摘要:

ISSN 0265-0568 NPRRDF 14(5) 433-558 (1997) Natural Product Reports A journal of current developments in bioorganic chemistry Volume 14 Number 5 CONTENTS ... 111 Hot off the press Robert A. Hill and Andrew R. Pitt Reviewing the recent literature on natural products and bioorganic chemistry 433 Natural products derived from unusual variants of the shikimate pathway Heinz G. Floss 453 Secondary metabolites from marine microorganisms bacteria protozoa algae and fungi Francesco Pietra 465 Coumarins Ana Estkvez-Braun and Antonio G. Gonzalez Reviewing the literature published between January 1995 and December 1996 477 Monoterpenoids David H. Grayson Reviewing the literature published in part of 1993 all of 1994 and part of 1995 523 Biosynthesis of polyketides Bernard J.Rawlings Reviewing the literature published from mid 1993 to the end of 1994 557 Book review Biochemical aspects of marine pharmacology eds. P. Lazarovici M. E. Spira and E. Zlotkin (reviewed by John Mann) 558 Corrigendum 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 (JuZy 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 11 1 The biosynthesis of C,-C, terpenoid compounds (2993 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 Number 4 309 Chemistry and biosynthesis of clavulanic acid and other clavams Keith H. Baggaley Allan Brown and Christopher J. Schofield 335 Biosynthesis of fatty acids and related metabolites (up to end 1994) Bernard J. Rawlings 359 Biosynthesis of plant alkaloids and nitrogenous microbial metabolites (1995)Richard B. Herbert 373 Steroids reactions and partial synthesis (1995)James R. Hanson 387 Phenethylamine and isoquinoline alkaloids (July 1995 to June 1996) Kenneth Bentley 413 Recent progress in chemistry of non-monoterpenoid indole alkaloids (July 1995 to June 1996) Masataka Ihara and Keiichiro Fukumoto 431 Book review Analysis of steroids by L.John Gould and Toshihiro Akihisa (reviewed by James R. Hanson) 432 Corrigendum Number 5 433 Natural products derived from unusual variants of the shikimate pathway Heinz G. Floss 453 Secondary metabolites from marine microorganisms bacteria protozoa algae and fungi Francesco Pietra 465 Coumarins (January 1995 to December 1996) Ana EstCvez-Braun and Antonio G. Gonzalez 477 Monoterpenoids (part 1993 all 1994 part 1995) David H. Grayson 523 Biosynthesis of polyketides (mid 1993 to end 1994) Bernard J. Rawlings 557 Book review Biochemical aspects of marine pharmacology eds. P. Lazarovici M. E. Spira and E. Zlotkin (reviewed by John Mann) 558 Corrigendum Articles that will appear in forthcoming issues include Triterpenoids (July 1995 to June 1996) Joseph Connolly and Robert Hill Isopentenyl diphosphate isomerase a core enzyme in isoprenoid biosynthesis. A review of its biochemistry and function Robert van der Heijden Ana C. Ramos-Valdivia and Robert Verpoorte Recent progress in the chemistry of the monoterpenoid indole alkaloids (1996) J. Edwin Saxton Pyrolizidine alkaloids (1995) J. Richard Liddell Indolizidine and quinolizidine alkaloids (July 1995 to June 1996) Joseph P. Michael Quinoline quinazoline and acridone alkaloids (July 1995 to June 1996) Joseph P. Michael Pyrrole pyrrolidine pyridine piperidine azepine and tropane alkaloids (1994 to 1996) David O’Hagan

ISSN:0265-0568    

DOI:10.1039/NP99714FP009

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 University of Glasgow Dr C. J. Schofield University of Oxford Dr D. A. Whiting University of Nottingham 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 Tech n icaI Ed it or Telephone +44 (0) 1223 420066 Mrs Dawn J. Webb Facsimile +44 (0) 1223 420247 Miss Karen L. White E-mail perkin @ rsc.org Editorial Secretaries RSC Server h tt p ://chemistry.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 Surface 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/NP99714FX021

出版商:RSC    

年代:1997

数据来源: RSC

ISSN:0265-0568    

DOI:10.1039/NP99714BX023

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

Natural products derived from unusual variants of the shikimate pathway ~~ ~ Heinz G. Floss Department of Chemistry Box 351700 University of Washington Seattle WA 981 95-1 700 USA 1 Introduction-the shikimate pathway as a source of natural products 2 Biosynthesis of cyclohexane and cycloheptane rings 2.1 Ansatrienin 2.1.1 Feeding with labelled precursors 2.1.2 Enzymes and encoding genes 2.2 Rapamycin and FK 520 2.3 Cyclohexyl fatty acids 2.4 Cycloheptyl fatty acids 3 Biosynthesis of C,N units 3.1 3-Amino-5- hydroxybenzoic acid (AHBA) as a precur- sor of C,N units 3.1.1 Origin of C,N units in ansamycins and mitomycins 3.1.2 Identification of AHBA as a specific precursor 3.1.3 Formation of AHBA in cell-free extracts 3.1.4 AHBA synthase from Amycolatopsis mediterranei 3.2 Molecular genetics of rifamycin biosynthesis 3.3 Cloning and analysis of other AHBA synthase gene clusters 3.4 C,N Units not derived from AHBA 3.4.1 Acarbose and validamycin 3.4.2 Asukamycin and manumycin 4 Biosynthesis of phenazines 4.1 Background 4.2 Biosynthesis of saphenamycins and esmeraldins 4.3 Mode of formation of the aromatic ring and of the extra methyl group 5 Conclusion 6 Acknowledgements 7 References ~ ~~ ~ Heinz Floss studied chemistry at the Technical University in Berlin and completed his Dr rer.nat. degree in Organic Chemis- try at the TU Munich under the direction of F.Weygand. At this time his life-long interest in the biosynthesis of natural products was re-awakened by a collaboration of the Weygand laboratory with the group of Kurt Mothes at the Institute of Plant Biochem- istry in Halle. After a postdoctorate with E. E. Conn at the University of California Davis in 1964/65 and his 'habilitation' in biochemistry at the TU Munich he followed an invitation of newly appointed Dean Varro E. Tyler Jr. to join the faculty of the Department of Medicinal Chemistry and Pharmacognosy at the Purdue Univeristy School of Pharmacy in 1966. After 16 happy years at this institution where he rose through the ranks to become the Lilly Distinguished Professor and Head of the Department he joined the Ohio State University as Professor and Chair of the Chemistry Department.In 1987 his love of sushi and mountains brought him west to Seattle where he is now Professor of Chemistry at the University of Washington. His research continues to deal with the biosynthesis of micro- bial and plant secondary metabolites particularly anti-biotics and Taxol and with the stereochemistry and mechanism of enzyme reactions. 1 Introduction-the shikimate pathway as a source of natural products The shikimate pathway' (Scheme 1) is an essential metabolic route by which microorganisms and plants synthesize the aromatic amino acids phenylalanine tyrosine and tryptophan as well as a number of other aromatic compounds which are critical to sustaining the primary functions of living organisms.The absence of this pathway in animals makes it an attractive target for metabolic intervention in the development of chemo- therapeutic agents as well as herbicides. However the signifi- cance of the shikimate pathway extends well beyond the manufacture of these primary cellular metabolites. The path- way is also the source of a vast number of secondary metabo- lites of plants and microorganisms natural products which although not critical to the immediate survival of the produc- ing organism help it in a variety of ways to maintain its position in its ecological environment. The majority of these shikimate-derived natural products are formed from the end products of the shikimate pathway i.e. the aromatic amino acids.Of these phenylalanine in particular gives rise to a tremendous variety of different phenylpropanoid compounds e.g. the flavonoids or the lignans,',2 although all three aro- matic amino acids along with anthranilic acid are the precur- sors of a vast number of alkaloids.' A much smaller number of natural products are derived from variants of the shikimate pathway which branch off at different points along the main metabolic sequence. Such diversions from the normal shiki- mate pathway are seen more frequently in microorganisms than in higher plants. As part of our long-standing interest in the reactions of the shikimate pathway we have studied some of these unusual variants leading to unique natural products. Some of these diversions from the main pathway are rather simple as in the biosynthesis of 2,5-dihydrophenylalanine,a common Strepto-myces metabolite.In this case it seems an extra reduction step has simply been inserted into the normal rearrangement sequence leading from prephenic acid to phenylpyruvic acid (Scheme 2).4 In other cases as in the formation of cyclo- hexanecarboxylic acid by reduction of shikimic acid the modifications are substantial and must have required the recruitment of a number of extraneous enzymes to this new pathway. In yet other examples as in the formation of 3-amino-5-hydroxybenzoicacid (AHBA) or of the phenazine ring system an apparently minor modification of the pathway sets the metabolism on a substantially different course. This review will illustrate some of these unusual variants of the shikimate pathway by examples investigated in the author's laboratory.2 Biosynthesis of cyclohexane and cycloheptane rings 2.1 Ansatrienin The ansatrienin~,~ also called myc~trienins,~-'~ are a small set of macrocyclic lactam antibiotics of the ansamycin type. They show a variety of interesting biological activities includ- ing antifungal and antitumor activity. Ansatrienin A 10 produced by Stveptomyces collinus Tiil892 first attracted our attention because it contains a biosynthetically rather unusual structural element a cyclohexanecarboxylic acid moiety linked 433 Floss Natural products derived from unusual variants of the shikimate pathway 7 7 Phosphoenolpyruvate COOH COOH HO d + ___) H CHO OH OH HO” OH @o-’ HtOH OH OH OH OH CH20@ I 3-Deoxy-D-arabino-CH20@ heptulosonic acid 3-Dehydroquinic 3-Dehydroshikimic Erythrose 4-phosphate (E4P) 7-phosphate (DAHP) acid (DHQ) acid (DHS) Shikimic acid Shikimate 3-phosphate 2 3 4 5 6 COOH d’H2 kPEP HOOCJ COOH &o -1 6 \ ‘COOH @O,’ O‘COOH L-Phenylalanine OH OH OH a / Prephenic acid Chorismic acid (CA) \5-Enolpyruvylshi kimate 3-phosphate (EPSP) GH pyruvate glu YOOH I I R L-Tyrosine R = OH phydroxybenzoic acid OH ONH2 Qj-flNroH-R = NH2 paminobenzoic acid H L-Tryptophan Scheme 1 The shikimate pathway 0 1 OH .H20 -COz Prephenic acid COOH Po Ansatrienin A 10 L-2,5-Dihydrophenylalanine 9 Scheme 2 Proposed biosynthesis of 2,5-dihydrophenylalanine via a D-alanine to the ansa macrocycle.Such a cyclohexane- carboxylic acid moiety is quite rare in nature occurring only in a few other antibiotics e.g. the trienomycins11p’4 and 0 thiazinotrienomycins. Singly substituted cyclohexane rings in general are only found in a few other compounds where they NH I serve as starter units for the synthesis of polyketide chains as in the antibiotic asukamycin 1116 from S. nodosus ssp. asu-kuensis (see section 3.4.2) and notably in the w-cyclohexyl Asu kamycin 11 fatty acids of certain thermophilic and mesophilic bacterial’ (see section 2.3) exemplified by o-cyclohexylundecanoic acid 12. Ansatrienin contains another structural feature of biosyn- ShooH thetic interest a so-called mC,N unit which makes up the benzenoid ring of the macrocycle.This will be discussed in o-Cyclohexylundecanoic acid section 3.1. 