ene (Section 3.2) and thence the which [I P,2P-2H,]GA was converted into [2H2]GA and fungal GAS. First the 4-pro-R hydrogen of MVA is retained at [2H2]GA [Scheme 17(a)]. C-5 and C-9 in GA and both the 2-pro-R and 2-pro4 hydrogens of MVA are retained at C-1 in GA,. It can be concluded therefore that the loss of C-20 in the conversion 5.3.2 Higher plants of the C,,-GAS to the C,,-GAS does not involve the loss of In the conversion of the C2,-GAS to the C,,-GAs by the hydrogen from C-1 C-5 or C-9. Second the 2-pro-R and the stepwise oxidation of C-20 it has already been noted that some 5-pro-S hydrogens of MVA are retained in GA and the 20-oxidases utilise the lactone form of the intermediate and 2-pro-S and the 2-pro-R hydrogens are lost at C-1 and C-2 others only accept the open lactone.This distinction has been H0yC02H I A H ’c M.macrocarpus O3 DP GA5 R=OH GA3 R=OH A2-GAg R=H GA7 R=H ow co --J M.macrocarpuslo3 .-.d DP C02H D C02H GA5 R =OH GAG R=OH A2-GAg R=H 2,3-epoxy-GAg R = H Scheme 17 Formation of GA,; (a) in cultures of G. fujikuroi; and (b) in M. macrocarpus cell-free system MacMillan Biosynthesis of the gibberellin plant hormones A. thaliana clone At 2353 GA15 open lactone R = H GA24 R= H Gh4 open lactone R = OH GA19 R=OH hornogenate from -S. oleracea leaves Scheme 18 Stereospecificity of oxidation at C-20 E.Fe3+-Q 0H L HO 'CH2 0E.Fe4+=0 GA12 R=H GA53 R=OH __I___ shown to have stereochemical implications (see Scheme 18).By deuterium labelling it has been shown'98 that the recombinant C-20 oxidase from A. thaliana clone At2353 only accepts the open lactone form of GA, and GA,, respectively forming GA24 and GA, with stereospecific loss of the 20-pro-R hydrogen. In contrast cell-free homogenate from leaves of S. oleracea converts GA to GA, with stereospecific loss of the 20-pro-S hydrogen. Using 2H-labelled substrates and enzyme preparations from seeds of P. vulgaris it has been shown"2 that the 2o-hydroxylation of GA to GAS (Scheme 8) and the 3P-hydroxylation of GA, to GA (Scheme 8) proceed with retention of stereochemistry and that GA is formed from GA, (Scheme 8) by the loss of the 2p- and 3P-hydrogens.The stereochemistry of the conversion of GA to GA and 2,3- dehydro-GA to GA has been e~amined"~ in an unfraction- ated enzyme preparation from seeds of M. macrocarpus [Scheme 17(b)]. These conversions involve the loss of the lo-hydrogen in a step that seems to be rate-determining from the observation that the 1P-2H-labelled substrates yield traces of the 2,3-epoxides which are not formed from the unlabelled E.Fe2+ HO 0 E.Fe4+=0 E.Fe2+ LH 0-C'-H C02H GA15 R=H I GA44 R=OH E.Fe3+~ 0OH E.Fe2+ I GA24 R = H (~actots) or GA1g R=OH 0 E.Fe3+- OH I E.Fe2+ p C02H C02H LupH GA24 R=H GA1g R =OH Scheme 19 Formal mechanism for the conversion of GA,,/GA, to GA,,/GA,, GA,,/GA,9 and GA,,/GA,9 lactols 238 Natural Product Reports substrates presumably because of the absence of a primary isotope effect.Since GA is formed from GA both in (Scheme 9) it may vitro103,106,I 14 (Scheme 8) and in vivo'28>130 be deduced that the overall stereochemistry of the introduction of the 1,2-double bond is by lp,2p-abstraction of hydrogen. Thus the pathways to GA and GA, and the overall stereo- chemistry of the formation of the 1,2-double bond are differ- ent in higher plants from similar transformations in the fungus G. fujikuroi. 5.4 Mechanistic considerations As noted in Section 5.2.3 the 20-oxidases catalyse some or all of the successive steps from GA, to GA,, GA24 GA25 and A, and from GA, to GA, GA19 GA, and GA,,.Formal mechanisms for these steps are shown in Schemes 19 and 20. In 0 E.Fe4+=0 E.Fe4+70 HO HJ CO2H GA24 R=H GA1g R = OH 0 E.Fe3+y OH E.Fe2+ H20 E-Fe2 + cp. o/c=o .L $-y-(-& Scheme 19 possible mechanisms for the formation of GA,,/ GA, and their corresponding lactols and of GAJGA,, are shown via either the C-20 alcohols GA, and GA,, or the GA,,/GA open lactones. For cultures of G. fujikuroi there is the following evidence'99 that the lactone GA, is not an intermediate in the formation of GA2 and GA from GA,,. Incubation of [19-'s0,]GAl gave. GA24 GA,, and GA in which both oxygens of the C-19 acid were retained resulted in the isolation GA, containing only half of the '80-content.Thus for the fungus the open lactone (C-20 alcohol) and not the &-lactone must be the intermediate. Similar experiments using [19-180,]GA,,,, and the available recombinant 20-oxidases would confirm the inference that the C-20 alcohol is the intermediate for the non-vegetative 20-oxidases and provide information on the role of the &-lactone in the case of the vegetative 20-oxidases. E.Fe3+-0-0' E.Fe4+=0 f GA24 R=H GA24 R= H GAig R=OH GAig R=OH ic n E.Fe3+-O-OH f c n // '0 E.Fe3+,0-C H __-J C02H GAg R=H GA20 R=OH MacMillan Biosynthesis of the gibberellin plant hormones H-\b H Scheme 21 Possible mechanism for the formation of GA and GA, Four possible mechanisms are shown in Scheme 20 for the formation of the y-lactones GAJGA,,.In considering them it is important to note that the mechanism may not be the same in the fungus and higher plants. None of the four mechanisms involve the 1-H 5-H or 9-H in agreement with the findings for cultures of G. fujikuroi (Section 5.3.1) but this information is lacking for higher plants. Mechanisms A and B are similar and proceed respectively from the lactol via the anhydride hydrate and from the aldehyde via the anhydride. However the anhydride of mechanism B does not serve as an intermediate in fungal cultures,'73 or in incubations with a soluble enzyme fraction from P. sativum embryo^.'^ Neither mechanism C nor D conforms to the findinglo8 for the 20-oxidase from C.maxima endosperm (Scheme 14) that 2-oxoglutarate is oxidised stoichiometrically to succinate and CO, and that the CO is not formed via formate. Mechanism C proceeds via a 20-hydroperoxide similiar to a suggestion by Dockerill and Hanson,200 following their observation that ent-kaur- 16-ene 14C-labelled inter alia at C-20 was metabolised to [14C]C19- GAS and [14C]C0, but not 14C-labelled H,C=O or HC0,H. However attempts2" to detect H13C02H by NMR spectros- copy from incubations of [3'-13C]MVA in cultures of the fungus G. fujikuroi were unsuccessful; ',CO was detected but it was shown that formate was partially converted to CO by the fungal cultures. It will be of interest to determine if the C-20 is released directly as CO in all cases.If so of the four mechanisms considered mechanism A appears to be the most likely. The conversions of GA to GA and 2,3-didehydroGA9 to GA [Scheme 17(b)] extend the functional diversity of the 2-oxoglutate-dependent GA-oxidases. It has been shown (L. J. Benjamin J. MacMillan and L. N. Mander unpublished) that GA,, the 1,2-double bond isomer of GA, is also metabolised to GA,. Thus as shown in Scheme 21 the formation of GA probably proceeds through an allylic radical. Delivery of the hydroxyl radical at C-3 appears to be preferred. Delivery of the hydroxyl radical at C-1 occurs to a minor extent to give GA,, found in T. aestivum grain.202 6 Concluding remarks Studies on the biosynthesis of the GAS are at an advanced stage. Step-by-step details of the biosynthesis of most of the 108 natural GAS are now known both in vitro and in vivo.However the origin of the 9,11-dehydro- the 11- 12- and 15-hydroxy-GAS and of the 9,15-cyclo-GAs remain to be determined. A knowledge of the pathways combined with studies on GA biosynthesis mutants has identified GAS that trigger plant growth responses without further metabolism. The types of enzymes involved in stages A B and C (see Scheme 1) have been characterised and genes for the three different enzyme types from GGPP have been cloned namely the cyclases (stage A) a P450 oxidase (stage B) and several 2-oxoglutarate-dependent oxidases (stage C). 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ISSN:0265-0568    