12 434 Natural Product Reports 1997 2.1.1 Feeding with labelled precursors Inspection of the structure of ansatrienin A in light of general biochemical knowledge suggests that the macrocyclic core of this compound is a polyketide assembled from a carboxylic acid starter representing an mC,N unit (see section 3) by iterative addition of acetate and propionate units in the form of malonyl CoA and methylmalonyl CoA respectively. This expectation was borne out by the appropriate feeding exper-iments with labelled acetate and propionate samples." By analogy with the biosynthesis of erythromycin," rapamycin2' and similar compounds,2' the polyketide core of ansatrienin is probably assembled on a modular polyketide synthase.The D-alanine and the cyclohexanecarboxylic acid moieties are added to the macrocycle in a sequential fashion as opposed to pre-assembly of N-cyclohexylcarbonyl-alanine and its attach-ment.22 The cyclohexanecarboxylic acid moiety interestingly was found to arise directly from shikimic acid which is incorporated with retention of its carboxy group.22 The detailed pathway from shikimic acid to cyclohexanecar-boxylic acid was explored by synthesizing and feeding shikimic acid samples with I3C or deuterium incorporated at various ring positions. Most of these compounds were prepared by adaptations of the excellent route of Fleet et al.23The resulting ansatrienin samples were then analyzed by 13C or deuterium NMR spectroscopy to determine the isotope distribution.Analysis of a sample of 10 biosynthesized from [2-'3C]shikimic acid showed that of the two diastereotopic carbons C-32 and C-36 in the cyclohexane ring only one was enriched. The absolute configuration was determined by hydrolysis of 10 to cyclohexanecarboxylic acid 13 and further reduction to cyclohexanol which was compared as its optically active mandelate ester to an independently synthesized stereospecifi-cally deuterated sample of known absolute configuration. This analysis located the 13C in the original sample to the pro-S position ie. C-36.24Interestingly the sample of 13 from the hydrolysis of this 13C-labelledansatrienin was found to con-tain a small amount of cyclohex-1-enecarboxylicacid 14 and this material carried the I3C label at C-6 rather than C-2 (Scheme 3).24Deuterium NMR analysis of 10 biosynthesized H--Da 6 14 13 Scheme 3 Labelling of the cyclohexane-and cyclohex-1-ene-carboxylic acid moiety of 10 from various positions of shikimic acid from the various deuterated shikimic acid samples revealed that the hydrogens from C-2 C-3 C-4 and C-5 of shikimic acid were retained in the conversion occupying respectively the pro-36R pro-35R 34E and pro-33R positions whereas both hydrogens from C-6 of shikimic acid were replaced by un-labelled hydrogens.Thus the transformation of 6 into the cyclohexanecarboxylic acid moiety of 10 must involve two proton eliminations from C-6 which remove stereochemically opposite hydrogens.Since the hydrogens at C-3 and C-5 were retained dehydration by removal of the C-4 hydroxy group cannot be the first step in the reaction sequence. Furthermore mass spectral analysis of a sample of 10 obtained from feeding [2,3,4,5-*H4]shikimicacid revealed that the reaction sequence does not involve any intermolecular hydrogen transfers.25 While these results established important boundary con-ditions they were insufficient to define a pathway completely. Delineating the pathway further required a substantial amount of detective work involving mainly the synthesis and feeding of a substantial number of potential intermediates in I3C-or deuterium-labelled form.The results supplemented by conclu-sions from the study of the biosynthesis of a-cyclohexyl fatty acids (see section 2.3) allowed the complete step-by-step for-mulation of the pathway as shown in Scheme 4. Since benzoic H CO-R CO-R CO-R 6:; -H20 +[2:] -bH6' -&i6' anti antil Si HO" OH OH OH OH OH OH 6 15 16 -H20 syn I 0 H CO-R + [2 A] -'OH 'L3'y' H4 H4 19 18 17 -H20 anti I dn 0 IU t fi\ COlR , H ,CO-.R CO-R fi\ COlR 10 &H P +[2;] -H++H+ & - 6; suprafacial H antil Si H5 H5 \\ Y 20 14 13 12 R = OH or SCoA Number on hydrogens refer to the original shikimate hydrogens Scheme 4 The pathway from shikimic acid to cyclohexanecarboxylic acid in the biosynthesis of 10 and 12 acid was not reduced although it was attached as such to the ansatrienin backbone the pathway does not proceed through an aromatic intermediate.The 1,4-dehydration of 6-15 was implicated as the first step because the dihydro derivative of 6 was not incorporated and the OH group remaining in 18 further down the pathway was shown to arise from C-5 not C-3 of 6. Labelled [2,3,4,5-2H,]-15 was efficiently incorporated into 10 and gave the same deuterium distribution as the correspondingly labelled shikimic acid. Compound 16 is the only compound not directly evaluated for its incorporation into 10; it is however clearly implicated as the next intermedi-ate by substantial indirect evidence. Crucial was the finding that 18 deuterated at C-5 was efficiently incorporated and showed labelling at an axial hydrogen (H-33R) rather than an equatorial hydrogen (H-35R) in 10.This identified the labelled position in the precursor 18 as corresponding to C-5. not C-3 of 6. The 4-hydroxy isomer of 18 carrying deuterium at C-4 was also incorporated but since it labelled the 342 hydrogen rather than the 34E position as did [4-2H]-6 it cannot be a normal pathway intermediate.25For the conversion 18+20 we initially favored a sequence involving dehydration prior to double bond reduction i.e. the intermediacy of 21 (Scheme 5).26 13C-Labelled21 was efficiently incorporated into 10 and its coenzyme A ester was reduced to that of 13 by a cell-free extract of S.collinus Tii1892.27A distinction between 19 and 21 as intermediates only became possible through studies on the biosynthesis of a-cyclohexyl fatty acids (see section 2.3). Analysis of a blocked mutant accumulating 19 unequivocally identified this compound rather than 21 as the true pathway intermediate.28 The reaction sequence for the conversion of 6 into the 13-moiety of 10 is remarkable in that it consists of an alternat-ing array of dehydrations and reductions arranged in such a way that no intermediate ever becomes aromatic. It is also remarkable in that two of the three dehydrations 6+15 and 435 Floss Natural products derived from unusual variants of the shikimate pathway CO-R I A uOH 19 Scheme 5 Alternative pathways from 18 to 20 19+20 involve the elimination of unactivated hydrogens.The initial dehydration 6+15 very much resembles the well known chorismate synthase reaction of the shikimate pathway raising the question whether the true branch point for this pathway might be at chorismic acid 7. However deuterium-labelled 7 was clearly not incorporated into yet the stereochemistry suggests the possibility of a mechanistically analogous enzy- matic reaction. The steric course of all the reactions except the first step follows from the experiments described above.25 28 However the question of which hydrogen is lost from C-6 of 6 could not be answered in this system because both hydrogens are ultimately replaced by hydrogens from other sources. Again in the parallel studies on a-cyclohexyl fatty acid biosynthesis the availability of blocked mutants made this determination possible.It was found that the conversion of 6+15 proceeds by an anti elimination of H6R,30 the same stereochemistry that had been established earlier for choris- mate syntha~e.~'? It still remains to be explored whether this 32 reaction as the chorismate synthase reaction requires the 3-phosphate as substrate. At some point along the sequence from 6 to 13 the carboxy group must be activated to its coenzyme A thioester because the enzymes catalyzing the last two steps in the sequence 20-+14 and 14-+13 require the CoA esters rather than the free acids as The exact stage at which this activation occurs is not known. Since many free carboxylic acids along the pathway are efficiently incorporated it seems likely that the activating system is rather promiscuous.There is however considerable evidence that the cyclohex- 1-enylcarbonyl CoA reductase catalyzing the conversion 14+ 13 also catalyzes the analogous reductions 18+19 and 15+16.33Since this enzyme does not accept the free acids as substrates it is likely that the activation may occur very early in the pathway at the level of 15 or possibly even 6. In fact the reductase could act as a 'gatekeeper' enzyme by enforcing activation at an early stage in order for compounds to be processed further. 2.1.2 Enzymes and encoding genes With the elucidation of the pathway from 6 to 13 the stage was set for the isolation of some of the enzymes catalyzing these reactions.The enzymology of the S. collinus system was largely investigated by Reynolds and co-workers. Cell-free extracts of S. collinus Tu1892 were found to catalyze both the isomeriz- ation of cyclohex-2-enylcarbonyl CoA to the A1 isomer and the reduction of the latter to cyclohexylcarbonyl COA.~~ For the double bond isomerization reaction Reynolds and co-workers independently established the suprafacial stereo- chemistry shown in Scheme 4 and the double bond reduction was shown to proceed by addition of the pro-4S hydrogen of NADPH to the Si face of C-2 of the cyclohexene double bond.34 Purification to homogeneity revealed the enzyme to be a homodimer of 36 000 dalton subunit molecular mass which showed an absolute requirement for NADPH as cofactor and for the coenzyme A ester of its substrate the free acid or the N-acetylcysteamine derivative being inactive.The purified enzyme also reduced the CoA esters of 1835and of diene 21,33 436 Natural Product Reports 1997 supporting our original notion26 that 21 was the pathway intermediate between 18 and 20 and suggesting that a single enzyme is employed repetitively to carry out several reduction steps in the pathway. The gene encoding cyclohex- 1-enylcarbonyl CoA reductase chcA was cloned by reverse genetics from a genomic library of S. collinus Tu1892 using sequence information from two internal cleavage peptides generated from the homogeneous protein. Sequence analysis revealed that the gene encodes a protein of 280 amino acids which shows no homology to other enoyl CoA reductases but was homologous to members of the short-chain alcohol dehydrogenase ~uperfamily.~~ Expression of chcA in E.coli gave a protein with characteristics identical to those of the native enzyme. Inactivation of the gene by deletion of the 5'-terminal region led to a mutant which was unable to produce either 10 or o-cyclohexyl fatty acids. Supplementation of the mutant with cyclohexanecarboxylic acid restored o-cyclohexyl fatty acid formation but surpris- ingly not the synthesis of The reason for the failure of this mutant to produce ansatrienin upon supplementation with 13 is not clear; it is possible that the gene disruption has also inactivated another gene essential in the biosynthesis of 10.Nevertheless this experiment strongly suggests the involvement of chcA in 10 biosynthesis setting the stage for the analysis of the surrounding DNA for other ansatrienin biosynthesis genes. 2.2 Rapamycin and FK 520 Although cyclohexanecarboxylic acid has been encountered only rarely in natural products either as such or as the starter unit of polyketide chains a more oxygenated analog (1R,3R,4R)-3,4-di hydroxycyclo hexanecarboxylic acid 25 has been implicated as the polyketide starter unit in the biosyn- thesis of the import ant immunosuppressant antibiotics bROH 0'0 OMe 0 Me Me ,I v.., 22 R = CH2Me Ascomycin (FK520) 23 R = CH2-CH=CH2 (FK506) mMe / b-J I' 0 A0 Me.. Me OMe Me Me 24 Rapamycin ascomycin (FK 520) 22,36-38FK 506 2339,40and rapamycin 24.41These three compounds all Streptomycete metabolites are of major clinical interest.42 In addition to the unusual polyketide starter unit these antibiotics contain another intriguing feature.The polyketide chain ends at its carboxy terminus in an amide linkage to a pipecolic acid which in turn forms an ester linkage back to the polyketide backbone. Thus these antibiotics are both macrolides and macrolactams. Their polyketide nature has been confirmed both by feeding exper- iment~~~’ 44 and in the case of rapamycin by extensive genetic studies.20,45,46 The latter have resulted in the cloning and complete sequence analysis of the biosynthetic gene cluster of 24.This cluster encodes a modular polyketide synthase which also includes a single module of a peptide synthase presum- ably responsible for the incorporation of the pipecolic acid moiety as well as additional genes encoding individual processing enzymes. Both in rapamycin 24 and ascomycin 22 the origin of the dihydroxycyclohexane moiety has been traced to shikimic acid 6 which is incorporated with all seven carbon atoms.44’47 The pathway from 6 to the presumed and in the case of 24 c~nfirmed,~~ starter unit 25 has been studied in more detail in the case of as~omycin.