DOI:10.1039/NP9971400221

出版商:RSC    

年代:1997

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Diterpenoids James R. Hanson School of Molecular Sciences University of Sussex Brighton Sussex UK BNI 9QJ Covering 1995 Previous review 1995 13 59 1 2 3 3.1 3.2 4 4.1 4.2 5 5.1 5.2 5.3 5.4 6 6.1 6.2 7 8 Introduction Acyclic and related diterpenoids Bicyclic diterpenoids Labdanes Clerodanes Tricyclic diterpenoids Pimaranes Abietanes and related diterpenoids Tetracyclic diterpenoids Kaurenes Beyerenes Aphidicolanes Gibberellins Macrocyclic diterpenoids and their cyclization prod- ucts Taxanes Cembranes and other cyclization products Miscellaneous diterpenoids References 1 Introduction This review follows the pattern of its predecessors.' The period under review has been dominated by publications on the taxanes and a separate section has been devoted to them.Diterpenoids are often considered as taxonomic markers. The chemosystematic potential of 15 of the more common diter- pene types has been evaluated2 in the classification of the angiosperms. Although there is a structural diversification which increases with evolutionary advance closely related families reveal disjunctions thus apparently restricting the taxonomic value of their diterpenoid content. 2 Acyclic and related diterpenoids Phytyl heptanoate has been found3 in the medicinal plant Bidens pilosa (Compositae) from the Philippines. A review4 of the chemotaxonomy of the marine brown algae of the family Cystoseiraceae has concentrated on their tetraprenylquinol content.The presence of geranylgeranylglycerol in the brown alga Taonia lacheana and T. atomaria may have chemotaxo- nomic ~ignificance.~ The tetraprenyltoluquinone 1 has been isolated6 from Cystoseira crinata. Geranylgeranylhydro-quinone has been obtained7 from the fungus Luctarius lignyotus. The diterpenoid composition of the brown alga Bifurcaria bifurcata shows marked geographical variation suggesting that there are different chemotypes. The (9-1 3- hydroxygeranylgeraniol derivatives 2 and 3 have recently been isolated' from this source. The furanoditerpene 4 was obtained' from the Australian sponge Thorectandra choanoides whilst the Indian sponge Callyspongia spinosissima yielded" callyspinol 5. 3 Bicyclic diterpenoids 3.1 Labdanes Labdanes are often found in the resinous exudates of plants particularly those that grow in semi-arid regions.13-0-0-Hanson Diterpenoids 0 1 0- 2 3 4 /5 Xylopyranosyl-ent-manool has been found' in the resin of Haplopappus diplopappus and the corresponding 3'-and 4'-acetates have been found'* in the exudate of Conyza linearis (Compositae). Some labdane glycosides including 6 have been isolated13 from the Japanese fern Gleichenia japonica. The widespread occurrence of labdanes is exemplified by the iso- lationI4 of (3-labd- 13-ene-8a 15-diol from Arnica angustifolia (Asteraceae) whilst the corresponding 15-aldehyde was obtainedI5 from the skin of the mollusc Pleurobranchaea meckelii. Examination of the various chemotypes of Halimium viscosum (Cistaceae) has revealed' that labd-7-ene-30 15-diol 7 is a major constituent of one form.16-Hydroxymanoyl oxide 8 and its 13-epimer have been from the rhizomes of Kyllingia erecta (Cyper-aceae). The 13-epimanoyl oxide derivative tarapacol 9 together with the tarapacanols A and B which possess C-11 and C-12 oxygen functions respectively have been isolated" from Grindelia tarapacana (Asteraceae). The more highly oxygenated ptychantins (e.g. 10) were obtained2' from the liverwort Ptychanthus striatus. The structure of the bis-lactone limonidilactone 11 obtained from Vitex limonifolia (Verbenaceae) was established2' by X-ray crystallography. The interesting cytotoxic lactone accu- minolide 12 from Neouvaria acuminatissima (Annonaceae) showed22 significant activity against a number of human cancer cell lines.A number of dimeric diterpenoids have been reported including the ether persianone 13 from the Iranian Labiate Ballota a~cheri.~~ The lactone leopersin A 14 and some relatives have been isolated24 from Leonurus persicus (Labiatae). Marrubinone A 15 was obtainedz5 from Marru-bium astracanicum (Labiatae). Its 13-epimer marrubinone B (polyodonine) was also isolated26 from M. polydon. These OH y GIYO~H~ 6 Gly = a-L-rhamnopyranosyl(1+2)-~-D-glucopyranosyl ~CH,OH HOA 7 8 AcO I OH 9 10 compounds are similar to isonitin 16 from Leonotis ocymifolia varn. r~ineriana~~ and lagochirzin 17 from Lagochilus hirsutissimus.28 The peroxide amoenolide K 18,29a number of labdane glyco~ides~~ and the modified labdane 1g3’have been isolated in the continuation of studies on Amphiachyris amoena (Compositae).The secolabdane maravuic acid 20 has been ob tained3* from Cro ton matourensis (Euphorbiaceae). The biotransformation of labdanes has continued to provide useful entry to the more highly hydroxylated derivatives. Several studies on the synthesis of some 1- 7- and 11-hydroxylated manoyl oxide and 13-epimanoyl oxide deriva- tives as potential forskolin analogues utilizing a combination of chemical and microbiological methods have been Anti-mutagenic activity has been associated36 with the pres- ence of polyalthic acid 21 in extracts of the Asian medicinal plant known as ‘Mankeishi’ obtained from Vitex rotundifloria (Verbenaceae).The I3C NMR signals of a number of andrographolide derivatives have been assigned37 whilst the optical rotation of a range of pure C-15 oxygenated labda-8( 17) 13-dienes has been recorded.38 The synthesis of some grindelane diter-penoids has confirmed3’ their absolute configuration. Aspects of the chemistry of lagochilin 22 including the degradation of its side chain have been examined.40341 The oxidative degradation of the side chain of labdane diterpenoids has continued to attract interest.42 The reaction of methyl 15-0- acetylisocupressate with selenium dioxide in methanol yielded43 in addition to the expected allylic oxidation products a selenium containing product 23.The naphthofuranone 24 was unexpectedly obtained in the course of the partial syn- thesis of nagilactone analogues. Intermediates in its formation have now been synthesized.u 3.2 Clerodanes The occurrence of neo-clerodanes in American Salvia species has been reviewed.45 ent-(E)-Clero-3,13( 14)-dien-15-oic acid 25 and some derivatives have been isolated46 from the leaves of Detarium microcarpum (Leguminosae) and shown to have anti- feedant activity against the termite Reticulitermes speratus. The related diacetate 26 has been from the 246 Natural Product Reports 11 12 0 13 14 -0_ co-0 15 16 HO.. HHOCH2O W 17 18 -OH 19 20 roots of Linaria saxatilis (Scrophulariaceae). The epoxide 27 and the related diols have been found48 in the liverwort Jungermannia hyalina along with the halimane 28.The cis-fused structure 29 has been assigned49 to crotonic acid from Croton chilensis. Continuation of studies on Cistus populifolius (Cistaceae) has led to the isolation5’ of 2a,3P-dihydroxy-4( 18)-neocleroden- 15-oic acid and the corresponding trio1 30.51The latter showed powerful insect antifeedant activity when tested against Spodoptera littoralis. Diterpenoids are often obtained from the Annonaceae. The simple clerodane 31 and some relatives have been obtaineds2 from the stem bark of the Indian species Polyalthia longifolia (Asoki). The corresponding acetate has been founds3 in the Sri Lankan tree Cyathocalyx zeylanica along with the acid 32. The extract of this tree is toxic to the mosquito Aedes aegyptii.The clerodane 31 and the desoxy-acid ent-cleroda-4( 1S) 13-dien-15-oic acid have been reported54 to be cytotoxic components of Polyalthia cheliensis. Heteroscyphone A 33 along with some Me02C R2 A,OH 34 R'=H; R2=OGlu 36 R' = OGIU; R2= H HO ($Ye po C02Me 23 24 R'.. Q$? Me02C OGlu 37 38 R' = H; R2 = flOH 0 OH 39 R'=OH; R2=H 25 26 0. 0 HO8 HO 0" 27 28 C02Me HO. AcOCH2 HO 29 30 0 ?OH 31 32 33 further clerodanes and verrucosanes have been isolated55 from the liverwort Heteroscyphus planus. A further member of the terpentecin series of tumour inhibitory antibiotics has been isolated56 from a Streptomycete. The medicinal plant Tinospora cordifolia (Menispermaceae) has been a rich source of clerodanes.It is a widely used component of Ayurvedic medicine. Cordioside 34,57 tino-sponone 3558 and cordifoliside D 3659 are compounds that have recently been isolated from this species whilst rum-phioside A 37 was obtained6' from the Philippine plant T. rumphii (Makabuhai). The tinosinesides were obtained61 from T. sinensis. Hanson Diterpenoids 'OAc 40 41 Examination of the perennial herb Ajuga decumbens (Labiatae) afforded62 ajugacumbin G 38. The Scutellaria are a large genus of the Labiatae and contain a number of powerful insect antifeedants. 2a-Hydroxyajugarin V 39 and scutedrum- monin 40 have been obtained63 from S. drummondii whilst the scutalpins (e.g. 41 scutalpin G) were obtained64 from S.alpina. Another rich source of clerodanes are Teucrium species (Labiatae). New compounds that have been isolated include teuracemin 42 from T. ~acemosum,~~ teucryemin 43 from T. yemense,66 teucorymbin 44 from T. ~orymbosum,~~ alysine A 45 from T. alyssifolium6* and the biogenetically unusual teubetonin 46 from T. betoni~um.~~ This structure arises from the overall transfer of formaldehyde from C-5 to C-7. The teubrevins (e.g. 47) from T. brevifolium7' may arise from a deep-seated series of ring cleavage and cyclization reactions shown in Scheme 1. The survey of Mexican Salvia species has continued7' with the isolation of some languidulane clerodanes (e.g. 48) from S. urolepsis. A number of furanoditerpenoids from the liverwort Jameso-niella autumnalis such as jamesoniellide D 49 may arise by cleavage of a clerodane precursor.72 Ring expansion of a clerodane may afford compounds of the portulal series such as pilosanone C 50 from Portulaca pilosa (Port~lacaceae).~~ 4 Tricyclic diterpenoids 4.1 Pimaranes Sandaracopimaric acid has been identified74 as the lipo-oxygenase inhibitor from Juniperus phoenicea.Extraction of CJ HO.. p 0 ‘OAc CH20H 0 43 40 49 H frn””0 ‘OAc 44 45 50 p the bark afforded7’ of the Endospermum diadenum (Euphorbiaceae) dolabrane 51 and some relatives. A large number of diterpenoid phytoalexins have been detected in rice (Oryza saliva) infected with various fungi. Recent work has led76 to the isolation of the phytocassanes A-D (e.g.52). The continuing search for novel antifungal agents has afforded77 the sonomolides A 53 and B 54 which were obtained from an unidentified coprophilous fungus. Cyclodione 55 is a dimeric 46 47 cassane isolated7’ from the stem bark of Cyclodiscus gabunen- sis (Mimosaceae). Some further cleistanthanes (e.g. 56) have been isolated79 from Vellozia flavicans (Velloziaceae). The preparation of some anti-inflammatory derivatives of 6a,7p- dihydroxyvoucapan- 17p-oic acid has been reported.80 OH R \ HO AcO 51 52 [OAc H+ t c- AcO 53 54 H+ H+ 0‘ 47 55 Scheme 1 248 Natural Product Reports 4.2 Abietanes and related diterpenoids The considerable chemistry of totarol much of which has been developed in New Zealand has been reviewed.81 The 13C and 'H NMR assignments for some dehydroabietic acid derivatives have been reported.82 The 15-(R)-configuration has been assigneds3 to 15,16-dihydroxyabieta-8,11,13-trien-l8-0ic acid which was produced as a metabolite of dehydro- abietic acid by the fungus Chaetomium cochliodes.Aerial autoxidation of abietic acid produced inter alia 15-hydroperoxydehydroabietic acid which is regardeds4 as the contact allergen in colophony produced in Pinus species. The epimeric 18- and 19-nor-4-alcohols (e.g. 57) their correspond- ing hydroperoxides and 7-ketones have been founds5 in &T TH I OH 57 58 @f C02H OH 59 60 61 62 Juniperus chinensis (Cupressaceae) whilst the 7-20 ether for- mosanoxide 58 was isolateds6 from the Taiwan red cypress Chamaecyparis formosensis (Cupressaceae).The abietane 59 has been foundx7 as a constituent of Calceolaria hypericina (Scrophulariaceae). The ent-abietane stereochemistry has been assigneds8 to a constituent 60 of the roots of Solidago rugosa (Asteraceae). The study of the abietane diterpenes of Trypterigium wilfordii (Celastraceae) has continueds9 with the isolation of wilforol E 61 and a study of the biotransformation of these abietanes by a Cunninghamella species." The isolation of some non-irritant diterpenoids (e.g. 62) from Euphorbia sequieriana has led to the suggestion" that bioactivity-guided investi- gations of these species may give only a partial indication of their diterpenoid content.Some disaccharide glycosides of the nagilactones have been isolated92 from Podocarpus nagi. Biologically active diterpenoid quinones (e.g. 63) continue to be isolated. The latter which showed both antibacterial and antiviral activity was obtained93 from Plectranthus heteroensis (Labiatae) whilst 64(sessein) was from Salvia regla (Labiatae). The 7a-(3P-hydroxyolean- 12-en-28-oate) ester known as reglin is a mixed diterpene-triterpene. A number of other Salvia species have yielded diterpenoids. Heldrichinic acid from a Turkish species S. heldrichiana has been assigned95 the unexpected stereochemistry 65. A number of relatives of ferruginol including 1 -oxo- and 6-0x0-ferruginol have been obtained96 from the roots of another Turkish Hanson Diterpenoids OH I 63 64 R=Ac R = oleanolic acid 0p0 C02H 65 66 HO 67 68 species S.napifolia whilst the aerial parts of S. candidissima afforded97 the naphthoquinone 66. The saturated ketone 3-oxosapriparaquinone which has been found in the Chinese medicinal herb S. prionitis has been synthesized from de- hydroabietic acid.98 Salprionin 67 has also been obtained99 from this plant. The absolute configuration of a sample of coleon A 68 obtained from Coleus igniarius (Labiatae) in which it is a major leaf pigment has been established"' by X-ray methods. The skeletal rearrangements of dehydroabietic acid have been investigated in a biogenetic context. The BF,-catalysed rearrangement of the alcohol 69 has been shown to afford'" the pygmaeocane skeleton 70.The ring contraction product 71 has been isolated102 from Taiwania cryptomerioides (Taxo- diaceae). Examination of the roots of Zhumeria majdae an Iranian medicinal plant has afforded" a number of re-arranged abietanes including 72 and 73. The chemotaxonomic studies of Mexican plants have afforded a number of icetexane diterpenoids including salviasperanol 74 from Salvia aspera'04 and 75 from S. ~andicans.''~ The relationship of these com- pounds to abietanes is emphasized by their co-occurrence with abietanes such as 76. Lanigeraol 77 was obtained'06 from the roots of S. lanigera. Extraction of taxanes from Tuxus brevi-folia gave rise to pigmented residues from which the diterpene lignan brevitaxin 78 was i~olated."~ The red alga Laurencia karlae has affordedlo8 laukarlaol 79 whilst the isoagathenediol80 was i~olated'~' from the lipids of the bacterium Rhodospirillium rubrum.'CHO 71 72 73 74 75 76 Ho\ 77 78 79 80 C02Me 81 The availability of abietic acid regularly provides a starting material for synthetic work. This has recently been explored in the conversion of dehydroabietic acid to pisiferic acid"' and in the preparation of the chiral synthon 81."' The reactions of the (q6-arene) tricarbonylchromium(0) complexes of podo- carpic acid have been examined' l2 in the context of the partial synthesis of steroid analogues. 5 Tetracyclic diterpenoids 5.1 Kaurenes Correlations have been noted' l3 between the 'H NMR spectra and the stereochemistry at C-16 in 16,17-dihydroxykauranes.250 Natural Product Reports The plant growth regulator paclobutrazol inhibits the oxi- dation of ent-kaur- 16-ene at C-19 leading to accumulation of ent-kaur- 16-ene ent-3P-hydroxykaur- 16-ene and ent- 17- hydroxykaur- 15-ene in treated wheat seedlings. 'I4 The termite antifeedant activity of extracts of Xylopiu aethiopicu (Annon- aceae) has been attributed"' to the presence of ent-kaur-16- en- 19-oic acid. A high regular consumption of coffee can lead to an increase of serum cholesterol levels associated with the presence of the cholesterol elevating diterpenes cafestol and kahweol. The extent to which these are extracted by different brews has been examined. 'l6 The 17-isovalerate of ent-16P-kauranoic acid has been found'17 in the stem bark of the Korean medicinal plant Acanthopanax koreunurn (Araliaceae).The lactone odolide 82 CH~OH 82 83 AcO' 86 Gly = P-D-glucopyranosyl-(1'~2)-P-D-glUCOpyranOSyl ' OAc 87 88 has been obtained' l8 from the roots of Gynocurdiu odorutu (Flacourtiaceae). The dihydroxyketone 83 and a similarly oxygenated pimarane have been isolated' I9 from Vernonun- thura amplexicaulis (Vernonaciae). The Chinese medicinal drug 'Ebeibeimu' derived from the bulbs of Fritilluriu ebeiensis (Liliaceae) has been shown12' to contain fritillebic acid 84 and the dimeric kaurenes the fritillebins A and B (85 R=H and OAc respectively). The glycoside subpubescensoside 86 which has been obtainedI2' from Stevia subpubescens (Compositae) contains an unusual llp-16-ether.Frutoic acid 87 from Xylopiufrutescens has been shown'22 to be a dimeric diterpene and is formed by a Diels-Alder type of reaction between a labdane and a kaurane unit. Highly oxidized kaurenes have continued to be found as constituents of Isodon (Labiatae) species many of which are Chinese medicinal plants. Recent isolates include the 89 90 OAc OAc 91 92 93 gesneriodins A-C (e.g. 88) from I. gesner~ides,'~~ the macro-calyxins (e.g. 89) from I. macrocaly~,'~~ loxothyrin A 90 from I. lo~othyrae,'~~ the laxoflorins (e.g. 91) from I. eriocalyx varn. laxlJlora126and longirabdolide D 92 from Rabdosia longit~ba.'~'A revised structure 93 has been proposed'24 for maoyerabdosin.The biotransformation of kaurenoid diterpenes has contin-ued to attract interest. The biotransformation of ent-15-oxokaur-16-ene to form 15-oxogibberellins by Gibberella fujikuroi has been explored. '28 This biosynthetically directed transformation was accompanied by reduction of the 16,17-double bond. The transformation of 15-oxokaur-16-en-19-oic acid by Cephalosporium aphidicola Rhizopus stolonifer and Mucor plumbeus has also been reported,'29 in which a similar reduction was found. The borderline between biosynthetically patterned and xenobiotic biotransformation has been exam-ined' 29 in the hydroxylation of ent-16P,19-dihydroxykaurane by C. aphidicolu. 5.2 Beyerenes Rearrangements of ent-1P-acetoxy-15a,16a-epoxybeyeranes with an axial or equatorial hydroxy group at C-12 catalysed by ruthenium acetylacetonate gavel3' ent-14-hydroxykaur-15-and -16-enes and a ring C noraldehyde.The formation of some of the products was accounted for by fission of ring C. Other rearrangements led to products with an antheridiogen-like ring system. 5.3 Aphidicolanes A number of synthetic studies in the aphidicolin series have been rep~rted.'~'-'~~ The results of incubation of 3a,16P-dihydroxyaphidicolane with Cephalosporium aphidicola showed'34 that the presence of a hydroxy group at C-3 blocked further hydroxylation at C-18 a step in the normal bio-synthetic sequence leading to aphidicolin. Some thyrsiflorin derivatives (e.g. 94) have been obtained' 35 from Calceolaria dentata (Scrophulariaceae).5.4 Gibberellins Gibberellin A, 95 has been isolated13 from a fungus Phaeospheriu species L487. The biosynthesis of gibberellin A Hanson Diterpenoids HO C02H 'CH20H 94 95 in this fungus from gibberellin A proceeded'37more efficiently via gibberellin A rather than gibberellin The combination of liquid chromato&aphy fast atom bombardment and electrospray ionization mass spectrometry has been applied'38 to the glucosyl esters of the gibberellins. A GCMS study of the endogenous gibberellins from callus cultures of maize has led'39 to the identification of the gibberellins characteristic of the early 13-hydroxylation pathway leading to gibberellin A 97 which proceeds via H02C C02H 96 97 13-hydroxygibberellinA, (GA,,) 96.Gibberellin metabolism in cultured cells of Raphanus sativus (Cruciferae Japanese radish) has been ~tudied.'~'Further evidence has been pre~ented'~'for the involvement of gibberellins in the development of fruit in peas. There has been doubt about the natural occurrence of 3-epigibberellinA,. A method has been developed'42 to distin-guish between its endogenous and artefactual formation. It appears to be formed naturally in lettuce. A selective method for the epimerization of the 3P-hydroxy group in gibberellins has been deve10ped.l~~3-Epigibberellin A, 98 has been qqr) -HO" H02C OH 98 99 R=H; a-andP-OH HO 100 as an antheridiogen in Anemia phyllitidis. The partial synthesis of antheridiogens of Lygodiurn species includ-ing 99 from gibberellic acid has been re~0rted.l~~ The conver-sion of gibberellic acid to the trachylobagibberellin analogue 100 has been de~cribed.'~ 6 Macrocyclic diterpenoids and their cyclization products 6.1 Taxanes Recent advances in the chemistry of the taxanes have been reviewed.147 The synthesis of Tax01 has been achie~ed'~~,'~~ and the synthetic effort in this area has been re~iewed.'~' 25 1 0% number of novel taxanes from T.~hinensis.'~~''~~ The enol- acetate taxuspine D 104 has been isolated'58 from T. cuspidata and it is reported to have some inhibitory activity against the depolymerization of microtubules. Other taxuspines including OH 105159and the abeotaxane 106160have been isolated from the same plant.Further investigations of the Himalayan yew T. wallichiana have yielded a number of taxanes including 101 107,16' 108,'62 109163 and 110 (R=Ac).'~~ A series of taxanes known as the taxayunnanines (e.g. E 111 and G 112)'653'66and the taxayuntins (e.g. 110 R=Bz)'~~ have been obtained from the Chinese species T. yunnanensis. Studies'68 on the biosynthesis of taxol have led to the AcO-AcO-* synthesis of the potential intermediate 113. Examination of the fluctuation of Taxol content with seed development has OAc' ' OAc suggested'69 that the immature seed may be a good source of a 103 cell free system with which to study the biosynthesis. The occurrence of taxane derivatives has also been examined'70 in this context.A number of X-ray studies have been on taxanes including studies to obtain the dimensions of biologi-cally active derivatives. AcO HO-An immense amount of work has been reported on 'OCinn the preparation of novel taxane analogues in the study of structure-activity relationships. Compounds based on (R)-9- 104 105 dihydro-Taxol showed'74 greater stability and water solubility and the preparations had a longer lasting biological activity. The stereoselective reduction of taxanes with samarium diiodide has been de~cribed.'~' Other modifications of C-9 and '1 C-10 have also been rep0~ted.l~~ The reduction of the A'' HO OAc /"'OH double bond in the unsaturated 13-ketones with zinc afforded'77 unstable dihydro derivatives. Cleavage of the 10 11 bond takes place by a retro-Michael reaction.The facile 106 cleavage of rings A and B in taxoids has been reported'78 in another study. A synthesis of 13-epitaxol has been de~cribed.'~~ Various methods for the selective hydrolysis of The search for novel taxanes from various Taxus species has taxane esters have been explored. 180-182 Many other modifi- A led to the identification of a number of new derivatives cations of these structures have been including the dione 101 from the roots of a Taxus hybrid.I5' number of modifications of ring C and in particular the ring Examination of T. baccata has affordedIS2 some contraction to form analogues such as 114 have been 10-deacetylbaccatin I11 analogues and compounds with the rearranged 1l(15-1)abeo-taxane skeleton (e.g.102).Is3 The extraction of T. canadensis has aff~rded"~~"' some further taxanes (e.g. 103). Structures have been established for a 114 AcO-'OH "OR2 described.'88-190 Water soluble derivatives containing a poly- 109 ethylene glycol unit attached at C-7 have been reported. 19' The preparation of paclitaxel and docetaxel photoaffinity labelsI9' and [3'-'4C]-labelled taxotere have been described. 193 6.2 Cembranes and other cyclization products The conformational analysis of the 10- and 13-hydroxy deriva- HO-AcO-tives of cembrene has been re~0rted.l~~ Sinugibberol 115 has been identified19' as a cytotoxic constituent of the soft coral Sinularia gibberosa. The uprolides (e.g. 116) are cembranolides which were ~btained'~~.'~~ from a Puerto Rican collection of 110 R =Ac; BZ 111 the gorgonian Eunicea mammosa whilst sinulariolone 117 was obtained'98 from a Philippine collection of the soft coral Sinularia jlexibilis.Many of the hydroxy epoxides in the cembrane series are accompanied by cyclic ethers. The syn- thesis of a number of these and their biological evaluation as cytotoxic agents has been reported. 199 The cyclization of cembrene diterpenoids has continued to be of intere~t.~~~~'~' The irregular cembranoid sarcotol 118 with a 13-membered 112 113 ring has been obtained202 from a Sarcophyton species. 252 Natural Product Reports 123 obtained209 from the soft coral Sarcophyton solidum represents another mode of cyclization of cembrene. The further cyclization products 124 and 125 have been obtained2'0'21' from Sinularia species of soft corals.Dissec- tolide A 124 may be a nor-derivative of isomandapamate. The Euphorbiaceae produce a number of biologically active 116 diterpenoid esters which have attracted interest for some time. 115 Compounds which have recently been isolated include the euphoreppines from Euphorbia aleppica which are esters of 126,212some myrsinol relatives (e.g. 127) from the roots of E. OH HO 117 HO 126 127 120 119 H, c OR2 CH20R1 0 121 R' = -C-CH2-,C,-CH2C02H; R2= H/OAc HO Me NMR and molecular mechanics methods have been applied to a study2" of the stereochemistry of the dolabellane diter- penoids. A number of these including 119204and 120205have been detected in Caribbean gorgonian octacorals such as Eunicea laciniata and E.tourneforti. Dolabellane diterpenoids are not restricted to marine organisms. The 3-hydroxy-3- methylglutaryl ester 121 has been obtained206 from Chrozo-phora obliqua (Euphorbiaceae). The funicolides (e.g. 122) are a series of briaranes which have been isolated207 from the coral Funiculina quadrangularis. Various conformational isomers of the ten-membered ring have been observed208 by NMR spectroscopy. Sarsolenone 0 122 HO Hgp123 0 0 128 129 pJ=O 130 131 R1 ,R2 = propionyl and butyryl prol$era213and the euphactins (e.g. 218)214and euphoractines (e.g. 129)215from the Chinese medicinal plant E. micractina. The structure of euphoractine B has been revised2I5 to 130.A series of propionyl and butanoyl esters of 131 which possess moderate anti-HIV-I reverse transcriptase activity have been obtained2I6 from E. myrsinites. 7 Miscellaneous diterpenoids Many diterpenoids with the xenicane skeleton have been isolated from corals. The natural product chemistry of West Indian corals has been reviewed.217 Compounds with the xenicane skeleton that have recently been isolated include deoxyxeniolide B 132 from Xenia elongata,218 the tsitsixenicins (e.g. 133) from Capnella thyr~oides,~'~ and the xeniatines (e.g. 134) from a Japanese Xenia species of soft cora1.220,221 Antheliatin 135 is a more highly oxidized member of this series which was obtained222 from the coral Anthelia glauca.Some further briarellins (e.g. 136) have been obtained223 from the cor a1 Briareum as bes t in um . The unusual structure 137 of garberic acid which was isolated224 from the evergreen shrub Garberia heterophylla (Asteraceae) may be derived by cleavage and rearrangement of a eunicellane diterpene. A number of diterpenoids have structures that are prenyl- ated versions of sesquiterpenoids. Compounds of this type that have been isolated include the lobane fuscol methyl ether 138 from the coral Lobophytum pauc~jlorurn~~~ and a more highly 124 125 oxidized compound 139 from another Lobophytum species.226 Hanson Diterpenoids -A I ~OAC 0 0 AcO 132 133 H oAc HoA -*F 'OAc 146 147 0 0 0 / '-OAc H been isolated23 from Plagiochila cristata (Hepaticae) along 134 135 with some known fusicoccanes.The plant growth regulatory activities of some structural variants of cotylenol have been examined.232 Epitaondiol 145 is a meroditerpenoid which was obtained233 from the Pacific alga Stypopodium flabelliforme. Spectroscopic VCozH C02H NOE studies confirmed that the B/C ring fusion in this epimer A 137 %oAc 138 139 140 141 ?w 142 1 43 The aromadendrene analogue emmottene 140 was obtained227 from the gorgonian Briareum polyanthes. The brown alga Stoechospermum marginatum afforded228 the spatane diter- penoid 141. The occurrence of these structural types is not restricted to marine organisms. The sacculatane diterpenoid 142 was obtained229 from the liverwort Pellia endiviifolia.The subglutinols (e.g. 143) are immunosuppressive diterpenoid pyrones which were obtained230 from an endophytic strain of the fungus Fusarium subglutinans. Endophytic organisms inhabit the intercellular spaces inside living plants in this case Tripterygium wilfordii. Verrucosane and related diterpenoids are quite widely dis- tributed in the liverworts. A 13-epihomoverrucosane 144 has 254 Natural Product Reports forced these rings to adopt a twisted boat conformation. A number of seco-diterpenoids such as dendrillin 146 from the Antarctic sponge Dendrilla membrano~a~~~ and a chlorinated compound 147 from Chromodoris h~miltoni~~~ have a similar origin. The trichoaurantiolides (e.g.149)236,237 and trichoaurantin 150238 which were obtained from the fungus Tricholoma 148 149 R=OH 150 R=H HO-TOM' 151 \ NHCHO Me02C-152 153 aurantium may arise biosynthetically by cleavage of a neo- dolastane precursor (e.g. 148). Isomandapamate 151 has been described239 as a constituent of Sinularia maxima. The anti- fungal activity of pseudolaric acid B 152 a major constituent of Pseudolarix kaempferi has been reported.240 Further anti- fouling kalihinenes (e.g. 153) have been obtained241 from the sponge Acanthella cavernosa and shown to be active against the larvae of the barnacle Balanus amphitrite. 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Chem. 1995 13 643. 197 A. D. Rodriguez J. J. Soto and I. C. Pina J. Nat. Prod. 1995,58 1209. 198 P. P. Guerrero R. W. Read M. Batley and G. C. Janairo J. Nut. Prod. 1995 58 1185. 199 A. D. Rodriguez I. C. Pina and C. L. Barnes J. Org. Chem. 1995 60 8096. 200 A. V. Shpatov V. A. Raldugin Y. V. Gatilov I. Y. Bagryanskaya and M. M. Shakirov Khim. Prir. Soedin. 1994 249. 201 A. V. Shpatov M.M. Shakirov and V. A. Raldugin Khim. Prir. Soedin. 1994 642. 202 T. Iwagawa S. Nakamura T. Masuda H. Okamura M. Nakatani and M. Siro Tetrahedron 1995 51 5291. 203 M. Piatelli C. Tringali P. Neri and C. Rocco J. Nut. Prod. 1995 58 697. 204 A. D. Rodriguez E. Gonzalez and C. Gonzalez J. Nut. Prod. 1995 58 226. 205 M. Govindan G. Govindan and D. G. I. Kingston J. Nut. Prod. 1995 58 1174. 206 K. M. Mohamed K. Ohtani R. Kasai and K. Yamasaki Phytochemistry 1995 39 15 1. 207 A. Guerriero M. D’Ambrosio and F. Pietra Helv. Chim. Acta 1995,78 1465. 208 G. Chiasera A. Guerriero M. D’Ambrosio and F. Pietra Helv. Chim. Acta 1995 78 1479. 209 M. Zhang K. Long K. Ma X. Huang and H. Wu J. Nut. Prod. 1995 58 414. 210 M. Kobayashi K. M.C. A. Rao M. M. Krishna and V. Anjaneyulu J. Chem. Res. (S) 1995 188. 21 1 K. Iguchi K. Kajiyama and Y. Yamada Tetrahedron Lett. 1995 36 8807. 212 Y. P. Zhi Z. J. Jin J. Jamil and S. Sadiq Phytochemistry 1995 40 1219. 213 D. Wu B. Sorg and E. Hecker J. Nut. Prod. 1995 58 408. 214 J. G. Shi Z. J. Jia and Y. X. Cui J. Nat. Prod. 1995 58 51. 215 J. G. Shi and Z. J. Jia Phytochemistry 1995 38 1445. 216 S. Oksuz F. Gurek R. R. Gil T. Pengsuparp J. M. Pezzuto and G. A. Cordell Phytochemistry 1995 38 1457. 217 A. D. Rodriguez Tetrahedron 1995 51 4571. 218 T. Miyamoto Y. Takenaka K. Yamada and R. Higuchi J. Nut. Prod. 1995 58 924. 219 G. J. Hooper and M. T. Davies-Coleman Tetrahedron 1995 51 9973. 220 T. Iwagawa Y. Amano T. Hase and M.Shiro Tetrahedron 1995 51 11 111. 221 T. Igawa Y. Amano T. Hase and M. Shiro Chem. Lett. 1995 695. 222 A. Rudi S. Ketzinel I. Goldberg Z. Stein Y. Kashman Y. Benayahu and M. Schleyer J. Nut. Prod. 1995 58 1581. 223 A. D. Rodriguez and 0.M. Cobar Chem. Pharm. Bull. 1995,43 1853. 224 H. Tak F. R. Fronczek and N. H. Fischer Phytochemistry 1995 40 185. 225 A. S. R. Anjaneyulu B. V. Rao K. V. S. Raju and M. V. R. Krishna Murthy Ind. J. Chem. Sect. B 1995 34,1074. 226 V. Anjaneyulu K. N. Rao and K. M. C. A. Rao Ind. J. Chem. Sect. B 1995 34 1071. 227 J. M. Cronan T. R. Daviau L. K. Pannell and J. H. Cardellina J. Org. Chem. 1995 60 6864. 228 Y. Venkateswarlu and M. A. F. Biabani Phytochemistry 1995 40 331. 229 T. Hashimoto Y.Okumura K. Suzuki S. Takaoka Y. Kan M. Tori and Y. Asakawa Chem. Pharm. Bull. 1995 43 2030. 230 J. C. Lee E. Lobkovsky N. B. Pliam G. Strobe1 and J. Clardy J. Org. Chem. 1995 60 7076. 231 S. Valcic V. Huch M. Veith and H. Becker Phytochemistry 1995 40 199. 232 N. Kato H. Okamoto and H. Takeshita Bull. Chem. SOC. Jpn. 1995 68,2679. 233 F. Sanchez-Ferrando and A. San Martin J. Org. Chem. 1995,60 1475. 234 B. J. Baker R. W. Kopitzka W. Y. Wensley and J. B. McClintock J. Nut. Prod. 1995 58 1459. 235 J. Pika and D. J. Faulkner Tetrahedron 1995 51 8189. 236 A. G. Invernizzi G. Vidari and P. Vita-Finzi Tetrahedron Lett. 1995 36 1905. 237 F. Benevelli 0. Carugo A. G. Invernizzi and G. Vidari Tetra-hedron Lett. 1995 36 3035.238 L. Knops M. Nieger B. Steffan and W. Steglich Liebigs Ann. Chem. 1995 71. 239 A. S. R Anjaneyulu K. S. Sagar and M. J. R. Venugopal Tetrahedron 1995 51 10 997. 240 E. Li A. M. Clark and C. D. Hufford J. Nut. Prod. 1995,58 57. 241 T. Okino E. Yoshimura H. Hirota and N. Fusetani Tetrahedron Lett. 1995 36 8631. 242 T. Pilati G. Cravotto and G. Palmisano Acta Crystallogr. Sect. G 1995 51 2676. 258 Natural Product Reports