~~ The pathway (Scheme 6) rn bHs bH6’ CO-R CO-R H,J&z6s CO-.R HO” -H20 HR 7 OH OH P+[2;1] syn 1 Si H3 I OH OH OH OH 6 15 16 H CO-R CO-R OH OH 1-epi-l6 + [2 HI anti 1 Re :I26 H CO-R +5 VOH OH 25 Scheme 6 Biosynthesis of the 3,4-dihydroxycyclohexanecarboxylicacid starter unit of FK520 shares the first step with the formation of cyclohexanecarboxy- lic acid 13.The 1,4-dehydration 6+15 proceeds with the same stereochemistry anti elimination of H, of 6 in both path- way~~~ and also in the biosynthesis of 24? The pathways to 13 and 25 however diverge at the stage of 15. Both 15 and 26 were incorporated into 22 implicating 16 or its C-1 epimer as a likely intermediate. However deuterium labelling revealed that the enoyl reductions in this pathway proceed with a different stereochemistry than those in the pathway from 6 to 13 (Scheme 4). Since the C-1 configuration of the inferred intermediate 16 is not known two alternative stereochemical pathways (Scheme 6) one proceeding through 16 and the other through its C-1 epimer cannot be di~tinguished.~~ Consistent with the difference in enoyl reduction stereochemistry the analysis of the rapamycin biosynthetic gene cluster suggests that an enoyl reductase which is part of the first PKS module is responsible for the last double bond reduction in the starter In contrast the cyclohex- 1-enylcarbonyl CoA reductase of the ansatrienin pathway is a distinct separate enzyme with no homology to enoyl redu~tases.~~ Interestingly the final 0-methylation of the dihydroxycyclohexane moiety only takes place after the assembly of the polyketide as has been shown in the case of FK 520.52 2.3 Cyclohexyl fatty acids The thermo- and acido-philic bacteria Alicyclobacillus acido- caldari~s~~ as well as the mesophile and A.a~idoterrestris,~~ Curtobacterium pusill~m,~~ contain a high percentage (79 to 90%) of o-cyclohexyl fatty acids in their membrane lipids particularly when grown at high temperature and low pH.55 56 Mutants of A. acidocaldarius deficient in a-cyclohexyl fatty acid biosynthesis grow poorly at high temperature and low pH,57 implicating these unusual fatty acids in membrane stabilization to withstand extreme environmental growth con- dition~.~~, 59 The pattern of naturally occurring compounds with a-cyclohexylundecanoic acid 12 as the major component and lesser amounts of a-cyclohexyltridecanoic acid suggested a fatty acid biosynthesis from cyclohexylcarbonyl CoA as starter unit which undergoes chain extension by addition of four to six acetate units.,’ a-Cyclohexyl fatty acids are also produced by S.collinus Tiil892 when ansatrienin biosynthesis is blocked by high temperature and/or acidic pH or when the cultures are supplemented with excess cyclohexanecarboxylic acid 13.,’ Cyclohexyl fatty acid production is also observed in other Streptomycetes upon supplementation with 13 but not upon cultivation at elevated temperatures or decreased pH,,’ suggesting that this phenomenon reflects a relatively broad substrate specificity of Streptomycete fatty acid synthases.62 The shikimic acid origin of the 13 starter unit had been demonstrated by several groups.60 63-65 The detailed pathway from 6 to 13 was unravelled by studies with I3C-and deuterium-labelled precursors paralleling those on the origin of the 13 moiety of The biosynthetic pathway revealed by these studies is identical to the one in S.collinus Tii189225 shown in Scheme 4. The investigations were complicated by the extreme growth conditions of A. acidocaldarius 50 “C and pH 4 which resulted in a number of artefactual transfor- mations of added precursors or intermediates. For example while in S. collinus chorismic acid 7 was clearly not incorpor- ated into 10 in A. acidocaldarius a modest incorporation into the cyclohexyl moiety of 12 was observed.29 However this was due to non-enzymatic hydrolysis of 7 to 15 a known pathway intermediate.The fact that glyphosate [N-(phosphonomethyl) glycine] an inhibitor of 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase at a concentration which completely inhib- ited protein synthesis did not inhibit 12-formation confirmed that the pathway to 13 must branch off from the main shikimate pathway prior to EPSP and 7.29 The investigations of 13biosynthesis in A. acidocaldarius were aided on the other hand by the availability of some mutants blocked in this biosynthetic pathway which were dependent upon supplemen- tation with 13for growth.57 One of these mutants was found to be blocked in the conversion of 17+18 (Scheme 4). This mutant excreted benzoic acid and 3-hydroxybenzoic acid presumably due to chemical aromatization of the accumulated intermediates 15 16 and 17 in the warm acidic culture medium.28 Two other mutants were found to be blocked in the formation of 20.These two mutants were crucial to the investigations because they were found to accumulate 19 rather than 21 in the culture medium revealing that the physiological pathway for the formation of 20 proceeds via 19 not 21 (Scheme 5). This mutant also accumulated smaller amounts of the 3-epimer of 19 as a result of epimerization of 18 by oxidation to the ketone and subsequent reduction with opposite stereochemistry. The availability of this mutant en- abled the stereochemical analysis of some of the pathway reactions because it allowed dissection of the pathway at a stage when one hydrogen from C-6 of 6 was still present.Feeding of (6R)-and (6S)-[6-2H,]-6to this mutant and analysis of the resulting 19 and its epimer revealed that the initial 1,4-dehydration step proceeds with loss of the pro-R hydrogen Floss Natural products derived from unusual variants of the shikimate pathway from C-6 of 6 in an anti fashion.30 Analysis of 19 from [6-2H]-6 also showed that the retained deuterium occupied the 2s position in 19 revealing the stereochemistry of both the initial double bond reduction of 15 and of the dehydration 19+20.28 These findings completed the stereochemical analysis of the pathway from 6 to 13. The experiments described above resulting in the pathway shown in Scheme 4 represent more or less all that can be learned by in vivo feeding experiments.Many interesting questions remain which can only be resolved by further studies at the enzymatic andlor genetic level. Examples include the nature of the derivative of 6 undergoing the 1,4-dehydration reaction i.e. whether the substrate is 6 itself its 3-phosphate and/or its coenzyme A thioester; the question in general of the point at which the pathway intermediates are converted to their CoA esters and the substrate specificity of this activating enzyme; and the detailed enzymatic mechanisms of the intrigu- ing dehydration reactions 6+15 and 19+20. At the genetic level of particular interest is the evolutionary relationship if any of the pathway in Alicyclobacillus to that in Strepromyces. We hope to address some of these issues in future research.2.4 Cycloheptyl fatty acids The presumed membrane-stabilizing function of the o-cyclohexyl fatty acids of various Alicyclobacillus species is uniquely replaced in one species A. cycloheptanicus by the homologous a-cycloheptyl fatty acids.66-68 The spectrum of fatty acids found is similar to that of the cyclohexyl fatty acids in the other Alicyclobacillus species except for the presence of a cycloheptane instead of the cyclohexane 69 This raises the intriguing question whether the functional equivalence of the two types of compounds is the result of a divergent or convergent biosynthesis. In other words are the cycloheptyl fatty acids produced by a modification of the pathway to the cyclohexyl fatty acids or is the cycloheptane ring generated in a completely different fashion.With this question in mind we studied the biosynthesis of the o-cycloheptyl fatty acids in A. cy~loheptanicus.~~ In specific feeding experiments we explored a number of different hypotheses. These included (i) the formation of cycloheptanecarboxylic acid from a hypothetical seven-membered ring homolog of shikimic acid which could arise from the lipopolysaccharide constituent 3-deoxy-~-?nanno-2- octulosonate 8-phosphate (KDO);71(ii) addition of a one carbon unit to an intermediate of cyclohexanecarboxylic acid formation followed by ring expansion; and (iii) o-oxidation of a straight chain fatty acid followed by cyclization to a terminal cycloheptane ring. All these hypotheses were disproved by the feeding experiments illustrated in Scheme 7.Acetate labelled only carbons 1 to 10 of o-cycloheptylundecanoic acid 27 the 13c00~ 1-27 o-Cycloheptylundecanoic acid 13700~ / I 8H3-800H 8H3-S-CHz-CH2 -CH -COOH I 28 HO,' O O H NH2 OH 6 Scheme 7 w-Cycloheptylundecanoic acid and its biosynthesis from labelled precursors main component of the fatty acids and cycloheptanecarboxy- lic acid 28 carrying I3C in the carboxy group was efficiently incorporated and labelled exclusively carbon 11 of 27. This eliminated hypothesis (iii). Hypothesis (ii) was ruled out by the fact that neither the methyl group of methionine nor an acetate carbon was incorporated into carbons 11 to 18 of 27; that carboxy-labelled cyclohexanecarboxylic acid was not incorporated although it produced the formation of the corresponding cyclohexyl fatty acids in A.cycloheptanicus without dilution of the isotope by endogenous material; and that [1,7-'3C2]shikimic acid 672 gave rise to 27 showing no 13C-13C coupling between carbons 11 and 12. The possible involvement of a hypothetical 'homoshikimic acid' was probed in feeding experiments with uniformly and positionally I3C- labelled glucose samples. The resulting labelling and coupling patterns in the cycloheptyl moiety of 27 were clearly not compatible with this hypothe~is.~~ While these studies were underway a paper from the labora- tory of D. E. Cane73 reported on the biosynthesis of thiotro- pocin 29 a metabolite of a Pseudomonas species containing a s=(=,? 29 Thiotropocin carbon framework identical to that of cycloheptanecarboxylic acid.When the very complex l3C-I3C coupling pattern of 27 derived from [U-''Cp]glucose had been unravelled (Scheme 8),70we realized that it was identical with that observed by the I D-[U-' 3C6]Glucose OH 6 30 27 27 Scheme 8 I3C-I3C Coupling pattern in 27 biosynthesized from [u-'3~,]glucose Cane group for 29 complicated only by the additional sym- metry of the cycloheptane ring in 27. As Cane and co-workers had recognized this coupling pattern arises from the ring expansion of phenylacetic acid 30 formed plausibly from 6 via phenylalanine 8. They confirmed this interpretation by demonstrating clean incorporation of [1,2-'3C2]-30 to give 29 with the expected labelling pattern.73 Similar feeding exper- iments with [I ,2-"C2]-30 to A.cycloheptanicus gave o-cycloheptylundecanoic acid showing 3C enrichment in carbons I1 and 12 (20-27%) and strong one-bond coupling between these two I3C nuclei. Thus 30 is clearly also the specific precursor of the 28 moiety of o-cycloheptyl fatty acids. The suspected origin of 30 from 8 was verified by a number of feeding experiments with deuterium-labelled samples of 8. 438 Natural Product Reports 1997 c 1 CO-R CO-R CO-R -50% Hs -50% H CO-R CO-R CO-R 6.-6R -hR +[2H] ---D 27 +[2H] -+[2H] -&R -Hoa:shim -Hm Hp 32 34 35 36 28 Scheme 9 Deuterium distribution in 27 from deuterated 30 and a possible pathway for the conversion of 30 into 28 To gain more information on the transformation of 30-+28 the fate of the various hydrogens of 30 in this conversion was traced by deuterium labelling.The results are summarized in Scheme 9 which also shows a hypothetical pathway for the conversion of 30 into 28 based on the available data. Of the two methylene hydrogens of 8 and hence 30 H is completely eliminated suggesting initiation of the reaction by an oxidative attack at the benzylic carbon. However mandelic acid 31 the hydroxylation product of 30 is not incorporated into 27 (Scheme 10). The other benzylic hydrogen of 30 H, migrates 31 27 Scheme 10 Non-incorporation of mandelic acid into the cycloheptyl moiety of 27 to carbon 3 of the cycloheptane ring where it becomes equiva- lent with one of the two meta hydrogens from the precursor.The two ortho hydrogens the para hydrogen and the other meta hydrogen of 30 appear in the expected positions in the ring of 28 at C-2 and C-6 at C-4 and at C-5 respectively. However the data also revealed that the biosynthesis must proceed through a symmetrical intermediate since the label- ling patterns on the two sides of the ring have been equili- brated. That is for example 50% of H at C-2 are found in the equatorial position and the other 50% in the axial position presumably at C-7. Likewise Hp is located 50% in the equa- torial position at C-4 and 50% in the axial position presum- ably at C-5. This is true for every labelled hydrogen on the cycloheptane ring. Finally half of H and half of the meta hydrogen with which it has become equivalent are eliminated.Whether this equilibration of H and the meta hydrogen is the result of the 'symmetrization' of the ring or whether it reflects a non-stereospecific hydrogen elimination as suggested in Scheme 9 is not clear at this point. The deuterium labelling data provide important boundary conditions for the formulation of a biosynthetic pathway but they are insufficient to define the pathway completely. The hypothetical route shown in Scheme 9 satisfies most of the boundary conditions but it is at this point entirely hypotheti- cal. Evidence is available to support some of the later steps in Floss Nuturul products derived from unusual variants of the shikimate pathway 13 C H2 -(C H2)g -COOH 33 Specific incorporation '3C 4 Yo 'H 0.4 Yo 3~~~~ 13CH2-(CH2)g -COOH DH 32 Specific incorporation d3-6;: 13Cand 2H 8% (Specific incorporation of [I ,2-W2]-30 20%) Scheme 11 Incorporation of labelled cycloheptatrienecarboxylic acids into 27 the pathway.70 Compound 32 (Scheme 9) and its double bond isomer 33 (Scheme 11) were synthesized and tested as precur- sors of 27.Compound 33 was incorporated to the extent of 34% versus 20% for 30 and with retention of only 10% of the deuterium from C-1 as opposed to 50% of H from 30. Most importantly the deuterium in the product although it had undergone the 1,3-migration was located exclusively in the axial position at C-3 of the cycloheptane ring. This makes it unlikely that the conversion of 33 is part of the natural biosynthetic pathway to 27.The data for 32 7-8% incorpor-ation with complete retention of deuterium at C-3 is more consistent with the intermediacy of this compound in 27 biosynthesis. The location of the incorporated deuterium half in the axial and half in the equatorial position is also consistent with the pattern of incorporation of 30 although since 32 was deuterated non-stereospecifically this distribution would also have resulted from a non-physiological conversion. The intermediacy of 34 35 and 36 (Scheme 9) is supported by direct feeding experiments with the 3C-labelled samples which gave specific incorporations between those of 30 and 28. Furthermore the enzymatic reduction of their CoA esters was demonstrated in cell-free extracts of A.cycloheptanicus. Neither compound 18 nor compound 21 (Scheme 12) both efficient precursors of cyclohexyl fatty acids in A. acidocal-darius were converted into cyclohexyl fatty acids when fed to A. acidocaldarius A. cycloheptanicus Me Me Me COOH (CH2)lo-COOH i:1 12 37 COOH 12 12 21 COOH 12 12 18 Scheme 12 Conversion of cyclohexyl derivatives into cyciohexyl fatty acids in A. acidocaldarius and A. cycloheptanicus A. cycloheptanicus. However compound 37 a mimic of 34 which is not converted into cyclohexyl fatty acids in A. acidocaldarius but gives rise to the cyclohex-3-enyl analogs was efficiently reduced in A. cycloheptanicus and converted into cyclohexyl fatty acids.However despite these initial results substantially more work particularly the synthesis and feeding of the other postulated intermediates will be necessary to evaluate our working hypothesis for the pathway from 30 to 27. 3 Biosynthesis of C,N units 3.1 3-Amino-5-hydroxybenzoic acid (AHBA) as a precursor of C,N units 3.1.1 Origin of C,N units in ansamycins and mitomycins As mentioned earlier ansatrienin A 10 in addition to the cyclohexanecarboxylic acid moiety contains another biosyn- thetically unique structural element a C,N unit consisting of a 6-membered carbocycle carrying an extra carbon and a nitrogen in a meta arrangement. Such C,N units are charac- teristic structural components of all ansamycin antibiotic^,,^ both of the naphthalenic type exemplified by rifamycin B 3875,76 from Amycolatopsis mediterranei and of the benzenic type exemplified by lo5 and by geldanamycin 3977,78 from Streptomyces hygroscopicus and of the related maytansinoids e.g.maytansine 4079 from plants in the family Celastraceae and ansamitocin 41g1* from Actinosynnema pretiosum. The '* C,N unit in these compounds represents the starter unit from which the ansa chain is assembled by a polyketide-type biosynthesis. In a different context combined with a carbohydrate-derived moiety the C,N unit is also found in the antibiotics of the mitomycin family e.g. mitomycin C 42.83 The C,N unit was first recognized as a biosynthetically unique structural element in studies on the biosynthesis of rifamy~in,~~, 85 representing the only part of the ansa structure not derived from acetate/propionate units.The shikimate pathway origin of the C,N unit in both the ansamycins86 and mitomycinsg7 was indicated by feeding experiments with labelled and by genetic e~perirnents,~~-'~ although attempts to incorporate shikimic acid 6,88,90,92 96 quinic acid9 or dehydroquinic acid 492 into the C,N unit of these compounds were uniformly unsuccessful. While the non-incorporation of these shikimate pathway compounds could be due to cell impermeability-the cells of the rifamycin producer A. mediterranei indeed do not take up 69s-such a possibility was ruled out in the case of ansatrienin 10. Labelled [2-13C]-6 was not incorporated into the C,N unit of 10 while efficiently labelling the 13 moiety.24 The conclusion that the biosynthesis of the C,N unit must branch off from the main MeCOOU o+b0 OCH~COOH Me 38 Rifamycin €3 0 MeO'-wO OH Me MeOANH2 39 Geldanamycin CI M," MeO Me ; OH OMe Maytansinoids 40 Maytansine R = COCH(Me)N(Me)COMe 41 Ansamitocin P-3 R = COCH(Me)2 0 CH20CONH2 H6a Me 2 N a eNMe 0 42 Mitomycin C shikimate pathway prior to 6 is consistent with genetic studies in A.mediterranei which showed that the branch point for rifamycin biosynthesis must lie before dehydroquinic 3.1.2 Identification of AHBA as a specific precursor A major breakthrough in the understanding of the biosynthesis of the C,N unit came with the identification of 3-amino-5- hydroxybenzoic acid (AHBA) 43 (see Scheme 13) as a specific precursor.This discovery was made independently in the laboratories of Ghisalba and Niiesch in Basel by genetic experiments and in the laboratory of Rickards in Australia by specific feeding experiments. The Swiss group used a 38-non- producing mutant of A. mediterranei blocked early in the pathway to carry out complementation experiments. They found that 43 was specifically and uniquely able to restore rifamycin production in this mutant.98 The Rickards group compared the structures of all the antibiotics containing a C,N unit and recognized that in each case the second meta position on the 6-membered ring carried an oxygen. Since the C,N unit precursor must be the starter for a polyketide synthesis they reasoned that C-7 should be a carboxy group.The simplest structure incorporating all these elements was 43 which they then synthesized in I4C- and 13C-labelled form.99 Feeding 440 Natural Product Reports 1997 experiments showed that this compound is very efficiently and specifically incorporated into the C,N unit of the naphthalenic ansamycins actamycin"' and rifamycin 38,''' ansamitocin 41'02 and mitomycin 42.lo3 Other laboratories subsequently also showed specific incorporation of 43 into the benzenic ansamycins geldanamycin 39,lo4 strept~varicin''~ and ansa-trienin and into naphthomycin. '06 These experiments left no doubt about the specific precursor role of 43 in the formation of the C,N unit of these antibiotics.The biosyn- thetic question therefore now was how AHBA arises from the shikimate pathway . A very important finding in this respect came from work by Hornemann and his co-workers on the biosynthesis of mito- The carbon carrying the amino group in 43 could be equivalent to either C-3 or to C-5 of cyclic shikimate pathway intermediates like 4 or 5. Since C-3 is present as a keto group in 4 and 5 the plausible assumption was that the nitrogen might be introduced by a transamination and would thus be attached to the carbon corresponding to C-3." However from the labelling patterns of 42 derived from feeding [l-'4C]pyruvic acid and [4-'4C]erythrose Hornemann and co-workers con- cluded that in contrast the nitrogen is attached to the carbon equivalent to C-5 of shikimate pathway intermediate^.'^ This conclusion was initially called into question when it was reported by Rinehart and co-workers that in the C,N unit of another antibiotic pactamycin the nitrogen is indeed attached to the carbon equivalent to C-3.'07 However the same group soon found that the labelling and coupling pattern of 39 derived from [U-'3C,]glucose confirmed the conclusion drawn from the mitomycin experiments i.e.that the carbon carrying the nitrogen of the C,N unit is equivalent to C-5 of shikimate pathway intermediates. log Subsequently we have also con- firmed this for the C,N units of ansatrienin cf ''' and of naphthomycin. These findings together with the conclusion that formation of 43 must branch off from the shikimate pathway prior to dehydroquinic acid 4 provided the basis for a variety of hypotheses for the biosynthesis of 43.92,lo93 'lo 3.1.3 Formation of AHBA in cell-free extracts Based on the results described above and on a report from the laboratory of Jiao' ' that the amide group of glutamine is the best source of the nitrogen in 38 we proposed the pathway shown in Scheme 13 for the formation of AHBA."' The key intermediate aminoDAHP 44,had already been proposed by Hornemann et al.as a precursor of 43 but was suggested to arise from DAHP 3.92Specifically our hypothesis envisioned a modified DAHP synthase containing an additional subunit or domain which hydrolyzes glutamine to generate in the active site of the enzyme ammonia which immediately condenses with the aldehyde group of the second substrate 2.Conden-sation of this imine with 1 then gives aminoDAHP 44,which carries the nitrogen at the appropriate carbon. Cyclization to aminoDHQ 45 and dehydration to aminoDHS 46 catalyzed either. by the normal shikimate pathway enzymes or by a separate set of enzymes then sets the stage for dehydration and enolization of 46 to 43. The idea of in situ generation of ammonia by hydrolysis of glutamine has precedent in enzymes like anthranilate synthase' l2 or p-aminobenzoate synthase,' l3 which also contain amidohydrolase activies functioning in a similar way.'I4 To test this hypothesis we prepared the three postulated intermediates 44 45 and 46 all of them unknown compounds at the time and tested their conversion into AHBA in cell-free extracts of the rifamycin producer A.mediterranei and the ansatrienin producer S. collinus.' 