ISSN:0265-0568    

DOI:10.1039/NP9971400245

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

Marine natural products D. John Faulkner Scripps Institution of Oceanography University of California San Diego La Jolla CA 92093-0212 USA Covering 1995 Previous review 1996 13 75 1 Introduction 2 Marine microorganisms and phytoplankton 3 Green algae 4 Brown algae 5 Red algae 6 Sponges 7 Coelenterates 8 Bryozoans 9 Molluscs 10 Tunicates 11 Echinoderms 12 Miscellaneous 13 References 1 Introduction This report is a review of the literature of marine natural product chemistry for 1995. Earlier reports published in this journal cover the period from 1977 to December 1994.’-12 For the first time during this series of reviews the number of new marine natural products and papers describing them appear to have decreased during the past year.While this may be a temporary slow down in productivity it may also be indicative that new compounds are becoming more difficult to locate. The format for this review is identical to that of the last reports in which compounds from cyanobacteria (or blue-green algae) were included in the section on marine microorganisms and phytoplankton. The review does not provide a comprehensive coverage of all research involving chemicals from marine organisms but concentrates on reports of novel marine natural products with interesting biological and pharmaceutical prop- erties. Biochemical studies involving marine organisms and reports of primary metabolites are specifically omitted. Research on the biosynthesis of marine natural products has been reviewed in detail el~ewhere’~,’~ and is not included in this report.Wherever possible the biological and pharmaco- logical properties of new marine natural products have been reported but papers detailing the pharmacological studies are beyond the scope of this review. In the area of synthetic organic chemistry the review focuses on reports of the total synthesis of marine natural products that confirm or redefine chemical structures and omits syntheses that are using marine natural products simply as a vehicle to illustrate the potential of a specific reaction. No attempt has been made to review the patent literature or conference abstracts. In addition to a plethora of articles in scientific and popular magazines several reviews of broad interest appeared during 1995.These include ‘New dimensions in natural products research cultured marine microorganisms’ ‘Marine fungi -a prolific source of biologically active natural products’ l6 ‘The natural products chemistry of West Indian gorgonian octa- corals’,I7 ‘Structural studies on chemical constituents of echinoderms’,’’ ‘Bryozoan secondary metabolites and their chemical ecology’ l9 ‘Marine sesquiterpene/quinones’,20 and ‘Structurally similar natural products in phylogenetically dis- tant marine organisms and a comparison with terrestrial species’.21 A more personal review is entitled ‘Marine natural products research a look into the dive bag’.22 An otherwise excellent overview of ‘Natural products of opistobranch molluscs a biological review’ regretably lacks structural dia- gram~.~~ A meeting report on the 1994 Japan-US Seminar on Bioorganic Marine Chemistry provides a synopsis of research in the two countries.24 2 Marine microorganisms and phytoplankton The metabolites reported from cultured marine bacteria many of which were isolated from marine invertebrates are rarely significantly different from those reported from terrestrial sources.However only a small percentage of marine bacteria can be grown in culture and the development of new culture media and techniques is expected to yield more unusual isolates and compounds. A FZuvobacterium species isolated from the bivalve mollusc Cristariu plicata produced two sulfonolipids flavochristamides A 1 and B 2 both of which inhibit calf thymus DNA polymerase a.25 Thiomarinols B 3 and C 4 are two additional antimicrobial metabolites from Alteromonas rava sp.nov. SANK 73390.26 A cultured marine actinomycete (CNB-880) isolated from the sediment of a -03sT’ OH 1 2 4,5-dihydro Faulkner Marine natural products 5 R=OH 7 R=H Me do8 / I HooCfTNH 14 R = -(CHZ)~CH(M~)C~H~ 0 R/ 15 R = -(CH2)7CH(Me)2 9 10 R=OH 16 R = -(CH2)9Me 11 R=H 17 R = -(CH2)7CH(Me)C2H5 18 R = -(CH2)8(Me)2 R I 12 R=a-OH 13 R = P-OH coastal lagoon in Southern California yielded elijopyrones A-D 5-8.27 An actinomycete from a deep-sea sediment produced four pluramycin antibiotics only one of which y-indomycinone 9 had not been described previously.28 Strep-tomyces sp.SA-3501 which was isolated from a marine sediment contained the N-acetyl-p-D-glucosaminidase inhibi-tors pyrostatins A 10 and B ll.29Maremycins A 12 and B 13 are unusual diketopiperazines from a Streptomyces sp. that 19 R=Me 20 R=H had been isolated from a marine sediment from Chile.30 Five surfactin-like cyclic depsipeptides bacircines 1-5 1418 were obtained from a culture of BaciZZus pumilus that was isolated from a sponge of the genus Ir~inia.~~ An isolate (SCRC-4h1-2) of BaciZZus cereus that was obtained from the surface of the snail Littorina sp. contained the cytotoxic agent homocereulide 19 and the known cytotoxin cereulide 20; B. cereus is normally regarded as a source of emetic food poisoning.32 The structure of alterobactin A 21 which is a siderophore having an extraordinary affinity for ferric ion from an open ocean bacterium Alteromonas Z~teoviolacea,~~ has been confirmed by total synthesis.34 The carotenoid myxol 22 was obtained from an orange Havobacterium sp.associated with the sponge HomaxineZla sp. from pa la^.^^ Two new carotenoid glycosides (3S,3'S)-astaxanthin-~-~-glucoside23 and 21 OH HO 22 R OH 23 R=O 24 R=H2 260 Natural Product Reports MeOOC MeOOC~o 40 41 0 CI 26 27 q=-=7qCH0 poH 0 I 42 43 Ho\ y I 's 45 N%H A A 29 ~=4; y=3 33 x=2 30 ~=3; y=3 34 x=3 31 ~=2; y=3 35 x=4 32 ~=2; y=4 FS OMe 24 were isolated from (3S,3'R)-adonixanthin-P-~-glucoside 46 Agrobacterium aurantiacum from a seawater sample.36 Phomactins E 25 F 26 and G 27 are additional platelet activation factor (PAF) antagonists from the marine fungus Phoma sp.37 A strain of Penicillium sp.(BM1689-P) that was four new alkaloids that were isolated together with the pre- isolated from a marine sediment from Uchiura Bay Japan viously reported4' compounds in this series from a strain of contained the neuritogenic compound epolactaene A Aspergillus fumigatus isolated from the gastrointestinal tract strain of the fungus Leptosphaeria sp. that was isolated from of the fish Pseudolabrus japoni~us.~~ the surface of the brown alga Sargassum tortile contained leptosins G 29 G 30 G 31 H 32 K 33 K 34 and K 35 which are closely related to metabolites described previously from the same so~rce.~~~~~ Fumiquinazolines D-G 36-39 are Me (c 6 48 R=H 50 R=Ac 0 0 AcO 36 37 R1 R2 OMe \ -------w-To N Me CI 0 49 OMe aCOOH 51 Faulkner Marine natural products 261 In addition to the major metabolite debromoaplysiatoxin a specimen of Lyngbyu mujuscula contained small quantities possibly artifacts of lyngbyacarbonate 40 the related lactone 41 1,7-dimethylindole-3-carboxaldehyde 42 and a monoterpene 43 that had previously been isolated from the red alga Desmiu (=Portieriu) hornem~nni.~~ Two new antimitotic metabolites curacins B 44 and C 45 were isolated from L.mujusculu from Curaqao4 and the absolute configuration of curacin A 46 was reported.45 The structure and absolute configuration of curacin A 46 were confirmed by total synthesis.46 The ichthyotoxic amides malyngamides H 47 and I 48 were isolated from speci- mens of L.mujusculu from Curaqao and Okinawa respect- i~ely.~~,~' Comparison of the spectral data of malyngamide I 48 with those reported for stylocheilamide which was isolated from the opisthobranch mollusc Stylocheilus longi~auda,~~ led to the suggestion that the structure of stylocheilamide be revised from 49 to 50.48 The absolute configuration of (4E,7S)-(-)-7-methoxytetradec-4-enoic acid 51 which is a metabolite of L. rn~jusculu,~~ has been confirmed by total ~ynthesis.~' Antillatoxin 52 which is a minor metabolite of I0 52 53 L. mujusculu from Curaqao has both a novel structure and ichthyotoxic and molluscicidal proper tie^.^^ A cyanophyte of the genus Synechocystis that was found overgrowing dead corals in Okinawa contained four cytotoxic compounds nakienones A-C 53-55 and nakitriol 56.53A laboratory culture of Hupalosiphon luingii which was isolated from the surface of dead coral in Papua New Guinea produced three new ichthyo- toxins 12-epi-hapalindole H 57 12-epi-hapalindole G 58 and 12-epi-hapalindoleQ isonitrile 59 in addition to known mem- bers of the hapalindole series.54 Total syntheses of hyellazole 60 from Hyellu c~espitosu,~~ and both oscillatoxin D 61 and 30-methyloscillatoxin D 62 from a mixed collection of Schizothrix culcicola and Oscillutoria nigr~viridis,~~ have been rep~rted.~~.~~ A dinoflagellate of the genus Amphidinium (strain S1-36-5) that was isolated from St.Thomas US Virgin Islands pro- duced the linear cytotoxins amphidinoketides I 63 and I1 ?H ?H I ?H I I H 57 58 59 (&-OMe Ph 60 61 R=H 62 R=Me 63 64 R 0 65 66 R=O 67 R=H2 64 the stereochemistries of which remain ~ndetermined.~~ The same organism also produced caribenolide I 65 which is a highly cytotoxic 26-membered macrolide.60 Amphidinolides 0 OH I OH OH OH 68 262 Natural Product Reports ROOC OH OH 69 R = -H ~ C 0 ~0~oso3H OS03H OS03H 70 R=H OH OH OH / !,),),+,oSo3~ 71 = -H2cT-o-0 0 OS03H 0 0 OSO~H 72 R = -HzC N*oso3H OH OH OH NH2 m;,;&oH OH HO OH HOWH 3H 'OH OH 73 one OH replaced by H 66 and P 67 are cytotoxic 15-membered macrolides from an NMR spectra of brevetoxin-3 7770and the use of the three- Amphidinium sp.cultured from the flatworm Amphiscolops dimensional pulsed-field gradient NOESY-HMQC spectrum Amphidinium klebsii isolated from the surface of a allowed complete assignment and confirmation of the structure The total synthesis of brevetoxin B 78 which seaweed contained amphidinol 2 68 which is a potent of mait~toxin.~' haemolytic agent.62 is a polyether toxin from Gymnodinium breve,72 has been A second synthesis of hemibrevetoxin B 79 Dinoflagellates are the source of many toxins with such ac~omplished.~~~~~ complex structures that the stereochemistry is very difficult to has been reported.75 assign.DTX-4 69 a water-soluble phosphatase inhibitor from 9'-Apo-fucoxanthinone 80 was shown to be a cytotoxic Prorocentrum lima is an ester of okadaic acid 70.63 Two constituent of the dinoflagellate Amphidinium while similar water-soluble esters of okadaic acid the homologues 10'-apo-fucoxanthinal 81 12'-apo-fucoxanthinal 82 12-apo-DTX-5a 71 and DTX-Sb 72 were isolated from a culture of fucoxanthinal 83 and 13'-apo-fucoxanthinone 84 were P. maculosum that had previouslya been described as P. reported as metabolites of the diatom Phaeodactylum tri-Ostreocin D 73 which was isolated from the cornutum that inhibit feeding by the copepod Tigriopus califor-~oncavum.~~ Three strains of the marine ciliate Euplotes raikovi two dinoflagellate Ostreopsis siamensis is an analogue of palytoxin nic~s.~~ in which two methyl groups at C-3 and C-26 and one hydroxy from the Mediterranean and one from California produced group the position of which is not known are replaced by epiraikovenal 85 and two of these strains also contained hydrogen.66 The vasoconstrictors zooxantellatoxins A 74 secoepiraikovenal 86.78 and B 75 were isolated from a symbiotic dinoflagellate Symbiodinium sp.(Strain Y-6) that was associated with the flatworm Amphiscolops sp.67,68Gymnodimine 76 is an unusual toxin from a Gymnodinium sp. that was responsible for neuro-3 Green algae toxic shellfish poisoning in New Zealand oysters.69 Improved A new polyunsaturated fatty acid (42,72,9E,l lE NMR techniques and particularly the use of a micro-detection l32,162,19Z)-docosaheptaenoic acid 87 was encountered in probe have enabled the complete assignment of the 'H and 13C Anadyomene stellata from Florida.79 A series of oxygenated Faulkner Marine natural products 263 OH OH OH OH Me OHC ' O76 W OH 79 77 R=CHzOH 78 R=CHO clerosterols 88-90 have been isolated from Codium arabicum." Some potentially ecologically relevant reactions of amines with caulerpenyne 91 which is a major metabolite of Caulerpa taxi$oliu,81 have been studied.82 4 Brown algae A review of the chemistry and chemotaxonomy of brown algae of the family Cystoseiraceae provides a critical evaluation of the relationships between the diterpenes meroditerpenes and other metabolites reported from this family.83 In a very surprising result it has been demonstrated that the thermo- labile [t,, (18 "C)=21 min] cis-cyclopropane 92 is significantly 264 Natural Product Reports more active than its Cope rearrangement product ectocarpene 93 as a pheromone of Ectocarpus siliculo~us.~~ The absolute configuration of desmarestene 94 which is the gamete-releasing and gamete-attracting pheromone of Desrnarestia aculeatu and D.Jirma,85 has been determined by total syn- thesis.86A synthesis of (+)-mutifidene 95 which is a phero- mone of Cutleria mult~jida,~' has been described.88 The structure of sargassumketone 96 which is a highly oxygenated metabolite of Sargassum kjellmanianum was determined by X-ray cry~tallography.~~ Taonia lacheana contained (+)-11-epispathulenol 97 and other aromadendrane sesquiterpenoids while T.atomaria f. ciliata contained cadinane sesquiter-penoids." Two new sesquiterpenes 4~,5a-dihydroxycubenol 98 and cuben-3-one 99 were isolated from Dictyopteris H AcOwo 80 / 81 88 / 82 CHO AcO 83 AcOp84 90 AcO,* 85 86 OAc 91 delicatula and were identified using a combination of shift reagent and 2D NMR methods." The synthesis of a-dictyopterol 100 which is a metabolite of D. div~ricata,~~ involves an intramolecular Diels-Alder cy~lization.~~ Bifurcaria bifurcata from Brittany contained two new diter- penoids bifurcane 101 which inhibited the development of fertilized sea urchin eggs and epoxyeleganolactone 102.94 A new spatane diterpene (5R)-17,18-epoxy-5,16-dihydroxyspat- 13( 14)-ene 103 was reported from Stoechosperrnum margi- naturn from the Indian Ocean.95 As a result of studies of the preferred conformations of dolabellane diterpenoids from Dictyota dichotoma the stereochemistry of three epoxides were revised from 104-10696 to 107-109.97 The structure and 95 HO ' 96 97 absolute configuration of (+)-acetoxycrenulide 110 which is a metabolite of D.cren~Iata,~*~~~were confirmed by total synthesis.loo Three new meroditerpenes 111-113 were obtained from Cystoseira crinata from the French Riviera coast as part of a zoogeographical study."' The total synthesis of the structure proposed for ( f)-tetramethylmediterraneol B 114 from C. rnediterraneaIo2clearly demonstrates that the proposed struc- tures for mediterraneol B and its permethylated derivative are 98 99 100 incorrect.lo3 Conformational analysis of epitaondiol which was first described as a metabolite of Stypopodium ona ale'^^ but was later isolated from S.j?abelliforrne revealed that the B and C rings were forced into the twist-boat conformation OH 101 which required a reassignment of the structure from 115 to 116.Io5 The absolute configuration of stypoldione 117 from S. zonaleIo6 has been determined by total synthesis.'07 A further 26 new polyphloroglucinol derivatives of the phlorethol fuhalol and deshydroxyfuhalol families were isolated from Faulkner Marine natural products 265 1 03 1 04 105 R=H 107 106 R=OH 108 R=H 109 R=OH 110 OH I OAC OR 111 R=Me 112 R=H 0 113 OMe Me0 114 OH I \ 115 OH HO 116 117 Sargassum spinuligerum and identified as their peracetyl derivatives the structures of which have been omitted because they are not strictly natural products.'08,'09 266 Natural Product Reports 5 Red algae Specimens of Corallina rnediterranea from Alicante Spain contained two long chained aldehydes one of which (2E,4E)- 2-(tridecyl)-heptadeca-2,4-dienal 118 was new. 'lo The abso- lute configurations of constanolactones A 119 and B 120 CHO C12H25 dC13H2 118 121 OMe OMe I 122 R' = R2 = OH 123 R1 = R2 = OMe 124 R1 = OAc; R2 = OMe 125 R1 =OH; R2 = OMe which are eicosanoids from Constantinea simplex,' ' ' have been confirmed by total synthesis.Il2 A simple synthesis of the dihydro-3(2H)-furanone laurencione 121 from Laurencia spectabilis' l3 has been disclosed.' l4 Polycavernosides A2 122 A3 123 B 124 and B2 125 are four new analogues of the toxic macrolide polycavernoside A 126 from Polycavernosa tsudai OMe OMe 126 OAc Br 127 128 (=Gracilaria edulis).' The relative configuration of POlY-cavernoside A 126 was determined and the sugar portion has been synthesized.'16-' l8 The absolute configurations of a series of halogenated furanones from Delisea pulchra (cJ fimbriata)' l9 were based on an X-ray crystallographic determi- nation of the absolute configuration of furanone 127.120 A simple synthetic route to fimbrolide 128 which is a metabolite of D. fimbriata,'21 and related furanones has been reported.12' Rr _../-r -Y Br-129 130 Br Br Br OH 131 Br 132 L OAc 133 + \ \\ 134 135 Two brominated C15 acetogenins 129 and 130 which had previously been reported from the Mediterranean sponge Mycale rotalis '23 were isolated from Laurencia paniculata from Turkey and a new brominated allene 131 was isolated from L. obtusa.I2 The structure of the known acetogenin 130 was confirmed by a single crystal X-ray analysis.'24 The total syntheses of ( -)-trans-kumausyne 132125and (+)-laurencin 133126have been reported and a biosynthetic conversion of (3E,6S,7S)-laurediol 134 into (E)-prelaureatin 135 using hydrogen peroxide sodium bromide and lactoperoxidase has been described.127 A new macrocyclic pyrone 136 and two known compounds were obtained from a specimen of Phacelocarpus peperocarpos from Victoria Australia and the 2 geometry at C-17 in the known macrolide 137 from P.labillardieri128was determined for the first time.129 Interest in the halogenated sesquiterpenes from Laurencia species may well be revived by the report of differential cytotoxicity of three such compounds 13S140 toward colon tumour cell lines.30 Seven new sesquiterpenes majapolenes A 141 which is a cytotoxic peroxide and B 142 majapolone 143 and majapolols A-D 144-147 were isolated from Laurencia rnajuscula from the phi lip pine^.'^^ Since majapolene A 141 majapolone 143 and majapolols A-D 144-147 were all isolated as diastereoisomeric mixtures it is possible that all are artifacts Faulkner Marine natural products Br p0CH2OH \ R2 Br kl // 138 R' = R2 = H 141 139 R1 = Br; R2= H 140 R1= H; R2 = Br R OH HO CI' 142 R=a 143 R=a 144 R=a 145 R=a OH 146 R=a 147 R=a resulting from air 0xidati0n.l~~ L.karlae from the South China sea contained the diterpene laukarlaol 148 together with known ~esquiterpenes.'~~ Enshuol 149 is a pentacyclic triterpene pol yether from L. omaezakiana from Japan and callicladol 150 is a cytotoxic triterpene polyether from a Vietnamese species of Laurencia.133'134 The structure of a new 3,6-diketosteroid 151 from Hypnea rnusciforrnis from India was determined by X-ray crystallography. 35Halichondria cylin- 148 Br OH 149 OH Br-OH 150 0 151 6 Sponges Sponges again provided the largest number of biologically active marine natural products and continue to be the most studied marine phylum.A new sphingosine derivative 152 was isolated from Spirastrella inconstans from India,136 and ten cytotoxic and antifungal glycosphingolipids halicylindrosides A,-A 153-156 and B,-B 157-162 were obtained from OH OH NH OH NH OH Ho -cllH23 C22H45 NH OH HO” ‘NHAc HO’ ‘NHAc 0 HO OH 152 153 m=16; n=10 157 m=16; n=8 154 m=16; n=ll 158 m=17; n=8 155 m=17; n=ll 159 m=16; n=9 156 m=18; n=ll 160 m=16; n=10 161 m=17; n=9 162 m=16; n=ll ?H O PNH R 1OH 165 R‘ = C19H39 C20H41 I c21H43 R2= various C11 to c16 alkyl chains 168 R’ = C17H35 to C21H43 R2= various C1 1 to c16 alkyl chains 167 R’ = C17H35 to C21H43 R2= various C13 to C16 alkyl chains 170 R’ = C1gH3g to C22H45 R2= various C12 to c16 alkyl chains H? H? -COOMe 171 R=H OH 173 174 172 R=Me Br -COOMe 175 COOMe 176 177 H OH OH Br COOMe 16 15 Br 1 78 HoH>;-+ 179 1 a0 0 OH H 183 R = -CSHll 184 R = -CH2CH=CHC2H5 (Z) OH OH 187 188 4,5-dihydro OH 189 190 23,24-dihydro 192 23,24-dihydro 193 194 195 23,24-dihydro 0 197 OH X COOH Br COOH 199 X=H 201 200 X=Br Br COOR1 COOH 206 R=H 207 R =Br B r I c o o H COOH& “ R r \\ r COOH Br Br R Br 208 R=H 210 211 R=-C6H13 209 R =Br 212 R =-(CH2)13CH=CHC3H (2) 213 R =-(CHZ)~~CH=CHC~H~(2) 214 R = -(CH2)1&H(CH3)2 215 R =-(CH2)16CH(CH3)2 Faulkner Marine natural products 269 14'species from Trinidad.Axinella Halichondria cylindrata from Japan.'37 A series of three papers has described the glycosyl ceramide composition of the Caribbean sponges Agelas clathrodes 163-166 '38 A. longissima 167-169139and A. conifera 164-167 and 17O.l4OThe two cyclic hemiketals raspailols A 171 and B 172 from the Palauan sponge Raspailia (Raspaxilla) sp. do not appear to fall into any previously established chemotaxonomic group. 14' Rottnestol 173 is a related hemiketal from a Western Australian species of Haliclona. 142 Dysidea fragilis from Pohnpei contained four azacyclopropene derivatives (4E,S)- dysidazirine 174 which is the enantiomer of the known metabolite dysidazirine 175,143(42)-dysidazirine 176 (4E,S)-antazirine 177 and (4Z)-antazirine 178.Ia The structure and absolute configuration of (R)-(-)-dysidazirine 175 have been confirmed by total synthesis.'45 As a result of the synthesis of three stereoisomers of penaresidin A 179 which is an acto- myosin ATPase activator from an Okinawan Penares SP.,'~~ it was concluded that the natural product must have either the (2S,3R,4S 15S,169 or (2S,3R,4S 15R,16R) stereochemis- try.'47 Axinellamide 180 is an unusual hydroxylactam from an The antimicrobial guanidine alkaloids aplysillamides A 181 and B 182 were isolated from Psammaplysilla purea (=purpurea?) from Okinawa and the absolute configuration of aplysillamide B 182 was established by total synthesis.149 The structures and absolute stereochem- istry of halicholactone 183 and neohalicholactone 184 which are eicosanoids from Halichondria okadai '5071 51 were con-firmed by total synthesis but a note added in proof revealed that there is still some controversy regarding the absolute stereochemistry at C-15 of neohalicholactone 184.152 Two new acetylenic alcohols (3S,4E)-3-hydroxyheneeicos-4-en-1-yne 185 and (SS,3Z)-5-hydroxy-l6-methyleicos-3-en-1-yne 186 were reported as part of a discussion of the antitumour activity and absolute stereochemistry of a series of acetylenic alcohols from Cribrochalina vasculum. '53 Twelve minor acetylenic alcohols 187-198 were isolated from the Mediterranean sponge Petrosia ficiformis and were screened for brine shrimp t0xi~ity.l~~ An Indian Ocean specimen of Xestospongia sp.that contained high proportions (ca. 50%) of eubacteria in its tissues yielded three new brominated acetylenic acids 199-201 one of which (201) was characterized as its methyl ester.'55 Seventeen brominated acetylenic acids of which fourteen 202-215 were new and three had previously been found as components of sponge phospholipid^,'^^^'^^ were reported as metabolites of an Okinawan specimen of Xestospongia sp. (for spectral data see the microfiche edition).15' A Plakortis sp. from Fiji contained two new cyclic per- oxides plakortolide E 216 which showed selective cytotoxicity against melanoma and breast tumour cell lines and plakoric acid 217.'59Manadic acids A 218 and B 219 are cytotoxic cyclic peroxides from a Plakortis sp.from Indonesia.I6' An Indonesian Plakinastrella sp. contained elenic acid 220 which is a topoisomerase I1 inhibitor. 