15 'l6 AminoDAHP 44 was synthesized by a 15 step route from 2-deoxyglucose in 3% overall yield.' I7 AminoDHQ 45 was prepared116 from 44 by enzymatic cyclization using recombinant DHQ synthase from E. coli,"* for which 44 was an excellent substrate. AminoDHS 46 was prepared similarly from 5-deoxy-5-aminoshikimic acid116,1 19 using recombinant shikimate dehydrogenase from CHO @OTCOOH + H-?-OH I H-C-OH I CH20@ 1 PEP COOH I c=o I CH=NH y e I NH2-C-OH H-C-OH H-C-OH H-C-OH I H-C-OH AH20@ 44 arninoDAHP -@-OH I COOH COOH HO COOH HOhNH*=obNH2=ob OH OH NH2 43 AHBA 46 arninoDHS 45 arninoDHQ Scheme 13 Proposed pathway for the formation of AHBA E.coli.'20 The latter oxidizes the amino analog at 86% of the rate of its normal substrate 6. Enzymatic AHBA formation from these unlabelled substrates was assessed by an inverse isotope dilution assay adding a known amount of [7-I3C]-43 to the reaction mixtures at the end of incubation reisolating 43 and analyzing its silyl derivative by GC-MS for the depletion in I3C due to dilution with the enzymatically formed un-labelled 43. The efficiency of enzymatic conversion into AHBA in crude undialyzed cell-free extracts of A. mediterranei increased the further advanced the substrates were in the pathway. PEP 1 plus erythrose 4-phosphate 2 gave 6% of 43 44 gave 45% 45 gave 41% and 46 was converted into 43 essentially quantitatively.On the other hand DAHP 3 plus glutamine as substrate gave no AHBA. Formation of amino- DAHP 44 was also detected from 1+2 (9%) but not from 3 plus glutamine. 'l6 Similar results were obtained with cell-free extracts of the ansatrienin producer S. collinus. These exper- iments thus confirmed our hypothesis that the formation of AHBA occurs by a novel parallel route to the normal shiki- mate pathway which branches from the regular pathway in the very first reaction. However whether the hypothesis is correct in every detail remains to be established. The cell-free experiments did not demonstrate any dependence of 43 for-mation on glutamine or for that matter on any other nitrogen source.When [amide-' SN]glutamine was included in an incu- bation with 1 and 2 as substrates the resulting 43 showed only 2.7% of the "N enrichment of the glutamine. This result casts some doubt on the role of glutamine as the specific nitrogen donor since it would require a rather high concentration of unlabelled glutamine (25 mM) in the cell-free extract to account for the observed dilution of "N. Clearly the biochemical and mechanistic details of the pathway need to be worked out at the enzymatic level. 3.1.4 AHBA synthase from Amycofatopsis rneditevvanei We started our investigation of the enzymes of 43 biosynthesis with AHBA synthase from A. mediterranei for two reasons. One is that this is the only enzyme in the AHBA pathway which does not have an equivalent in the normal shikimate pathway.Thus a gene probe derived from the amino acid sequence of this enzyme would give us the best chance of Floss Natural products derived from unusual variants of the shikimate pathway 441 unequivocally identifying the rifamycin biosynthetic gene cluster. Secondly the reaction catalyzed by this enzyme is mechanistically intriguing. When aminoDHS 46 is aromatized chemically by acid or base treatment or by warming in buffer it gives exclusively protocatechuic acid 47 (Scheme 14) COOH I HOT OH 47 Protocatechuic acid OH \ 46 HO 43 Scheme 14 Chemical vs. enzymatic aromatization of aminoDHS behaving just like normal dehydroshikimic acid. 12' However the enzyme completely redirects the aromatization chemistry to produce 43 from 46 quantitatively.The mechanism by which this occurs is not obvious. AHBA synthase was purified 180-fold from A. mediterranei strain S699 by ammonium sulfate precipitation successive chromatography on DE52 Phenylsepharose and Sephadex G-200 followed by FPLC on MonoQ and Phenylsuperose columns to give homogeneous protein in 5% overall yield.122 The native enzyme is a homodimer of about 39 kDa subunit molecular mass. Gas-phase microsequencing of the protein itself and of two cyanogen bromide cleavage peptides gave a 10 amino acid N-terminal sequence and two internal sequences of 16 and 26 amino acids. These were used to design degenerate oligonucleotides biased for high G +C DNA which served as primers for polymerase chain reaction (PCR) on A.mediter-ranei genomic DNA as template. DNA sequencing of the initial PCR products allowed the refinement of the primers to non-degenerate oligonucleotides two of which were then used to produce a PCR product of 717 bp. This encoded the N-terminal and both internal amino acid sequences of the enzyme. This PCR product was then used as a stringent probe to clone the AHBA synthase gene from an A. mediterranei S699 genomic DNA library size fractionated to 3040 kb in the cosmid vector pOJ446. Six cosmid clones hybridizing to the probe were isolated and the AHBA synthase gene was sub- cloned to a 2.3 kb Xhol fragment which was then sequenced. Analysis of the sequence revealed that AHBA synthase is encoded by an open reading frame (ORF) of 1164 bp desig- nated rifK corresponding to 388 amino acids with a predicted molecular mass of 42 28 1.I2* 123 Most importantly the deduced amino acid sequence of AHBA synthase showed homology to those of a family of genes involved in transfor- mations of sugar nucleotides for the biosynthesis of antibiotics or of bacterial cell wall polysa~charides.'~~ Although the function of most of these genes was entirely speculative the product of one gene ascC has been studied extensively by the group of L~u.'~~ This enzyme was known to contain pyridoxamine phosphate (PMP) as a cofactor providing the first clue as to the catalytic mechanism of AHBA synthase.Further sequence alignments demonstrated that AHBA syn- thase contains a typical pyridoxal phosphate (PLP) binding motif and that it belongs to the subgroup I1 of PLP/PMP- dependent aminotransferases.126 To establish whether the enzyme contains PMP or PLP as the cofactor and to study its mechanism further rlfiv was overexpressed in E. coli. A 1.6 kb EcoRI fragment carrying the entire rifK gene except for 23 nucleotides at the start of the coding region was cloned into the expression vector pRSET for expression as a (His)6 fusion protein under the control of the T promoter. Expression in the host E. coli BL21 (DE3)/ pLyS gave after optimization of the induction and culture conditions recombinant AHBA synthase as 2.7% of the total soluble protein. Homogeneous protein was obtained by a 38-fold purification involving ammonium sulfate precipitation chromatography on DE52 and adsorption on a Ni resin in 46% yield.Except for the predictable effects of the polyhistidine moiety higher molecular mass and PI the properties of the recombinant protein match those of the native enzyme. 122,123 Neither the truncation of the enzyme by eight amino acids nor the addition of the six histidines had a significant effect on K or V,,,. In conjunction with site-specific mutagenesis studies the expression clone has recently been reengineered to express the complete gene with and without the polyhistidine tag and in much higher yield,'27 but all the mechanistic studies so far were carried out with the truncated but fully active enzyme.Recombinant AHBA synthase was inhibited by the PLP- inhibitor gabaculine 48. Its UV spectrum showed an absorp- tion at 418 nm typical of PLP in a Schiffs base linkage which COOH 48 Gabaculine changed to 330nm upon reduction with sodium boro-hydride.'22-123 The presence of one mole of PLP per subunit was confirmed by electrospray-MS of the native and the borohydride-reduced enzyme. Reduction of the enzyme-substrate or the enzyme-product complex with tritiated sodium borohydride followed by hydrolytic workup gave N-pyridoxyl-AHBA 49 from both incubations (Scheme 15). H I 0 GCH OH [3H]NaBH4 \ H+ [3H]NaBH4/ Scheme 15 [3H]NaBH Reduction of the AHBA synthase-substrate and synthase-product complexes These results demonstrate that (i) the enzyme uses PLP rather than PMP as cofactor (ii) the reaction proceeds via a Schiff's base between PLP and the amino group of the substrate 46 and (iii) the predominant enzyme-intermediate species present at equilibrium is more product- than substrate-like.I2' Based on these results a plausible mechanism for the conversion 46j.43 can be formulated (Scheme 16) which involves Schiff's base formation followed by tautomerization with loss of the C-5 442 Natural Product Reports 1997 COOH H OH OH N//CH OH 46 I 49 enzyme "t?, H H H I COOH COOH HR H6S 0-50 enzyme H6S ...H-O ... enzyme 49 Scheme 16 Mechanism of the AHBA synthase reaction and a proposed structure of the PLP-substrate complex relative to an enzyme active site base hydrogen to give the quinoid intermediate 49 followed by elimination of the hydroxy group from C-4.These reactions are completely analogous to the a,p-elimination reactions of amino acids which are catalyzed by serine dehydratase and mechanistically related enzymes. 12* The resulting Schiff 's base of dienone 50 can then undergo enzymatic enolization fol- lowed by hydrolysis or the hydrolysis of the Schiffs base may occur first followed by spontaneous aromatization of the free dienone. To determine whether the aromatization of the dienone is still enzyme-controlled we synthesized the substrate 46 labelled stereospecifically with deuterium at C-6 with either 6R or 6s configuration. Conversion of both labelled substrates into 43 demonstrated that the aromatization proceeds cleanly with stereospecific loss of the pro-S hydrogen.123 This finding not only establishes that the proton removal from C-6 is enzyme catalyzed but it also fits the stereochemcial paradigm established for virtually all PLP-catalyzed reactions of amino acid i.e.that proton transfer steps occur on only one face of the relatively planar PLP-substrate complex allowing for extensive proton recycling by a single base.cf 129 130 In all the PLP reactions studied for their stereochemistry this is the si face at C-4' of the cofactor predicting an arrangement for the coenzyme-substrate complex in AHBA synthase as shown in Scheme 16 (the complexation of 49 with the enzyme). In this conformation a single base presumably the &-amino group of the active site lysine can successively catalyze deprotonation at C-5 protonation of the C-4 OH leaving group and abstraction of the pro-S hydrogen from C-6.3.2 Molecular genetics of rifamycin biosynthesis With the availability of the cloned rfK gene the stage was set for the investigation of the rifamycin biosynthetic gene cluster by analysis of the DNA surrounding this gene. This approach is based on the observation in all cases examined so far that in Actinomycetes the genes coding for a given antibiotic biosynthesis are arranged in a cluster.131 Before embarking on the analysis of the presumed rifamycin biosynthetic gene cluster it was necessary to prove that rfK is indeed involved in Floss Natural products derived from unusual variants of the shikimate pathway 38 biosynthesis.To this end an inactive version of the gene was created by insertion of the hygromycin resistance gene into the BglU site of r$K. Transfer of this modified gene into the integrative vector pSK -carrying a carbenicillin resistance marker and lacking a Streptomyces origin of replication allowed for the transfer into A. mediterranei S699 by electro- poration. Selection for a hygromycin - carbenicillin -pheno-type gave several transformants resulting from single crossovers. From these double crossover mutants were then obtained which showed the hygromycin - carbenicillin' phenotype and in which the functional AHBA synthase gene had been replaced by the non-functional copy.These mutants did not produce any 38 under normal growth conditions but 38 production was restored upon supplementation with AHBA. Supplementation with 13C-labelled 43 resulted in 13C-labelled rifamycin of virtually the same enrichment as the precursor. Thus the cloned gene r$K is clearly involved in 38 biosynthesis. 123 One of the cosmids from the original cloning of rfK covering a region of about 40 kb of surrounding DNA was chosen for further analysis. By end-sequencing of randomly generated DNA fragments from a 22 kb subclone of this cosmid and comparison of the deduced amino acid sequences with protein sequences in data bases we established that this region did indeed contain genes which were likely involved in 38 biosynthesis.These analyses revealed homologies to two P450 enzymes to sections of the erythromycin polyketide synthase from Saccharopolyspora erythrea and to DAHP and DHQ synthases.' 32 This justified the complete sequence analy- sis of the 22 kb fragment to give the map shown in Scheme 17.'33 Some additional sequencing was done on the remaining DNA of cosmid pFKN108 as also shown in the map. The region furthest upstream from r$K encodes the terminal region of a modular polyketide synthase. By an inactivation exper- iment it was confirmed that this region indeed encodes part of the rifamycin polyketide synthase. The mutant in which this region had been deleted was no longer able to synthesize 38 even when supplemented with 43.12' The gene immedi- ately downstream from the PKS is homologous to genes 1 = PKS (61 Yo similarity 44% identity eryA 7 Sac.ery.) 2 = Amide synthase (45.9% similarity 24.6% identity nhoA S. typh.) 3 = unknown 4 = Dehydroquinate synthase (63.5% similarity 41.6% identity aroB M. tuberc.) 5 = DAHP synthase (63.9% similarity 41.4% identity phzF P. fluor.) 6 = Shikimate 5-dehydrogenase (61 % similarity, 44% identity aroE P. aerug.) 7 = 3-Amino-5-dehydroxybenzoicacid synthase 8 = Oxidoreductase (48.6% similarity 28.1% identity ORF S. ann.) 9 = Phosphoglycolate phosphatase (53.9% similarity 32.9% identity pgpC A. eutr.) 10 = Glucose kinase (48.9% similarity 26.8% identity glk S. coelicolor.) 11 = Lincomycin biosythesis (53.0% similarity 31.5% identity lmbE S.linc.) 12 = Esterase 13 = Antibiotic export (81.2% similarity 66.1Yo identity ptr S. pristinaespiralis) 14 = Transcription repressor (51.9% similarity 25.9% identity act//orf7 S. coef.) 15 = unknown 16 = Cytochrome P450 monooxygenase (52.2% similarity 28.6% identity rif ORF17) 17 = CytochromeP450 monooxygenase (62.3% similarity 42.4% identity P450 S. thermo.) 18 = PMP dependent dehydratase (63.2% similarity 45.3% identity ribH S. typhimurium) 19 = Glycosyl transferase (53.6% similarity 36.0% identity snoT S. nodosus) 20 = dTDP-glucose 33 epimerase (64.5% similarity 46.0% identity sfrM S. griseus) 21 = Type Ill PLP dependent aminotransferase (52.6% similarity 32.6% identity argD Anabaena sp.) 22 = Oxidoreductase (similar to rifORF8) 23 = Alkanal monooxygenase (60.0% similarity 43.0% identity lmbY S.linc.) 24 = Cytochrome P450 monooxygenase 25 = Cytochrome P450 monooxygenase 26 = Type II Dehydroquinate dehydratase (64% similar 41Yoidentity to aroD Actinobacillus pleuro.) 27 = Transketolase of pentose phosphate pathway (70% similar 40% ident ity to tkt Musculus) Scheme 17 The rifamycin biosynthetic gene cluster from Amycolatopsis mediterranei which encode enzymes catalyzing the transfer of acyl groups from thioesters to aromatic amines. It is proposed that this gene codes for the enzyme which releases the assembled polyketide chain from the PKS by amide formation with the amino group of the starter unit to generate the macrolactam ring. Consistent with this notion the terminal region of this PKS lacks the thioesterase function which is normally present in modular polyketide synthases generating macrolide antibiotics.l9 Located immediately upstream from rifK are three genes showing strong homology to early shikimate pathway genes namely shikimate dehydrogenase DAHP synthase and DHQ synthase. Interestingly the homology of the DAHP synthase gene is strongest to plant-derived DAHP synthases not to the microbial enzymes. The presence of a possible shikimate dehydrogenase gene is surprising since such an enzyme is not required in the proposed AHBA pathway. On the other hand no gene homologous to dehydroquinate dehydratase an enzyme that should be required was found in this region. The nearest gene with strong homology to a type I1 dehydroquinate dehydratase ORF26 is located about 30 kb downstream of the AHBA synthase gene; its inactivation reduces 38 formation to 10% of wild-type levels with AHBA supplementation restor- ing full 38 production.126b Inactivation experiments showed that ORFs 4 and 6 encoding DHQ synthase and shikimate dehydrogenase homologs are not essential for rifamycin bio- synthesis.However a mutant in which all three genes had been deleted synthesized more than 10-fold lower levels of rifamycin than the wild-type and wild-type levels of production could be restored by supplementation with AHBA. 27 Expression of the DAHP synthase homologous gene in E. coli gave a protein which catalyzed the synthesis of DAHP 3 but no synthesis of aminoDAHP 44 has so far been demonstrated with this protein.134 Our tentative interpretation of these findings is that both the DAHP synthase and the DHQ synthase mutants are complemented by the products of homologous genes elsewhere in the genome but that the process for DAHP synthase is only inefficient possibly because this protein must combine with another protein in order to synthesize 44.444 Natural Product Reports 1997 Immediately downstream from the rifK there is a gene with homology to various oxidoreductases which is translationally coupled to rifK. The function of this gene is unknown but is essential for AHBA synthesis since a mutant in which this gene is inactivated shows the same phenotype as the AHBA syn- thase minus mutant.'35 Also part of the same transcription unit with ORFs 7 and 8 are three additional genes showing homology to a phosphatase a glucose kinase gene from S.coelicolor and to ImbE a gene of unknown function from the lincomycin biosynthesis pathway. Both ORF 9 and ORF 10 have been inactivated to give 38 non-producing mutants which can be complemented with AHBA to restore antibiotic syn- The next three open reading frames 12 13 14 and possibly 15 seem to be involved in regulation and possibly antibiotic resistance. ORFs 16 and 17 encode cytochrome P450 enzymes proposed to catalyze some of the postsynthetic hydroxylations of the macrocyclic lactam system. The enzymes encoded by ORFs 18 to 21 seem to be involved in the synthesis and transfer of a modified sugar moiety.Particularly intriguing is the presence of a close homolog of the genes ascC and rfbH which encode PMP-dependent dehydrases involved in the 3-deoxygenation of 6-deoxyhexoses. 25 Since rifamycin is not a glycoside the presence of these genes was surprising. Inacti- vation of ORF 18 resulted in a mutant with unimpeded ability to synthesize rifam~cin.'~~ We suspect that these genes encode the ability to produce a rifamycin-related glycoside which is not expressed under our culture conditions. The detailed biochemistry of AHBA formation is now being studied by expression of all the genes identified as essential for AHBA synthesis followed by characterization of their individual functions. 3.3 Cloning and analysis of other AHBA synthase gene clusters Using the cloned rifrv gene from A.mediterranei as a probe has made it possible to clone homologous genes from several other organisms providing access to gene clusters encoding the biosynthesis of other antibiotics containing AHBA-derived C,N units. In collaboration with the group of E. Leistner revised to one which no longer fits the pattern of a C,N unit.'50 University of Bonn we cloned and sequenced an AHBA In the other cases biosynthetic studies have demonstrated synthase gene from the ansamitocin producer Actinosynnema different biosynthetic origins of the C,N units. pretiosum presumed to be involved in maytansinoid biosyn- thesis. 137 Partial sequence analysis of the surrounding DNA revealed the presence of homologs of several other genes identified in the AHBA cluster of A.mediterranei e.g. ORFs 4 5 and 8. However except for the oxidoreductase gene the linkage arrangement of these genes is different from that in A. medzterrunei. 38 The ansatrienin producer S. collinus Tii1892 contains two AHBA synthase genes both of which were cloned and ~equenced.'~~ This is consistent with the fact that this strain produces a second ansamycin antibiotic the naphthoquinoid compound naphthomycin. I4O It suggests that separate gene clusters may encode the formation of 10 and naphthomycin respectively. The group of Sherman'406 at the University of Minnesota has used riflv to clone a homologous gene from the mitomycin producer 5'. lavendulae. Preliminary analysis of the surrounding DNA presumed to encode mito- mycin biosynthesis revealed the presence of sequences homologous to several of the genes encountered in the AHBA cluster of A.rnediterrannei including the oxidoreductase the phosphatase and the DHQ synthase and shikimate dehydro- genase homologs. Thus the availability of this key gene opens the way for the cloning of a number of important antibiotic biosynthesis gene clusters. 3.4 C,N Units not derived from AHBA When the biosynthetically unique nature of the C,N unit in ansamycin and mitomycin antibiotics was first discovered similar structural elements were recognized in a number of other microbial metabolites. As pointed out by H~rnemann'~' and others,'6 14* C,N units which may have the same biosyn- thetic origin are also present in the manumycin~,'~~-'~ e.g.manumycin A 51 and the related asukamycin 11 in the validamycins 145 e.g. validamycin A 52146and the a-amylase inhibitor acarbose 53,14,. 14' and in the structure originally proposed for kinamy~in.'~~ The latter structure has since been HO&O 51 Manumycin A 3.4.1 Acarbose and validamycin Feeding experiments with [U-'3C3]glycerol to the acarbose producer Actinoplunes sp.I5' and with [U-'3C6]glucose to the validamycin producer S. hygrosc~picus,'~~~ 153 revealed coupling and labelling patterns which were incompatible with a shikimate pathway origin of the C,N unit in these com- pounds. Consistent with this finding neither AHBA nor 3-aminobenzoic acid the precursor of pactamycin was incor- porated into 53.15'Rather the labelling and coupling patterns in both cases pointed to an origin of the aminocyclitol moiety in 53 and both cyclitol moieties in 52 from cyclization of a linear seven-carbon sugar generated by the pentose phosphate As shown in Scheme 18 for 53 this precursor tH20H CH20H I c=o I HO-G-H Cyclase I IH-C-OH 13CH20H 13CHOH I I I3CH20H 55 Valiolone \ \ r-------- 54 Sedoheptulose 7-phosphate [U-13C3]Glycerol 53 Acarbose HU Scheme 18 Biosynthesis of the C,N unit of acarbose viu the pentose phosphate pathway might be sedoheptulose 7-phosphate 54 which could undergo cyclization to valiolone 55 by a mechanism resembling that of dehydroquinate ~ynthase'~~ from the shikimate pathway.There is one problem with this otherwise plausible proposal 54 has the opposite configuration at C-5 from that of the cyclitol moieties of 52 and 53.However cyclization by a DHQ synthase-like mechanism would involve transient oxidation of C-5 to the ketone and in this process the stereochemical 'defect' could be corrected. Alternatively the cyclization may involve the 5-epimer of 54 ido-heptulose 7-phosphate as substrate or the cyclization could produce the 2-epimer of 55 which could then be epimerized by enolization. Some recent experiments in our laboratory indicate some incorporation of 7-tritiated 55 into 52,155but degradations are necessary to verify this preliminary result. 