16' The absolute configurations of 216-220 were all determined using variations on Mosher's method. Manzamenones J 221 and K 222 and plakoridine B 223 are three additional oxylipins from an Okinawan Plakortis species.'62 Total syntheses of both (+)-and ( -)-untenone A 224 which is a metabolite of the Okinawan Plakortis SP.,'~~ revealed that the natural product was essentially racemic.164,165 A Phakellia sp. from the coast of Maine contained small 1 quantities of 14,15-dihydrodinophysistoxin-225 together with two known dinoflagellate toxins.166 The known'67 dino- flagellate toxin pectenotoxin I1 226 was identified as the cytotoxic constituent of a two-sponge association consisting of an outer layer of a Poecillastra sp. overlaying a Jaspis SP.'~' A new halichondrin B homologue halistatin 3 227 was isolated in extremely small quantities from a Phakellia sp. collected at Chuuk (Tr~k).'~~ Full details of the total synthesis of swin- holide A 228 which is a cytotoxic dimeric macrolide from Theonella swinhoei I7O have been reported. '71 Phorboxazoles A 270 Natural Product Reports 216 217 COOH 218 COOH 219 HO -OH 220 0 221 222 O=?6H33 0 MeOOC**y c1 5H31 223 224 229 and B 230 which were isolated from a Western Australian specimen of Phorbas sp.are potent cytostatic macrolides with an unprecedented carbon ~ke1eton.I~~ The relative and absolute stereochemistry of hennoxazole A 231 which is an antiviral agent from Polyfibrospongia sp.,173 has been deter- mined by total synthesis of its enanti~mer.'~~ second A highly convergent and stereoselective total synthesis of disco-dermolide 232 which is an immunosuppressive agent from Discodermia di~soluta,'~~ has been described.'76 Although known to be ubiquitous diketopiperazines are still being reported as natural products rather than primary metabolites cyclo-[4-methyl-(R)-proline-(S)-norvaline]233 is a new example from Calyx cJ podatypa from the Caribbean.'77 Herbamide A 234 is a chlorinated amide that was isolated as a minor component of a Papua New Guinea specimen of Dysidea herbacea.17' Neosiphoniamolide A 235 from the New Caledonian sponge Neosiphonia superstes is an antifungal cyclodepsipeptide in the jaspamide/geodiamolide series.179 Cymbastela sp. (ex Pseudaxinyssa sp.) from Papua New Guinea contained four new cytotoxic peptides geodiamolide G 236 hemiasterlins A 237 and B 238 and criamides A 239 and B 240 as minor metabolites.'" An improved synthesis of arenastatin A 241 which is a cytotoxic agent from Dysidea arenaria,I8' has been reported. 182Three additional thrombin inhibitors cyclotheonamides C-E 242-244 were isolated from Theonella swinhoei collected in Japan.'83 Syntheses of cyclo- theonamides A 245 and B 246184have allowed further studies of the thrombin inhibition a~tivity,'~~"'~ The structure of the protein phosphatase inhibitor motuporin 247 which was iso-lated from Theonella SP.,''~ has been confirmed by enantio- specific total synthesis.'" Keramides E 248 G 249 H 250 and J 251 are additional cyclic peptides from an Okinawan HOOC 226 OMe OMe -v OMe 227 228 231 I cc13 0 0 LJ 233 234 235 236 OMe NH2 H 241 242 243 Ph HN [-..Po HN ,OoCh ’NHCOR VPh 0 NH ‘NAN, H 244 245 R=H 246 R=Me OH I R2 I O+ 4 250 R1 = Br; R2= OH 251 R’ = R2 = H 272 Natural Product Reports 0 Ph Ph 254 I OH 255 R1 = R2= Me k 258 256 R1 = CHMe2; R2 = Me 257 R1 = CHMe2; R2= H U 259 R' = -CH2Ph; R2 = -CHMeEt 260 261 A 262 R1 =OH; R2=Me; R3=H; X=p-D-gal 265 R1 = R2 = H; R3 = Br; X = p-L-ara 266 R1 = R2 = H; R3 = Br; X = P-D-gal N H 267 R1 = H; R2 = Me 'CONH2 268 R1 = Me; R2 = H 269 R1= R2=Me Faulkner Marine natural products Theonella sp.A specimen of Cribrochalina olemda from Pohnpei yielded kapakahine B 252 which is an unusual hexapeptide containing an .a-carboline moiety. 19' Stylopeptide 1 253 the structure of which was determined by X-ray crystallography is a cyclic heptapeptide from Stylo-tella sp. from Papua New guinea and Phakellia costata from Truk."' The same specimen of P. costata also contained very small quantities of the cytotoxic cyclic peptides phakellistatins 4 254 7 255 8 256 9 257 10 258 and 11 259.'92-'94Total syntheses of the proposed structures of axinastatin 1 260 from Axinella sp.195 ( = pseudoaxinellin from Pseudoaxinella massa)196 and malaysiastatin 261 from Pseudoaxinella sp.197 showed that the structure of axinastatin 1 260 was correct but that the structure of malaysiastatin must be revised from 261 to 260.19' A Theonella sp.from Japan contained five additional cytotoxic bicyclic peptides theonellamides A-E 262-266. 99 Halicylindramides A-C 267-269 are antifungal and cytotoxic depsipeptides from Halichondria cylindrata from Japan.200 Amphitoxin 270 is a poorly defined high molecular mass pyridinium salt from the Caribbean sponge Amphimedon compressa.201The absolute configuration of niphatesine C 271 270 X= XM$ or '$,-+ 271 272 273 274 275 X =OH 276 X=H which was isolated from an Okinawan Niphates SP.,~'~ was established by total synthesis of both enanti~mers.~'~ Five additional alkaloids ingenamines B-F 272-276 were isolated from the Papua New Guinea sponge Xestospongia ingens and the absolute stereochemistries of ingenamine 277 ingamine A 278 and ingenamine E 275 were determined using Mosher ester meth~dology.~'~ Three new P-carboline alkaloids xesto- manzamines A 279 and B 280 and manzamine X 281 were obtained together with known manzamine alkaloids from a Japanese Xestospongia sp.and a fourth manzamine Y 282 was isolated from a Haliclona sp. that also contained known man~arnines.~'~ The structures of xestomanzamine A 279 and manzamine X 281 were determined by X-ray crystal- 10graphy.~'~ An Indonesian Prianos sp.yielded an unusual manzamine dimer kauluamine 283 which demonstrated moderate immunosuppressive activity.206 274 Natural Product Reports 277 278 0-N N4 Me' 279 280 3,4-dihydro OH OH / 281 282 A species of Batzella from the Bahamas contained five new alkaloids batzelladines A-E 2W288 together with known alkaloids of similar structure.207 Batzelladines A 284 and B 285 inhibited the binding of HIVgp-120 to CD4 and are therefore potential inhibitors of AIDS.207 8P-Hydroxyptilocaulin 289 is a further alkaloid of chemotaxonomic interest from Monan-chora arbuscula from Brazil.20' The structure and absolute configuration of ( -)-ptilomycalin A 290 which was first isolated from Ptilocaulis spiculifer and Hemimycale sp.,209 were rigorously established by total synthesis.210 In a paper that discusses chemotaxonomic relationships involving the pyrroloiminoquinone alkaloids it was reported that Zyzzya fuliginosa from Pohnpei contained makalu-vamines H-M 291-296 and damirone C 297 together with known compounds in this series.211 Discorhabdin G 298 is an alkaloid from the Antarctic sponge Latrunculia apicalis that deters predation by the seastar Perknaster fuscu~.~~~ Further syntheses of cytotoxic pyrroloiminoquinone alkaloids have been rep~rted.~'~,~'~ Two additional lamellarins Q 299 and R 300 were isolated from a recollection of Dendrilla cactos from New South Wales.215 Lamellarin 0 301 which had earlier been isolated from D.cactos,216 has now been synthesi~ed.~'~ The isoquinoline quinone alkaloids cribrostatins 1 302 and 2 303 from Cribrochalina SP.~''have been ~ynthesized.~" Five new naamidine alkaloids 304-308 were isolated together with H NH 284 n = 8 (major),9 10 NH H NH 285 n = 6 (major),7 8 286 n = 6 (major),7 8 0 H NH H 287 n = 8 (major) 9 10 H 288 - 289 290 known compounds in this series and their zinc complexes from Leucetta sp. from New Caledonia.220 A synthesis of kealiiquinone 309 from a Leucetta sp.221 resulted in the 2-imidazolone tautomer 310 the structure of which was con- firmed by X-ray crystallography; since the spectral data of 309 are different from those reported for 310 there appears to be good reason to take a second look at the natural material which may crystallize in two tautomeric forms.222 A deep-water Orina sp.from New Caledonia contained four new bis- and tris-indole alkaloids gelluisines C-F 31 1-314 that were active in receptor binding assays.223 Isobromotopsentin 315 is a bis-indole constituent of Spongosorites sp. from Southern Australia.224 Chelonin A 316 which is an alkaloid from R' 0 0 + R2 OH 291 R1= R2=Me 293 R1 = H; R2=Me 292 R' = R2 = H 294 R1 = Me; R2 = H 297 295 R1=H; R2=Me 296 R1=R2=H 298 HO 300 HoQOOMe I HO 301 0 302 303 Me0 X 304 R=H; X=H,OH 305 R=Me; X=H,OH 306 R=H; X=H,OMe 307 R=Me; X=H,OMe 308 R=Me; X=O OMe OMe Me0 N -OH '> Me0 N\ 0 Me Faulkner Marine natural products 309 310 275 H 'Br 311 317 R'=R2=H 318 R1=Br; R2=H 319 R1 = R2 = Br H 31 2 320 321 R1 = R2=Br 322 R1 = Br; R2 = H 323 R1=R2=H H 31 3 324 325 Br OMe H 314 Meomcoo-HO' HO Me/ 'Me 326 327 315 Me2Na0 H Br & 328 NMe2 OMe 31 6 Chelonaplysilla sp.from pa la^,^^' has been synthesized as H the racemate.226 Stylotella aurantium from Yap contained 329 R= f styloguanidine 317 bromostyloguanidine 318 and dibromo- 0 styloguanidine 319 all of which inhibit moulting of the cyprid 330 R = -COClaH37 larvae of barnacles.227 Agelongine 320 is an antiserotonergic bromopyrrole alkaloid from Agelas longissima from the Br Bahamas.228 Axinella carteri from Java contained 3-bromohymenialdisine 321 in addition to known alkaloids HO in this series.229 The structures of hymenialdisine 322 and debromohymenialdisine 323 which have been isolated from Br sponges of several genera,230 were confirmed by total syn- 0 N thesis.231 A paper on the isolation and structural elucidation of 331 H debromoaxinohydantoin was withdra~n.~~~,~~~ As part of an analysis of the variation in the secondary metabolite content of several Caribbean specimens of Pseudo-ceratina crassa three new compounds 324-326 were were obtained might be a new species of P~eudoceratina.~~~ The isolation of two new metabolites 327 and 328 Lipopurealins D 329 and E 330 and purealidins H 331 and J-R led to the proposal that the Caribbean sponge from which they 332-340 are additional bromotyrosine alkaloids from the 276 Natural Product Reports OMe 332 HY o q N Z z OH OMe Br H H o g "OH 341 X = H; Y = Z = Br 342 X = Z = H; Y = Br 343 X=Y=Br; Z=H OMe OH OH OR 333 OMe Br Br Br 344 R=H 346 R=H 345 R= Br 347 R=Me N-NH2 OH o 334 Br Br -Q"'Br Br& / OH Br Meo.l'O..NyR Br Br 348 349 OH 0 N 335 R=NH2 H diphenyl ethers 344348 and 5,6-dibromocatechol 349 were 336 R=H obtained from four Indo-Pacific specimens of the genus Dysidea and were screened as enzyme inhibit01-s.~~' Aplysillin A 350 the double bond geometries of which are unknown is a Br OH H OH 0 OH 337 Br HO Br 350 HO H AcOfloAc 0 NMe2 338 OMe OAc 351 NMe2 N-352 R=H Br '-'CONH2 353 R=Me 339 340 weak thrombin receptor antagonist from Aplysina fistularis ji~lva.~~~ Okinawan sponge Psammaplysilla purea (=purp~rea?).~~~,~~~ Kirkpatrickia variolosa from Antarctica contained the The (6S)-stereochemistry of bastadins 8 341 10 342 and 12 simple stilbene derivative 351.243Anthosamines A 352 and B 343 which are known metabolites of Ianthella basta,238,239 was 353 which exist as lactones in aprotic solvents and iminium determined by Mosher's method.240 Five new polybrominated salts in protic solvents are metabolites of Anthosigmella Faulkner Marine natural products 277 Br NH2 Me dimethylisoguanine 356.246 Trachycladine A 357 which is cytotoxic and trachycladine B 358 are very unusual nucleosides from Trachycladus laevispirulifer from Western Australia.247 Kumusine from an Indonesian Theonella sp.pH / appears to have the same structure (357) as trachycladine A but may be the enantiomer (see note in ref. 245).248 HO 354 355 356 Wiedendiols-A 359 and -B 360 are inhibitors of cholesteryl ester transfer protein a protein of potential importance in the 0 control of atherosclerosis that were isolated from Xesto-spongia wiedenmayeri from the Bahamas.249 Wiedendiol-A 359 was synthesized from (+)-sclareolide which allowed the absolute configuration to be determined.250 An Okinawan sponge of the family Spongiidae has yielded two additional sesquiterpene quinones nakijiquinones C 361 and D 362 which inhibit c-erbB-2 kinase and a related phenol nakijinol The structure of ( -)-ilimaquinone 364 originally HO OH 363.25',252 has been con-HO OH isolated from Hippospongia rnetachr~rnia,~~~ 357 358 firmed by total Arenarol 365 from Dysidea arenari~~~~ has been synthesized in racemic form.256 The structures of 5'-acetylavarol 366 and 2',5'-diacetylavarol 367 A deep-water aff.raromicrosclera that induce larval metamorphosis in were confirmed by X-ray ~rystallography.~~~,~~~ Phycopsisenone 354 is a simple aromatic com- collection of Siphonodictyon coralliphagum yielded bis(su1fato) a~cidians.~~~ pound from an Indian Phycopsis sp.245 Agelas longissima cyclosiphonodictyol A 368 which inhibits binding of LTB from the Caribbean contained longamide 355 and 3,7-OH OH 0 HO HO &e &e 369 370 371 359 360 COOH OH @O 0 372 R=a-Me 374 373 R=P-Me 361 R=H 363 362 R=Me O& R2 OMe / &O 0 375 376 R1= H; R2= OMe 377 R1=OMe; R2=H 378 R'=CI; R2=OH 364 365 379 R'=OH; R2=CI -OS03Na Nag8: AcoQoR \ 0 &?H \ 0 366 R=H 368 367 R=Ac 380 381 278 Natural Product Reports Two to human ne~trophils.~~~ undescribed Hawaiian species of Hyrtios contained 21-chloropuupehenol 369 15-oxopuupehenol 370 which showed significant differential antitumour and antimalarial activity and molokinenone 371.260Cyclorenierins A 372 and B 373 were isolated as an inseparable mixture from a Huliclonu sp.from Vanuatu.261 A Xestospongiu sp.from the Philippines contained six new topoisomerase I1 inhibitors secoadociaquinones A 374 and B 375 14-methoxyxestoquinone 376 1 5-methoxyxestoquinone 377 15-chloro- 14-hydroxyxestoquinone 378 and 14-chloro- 15- hydroxyxestoquinone 379.262 The absolute configurations of (+)-adociaquinones A 380 and B 381 from a Trukese Adociu sp.263 were determined by total Irciniu spinulosu from the Adriatic Sea contained three sulfated 2-prenylhydroquinones 382-384 that are toxic to brine shrimp.265 An Irciniu sp. from New Caledonia con-tained a sulfated 2-prenylhydroquinone 385 three 2-prenylhydroquinones 386-388 three chromenols 389-391 and a sulfated furanoterpene 392 in addition to previously reported meroterpenoids.266 0SO3Na 382 n=7 I OS03Na 384 383 n=a 385 n=5 OH I OH 386 n=5 388 387 n=6 HO 389 n=4 390 n=5 391 n=6 Oceanapamine 393 is an unusual sesquiterpene alkaloid from a Philippine Oceunupiu sp.267 Dysideu urenuriu from Thailand contained two sesquiterpene ethers arenarans A 394 and B 395.268The sesquiterpene alcohol 396 was isolated from 393 396 0 HO 394 395 397 398 399 402 R=NHCHO 404 405 406 R=-NC 407 R = -NHCHO absolute configurations of axisonitrile-4 406 and the corre- sponding formamide 407 from Axinellu c~nnubinu~~* have been determined by synthesis of the unnatural enanti~mers.~~~ A specimen of Acanthellu cuvernosu from Japan yielded three additional diterpene formamides kalihinenes X-Z 408-410 that inhibit settlement and metamorphosis of barnacle larvae.280 Callyspinol 411 is a diterpene alcohol from an Indian specimen of Cullyspongiu spinosissimu.28' Thorectandru chounoides from Australia contained one new diterpene furan 412.282As part of an ecological study of the Antarctic sponge Dendrillu membrunosu an additional diterpene dendrillin 413 was described.283 The structures and absolute configurations of gracilins B 414 and C 415 from Spongionellu gru~ilis~~~,~~~ were confirmed by total synthesis.286 The syntheses of several an unidentified Antarctic sponge and its absolute configuration Spongiu furanoditerpenes have been dis~losed.~*~-~~~ The com- was tentatively assigned by interpretation of the CD spec- plete NMR assignments for diisocyanoadociane 416,290which tr~m.~~~ Dictyodendrillins A-C 397-399 were isolated together is a metabolite of Cymbustelu sp.and ent-3P,16a-atisanediol with the known sesquiterpene furan dendrolasin from an 417291 from Spirustrellu cunctutrix have been reported; Australian Dictyodendrillu species.270 The total synthesis of both compounds were originally reported from different ( -)-furodysin 400 from Dysideu tuphu and D. herb~ceu,~~'.~~~ The absolute configuration of ( -)-agelasine has been reported.273 ( -)-Furodysin 400 was incorrectly reported as a novel metabolite of D.frugilis from the South China Sea.274 A specimen of Axinyssu uplysinoides from Palau contained the sesquiterpene isonitrile 401 the corresponding formamide 402 (4E)-(4-hydroxystyryl)trimethylammonium chloride 403 and two diterpenes ( -)-neoverrucosan-5~-o1 404 and (+)-homoverrucosan-5~-ol405,275,276 that had not been isolated previously from a marine organism.277 The Faulkner Marine natural products A 418 from Agelus nuk~murui~~~ confirmed by total was and a synthesis of racemic agelasirnine-A 419 and agelasimine-B 420 from A.mu~ritiunu~~~ has been reported.297 A novel brominated diterpene 421 was obtained from Humigeru turugensis from New Zealand.298 The South African sponge Psummoclemu sp. contained three cytotoxic terpenoids durbinals A-C 422-424 that may be of diterpenoid or sesquiterpenoid origin.299 279 NHCHO NHCHO *-q 80-H CN-H ,, I HO'* 0 H '\ ::it:cHo 0 CI CI El 408 409 410 41 1 OAc 0 'OAc "OAc 41 4 415 An Australian specimen of Psammocinia sp.contained an additional sesterterpene tetronic acid 425.300(18R)-Variabilin 426 the antipode of that found previously and variabilin 11-methyloctadecanoate 427 have been isolated from Ircinia felix from Col~mbia.~~',~~~ A species of Igernella from Palau contained the sesterterpene alcohol igernellin 428 together with the known metabolite halisulfate 3.303 The absolute stereochemistry of luffariolide E 429 and related metabolites which were isolated from an Okinawan Lufariella SP.,~'~ was shown by total synthesis and chemical interconversion to be OH 425 0 0 \ NC 416 417 pY ,. 41 8 41 9 422 R=Ac 424 423 R=H (3S,4R).305,306 Three additional ichthyotoxic and antifeedant sesterterpenes 430-432 were isolated from the Caribbean sponge Cacospongia cJ linteiformis.307 Cacospongionolide B 433 25-deoxycacospongionolideB 434 the structure of which was determined by X-ray crystallography and cacospongio- nolide C 435 were obtained from Fasciospongia caverrzosa 426 R=H 427 R = -CO(CH2)9CHMeC7Hl5 0 L.0' 429 430 0 432 280 Natural Product Reports 433 R=OH 434 R=H 0 435 0 0 430 439 0 440 I 441 442 from the Adriatic Sea.308,309 Heteronema erecta from India contained 25-dehydroxy- 12-epi-deacetylscalarin 436 as a minor metab~lite.~" The structure of scalaradial 437 from Cacospongia mollior was confirmed by X-ray crystallogra- ~hy.~" Suberitenones A 438 and B 439 are two very unusual sesterterpenes from an Antarctic Suberites sp.312 Three additional cytotoxic triterpenoids sodwanones G-I 440-442 have been identified as metabolites of Axinella weltneri from South Africa; the structure of sodwanone G 440 was determined by X-ray analysis.313 An Ircinia sp.from Okinawa has yielded an additional pentacyclic steroid xestobergsterol C 443.314 In addition the structures of xestobergsterols A 444 and B 445 were revised by inversion of the configuration at C-23 and the absolute configuration of xestobergsterol A 444 was detem~ined.~ l4 Incrustasterols A 446 and B 447 are new steroids from Dysidea incrustans from the Mediterranean.31 Syntheses of araguster- 01s A-D 448-451,which were isolated from an Okinawan Xestospongia SP.,~'~ the structural elucidations of (23R)- and (239-isocalysterols 452 and 453 from Calyx niceaensi~~'~ and C.p~datypa,~" respectively and (17R)-17-methylincisterol454 from Dictyonella incisa319 have been The Faulkner Marine natural products OH 443 R' = H; R2=OH 444 R' = R2= H 445 R'=R2=OH Pacific deep-water sponge Poecillastra laminaris contained annasterol sulfate 455,which inhibited glucanase Desulfation and acetylation of the polar steroids from Trachy- opsis halichondroides and Cymbastela coralliophila yielded four undescribed sterol triacetates but the structures of the natural products were not rigorously established.324 7 Coelenterates Terpenoids continue to comprise the majority of new metabo- lites from coelenterates although there are a few interesting exceptions.Four ceramides 456459 have been isolated from the gorgonian Acabaria undulata and their absolute stereo- chemistries were determined by synthesis.325 A brominated fatty acid of unknown geometry 6-bromo-5,9-eicosadienoic acid 460,was obtained from the phospholipids of both the anemone Condylactis gigantea and the zoanthid Palythoa ~aribaeorum.~~~ Two new prostanoids clavulactones I1 461 and I11 462 were obtained from the Okinawan soft coral Clavularia ~iridis.~~~ The stony coral Tubastrea faulkneri was reported to contain the sponge metabolite mycalolide C 463 and two new macrolides mycalolides D 464 and E 465,but this unusual chemotaxonomic relationship seems to be in need of confirmation.328 A new UV-absorbing mycosporine- like amino acid 466 was isolated from the reef-building corals Pocillopora damicornis and Stylophora pi~tillata.~~~ Mycosporin-gly 467 from the zoanthid Palythoa tuberc~losa~~~ has been synthesized from quinic Three sesquiterpene hydroquinones rietone 468 8'-acetoxyrietone 469 and 8'-desoxyrietone 470 that were obtained from the South African soft coral Alcyonium fauri showed mild cytotoxic In addition to known cur- cuquinone sesquiterpenes curcuhydroquinone l -monoacetate 471 was isolated from Pseudopterogorgia americana from Barbados.333 Curcudiol 472 and curcuphenol 473 which was isolated from the Caribbean gorgonian P.rigid^,^^^ have been synthesized both as racemates and in the optically Four new active forms ill~strated.~~~,~~~ sesquiterpenes (1S,3Z)-acetoxygermacra-3,10(15)-diene 474 (1 S,32,5E)- acetoxygermacra-3,5,10( 15)-triene 475 (1 S,4R,5S)-guia-6,9- dien-4-01 476 and (1 S,SR,6R)-guia-3,1 O( 15)-dien-7,11 -epoxy- 6P-acetate 477,together with the diterpenes nephthol-A 478 and nephthol-B 479,were isolated from an Indian specimen of Nephthea ~habroli.~~~ A further nardosinane sesquiterpene 12-hydroxynardosin- 1 (lo) 1 1 (13)-diene 480 was isolated from the Brazilian gorgonian Phyllogorgia dilatata.338 Two sesquit- erpene methyl ethers 481 and 482 were obtained from an Indian specimen of Sinularia maxima that had been extracted with A synthesis of (f)-erythrodiene 483 a sesquiterpene from Erythropodium ~aribaeorum,~~' has been described.341 Sinularia gibberosa from Taiwan contained a new cytotoxic cembranoid sinugibberol 484 the structure of which was determined by X-ray ~rystallography.~~~ Sandensolide 485 is a new dihydroxycembranolide from an Indian Ocean specimen of S.sandensi~.~~~ Sinulariadione 486 is an unusual 1,3-diketone from an unidentified Indian species of Sin~laria.~~~ A Philippines specimen of S. jlexibilis provided a new lactone 281 HO ‘OH HO OH 446 RdH HN rcl4H29 0 456 R = -CH=CHCgHiS (€) 457 R = -CH=CH-CH=CH-C7H15 (€,€) 458 R = -CH=CH(CH2)7CH(CH& (E) 459 R = -CtlH23 Br HOOC 460 Po4 OAc \ OAc \ C5H11 C5H11 461 462 sinulariolone 487 the structure of which was determined by X-ray ~rystallography.~~~ Three new ring-contracted cembranoids sarcotol 488 sarcotol acetate 489 and sarcotal acetate 490 were isolated from a Sarcophyton sp.from Japan.346 The structure of sarcotol 488 was determined by X-ray analysis of the corresponding 11-0-p-brom~benzoate.~~~ The Caribbean gorgonian Eunicea mam- mom has yielded a total of 18 cembranolides uprolide A acetate 491 8-epi-uprolide A acetate 492 uprolide B 493 8-epi-uprolide B 494 uprolide C 495 7-epi-uprolide C acetate 496 uprolide D 497 uprolide E acetate 498 uprolide F diacetate 499 uprolide G acetate 500 and various other acetate derivatives 501-508.347,348There have been a number of syntheses of cembranoids that illustrate methods of closing medium sized rings.349-352 282 Natural Product Reports OHC I Me0 OH 463 0 OMe 465 Five additional dolabellanes edunone 509 eduenone 510 edudione 511 edunol 512 and isoedunol 513 have been reported from the Caribbean gorgonian Eunicea la~iniata.~’~ The related gorgonian E.tourneforti contained five new diter- penes 514-518 four of which are do label lane^.^^^ The total 466 467 MeOOC OH 468 R=OH 469 R=OAc 470 R=H 471 472 473 -4 474 475 476 477 478 R=OH 480 479 R=OOH HO’ 481 482 483 synthesis of ( f)-~-neodolabellenol519 which was found in a soft coral of the genus Ce~pitularia,~~~ has been reported.356 Several diterpenes with very unusual carbon skeletons have recently been isolated.Isomandapamate 520 was isolated from Sinularia maxima from the Indian Ocean.339 The soft coral Sarcophyton solidum from the South China Sea contained the unusual diterpene sarsolenone 521 .357 Emmottene 522 from the gorgonian Briareum polyanthes has a carbon skeleton that has not previously been encountered in marine organisms.358 A new tricyclic carbon skeleton is found in yonarolide 523 from an Okinawan species of Sin~laria.~~~ A specimen of Sinularia dissecta from India contained a novel tetracyclic norditerpene dissectolide A 524.360 The gorgonian Pseudop-terogorgia acerosa from Tobago contained a new diterpene of the pseudopterane class 11-goriacerol 525.361 Kallolide B 526 which is a diterpene from the Caribbean gorgonian P.kallo~,~~~ has been synthesized in racemic form.363 Faulkner Marine natural products 484 485 OH 486 487 488 R =-CH20H 490 489 R =-CH~OAC 491 492 493 R’= R2=H 494 R’ = R2 = H 501 R’=H; R2=Ac 503 R’ =H; R2=Ac 502 R’ = R2= AC 504 R’ = R2= AC 495 R‘ = R2= H 496 R1= H; R2= AC 505 R’ = H; R2=AC 507 R’ = R2 = AC 506 R’ = R2=AC ‘0 497 R= H 498 508 R=Ac 499 R=Ac 500 R=Me 283 0 The eunicellin-type diterpene 527 which is toxic to fish and brine shrimp was isolated from the Mediterranean gorgonian Eunicella cav01ini.~-The structure and absolute stereochemistry of ( -)-7-deacetoxyalcyonin acetate 528 509 510 0 527 528 51 1 51 2 V 529 530 R=H 531 R=COC3H7 51 3 514 R'=OAc; R2=H 515 R1 =OH; R2 = AC U 532 533 51 6 :W:7H15c7H150c0@-0 0 -OH 534 535 518 519 HO HO- MeOOC A 536 .-H3::s"' 537 0 520 521 HO AcO OCOEt .CHO 0 0 522 523 538 539 R=H R2 AcO p -OHv0 0 541 R=OAc AcO OCOEt 524 525 R1 =OH; R2 = COOMe 540 R=OCOEt 0 0 526 R1 = H; R2 = Me 543 R=OAc 542 544 284 Natural Product Reports which was obtained from an Okinawan Cladiella SP.,~~' have been confirmed by total synthesis.366 A total of ten eunicellin- base diterpenoids briarellins A-I 529-537 and secobriarellin 538 have been described as minor metabolites of Briareum asbestinum from Puerto Rico.367,368 The Mediterranean sea pen Funiculina quadrangularis contained six briarane-type diterpenes funicolides A-E 539-543 and 7-epifunicolide A 544 some of which were shown to exist as mixtures of conformers.369,370 545 546 547 548 kH 549 550 551 R=OH 552 R=H HO/-.. -H MeO..lQ -H 0@ 554 555 R =OH 556 R=H 557 R=OMe -H / -H / -H / 0&&& 0 H 0 0 H 0 0 H 558 559 560 Faulkner Marine natural products Five diterpenes of the lobane class have been reported from Indian soft corals; Lobophytum hirsutum produced 13- hydroxyloba-8,10,15,17-tetraen-19-a1 545 and two diastereo- meric epoxides of 13,15-epoxyloba-8,10,16-trien-18-01 546 while L. pauclforum contained (1 3E 16E)- 18-methoxyloba- 8,10 13( 15),16-tetraene 547 and the oxidized derivative 548.371,372 Nine diterpenes of the xenicane class xeniatine A 549 xeniatine A epoxide 550 isoxeniatines A 551 and B 552 xeniaethers A 553 and B 554 and three probable artifacts the methoxyacetals 555-557 were isolated from a Japanese Xenia sp.373,374 An undescribed Xenia sp.from the Philippines con- tained the antibacterial diterpenes 9-deoxyxeniolide-A 558 and 9-deoxyxeniolide-B 559.375Deoxyxeniolide B 560 is an ichthyotoxic constituent of X. elongata from Japan.376 An investigation of two South African soft corals revealed that Anthelia glauca contained antheliatin 561 and Alcyonium 561 562 R= H 563 R=OH 0 @ o@ AcO Acd 564 565 AcO AcO 566 567 568 569 aureurn produced zahavins A 562 and B 563.377The South African soft coral Capnella thyrsoidea which occurs in two colour variants contained four xenicane diterpenes tsitsixen- icins A-D 564-567 together with two new pregnane sterols 568 and 569.378 Three aromatic secosteroids calicoferols C-E 570-572 one of which calicoferol D 571 exhibited potent antiviral activity were isolated from a Korean gorgonian of the genus Muri-cell^.^^^ Three new 9,ll-secosterols 573-575 that show moder- ate inhibitory activity against protein kinase C were isolated 0 577 HO 570 HO 571 HO 572 573 574 from a Pseudopterogorgia sp.from Florida using a bioassay- directed fractionation scheme.380 One of these secosterols 573 was subsequently reported as a metabolite of P. americana from Barbados.381 The structure of (22R,23R,24R)-22,23-rnethylene-23,24-dimethyl-9,11 p 1 1--secocholest-5-en-9-one-3 diol 576 from the Caribbean gorgonian P.hummelinkii was determined by X-ray ~rystallography.~~' Five new sterols 577-581 two of which 578 and 579 exhibited moderate cyto- toxicity and antiviral activity were isolated from a Korean specimen of Alcyonium gracillimum. 383 A. patagonicum from 286 Natural Product Reports 04 ) 578 4,5-dihydro 579 7 h OH 582 the South China sea yielded the cytotoxic sterol 24-methylenecholest-4-ene-3p,6p-diol 582.384 Stolonolides I 583 and I1 584 are interesting 1,lO-secosterols from the Okinawan soft coral Clavularia viridi~.'*~ Two new sterol peroxides 585 and 586 were isolated from the Indian soft coral Sinularia maxima386 and (24S)-3p 16p-dihydroxy-24- methylcholest-5-ene 587 was obtained from Cladiella krempfi from Minicoy Island in the Lakshadweep 4-Dehydroecdysterone 588 was isolated from the zoanthid Parazoanthus sp.from Australia.388 8 Bryozoans The bryozoan Amathia convoluta from the Gulf of Mexico coast of Florida contained convolutamydines A-D 589-592 that exhibit potent activity in the differentiation of HL-60 cells.3893390 Debromoflustramides B 593 and E 594 and debromoflustramines B 595 and E 596 from Flustra foliacea have been synthesized as their race mate^.^^^ Murrayanolide 597 is an unusual terpenoid from Dendrobeania murrayana from the Atlantic coast of Canada.392 The major cytotoxic component of Bugula neritina from Japan is bryostatin 10 598.393 9 Molluscs Although there is still considerable interest in the defensive roles of metabolites of marine molluscs recent studies have 0 Br H L 589 R = -CH2COCH3 593 0 590 R = -CH2CH&I 583 584 22,23-dihydro 591 R=-CH3 592 R = -CH=CH2 R k 585 R' = Ac; R2 = Me 586 R' =R2=H 594 595 I Y e0 CH20Ac H Me HO 587 596 597 HO oH 588 \COOMe also stressed the potential biomedical uses of these com-598 pounds.The structure of aplyparvunin 599 which is an ichthyotoxic acetogenin from Aplysia parvula was determined by single crystal X-ray analysis.394 A synthetic route to both HO Br (32)-and (3E)-dactylomelynes 600 and 601 which are metabo- lites of A. da~tylomela,~~~ has been communicated.396 Three additional metabolites of A. dactylomela (+)-dactyl01 602,397 dactyloxene-B 603398 and dactyloxene-C 604,399 have also been synthesized in an enantioselective manner.400,401 The absolute stereostructures of aplyronines B 605 and C 606 which were isolated from A.k~rodai,~'~ have been determined by total synthesi~.~'~ Elucidation of the stereochemistry of dolabelides A 607 and B 608 which are cytotoxic macrolides from a Japanese speci- men of Dolabella auricularia required a complex chemical degradati~n.~'~ The same animals also yielded dolabellin 609 which is a cytotoxic bis-thia~ole,~'~ and a cyclic hexapeptide dolastatin E 610.406 The stereochemistry of dolastatin E 610 was determined by chemical degradation and total synthesis.407 Chemical studies of the Atlantic nudibranchs Cadlina laevis 602 603 604 and C.pellucida demonstrated the dietary relationship between C. pelhcidu and the sponge Spongia agaricina and resulted in the isolation of one new sesquiterpene laevidiene 611 from C. laevi~.~'~ (+)-Albicanol612 and the corresponding acetate 613 isomeric labdane aldehydes 614 and 615 were isolated from from Cadlina l~teornarginata~'~ are among several drimane the skin of the Mediterranean notaspidean mollusc Pleuro-sesquiterpenes whose syntheses have been rep~rted.~",~~ The glyceryl ester of a halimane Two branchaea me~kelii.~~ Faulkner Marine natural products 287 0 OH 605 R = COCH(NMe2)CH20Me 606 R=H CI CI i( 609 614 615 OLOH 616 'V \OAc 617 R=Ac 618 R=H diterpenoic acid austrodorin 616 which was identified after acetylation was found in the dorsal mantle of Austrodoris kerg~elenensis.~~~ The structures of tanyolides A 617 and B 618 from the dorsal mantle of Sclerodoris tanya from La Jolla were determined by spectral analysis and the total synthesis of both enantiomers of tanyolide B 6X414 Both the natural and unnatural enantiomers of 618 were effective fish feeding inhibitors when tested in an appropriate assay.414 Four unusual chlorinated homo-diterpenes hamiltonins A-D 619-622 and the sesterterpene hamiltonin E 623 were isolated from the South African nudibranch Chromodoris hamilt~ni.~~ The North Sea nudibranch Adalaria loveni con-tained a degraded triterpenoid lovenone 624 as its cytotoxic c~nstituent.~~' Pteroenone 625 is a defensive metabolite of the Antarctic pteropod Clione antactica which is abducted by the amphipod Hyperiella dilatata and positioned such as to prevent predation by fi~hes.~~~,~~' The structure of onchitriol I which was obtained from Onchidium has been revised from 626 to 627 as a result of total Syntheses of isopulo'upone 628 from Navanax inermis and Bulla go~ddiana,~~~ and hami-nols A 629 and B 630 from Haminoea have been rep~rted.~~~,~~' 288 Natural Product Reports 6R 607 R=Ac 608 R=H 610 611 \ 612 R=H 613 R=Ac 619 620 0 621 622 0 // 623 HO 624 One of the toxic constituents from the viscera of the Okinawan bivalve Pinna muricata has been identified as the calcium channel activator pinnatoxin A 631.426Two some-what similar macrolides spirolides B 632 and D 633 were isolated from the digestive glands of both the mussel Mytilus edilis and the scallop Placopecten magellani~us.~~~ During studies of the causes of shellfish poisoning in New Zealand it was shown that the cockle Austrovenus stutchburyi contained brevetoxin B 634 and the greenshell mussel Perna canaliculus contained brevetoxin B 635 both of which had not been described previo~sly.~~~,~~~ 10 Tunicates Tunicates (ascidians) continue to be a fertile source of new biologically active marine natural products.Four ";'2 W R H2N 'R OH OH 625 636 R = -(CH&CH=CH2 638 R = -(CH&CH=CH;! 637 R = -C*H17 639 R =-C8Hi7 0 0 626 0 0 627 0-628 OR 629 R=H di~torna.~~' The absolute stereochemistry at C-4 and C-5 of 630 R=Ac pseudodistomin C 640 which is a piperidine alkaloid from the Okinawan tunicate P.kanoko is opposite that reported for earlier members of this The structure of pseudodis-antimicrobial amines 63G39 isolated as their acetyl deriva- tomin A was revised from 641 to 642 as a result of chemical tives were obtained from a South African species of Pseudo-degradation.432 Four cytotoxic alkaloids lepadins B 643 and C 631 632 R=H 633 R=Me COOH Faulkner Marine natural products 654 644 and villatamines A 645 and B 646 were discovered in both Clavelina lepadiformis and the flatworm Prostheceraeus villatus that consumes it.433 Four additional alkaloids cylindricines H-K 647-650 have been isolated from Claveiina cylindrica from Tasmania.434 Piclavine A 651 which is an antimicrobial constituent of C.pi~ta,~~~ has been synthesized in an enantio- specific manner.436 The absolute stereochemistry of the cyto- toxins didemnenones C 652 and D 653 from Didemnurn voeltzkow~~~~ have been determined by total synthesis.438 RH2C k 647 R=SCN 649 648 R=NCS CH2CI 650 651 II LOH bOH &OH -OH '-OH 652 653 Didemnun (=Didemnum) rodriguesi from New Caledonia contained the unusual peptidyl alkaloid caledonin 654 that complexed Zn2' and Cu' ions between the thiol and primary amine Patellamide F 655 is an additional cytotoxic cyclic peptide from Lissoclinum patella from north-western Australia.440 The solution conformations of patellamides B 656 and C 657 have been determined by NMR spectroscopy and molecular dynamics.44' A total synthesis of lissoclinamide 4 658 which was isolated from L.patella,442,u3 has been des~ribed.~~ Cyclodidemnamide 659 is a weakly cytotoxic cyclic heptapeptide from Didemnum molle from the phi lip pine^.^^ Seven additional cyclic peptides didemnins M 660 N 661 X 662 and Y 663 nordidemnin N 664 epididemnin A 665 and acyclodidemnin A 666 have been reported from the Caribbean tunicate Trididemnum solidum.u6 290 Natural Product Reports 656 R = -CH*CH(CH3)2 657 R = -CH(CH& A 658 659 [Tyr'IDidemnin B (=didemnin N) 661 and [~-Pro~]didemnin B 667 were isolated from Trididemnum cyanophorum from Guadeloupe Island.447 Three additional antimicrobial polysulfides 668-670 of the varicin family have been isolated from a Polycitor sp.collected by dredging in the Sea of Japan.448 A new synthesis has of varacin 671 from Lissoclinum va~eau~~~been described.450 Lukaniol A 672 from an unidentified ascidian from Palmyra atoll,451 and polycitrin A 673 from a Polycitor sp. ,452 have both been synthe~ized.~~~.~~~ The P-carboline dimer 674 previously known only as a synthetic product was isolated from a Great Barrier Reef Didemnum sp.454 A Didemnum sp. from Rota in the Northern Mariana Islands contained four new P-carbolines didemnolines A-D 675-678 together with known ascidian and sponge metabolites.455 The structure of isoeudistomin U which is a metabolite of Lisso-clinum fragile,456 has been revised as a result of total synthesis from 679 to 680.457The structure of eudistomin U 681 also reported from L.fragile,456 has been confirmed by total synthesi~.~~~,~~' Both grossularine-1 682 and grossularine-2 683 which are alkaloids from Dendrodoa gros~ularia,~~~ have been ~ynthesized,~~~,~~~ as have a number of pyridoacridine alkaloids from as~idians.~~~,~~ Botryllamides A-D 684687 are brominated tyrosine deriva- tives from specimens of Botryllus sp. from Siquijor Island Philippines and B. schlosseri from the Great Barrier Reef.465 An Aplidium sp. from southern Australia contained aplidites A-G 688-694.466The proposed structures of aplidites A-G 688-694 which incorporate the very unusual orthonitrite moiety have been questioned.A reinvestigation of a Didem-num sp. from pa la^^^^ revealed that didemnaketals A 695 and B 696 were oxidation or hydrolysis products of the true natural product didemnaketal C 697.468Twelve additional cytotoxic steroidal alkaloids ritterazines B-M 698-709 have been Gq;.0R3 OI 0 OH Hydec = 660 R1 = R2 = R3 = Me; R4 = Gln-pGlu 661 R1 = Me; R2 = R3 = R4 = H 662 R' = R2= R3 = Me; R4 = Gln-Gln-Gln-Hydec 663 R1 = R2 = R3 = Me; R4 = Gln-Gln-Gin-Gln-Hydec 664 R' = R2 = R3 = R4 = H G%;ooMe 0 COOH MeoqL: OMe 0 674 675 X=Br Meoq;s S/s 676 X = H NH2 NH2 668 669 OMe 677 X=Br 679 s H 0 678 X=H MeoTs NH2 NMe2 670 HO 672 Br OMe 680 3,4-dihydro 682 681 NMe2 I HO' N4 NH2 671 Br 673 Faulkner Marine natural products 29 1 R 684 R=Br 686 R=H 688 R1=R2=H; EorZ 689 R1= R2 = H; Zor E 690 R' =OH; R2 = H; EorZ.691 R' =OH; R2= H; Zor E 692 R' = OH; R2 = Ac; €or Z 693 R' = OH; R2 = Ac; Zor E 694 R' = OAc; R2 = H; Zor E HO-I' H HO-700 R=H 701 R=Me 707 R' = H; R2 = OH; 22R 708 R' = R2 = H; 22R 709 R' = R2= H; 225 OH I R 685 R = Br 687 R=H A I l& l \ )'o I- HOAR 695 R =-COMe 696 R=# 698 R' =OH; R2 = H 704 R',R2=O c'"". H0-d" 699 .OH HO 703 R' =OH; R2 = H; A14 705 R',R2=0; R3= OH 292 Natural Product Reports 712 R'=H; R2=OH 713 R' =OH; R2 = H HO 710 major homologue ,-- 714 F N H 2 HOVN-NH2 0 715 71 1 major homologue OS03Na OS03Na 1 HO NaO3SO'' OH 716 Na03SO' 71 7 71 8 OS03Na HO> NaO3SO" w 71 9 OH 720 HO OH 721 OS03Na 722 723 OH 724 R = H 725 R = Me ?H % (\OS03Na <0s03Na 730 727 729 reported from Ritterella tokioka and the structure of ritterazine K 707 has been confirmed by 11 Echinoderms Two gangliosides LG-1 710 and LG-2 711 which occur as mixtures with various alkyl chains were isolated from the Japanese seastar Astropecten latespinosus and it was shown that LG-2 711 was cytot~xic.~'~ The major constituent of LG-2 71 1 was identified as its permethylated derivative LG-2M-5.473 One of the more interesting reports is that the seastars Fromia monilis and Celerina heflernani from New Caledonia contained relatively small quantities of the known sponge metabolites ptilomycalin A 290474and crambescidin 800 712,475together with new compounds celeromycalin 713 fromiamycalin 714 and the alcohol 715 although it seems reasonable to question the biosynthetic origin of these metabo- lite~.~~~ Eight known and four new sulfated polyhydroxy- steroids 716719 have been isolated from various ophiuroids as part of a comparative study of Ophiocomina nigra Ophiotrix fragilis Ophiuva texturata Ophiocoma echinata 0.scolopend-rina 0.wendti Ophionereis reticulata Ophiozona impressa and Ophionotus victori~e.~~~ Together with known metabolites one new steroidal sulfate Sa-cholestane-3P,5,6P 15a,26-pentol 26-sulfate 720 has been isolated from the seastar Luidia ludwigi from the Patagonian Ten new polyhydroxy- steroids 721-730 and several known polyhydroxysteroids and saponins were isolated from Luidia clathrata from the Gulf of Mexico.479 A reinvestigation of the Caribbean seastar Oreaster reticulatus resulted in the identification of one new polyhydroxysteroid 731 eleven steroidal oligoglycosides oreasterosides A-K 732-742 and two asterosaponins reticul- atosides A 743 and B 744.480Downeyosides A 745 and B 746 OH OS03Na 731 OR2 OH a R3=Me e R3=H OH OR1 732 R1= H; R2 =a 733 R1= H; R2= b 734 R' = H; R2= c 735 R1 = H; R2= d 736 R1= S03Na; R2= a ~40 OH 737 R1= S03Na; R2 = e b R3=Me; R4=H c R3=H; R4=Me d R3=R4=Me are sulfated steroid glucuronides from Henricia downeyae from the Gulf of The sea cucumber Holothuria scabra from India contained the new steroidal glycoside 747 and some known triterpene~.~~~ 294 Natural Product Reports OH 738 Ho-d-p"e 0 I HO \ OR 739 R=S03Na 740 R=H %..A 0'.OqoH 'OMe -OS03Na 741 HOWoH 'OH OH OS03Na A 742 12 Miscellaneous Four additional cytotoxic steroidal dimers cephalostatins 12 748 and 13 749 which were inadvertently omitted from the previous review,'* and 16 750 and 17 751 have been obtained in very small quantities from the South African tube worm Cephalodiscus gil~hristi.~~~,~~~ Cephalostatins 7 752485and 12 748 have been synthesized in a biomimetic manner.471 Eight additional lumazine derivatives 753-760 have been reported from the bioluminescent polychaete worm Odontosyllis ~ndecimdonta.~~~ The structure of a neurotoxic agent isolated from a hoplonemertine worm Aphiporus angulatus in 1976487 has been revised from 761 to 762 as a result of total syn- thesis.488,489Both enantiomers of bromoxone 763 which is a metabolite of the acorn worm Ptychodera SP.,~~' have been synthesi~ed.~~' The flatworm Prostheceraeus villatus con-tains alkaloids 64M46 obtained from the tunicate Clavelina lepudif0 rm is.433 The Japanese puffer fish Fugu poecilonotus contained two new tetrodotoxin derivatives 5,6,11 -trideoxytetrodotoxin 764 and its 4-epimer 765 which may be of biosynthetic signifi- ~ance.~~~ The (24R)-stereochemistry of squalamine 766 which is an antimicrobial and antiviral agent from the dogfish shark Squalus ac~nthius,~~~ has been established by synthesis of squalamine de~sulfate.~~~ A synthesis of 245-squalamine has been Some remarkable NMR spectra that were R3 R2N%>+ MeN%N;F OAN R' OAN Me N OH 744 24,25-dihydro HO..761 762 OH OS03Na 745 R1 = Me; R2 = H 0 0 746 R1= H; R2 = Me ooBr HN OH R2 763 764 R' = H; R2 =OH 765 R'=OH; R2=H HO 747 OH 748 R=H 750 749 R=OH - HO 'OH 751 Faulkner Marine natural products 295 obtained on < 100 pg of compound using micro-inverse probes and micro-cells have shown that the Caribbean ciguatoxin is similar in nature but different in structure to the Pacific cigua toxin.496 13 References 1 D. 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ISSN:0265-0568    

DOI:10.1039/NP9971400259

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

Amaryllidacae alkaloids John R. Lewis Department of Chemistry Aberdeen University Old Aberdeen UK AB9 2 UE Covering 1995 Previous review 1996 13 171 1 Introduction 2 Occurrence and structural studies 3 Synthetic studies 4 References 1 Introduction In this annual review 23 new alkaloids have been reported as well as known alkaloids that have been found in new sources. Table 1 lists those alkaloids that are reported upon in this review. The identity of the alkaloidal contents of a plant not only has important chemical implications but taxonomic deductions can also be implied. The identification of new skeletal arrangements which are often associated with specific pharmacological properties can lead to a deeper understand- ing of structure-activity relationships with the development of the synthesis of simpler analogues suitable for clinical evalu- ation.Alkaloids and other naturally occurring compounds can provide useful information on taxonomic classification; thus Narcissus primigenius has been classified as a new taxon of the Narcissus pseudonarcissus DC section as a result of the four alkaloids homolycorine 1 8-O-demethylhomolycorine 2 haemanthamine 3 and 8-O-demethylmaritidine 4 which were found in extracts of the whole plant.' 0 1 Homolycorine R1= R2 = R4 = Me; R3 = H 2 8-ODemethylhomolycorine R1 = R3 = H; R2 = R4= Me 37 9-0-Demethylhomolycorine R1 = R4 = Me; R2 = R3 = H 41 5-Methoxy-9-O.demethylhomolycorine R1 = R4 = Me; R2 = H; R3 = OMe 48 Hippeastrine R1R2= CH2; R3 = OH; R4= Me 63 Mansonine R1R2= CH2; R3 = H; R4 = Me 68 N-Demethylmansonine R1R2= CH2; R3 = R4 = H Lewis Arnavyllidacae alkaloids Microanalytical techniques for the analysis of the alkaloidal content of a plant depends a great deal on their stability under gas chromatography conditions.A new study2 using capillary gas chromatography coupled with mass spectrometry has shown that many underivatised Amaryllidaceae alkaloids were stable under the conditions employed. Thus an extract of the bulbs of Narcissus pseudonarcissus sub-sp. pseudonarcissus was shown to contain 30 alkaloids using direct insertion mass spectrometry. If gas chromatography preceded mass measure- ment only two alkaloids haemanthamine 3 and lycorine 5 R3 5 Lycorine R1R2= CH20; R3 = R4 = OH 22 Sternbergine R1 = Me; R2 = R4 = OH; R3 = OAc 55 Pseudolycorine R1 = H; R2 = R4 = OH; R3 = OMe 57 10-Norpluviine R1 = Me; R2 = R3 = OH R4 = H 58 1-0Acetyl-1 O-norpluviine R1 = Me; R2 = R3 = OH; R4 = OAc 59 1,lO-Diacetyl-10-norpluviine R1 = Me; R2 = R4 = OAc; R3 = OH 60 9-Norpluviine R1 = H; R2 = OMe; R3 = OH; R4 = H were found to partly decompose thermally in the column.This method was however found to be better than if the alkaloids were derivatised prior to gas chromatography. 2 Occurrence and structural studies Pancratistatin 6 has been found to exhibit a highly character- istic differential cytotoxicity profile against 60 human cancer OH R~OH 0 6 Pancratistatin R = OH 9 7-Deoxy-trans-dihydronarciclasineR = H lines.3 Unfortunately an efficient synthetic route has not been established and so an investigation into its production within the plant has taken place.4 Monthly analyses of the bulbs of cloned plants from Hymenocallis littoralis showed that the alkaloid 6 content varied when these plants were grown in an irrigated semi-arid Arizona desert location.Normally Hymenocallis littoralis is found in tropical or sub-tropical regions. Highest yields of the alkaloid were obtained in October after the plant had undergone six months of growth in the period following flowering. On average a yield of 0.015% of pancratistatin 6 was achieved; in addition narciclasine 7 Table 1 Isolation of Amaryllidaceae alkaloids Table 1 Continued Species Alkaloid (structure) Ref.Species Alkaloid (structure) Ref. Brunsvigia gregaria Crinamidine 10 7 Narcissus primigenius Homolycorine 1 1 Crinine 11 (whole plants) 8-O-Demethylhomolycorine2 Flexinine 12 Haemanthamine 3 Haemanthidine 13 8-O-Demethylmaritidine 4 Lycoramine 14 Narcissus pseudonarcissus Lycorine 5 2 8-Methylenedioxyphenthridine 15 (bulbs) 29 other alkaloids identified Tazettine 16 by GC-MS Undulatine 17 O-Acetylgalanthamine* 56 Brunsvigia josephinae 3-O-Ace t ylhamayne 18 8 10-Norpluviine* 57 Hamayne 19 1-0-Acetyl-lO-norpluviine*58 Crinamine 20 1,l O-Diacetyl- 10-norpluviine* 59 Ambelline 21 9-Norpluviine 60 Sternbergine 22 Narcissus pseudonarcissus Lycorenine 61 19 1 1-0-Acetylambelline* 23 subsp. pseudonarcissus Oduline 62 Brunsbelline* 24 Hippeastrine 48 Crinine 11 Mansonine 63 Buphanisine 25 Homolycorine 1 Buphanidrine 26 Haemanthamine 3 Undulatine 17 Vittatine 64 Hippadine 27 Galanthamine 30 Crinium kirkii Kirkine* 28 9 N-Demethylgalanthamine 35 (bulbs) 8-O-Demethylvasconine* 29 Lycoramine 14 Crinine 11 Epi-N-demethyllycoramine 65 Hamayne 19 Narwedine 34 3-O-Acetylhamayne 18 O-Methyllycorenine 66 Galanthus elewesii ( -)-Galanthamine 30 O-Methyloduline* 67 (whole plants) ( -)-Sanguinine 31 N-Demethylmansonine* 68 ( -)-Leucotamhe 32 Narcissus tortuous Lycorine 5 20 ( -)-O-Methylleucotamine 33 (whole plants) Tortushe* 69 ( f)-Narwedine 34 Vasconine 70 ( -)-N-Demethylgalanthamine 35 Pancratinum maritimum ( -)-Lycorine 5 21 (+)-1 l-Hydroxyvittatine 36 (bulbs) (+)-O-Demethylhomolycorine 37 (+)-9-O-Demethylhomolycorine 37 Trisphaeridine 15 ( -)-Lycorine 5 (+)-Haemanthamine 3 ( -)-Galanthine 38 (+)-Buphanisine 25 Hordenine 39 ( -)-Crinine 11 (E)-N-Feruloylytramine 40 ( -)-3P-1 la-Dihydroxy-1,2- (+)-5-Methoxy-9-O-dehydrocrinane 71 demethylhomolycorine* 41 ( -)-8-Hydroxy-9-(+)-Galwesine* 42 methoxycrinane 72 (+)-9-O-Demethylgalwesine*43 ( -)-3P-Methoxy-6a,lI P-dihydroxy- (+)-16-Hydroxygalwesine* 44 1,2-dehydrocrinane 73 (+)-16-Hydroxy-9-O-* New alkaloids demethylgalwesine* 45 Galasine* 46 Hippeastrum equestre Phamine* 47 12 (bulbs) Lycorine 5 Tazettine 16 Hippeastrine 48 N-Methylcrinasiadine 49 13 8,9-Methylendioxyphenanthridine15 Hymenocallis expansa Pancratistatin 6 14 (whole plant) Narciclasin 7 Hymenocallis littoralis Pancratistatin 6 14 RO (whole plant) 7 Narciclasine R =OH Hymeniocallis Pancratistatin 6 14 8 7-Deoxynarciclasine R = H maritimum Hymenocallis sonorensis Pancratistatine 6 14 7-deoxynarciclasine 8 and 7-deoxy-trans-dihydronarciclasine9 (whole plant) Hymenocallis speciosa Pancratistatin 6 14 were also produced.An extraction-separation procedure was (whole plant) consequently developed to allow a scale up of production of Leucojium autumnule Narcissidine 50 15 the alkaloids. (bulbs) Lycorine 5 An alkaloid extract obtained from the bulbs of Haemanthus 3-O-Acetylnarcissidine* 5 1 albijlos was reported5 to have an antiviral effect on RNA 3-O-Acetylnarcissidine N-oxide* 52 viruses. Further studies have now indicated that its activity is Lycoris sunguinea Norsanguinine* 53 (bulbs) Norbutsanguinine* 54 involved at an early stage in the virus’ development.6 The Sanguinine 31 identity of the active agent(s) remain unknown.Lycorine 5 Crinamidine 10 crinine 11 flexinine 12 haemanthidine 13 Pseudolycorine 55 lycoramine 14 8,9-me thylenedioxyphenan thridine 15 taze ttine Galanthamine 30 16 and undulatine 17 were all isolated from Brunsvigia Galanthine 38 greguria. Confirmation of their identities was made by their 304 Natural Product Reports R 0av R6 \ R20 II R3 R4 11 Crinine R1R2 = CH,; R3 = R4 = R6 = H; R5 = a-OH 13 Haemanthidine R1R2= CH2; R3 = H; R4 = R6 = 0-OH; R5 = a-OMe 21 Ambelline R1R2 = CH2; R3 = OMe; R4= H; R5 = a-OMe; R6 = a-OH 23 1 1 -0-Acetylambelline R1 R2 = CH2; R3 = OMe; R4 = H; R5 = a-OMe; R6 = u-OAC 24 Brunsbelline R1R2= CH2; R3 = OMe; R4 = H; R5 = P-OMe; R6 = P-OH 25 Buphanisine R1R2= CH2; R3 = R4 = R6 = H; R5 = P-OMe 26 Buphanidrine R1R2= CH2; R3 = OMe; R4 = R6 = H; R5 = a-OMe 71 (-)-3P 11a-dihydroxy-l,2dehydrocrinane R1R2= CH2; R3 = H; R4 = H; R5 = P-OH; R6 = a-OH 72 (-)-8-Hydroxy-9-methoxycrinane R1 = Me; R2 = R3 = R4 = R5 = R6 = H 73 (-)-3P-Methoxy-Ga,ll j3-dihydroxy-l,2-dehydrocrinane R1R2= CH2; R3 = H; R4 = a-OH; R5 = P-OMe; R6 = P-OH Xb 14 Lycoramine R1 = lone pair; R2 = R5 = Me; R3 = H; R4 = OH; ab sat.30 Galanthamine R1 = lone pair; R2 = R5 = Me; R3 = H; R4 = OH; ab unsat. 31 Sanguinine R1 = lone pair; R2 = Me; R3 = R5 = H; R4 = OH; ab unsat.32 Leucotamine R1 = lone pair; R2 = Me; R3 = R5 = H; R4= OCOCH2CH(OH)Me; ab unsat. 33 OMethylleucotamine R1= lone pair; R* = R5 = Me; R3 = H; R4= OCOCH&H(OH)Me; ab unsat. 34 Narwedine R1 = lone pair; R2 = R5 = Me; R3 = R4 = 0; ab unsat. 35 N-DemethylgalanthamineR1 = lone pair; R2 = R3 = H; R4 = OH; R5 = Me; ab unsat. 53 Norsanguinine R1 = lone pair; R2 = R3 = R4 = R5 = H; ab unsat. 54 Norbutsanguinine R1 = lone pair; R2 = R4 = R5 = H; R3 = OCOCH2CH(OH)Me; ab unsat. 56 OAcetylgalanthamine R1 = lone pair; R2 = R5 = Me; R3 = H; R4 = OAc; ab unsat. 65 Epi-N-demethyllycoramineR1 = lone pair; R2 = R4 = H; R3 = OH; R5 = Me; ab unsat. chromatographic behaviour and UV ~pectra.~ An original in- vestigation into the alkaloids present in the bulbs of Brunsvigia josephinae established the presence of 3-0-acetylhamayne 18 Lewis Amaryllidacae alkaloids (Om 0 /N 15 8,9-Methylenedioxyphenanthridine (trispharidine) hamayne 19 crinamine 20 ambelline 21 and sternbergine 22.A re-investigation of this plant bulb extract’ has produced two new alkaloids 1 1-0-acetylambelline 23 and brunsbelline 24. Also present were crinine 11 buphanisine 25 buphanidrine 26 undulatine 17 and hippadine 27. OMe I 16 Tazettine 27 Hippadine 28 Kirkine R2 A 29 8-ODemethylvasconine R1 = R2 = H 69 Tortusine R1 = Me; R2= OMe 70 Vasconine Ri =Me; R2=H Two new alkaloids have been characterised as being present in the bulbs of Crinurn kirkii.’ Both belong to the crinan group and are named kirkine 28 and 8-0-demethylvasconine 29.Also found in this extract were crinine 11 hamayne 19 and 3-0-acetylhamayne 18. Six new lycorenine-type alkaloids have been isolated from whole plant extracts of Galanthus elwesii.Also present were the 12 known alkaloids ( -)-galanthamine 30 ( -)-sanguinine 31 ( -)-leucotamine 32 ( -)-0-methyleucotamine 33 ( f)-narwedine 34 ( -)-N-demethylgalanthamine 35 ( f)-1I -hydroxyvittatine 36 (+)-9-O-demethylhomolycorine 37 ( -)-lycorine 5 ( -)-galanthine 38 hordenine 39 and (Q-N-feruloyltyramine 40. Galanthamine 30 and lycorine 5 had previously been found in this plant. The new alkaloids were (+)-5-methoxy-9-0-demethylhomolycorine 41 (+)-galwesine 42 (+)-9-O-demethylgalwesine 43 (+)-16-hydroxygalwesine 44 (+)-16-hydroxy-9-0-demethylgalwesine 45 and galasine 46.All compounds were characterised by spectroscopic analysis and in the cases of 33 43 and 46 confirmation was made by X-ray crystallography. Galasine 46 is the first spirocyclic lycoreine-type ring system to be reported to date. Five seem- ingly different colourless crystalline samples were obtained from five different subfractions of the alkaloidal extract obtained from the whole plant Galanthus elwesii. ’ ’ All were shown to be (+)-9-O-demethylhomolycorine 37 having identi- cal CD UV ‘H and 13C NMR spectral data. They were chromatographically identical but differed in melting point HO.. r N M e 2 M e Me0 \ O m v HO \ 38 Galanthine 39 Hordenine HOW U -O H OMe 40 (€)-N-Feruloyltyramine Mea 0 42 Galwesine Ri = Me; R2 = H 43 9-ODemethylgalwesine R1 = R2 = H 44 (+)-16-HydroxygaIwesineR1 = Me; R2 = OH 45 (+)-16-Hydroxy-9-Odernethylgalwesine R1 = H; R2 = OH Med !/? 0 0 46 Galasine 47 Phamine 0 49 N-Methylcrinasiadine and in optical activity.X-Ray data were obtained from four of these samples and it was seen that solvent molecules were or were not packed into the crystal and that the N-methyl- conformation was either axial or equatorial and this depended upon the conditions of crystallisation. Phamine 47 is a new phenanthridone alkaloid isolated from the bulbs of the Vietnamese plant Hippeastrum equestre.12 Also present were lycorine 5 tazettine 16 and hippeastrine 48. In a second publication by the same group N-methylcrinasiadine 49 and 8,9-methylendioxyphenanthridine15 were extracted and identified as alkaloidal constituents of the bulbs of Hip-peastrum equestre.’ The newly reported alkaloidal constituent phamine (loc cit) was also present. Following on from the earlier investigations on Hymeno-callis littoralis4 (loc cit) coupled to the efficacy of pancratistatin 6 as an antitumour agent the Pettit group has initiated a survey of the alkaloidal content of bulbs of the genus Hymenocallis to ascertain the extent of the occurrence of the active alkaloid 6 and its related isocarbostyrils narciclasine 7 7-deoxynarciclasine 8 and 7-deoxy-trans-dihydronarciclasine 9.l4 Hymenocallis speciosa from Singapore contained 6 and Hymenocallis expansa from Bermuda contained 6 and 7 while 306 Natural Product Reports Hymenocallis sonorensis from Mexico a variegated variety of Hymenocallis from Singapore Hymenocallis pedalis from the Seychelles and Pancratium maritimum from Israel all con- tained only 6.7-Deoxynarciclasine 8 was found in the Ismene variety ‘Sulphur Queen’ from the Netherlands but the variety Ismene ‘Advance’ did not contain any of the sought after alkaloids neither did a ‘Tropical Giant’ variety of Hymeno-callis from Singap~re.’~ Fresh bulbs of Leucojium autumnale contain four alkaloids narcissidine 50 lycorine 5 and hitherto unreported 3-0-acetylnarcissidine 51 and 3-0-OMe HO..A,.OR~ MeOa% \ Me0 I R2 50 Narcissidine Ri = H; R2 = lone pair 51 3-OAcetylnarcissidine R1 = Ac; R2 = lone pair 52 3-OAcetylnarcissidine-N-oxide R1 = Ac; R2 = 0 acetylnarcissidine N-oxide 52.Seven alkaloids have been found to be constituents of the bulbs of Lycoris sanguinea;16 two are new namely norsanguinine 53 and norbutsanguinine 54; the others were sanguinine 31 lycorine 5 pseudolycorine 55 galanthamine 30 and galanthine 38. The identities of four alkaloids obtained from the bulbs of the daffodil Narcissus pseudonarcissus were determined by GC-MS.I7 They are the known 0-acetylgalanthamine 56 which is reported here as a natural product for the first time 10-norpluviine 57 1-0-acetyl- 10-norpluviine 58 and 1,lO-diacetyl-10-norpluviine 59. A synthesis of 56 and 59 was accomplished by acetylation of galanthamine 30 and 10-norpluviine 57 respectively.This investigation enabled the alkaloid that was previously called 1O-norpluviine’8 to be redesignated as 9-norpluviine 60. The large cultivated daffodil Narcissus pseudonarcissus sub-sp. pseudonarcissus variety ‘Carlton’ has only been exam- ined once previously and narciclasine 7 was identified as a constituent. The new investigation” of the bulbs has resulted in the isolation of 15 alkaloids namely lycorenine 61 RIOW \ R20 R3 61 Lycorenine R1 = R2 = Me; R3 = P-OH 62 Oduline RiR2 = CH2; R3 = a-OH 66 OMethyllycorenine R1 = R2 = Me; R3 = p-OMe 67 OMethyloduline RiR2 = CH2; R3 = a-OMe oduline 62 hippeastrine 48 mansonine 63 homolycorine 1 haemanthamine 3 vittatine 64 galanthamine 30 N-demethylgalanthamine 35 lycoramine 14 epi-N-demethyllycoramine 65 narwedine 34 and 0-methyllycorenine 66.The two new compounds were 0-methyloduline 67 and N-demethylmansonine 68. Whole plants of Narcissus tortuosus contain two alkaloids lycorine 5 and a new anhydrolycorinium salt tortusine 69 whose structure was deduced by comparison with related vasconine 70.*’ R R I +-ifoH -N- \I CHO Me0v A\ OH Me0 74 R=H 76 R=Br iL75 R=Br iiiE77 R=H 1 resolution 30 (-)-Galanthamine Scheme 1 Reagents i Br, CHCl, MeOH room temp.; ii K,Fe (CN), H,O NaHCO, 60 "C then 75 stir 2 h; iii Zn (active) EtOH reflux 15 h H3$ i ii iii -OJO 78 79 80 J iv HH N' 'SnBu3 83 74% 82 15% 81 1 I vi vii-ix t c Me HOJ 0 84 (-)-Anabiline 85 (-)-Augustamine Scheme 2 Reagents i 3,4-methylenedioxy-4-bromobenzene, Bu"Li; ii CH,-CPPh, THF; iii Tf,O py C,H,,N=CHCH,Li dil.acid; iv Bu,SnCH,NH (1 equiv.) 4 8 mol. sieve Et,O; v BuLi (1.9 equiv.) THF -78 "C then H,O; vi Me,N'=CH,I- MeCN heat then HC1 MeOH; vii BuLi ( 1.9 equiv.) THF -78 "C then Me1 (1.9 equiv.); viii conc. HCl MeOH then CH(OM,),; ix MeS0,H (10 equiv.) CH,Cl, room temp. Pancratium mavitimurn found in Turkey has been investi- gated extensively over the past three years. The bulbs were first shown to contain two alkaloids (-)-lycorine 5 and (+)-9-O-demethylhomolycorine 37 and the ariel parts contain trisphaeridine 15.2' The latest publication22 has identified six alkaloids in its bulbs namely (+)-haemanthamine 3 (+)-buphanisine 25 crinine 11 ( -)-3P,1 la-dihydroxy-l,2- dehydrocrinane 71 ( -)-8-hydroxy-9-methoxycrinane72 which were accompanied by the new alkaloid (-)-3P-methoxy-6a 11P-dihydroxy- 1,2-dehydrocrinane 73.3 Synthetic studies The biomimetic synthesis of galanthamine 30 first proposed and achieved by Barton and his group in 1962 has been re-examined several times and the yield of the critical oxidative Lewis Amaryllidacae alkaloids coupling step has been increased from 0.2 to 5%. In a recent p~blication~~ this oxidative coupling process has been increased to 39% but only through modification of the amide precursor 74 (R=H) derived from isovanillin and tyramine by using an N-formyl- and dibromo-substituent 75 (Scheme 1).Debromination of the oxidatively coupled product 76 gave racemic galanthamine 77 which on resolution gave (-)-galanthamine 30. A short and efficient total synthesis of anabiline 87 has been accomplished using as a key step a cycloaddition proce- dure involving a 2-azaallyl anion with an alkene (Scheme 2). Starting with lactone 78 introduction of the 3,4-methylenedioxyphenyl substituent was followed by a Wittig reaction to create hydroxy alkene 79 and hence aldehyde 80 prior to stannane formation 81. Addition of butyllithium in THF at -78 "C to this stannane produced by transmetalla- tion a 2-azaallyl anion which cyclised intramolecularly to give two diastereoisomers 82 and 83. Treatment of 83 with Eschen- moser's salt in boiling acetonitrile with subsequent removal of acetonide resulted in the formation of (-)-anabiline 84 in 92% yield.The eight steps involved in this synthesis proceeded in a 43% overall yield.24 (-)-Augustamine 85 was synthe- sised from the same 2-azaallyl anion by working up the cycloaddition product in the presence of iodomethane. SiMe3 (0 OeQ ill + SiMe3 0 Iu v L I R' I ii iii 0 0 88 86 R=SiMe3 "'87 R=H Scheme 3 Scheme 3 Reagents i CpCo(CO), THF 25°C or reflux hv; ii Bu,NF THF 25 "C; iii Fe(NO3),*9H,O THF-H,O 0 "C; iv CF,CO,H CDCl, 55 "C; v H, ClRh(PPh,), PhMe 25 "C The attractive concept of using a cobalt-mediated [2+ 2+2] cycloaddition reaction of the double bond of an enamine with two alkynes has been explored and has culminated in the construction of the galanthan skeleton as exemplified by the synthesis of 88 which is a known precursor of y-lycorane 89 (Scheme 3).25 The mild protodesilylation sequence is to be noted.11 A. Latvala M. A. Onur T. Gozler A. Linden B. Kivcak and M. Hesse Tetrahedron Asymmetry 1995 6 361 (Chem. Abstr. 1995 122 291 230). 89 4 References 1 J. Bastida S. Bergonon F. Viladomat and C. Codina Planta Med. 1994 60 96 (Chem. Abstr. 1994 120 294 172). 2 M. Kreh R. Matusch and L. Witte Phytochemistry 1995 38 773. 3 G. R. Pettit G. R. Pettit 111 R. A. Backhaus M. R. Boyd and A. W. Meerow J. Nat. Prod. 1993 56 1682. 4 G. R. Pettit G. R. Pettit 111 R. A. Backhaus and F. E. Boettner J. Nat. Prod. 1995 58 37.5 J. R. Lewis Nat. Prod. Rep. 1996 13 171. 6 G. P. Husson P. H. Vilagines B. Sarrette and R. Vilagines Ann. Pharm. Fr. 1994 52 31 1 (Chem. Abstr. 1995 122 23 285). 7 0. R. Queckenberg A. W. Frahm D. Mueller-Doblies and U. Mueller-Doblies Planta Med. 1995 61 581 (Chem. Abstr. 1996 124 112 459). 8 F. Viladomat C. Codina J. Bastida S. Mathee and W. E. Campbell Phytochemistry 1995 40 961. 9 J. Bastida C. Codina P. Peeters M. Rutiralta M. Orozco F. J. Lugue and S. C. Chhabra Phytochemistry 1995 40 1291. 10 A. Latvala M. A. Onur T. Gozler A. Linden B. Kivcak and M. Hesse Phytochemistry 1995 39 1229. 12 W. Doepke L. H. Pham E. Grundemann M. Bartoszeck and S. Flatau Planta Med. 1995 61 564 (Chem. Abstr. 1996 124 112 454). 13 W. Doepke H.P. Lam E. Grundemann M. Bartoszek and S. Flatau Pharmazie 1995 50 51 1 (Chem. Abstr. 1996 123 165 076). 14 G. R. Pettit G. R. Pettit 111 G. Groszek R. A. Backhaus D. L. Donbek R. J. Barr and A. W. Meerow J. Nat. Prod. 1995 58 756. 15 M. Kihara T. Ozaki S. Kobayashi and T. Shingu Chem. Pharm. Bull. 1995 43 318. 16 0. M. Abdallah Phytochemistry 1995 39 477. 17 M. Kreh R. Matusch and L. Witte,. Phytochemistry 1995 40 1303. 18 S. Uyeo and N. Yanaihara J. Chem. Soc. 1959 172. 19 M. Kreh and R. Matusch Phytochemistry 1995 38 1533. 20 J. Bastida J. M. Fernandez F. Viladomat C. Codina and G. de la Fuente Phytochemistry 1995 38 549. 21 J. R. Lewis Nat. Prod. Rep. 1995 12 339. 22 B. Sener S. Konukol C. Kruk and U. K. Pandit J. Chem. SOC. Pak. 1994 16 275 (Chem. Abstr. 1996 123 5631). 23 J. Szewczyk J. W. Wilson A. H. Lewin and F. I. Carroll J. Heterocylc. Chem. 1995 32 195. 24 W. H. Pearson and F. E. Lovering J. Am. Chem. Soc. 1995 117 12 336. 25 D. B. Grotjahn and K. P. C. Vollhardt Synthesis 1993 579. 308 Natural Product Reports

ISSN:0265-0568    

DOI:10.1039/NP9971400303

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

Hot off the press Robert A. Hill“ and Andrew R. Pittb aDepartment of Chemistry Glasgow University Glasgow UK G12 8QQ. E-mail bobhachem. gla. ac. uk ‘Department of Pure and Applied Chemistry Strathclyde University Thomas Graham Building 295 Cathedral Street Glasgow UK GI I XL. E-mail a. r.pitt@strath. ac. uk Reviewing the recent literature on natural products and bioorganic chemistry The first branched glycerol enol ether hanishenol B 1 has been isolated from the sponge Acanthella carteri (I. Mancini et al. Tetrahedron 1997 53 2625). Thyrsenol A 2 from the red alga 2 Laurencia viridis is a polyether squalene derivative containing an unusual enol ether functionality (M. Norte et ul. Tetra-hedron Lett. 1997 38 3137). The first natural steroid oximes 3 and 4 have been isolated from Cinachyrella sponges R “CH 3R=H 4R=Et (C.Jiminez and co-workers Tetrahedron Lett. 1997,38 1833). Polygonapholine 5 has an interesting new alkaloid skeleton isolated from Polygonatum alte-lobatum (C.-N. Lin et al. Tetrahedron 1997 53 2025). Alboinin 6 from the Ascidian Dendrodoa grossularia contains an intriguing oxadiazinone system (B. Steffan and co-workers Tetrahedron 1997,53 2055) Hill and Pitt Hot 08the press 5 4 i-r 6 7 and alkaloid 223A 7 from the frog Dendrobatespumilio is the first trisubstituted indolizidine to be isolated (J. W. Daly and co-workers J. Nut. Prod. 1997 60 2). Isoharzandione 8 a fungal growth inhibitor from Tricho-derma viride (L. Mannina et al. Tetrahedron Lett. 1997 38 3135) and paralinone 9 from Euphorbia paralias (S.Oksiiz OAc / o=c 8 OAc 9 et al. Tetrahedron Lett. 1997 38 3215) both contain new diterpenoid skeletons. Scapaundulin B 10 is a dimeric labdane diterpenoid from the liverwort Scapania undulata (Y. Asakawa and co-workers Tetrahedron Lett. 1997 38 1975). The units of scapaundulin B 10 are joined by two hemiacetal linkages. Bismagdalenic acid 11 from Vellozia magdalenae appears to be a Diels-Alder adduct between eunicellane and labdane ... 10 11 0- 12 13 diterpenoids (A. C. Pinto et al. Tetrahedron 1997 53 2005). Olivacene 12 from the liverwort Archilejeunea ofivacea has an interesting new sesquiterpenoid skeleton (Y. Asakawa and co-workers Phytochemistry 1997 44 1261).The biogenesis of olivacene 12 must involve a methyl migration associated with the aromatisation. An interesting zwitterionic benzyl hydroxyadenine aplidiamine 13,has been isolated from an Aplidiopsis species (H. Kang and W. Fenical Tetrahedron Lett. 1997 38 941). The structures of the diastereoisomeric cynandiones from Cynanchum taiwanianum have been revised from the rather unlikely 14 to the more plausible 15 (C.-N. Lin et al. 0 HO 14 0 15 iv Natural Product Reports Phytochemistry 1997 44 1359). The structures of the can- gorosins triterpenoid dimers from Maytenus ilicifofia have also been revised (H. Itokawa and co-workers J. Nat. Prod. 1997 60 111). The cangorosins were originally thought to be triterpenoid dimers with an ether linkage between the two A rings.Detailed spectroscopic studies have now established the correct structures for cangorosin A 16 and cangorosin B 17. 16 17 CO*& Atropcangorosin A (now named isocangorosin A) has been shown to be analogous to congorosin A 16 but with C-2' C-6 and C-3' (2-7 ether linkages. M. Ishibashi and J. Kobayashi have recently reviewed their work on the amphidinolides macrolides from the marine dinoflagellates Amphidium sp. (Heterocycles 1997 44 543). A review of the utility of natural products in drug discovery and development has been published by D. J. Newman and co-workers who note that over 60% of the anticancer and antiinfective drugs approved in the last decade are of natural origin (J.Nat. Prod. 1997 60 52). Nelson J. Leonard has reviewed his work on a wide range of nitrogen-containing organic compounds including alkaloids cytokinins and probes for the study of enzyme-coenzyme interactions and nucleic acid structure and function (Tetrahedron 1997 53,2325). An interesting review of the novel compounds that have been found in spider toxins and webs has recently been published by S. Schulz (Angew. Chem. Int. Ed. Engf. 1997 36 314). A broad based review of the cysteine proteases and their inhibi- tors including the sources of the proteases and proteinaceous and low-molecular weight inhibitors has also recently been published (H.-H. Otto and T. Schirmeister Chem. Rev. 1997 97 133). The ability of the primitive invertebrates the cnidaria to synthesise 18 or more amino acids has been demonstrated using I4C labelling studies on five species (L.M. Fitzgerald et al. Biochem. J. 1997,322,213). This strengthens the argument for a separate evolutionary history from the rest of the metazoa. T. J. Simpson and his group have used extensive labelling experiments to unravel the complex story of the biosynthetic route to andibenin B 18 and andilesin A in Aspergillus HO. 0 18 variecolor (Tetrahedron 1997 53 4013). The structures turn out to be meroterpenoids of mixed terpenoid and polyketide origin. Studies using isotope labelling on the biosynthesis of murayaquinone 19 from Streptomyces murayamaensis have 19 shown that it is of polyketide origin but that it is not formed by simple folding of the polyketide chain but via a cyclisation bond cleavage and recyclisation route (S.J. Gould et al. Tetrahedron 1997 53 4561). Labelling studies have also been used to show that the initial step in the biosynthesis of a-cyclohexyl fatty acids the 1,4-conjugate elimination of water from shikimic acid 20 occurs with an overall anti stereochemistry in Alicyclobacillus acidocaldarius (Scheme 1) (S. Handa and H. G. Floss Chem. Cornmun. 1997 153). This Scheme 1 is analogous to the 1,4-elimination that forms chorismate from 5-enolpyruvylshikimate in the 'normal' shikimate pathway and suggests an evolutionary relationship between the two pathways. Using deuterium and carbon labelling and NMR spectroscopy the biosynthetic origin of the senecic acid por- tion of rosmarinine 21 and senecionine has been shown to be from two aminobutyric acid moieties (D.J. Robins and co-workers J. Chem. SOC.,Perkin Trans. I 1997 677). The aminobenzo[b]fluorene stealthin C 22 has been shown to be an intermediate in the biosynthesis of kinamycin C 23 in Streptomyces murayamaensis (S. J. Gould et al. J. Org. Chem. 1997 62 320). Hill and Pitt Hot oflthe press 21 22 23 The incorporation of oxygen into the carotenoid spheroid- enone 24 in Rhodobacter spheroides 2.4.1 under anaerobic conditions has been investigated using labelling (A. A. Yeliseev and S. Kaplan FEBS Lett. 1997,403 10) (Scheme 2). Water was found to be the source of the methoxy group presumably via hydration of the double bond followed by methylation but neither water nor CO were the source of the ketone oxygen.The authors suggest that there must be some other metabolite that can act as an oxidant. The metabolic cleavage of ascorbic acid to oxalic acid and threonic acid (Scheme 3) in plants has been investigated using I8O2 and H,"O labelling studies (K. Saito et al. Phytochemistry 1997 44 805). The results indicate that an oxygenase and a hydrolase are involved in the cleavage reaction. An efficient chemoenzymic route to N-glycolylneuraminic acid 25 an important member of the sialic acid family has been developed using three enzymes in a seven step route giving a 25% overall yield (A. Kuboki et al. Tetrahedron 1997 53 2387). K. Faber and co-workers have published details of an efficient deracemisation of 2,2-disubstituted epoxides using Nocardia EH 1 epoxide hydrolase (Tetrahedron Lett..1997 38 1753). The epoxide hydrolase opens the S epoxide 26 by nucleophilic attack at the less hindered carbon to produce the S diol27 leaving the R epoxide 28. When the crude mixture is treated with sulfuric acid an acid catalysed opening of the R epoxide 28 at the tertiary centre with inversion also produces the S diol 27 (Scheme 4). Yields and ees well in excess of 90% have been obtained using this procedure. T. Funabiki et al. have challenged the view that the ortho hydroxylation catalysed by tyrosine hydroxylase occurs by a peroxytetrahydropterin intermediate (Chern. Commun. 1997 151). Model studies have indicated that a more likely mechan- ism for the hydroxylation involves an iron-oxygen species.The rate enhancement of mandelate racemase has been measured by S. L. Bearne and R. Wolfenden to be 1.7 x lOI5 fold and the transition state affinity to be 2 x 10-l9 M (Biochemistry 1997 36 1646) which are remarkably high levels of catalysis. By comparing these data to the rate enhancement by the small molecules methylamine imidazole and acetate they estimate that the effective concentration of the catalytic residues in the active site are lysine 2622 histidine 23 x lo3 and glutamate 2 3 x lo5M. Hence they suggest that although general acid and base catalysis is inefficient in simple model systems it can be very efficient when there is strong structural complementarity to the transition state.The use of time resolved optical absorp- tion spectroscopy to measure the species present 50 ns to 50 ms after the photolytic decomplexation of CO from CO inacti-vated cytochrome c oxidase has shown the presence of seven from water I t 1 0 \ origin unknown 24 Scheme 2 "opco2H 0 \OH 25 intermediates including a peroxy ferrous species which decays to the ferry1 species followed by electron distribution to the copper ions (A. Sucheta et al. Biochemistry 1997 36 554). A single point mutation of Phe-87 of the Bacillus mega-terium cytochrome P450 BM3 causes no measurable change in the binding of the substrates laurate and myristate in the first intermediate but there are significant changes in the second (reduced) intermediate resulting in a-hydroxylation of these substrates that is not observed for the wild type enzyme (C.F. Oliver et al. Biochemistry 1997 36 1567). Site directed mutagenesis and the determination of the X-ray structure of the mutant have identified Ser-229 as the general acid in the catalytic reaction of MurB in cell wall biosynthesis (T. E. vi Natural Product Reports R' 26 RO ,* 28 Scheme 4 Benson et al. Biochemistry 1997 36 796 and 801). The Ser-229 to Ala mutant shows a lo7 fold decrease in the rate of the second step of the catalytic cycle the reoxidation of the flavin. The X-ray structure confirms that no significant changes have taken place in the active site except for the loss of the water molecule usually bound to the serine and a conse- quent reorganisation of the active site interactions.Muta-genesis of the putative active site base of cholesterol oxidase from Streptomyces results in the stalling of the isomerisation process after the initial oxidation at the 5-ene-3-one stage (N. S. Sampson and I. J. Kars J. Am. Chem. Soc. 1997,119 855). A number of autoxidation products were isolated as a result of intermediite sequestration. The inhibition of cysteine proteases by terminal peptidyl epoxides has been shown to proceed to give alkylation of the thiol group (A. Albeck and S. Kliper Biochem. J. 1997 322 879). The reaction is 5.5 orders of magnitude faster than the solution chemistry using an appropriately protected cysteine at pH 10 (corresponding to ca.a 10' increase in rate at pH 7.0). This suggests that these are mechanism based inhibitors with the active site histidine responsible for protonating the epoxide Scheme 5 (Scheme 5). L. R. Scolnick et a/. have studied the binding of hydroxamate inhibitors to carbonic anhydrase and discovered a novel mode of binding mimicking the binding of bicarb- onate where the hydroxamate nitrogen is deprotonated and binds to the zinc (J. Am. Chem. Soc. 1997 119 850) (Scheme 6). p-Aminobenzamidine 29 a well known inhibitor of serine proteases has also been found to be a competitive inhibitor but not a substrate of mouse brain NO synthase at PM levels (G. Venturini et al. Biochem. Biophys.Res. Commun. 1997 0 HOxNACF3 H 0 Zn His’ I ‘His His Scheme 6 30 232 88). N-[3-(Aminomethyl)benzyl]acetamidine 30 is a slow tight binding or irreversible inhibitor of human NO synthase (E. P. Garvey et a/. Biochern. J. 1997 4959). It has a binding constant of ~PM,but more importantly is selective for the inducable NO synthase. A new approach to cancer therapy has been successfully developed by G. Ragupathi et a/. (Angew. Chem. Int. Ed. Engl. 1997 36 125). The combination of the construction of a synthetic carbohydrate containing the tumour associated antigen GloboH which is then conjugated to a protein and introduced in conjunction with an adjuvant was used to vaccinate a mouse which subsequently produced anti-bodies that bound to MCF-7 tumour cells and caused their destruction in vivo.The contribution to the stabilization of a peptide helix by the interaction of a tryptophan and a charged histidine has been calculated using NMR spectroscopy and CD studies as about 1 kcal mol -(J. Fernandez-Recio et a/. 1997,267 184). There appears to be little difference between the second residues being in the i + 3 or i +4 position. The controversy over the relationship between the solution and gas phase association of non-covalent enzyme ligand complexes during electrospray mass spectrometry continues. Whitesides and his group have looked at the interaction of carbonic anhydrase and seven inhibitors both in the solution and the gas phase (Q. Wu et a/. J. Am. Chern. Soc. 1997 119 1157). They were unable to see the same correlation between hydrophobicity and binding in the gas phase that is apparent in the solution phase but a close relationship between tail length and contact of polar surfaces and binding in the gas phase was apparent. Hill and Pitt Hot ofl the press Vii

ISSN:0265-0568    

DOI:10.1039/NP997140iiic

出版商:RSC    

年代:1997

数据来源: RSC