3.4.2 Asukamycin and manumycin Feeding experiments with [7-' 3C]-43showed no incorporation into either 11 or 51.'56 Analysis of the labelling and 13C coupling patterns of 11 and 51after feeding [U-'3C,]glycerol also ruled out a shikimate pathway origin.Rather these patterns together with those seen after feeding various specifically labelled precursors indicated an assembly of this C,N unit from a four-carbon dicarboxylic acid like succinic Floss Natural products derived from unusual variants of the shikimate pathway acid and a triose phosphate (Scheme 19).156 The orientation of the triose unit in the C,N moiety was determined by feeding + -6; 5OOHD COOH &NH2 f '80; 57 / 1 I H I CONH2 11 56 4-Hydroxy-3-nitrosobenzamide Scheme 19 Biosynthetic origin of the C,N unit in asukamycin and manumycin and of 4-hydroxy-3-nitrosobenzamide stereospetifically labelled samples of glycerol.The pro-R hydroxymethyl carbon of glycerol which is phosphorylated in metabolism gives rise to C-3 whereas the pro-S carbon of glycerol labels the carbonyl carbon C-1 in the product.157 Recent work in the laboratory of S. J. Gould and co-workers led to the isolation of a new iron chelator 4-hydroxy- 3-nitrosobenzamide 56 from cultures of S. murayamaensis which was shown to arise from an isomer of AHBA 3-amino- 4-hydroxybenzoic acid (3,4-AHBA) 57. 15* The same group subsequently showed that 57 is not formed via the shikimate pathway but showed labelling patterns from various general precursors which indicated a biosynthetic origin similar to that of the C,N units in 11and 51.This led them to propose that 57 might also be a precursor of these C,N units.159 This was quickly confirmed in joint work with our laboratory first by demonstrating incorporation of [2-2H]-57 into 11 carrying deuterium at C-3 (Scheme 19). Subsequently [7-I3C]-57 was synthesized and shown to be incorporated specifically into the C,N units of both 11 and 51 validating Gould's hypothesis.16' Evidently just as 43 in the biosynthesis of ansamycins 57 serves as the starter unit for a polyketide biosynthesis and its amino group as that in the ansamycins is linked to the carboxy terminus of a polyketide chain presumably in a reaction catalyzed by an amide synthase. It will be interesting to see whether at the genetic level there are similarities between these processing reactions despite the different origins of the starter units.It is likely particularly in light of the precursor-directed biosynthesis work of Zeeck and co-workers'61 on the manumycins that the final oxidative modi- fication of the aromatic ring to the epoxyquinol structure of 11 and 51 in which both oxygens are derived from atmospheric oxygen,156 occurs late in the biosynthesis. A plausible mech- anism based on work by the laboratories of Gould and Dowd162 is shown in Scheme 20. However this very attractive mechanism presents one problem it dictates that the epoxide and the quinol oxygen must be syn oriented. While 11and the majority of the manumycins and related compounds meet this requirement two prominent compounds manumycin A 51 and manumycin B carry these two oxygens in an anti arrange-ment.Since 51 as well as manumycin B were nevertheless formed from 5716' one must invoke one of three possibilities (i) the stereochemistry of the epoxyquinol moiety of manu- mycins A and B is altered after its initial formation; (ii) the stereochemical assignment of these two compounds is in error (very unlikely); or (iii) the dioxygenase mechanism of epoxy-446 Natural Product Reports 1997 ,OH 70 Jvvv. -0-0 I > I I 0 @[\{ cLO' / Scheme 20 Proposed mechanism for the oxidative formation of the epoxyquinol moiety of asukamycin quinol formation shown in Scheme 20 does not apply to these compounds. Work is in progress to solve this interesting puzzle.4 Biosynthesis of phenazines 4.1 Background Since the discovery of pyocyanin 58,163 the first naturally occurring phenazine which was commonly observed as a blue pigment on infected wound dressing^,'^^ some 75 different Me I OH 58 Pyocyanin phenazines have been reported from various bacterial cul- tures. l 65 Different species of Pseudomonas were originally the most prominent sources of these compounds but a number of other bacteria also produce phenazines and with the beginning of intensive screening of Actinomycetes for antibiotic activity a number of phenazines,'66 many of them of unique struc- tures,'67* were isolated from Streptomyces species and related genera. Our interest in this class of compounds arose from the reported isolation of a unique class of dimeric phenazines the esmeraldins from Streptomyces antibioticus strain Tii2706 by the group of Zahner.'69 170 The esmeraldins are found as a mixture of different congeners.Esmeraldin A 59b is a mixture of long chain fatty acid esters of esmeraldic acid 59a at the 22-hydroxy group. Esmeraldin B 59c carries a 6-methylsalicylic acid moiety in the same position. Free esme- raldic acid 59a has so far not been isolated from natural sources. Cooccurring with the esmeraldins are their mono- meric subunits saphenic acid 60aI7l and saphenamycin the ester with 6-methylsalysilic acid both of which had been isolated before from other Streptomycetes and the saphenyl esters 60b which again carry a long chain fatty acid rnoiety.l7' An interesting facet of the structure of the esmeraldins was discovered during our biosynthetic studies of these com-pound~.'~~ In the course of this work the isolated mixture of esmeraldins was subjected to alkaline hydrolysis and the resulting esmeraldic acid 59a was treated with diazomethane.During purification of the resulting dimethyl ester the sample was resolved by chromatography into two compounds present in about equal amounts. The two compounds had the same molecular mass and showed almost identical NMR spectra suggesting that they were diastereomers. The CD spectra of the 18 19 A17 13 10 COOR 21 59a Esmeraldic acid R = R' = H 59b EsmeraldinA R = H; R' = a 12-Methyltridecanoyl b Tetradecanoyl c 12-Methyltetradecanoyl d 14-Methylpentadecanoyl e Hexadecanoyl f 14-Methylhexadecanoyl g 15-methylhexadecanoyl h Heptadecanoyl i Unassigned C-18 acyl j 16-Methylheptadecanoyl 59c EsmeraldinB R = H; R' = 2-Hydroxy-6-methylbenzoyl 13 Me 12 OR' Y 60a Saphenic acid R = R' = H 60b Saphenyl esters R = H; R' as in 59b 60c Saphenamycin R = H; R' as in 59c 60d Saphenic acid methyl ester R = Me; R' = H two compounds (61 and 62) were almost but not exactly mirror images of each other indicating that they were epimeric at the stereocenter dominating the spectrum.They showed rather high molar elipticities 2 x lo5 at 254 nm 1.2 x lo5 at 362 nm suggesting that this stereocenter has significant helical character.Compounds 61 and 62 were derivatized to their A COOMe 61 (or enantiomer) COOMe 62 (or enantiomer) Mosher esters at the 22-OH group and their spectra revealed that each compound again represented a mixture of at least two diastereomers. The larger Ad of the C-23 compared to the C-26 methyl protons suggests that the two isomers in each sample are epimeric at C-22. Similarly the methyl ester of saphenic acid 60a obtained from hydrolysis and esterification of the isolated 60b and 60c was found to be racemic. Control experiments showed that under the conditions of the alkaline Floss Natural products derived from unusual variants of hydrolysis no deuterium from the solvent methanol was incor- porated at C-22 of 61 and 62 and no conversion of pure 61 into 62 or vice versa was observed under these conditions.We conclude from these results that the esmeraldins occur in nature as a mixture of diastereomers of a single configuration at C-25 and two epimeric configurations at N-15 61 and 62 each of which is again a mixture of two epimers at C-22. This arrangement has interesting implications for the mode of dimerization leading to the esmeraldins. 4.2 Biosynthesis of saphenamycins and esmeraldins The biosynthesis of phenazines has been extensively studied in Pseumonas species particularly by the groups of Herbert and of Hollstein. The shikimate origin of the phenazine skeleton in these organisms was amply demonstrated by feeding exper- iments with labelled precursors. These gave good incorpor- ations of 6 into the phenazine skeleton they showed that both halves of the phenazine ring are labelled by 6 and they demonstrated the coupling mode shown in Scheme 21 by YOOH YOOH OH COOH COOH 2x6 63 Phenazine-l,6-dicarboxylic acid Scheme 21 Pairing scheme of shikimate units in the biosynthesis of phenazines which two molecules of 6 are linked to each other to give the phenazine ring system.17u79 No phenazines carrying the extra methyl group characteristic of the saphenamycins and esmer- aldins had been studied biosynthetically and thus the origin of this extra carbon was not known nor was it known whether the carbon framework of these phenazines in Streptomyces has the same origin as demonstrated for the Pseudomonas phena-zines.We therefore fed [U-14C]-6 and [1,7-'3C,]-6 to cultures of S. antibioticus Tu2706 but observed no incorporation of label into 59 or 60. Isolation and hydrolysis of the cellular protein from the cultures fed [U-14C]-6 showed little incorporation of radioactivity into the amino acid fraction suggesting the possibility that the cells of this organism are impermeable to 6. The origin of the aromatic rings in 59 and 60 was then probed in a series of feeding experiments with differently 13C-labelled glycerol samples. The observed labelling pattern is shown in Scheme 22. Although not all the expected enrichments and couplings were seen unequivocally due to the complexity and poor quality of the spectra the results nevertheless demon- strate the shikimate pathway origin of these structures.They clearly show that all seven carbon atoms of a shikimate pathway intermediate are incorporated ruling out a pathway via the aromatic amino acids. Finally they reveal a pairing scheme for the shikimic acid 6-derived rings identical to that demonstrated for the Pseudomonas phenazines (Scheme 2 1). 73 In further feeding experiments it was also found that the methyl group of methionine is not incorporated into 59 and 60 whereas C-2 of acetate is significantly incorporated labelling exclusively the extra methyl groups C-13 in 60 and C-23 and C-26 in 59. Thus the extra methyl groups of 59 and that of 60 are generated by chain extension of a suitable precursor with an acetate unit followed by loss of the acetate carboxy group.The process of addition of the extra carbon atom presum- ably requires a carboxy group activated as its thioester for a Claisen condensation with acetyl CoA or malonyl CoA. A general precursor of phenazines in Pseudomonas sp. which would meet this requirement is phenazine- 1,6-dicarboxylic the shikimate pathway COOH COOH efficient incorporation into 59 labelling the expected positions CH20H revealed the next two intermediates on the pathway to the -& --+vlb CHOH esmeraldins.’ An attractive mechanism by which saphenic CH20H HO’. OH OH COOH 6a 63 Phenazine-l,6- 0,- [U-13C3]Glycerol A [l ,3-13C2]GlyceroI dicarboxylic acid I 0 [2-13C]Glycerol [2-13C]Acetate I COOH COOH 64 6-Acetophenazine -1-carboxylic acid J I $OOH 596 60b aPredictedlabeling pattern bObserved labeling pattern Scheme 22 Labelling patterns of 59 and 60 after feeding general precursors and intermediates in their biosynthesis acid.This compound was therefore synthesized carrying 13Cin the carboxy group and fed to the esmeraldin producer. A specific incorporation of 13% into 59 was observed and the label resided in the expected positions C-21 C-22 C-24 and C-25. Next [13-2H3]-6-acetophenazine-1-carboxylic acid (64) and [12,13-2H,]saphenic acid 60a were prepared and fed. Their HO Ho\/ HY COOH HOOC H HOOC acid might dimerize to the esmeraldin ring system is shown in Scheme 23. 173 This process invokes dihydrophenazine inter-mediates which as vinylogous carbinolamines can undergo dehydration; it may involve either radical or ionic intermedi-ates.Importantly the stereospecificcoupling between C-12 of one unit and C-4 of the other must be the first of the two coupling reactions to take place in order to account for the formation of two diastereomers in the subsequent ring closure between N-5 of one unit and C-3 of the other. Quite possibly this second coupling reaction may not be strictly enzyme controlled. 4.3 Mode of formation of the aromatic ring and of the extra methyl group The branch point at which phenazine formation diverges from the main shikimate pathway in Pseudomonas has not been clearly established. Chorismic acid has been suggested as an intermediate based on indirect evidence,lS1 182 but no incor-poration of anthranilic acid into phenazines has been observed.On the other hand genetic studies have implicated a second copy of an anthranilate synthase gene in phenazine biosynthesis by P~eudomonas,~~~, lS4 suggesting either that the lack of incorporation of anthranilic acid is a permeability problem or that an intermediate in anthranilate biosynthesis e.g. compound 65 (Scheme 24),18’ 186 is the actual molecule undergoing the dimerization reaction. In light of the lack of incorporation of shikimate into 59 and 60 a situation remi-niscent of the biosynthesis of the C,N unit in ansamycins and mitomycins (see section 3.1.l) we considered an alternative hypothesis invoking 5-deoxy-5-aminoshikimic acid 66 as the phenazine precursor in Streptomyces antibioticus (Scheme 24 path b).This possibility was explored by synthesizing [1,6-13C2]-66and feeding it to S. antibioticus. No incorporation of 13C label into either 59 or 60 was observed. Although we cannot rule out the possibility that 66 like 6 cannot enter the cells this result does render the path b shown in Scheme 24 less likely. We are presently attempting to subject the hypothesis shown as path a in Scheme 24 to experimental scrutiny by / H Scheme 23 Hypothetical mechanism for the dimerization of saphenic acid into the esmeraldins 448 Natural Product Reports 1997 COOH /-\ 65 \ 1 COOH path a inversion retention I 1 1 COOH HOOC H COOH H COOH 63 path b I COOH I 66 5-Deoxy-5-aminoshikimic acid Scheme 24 Hypothetical pathways to phenazines via 65 and via 5-deoxy-5-aminoshikimic acid 66 synthesizing and feeding chorismic acid 7 compound 65 and anthranilic acid.Extension of carbon chains by methyl groups derived from C-2 of acetate is encountered in a number of antibiotic biosynthetic pathways (e.g.myxo~irescin,'~~ virginiamycin,Is8 elaiomycin '89 brevetoxin B190 and pulv~mycin'~~); yet little mechanistic information about this process is available. The availability of acetic acid carrying a chiral methyl group made it possible to study the stereochemistry of this process in the case of 59 biosynthesis. Thus (R)-and (S)-[2-2H,,3H]acetic acid192 was fed to S. antibioticus Tii2706 and the resulting esmeraldin samples were degraded by a Lemieux-von Rudloff degradation.193 Chirality analysis''.194 of the resulting acetic acid samples showed that the (R)-acetate gave a methyl group in the product of 45% ee S configuration whereas the sample derived from (@-acetate showed 42% ee R config~ration.'~~' This result was rather surprising because we had expected to find retention of configuration accompanied by ca. 50% loss of the optical purity. As detailed in Scheme 25 the condensation reaction would produce two tritiated species with one of these containing 'H and the other 2H,in the second methylene hydrogen. The first species upon further decarboxylation will give an achiral methyl group whereas the second species will give an optically active methyl group.Enzymatic Claisen condensations are known to proceed with inversion of config- uration whereas b-decarboxylations seem to proceed most frequently with retention of configuration. 194-196The replace- ment of C-1 of acetate by the incoming substituent is the equivalent of another inversion step therefore we expected to see overall retention of configuration. The fact that the experiments demonstrate the opposite most likely means that the decarboxylation step also proceeds with inversion stereo- chemistry. This seems to be the first case in which the stereochemistry of such a carbon chain methylation from C-2 of acetate has been investigated. 5 Conclusion The work described here illustrates that the shikimate path- way is a rich source of structural diversity in biochemical compounds.It not only generates end products which serve as the starting materials for the biosynthesis of countless natural (+X2T) achiral Scheme 25 Stereochemistry of the introduction of the extra methyl group from C-2 of acetate products but as a very complex metabolic pathway it also provides ample opportunities for 'derailments' along the path- way which through often very intriguing chemistry lead to additional unique secondary metabolites. With an increased understanding of these unusual variants of the shikimate pathway it should become possible to cause through genetic engineering other diversions in this metabolic route leading to the formation of novel metabolites which can be evaluated for potential biological activities.6 Acknowledgements One of the joys and rewards of an academic career is the opportunity to interact with many bright young people and to observe and foster their development into creative and produc- tive independent scientists. It is therefore both a pleasure and a desire to acknowledge the many co-workers whose dedicated and enthusiastic work forms the basis of this report. Their names appear in the appropriate references. The author also wishes to thank Mrs Kay B. Kampsen for her invaluable help in preparing this manuscript and the National Institutes of Health for the support of this research through grant A1 20264. 7 References 1 E. 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ISSN:0265-0568    

DOI:10.1039/NP9971400433

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

Secondary metabolites from marine microorganisms bacteria protozoa algae and fungi. Achievements and prospects ~~ ~ Francesco Pietra Laboratorio di Chimica Bioorganica Universita di Trento 38050 Povo- Trento Italy 1 Introduction 2 Bacteria (Eubacteria) 2.1 Actinobacteria 2.2 Cyanobacteria 2.3 0ther bacteria 3 Archaebacteria 4 Euglenoids (Euglenoidea Euglenozoa Protozoa) 5 Dinoflagellates (Dinoflagellatea Dinozoa Protozoa) 6 Ciliates (Ciliophora Protozoa) 7 Chrysophytes (Chrysophyceae Phaeophyta Chro-mista) 8 Diatoms (Bacillariophyceae Diatomae Chromista) 9 Eustigmatophytes (Eustigmatophyceae Phaeophyta Chromista) 10 Raphidophytes (Raphidophyta Chromista) 11 Prymnesiophytes (Prymnesiophyta=Haptomonada Chromista) 12 Cryptophytes (Cryptophyceae Cryptophyta Chro-mista) 13 Prasinophytes or grass-green scaly algae (Prasino- phyta Plantae) 14 Green microalgae (Chlorophyta Plantae) 15 Red microalgae (Rhodophyta Plantae) 16 Fungi (Eumycota) 16.1 True marine ascomycetes 16.2 True marine deuteromycetes 16.3 Terrestrial-genera deuteromycetes 17 Products of proven or alleged microbial origin isolated from marine macroorganisms 18 References 1 Introduction Advances in natural product chemistry are expected to rely ever more on interdisciplinary research and this is particularly important for marine microbial products where the marine microbiologist must provide material for study.The few tropical blooms of cyanobacteria or mass of microalgae which may be collected in wild form offer sporadic exceptions.However competent microbiologists for taxonomy and bio- mass growth of marine microorganisms are rare at least in academia. Such a gathering of complementary expertise is more common in industry although research plans in industry are often strongly biased by economic pressures and may not last long enough to turn out to be productive in new areas. The history of the early approach of the pharmaceutical industry to marine natural products is illustrative of this point. These are some reasons why marine natural products from microbes have lagged behind those from macroorganisms. Nonetheless the first successful economic ventures with marine natural products were based only on microbial prod- ucts,' mostly in the Japanese industry for work on dinoflagel- lates and actinobacteria.Therefore marine microorganisms deserve more and more organized attention by the natural products chemist. To provide a background this review article evaluates achievements and prospects in this area; it is com-prehensive as to the coverage of the groups of marine micro- bial organisms that have yielded to natural products chemistry but very selective because of space limitation in terms of examples. The primary criterion of choice was the originality Pietra Secondary metabolites from marine microorganisms of the molecular structure i.e. exclusive to the sea and significant in a wide biological context. To save space for the dinoflagellate polyethers the smallest representative molecules for each chemical class have been given.Only the latest references are given and regrettably pioneers in the field may therefore not be properly acknowledged. This account of marine microbial products follows the largely agreed view that along the lineage from the elusive progenote to the Eubacteria (from here on called bacteria and for which a much simplified taxonomic classification is used) branching occurred to the Archaebacteria and the Eukaryotes. The latter first appeared as amitochondriate protists (micro- sporidians diplomonads and trichomonads) which are now found mostly as parasites and for which secondary metabolites are not known followed by the crown group protists. These comprise uni- and multi-cellular organisms perhaps with the euglenoids and amoeba1 lineages as initial stages ending in plants animals and fungi.Evolutionary views on fungi dino- flagellates ciliates and red algae are changing these organisms are now thought to have emerged late,2 although extrapolation of molecular data to the distant past may be subjected to much ~ncertainty.~ For the fungi a commodious classification has been adopted while for the formerly called protists the latest interim taxonomic classification4 has been used. However only groups that have yielded natural products are discussed. 2 Bacteria (Eubacteria) At this stage of our knowledge subdivision into Actino- bacteria Cyanobacteria and all other Eubacteria may be detailed enough to allow some understanding of the patterns of natural products chemistry.2.1 Actinobacteria Species in the family Streptomycetaceae which sparsely thrive on coastal sea mud and invertebrates are the most productive of all marine actinobacteria. Unlike the land actinobacteria macrolides from marine actinobacteria are rare represented by only the aplasmomycins of Streptomyces griseqs which are in fact of the terrestrial boromycin type. Originality is found in medium-ring acetogenins like octalactin A 1 isolated from cultures of Streptomyces sp. taken from the surface of a gorgonian of the Gulf of California which shows cytotoxicity towards B- 16-F10 murine melanoma and HCT- 116 human colon cancer cell lines.6 In the alkaloids the most unusual example is an acaricidal monoterpene derivative altemicidin 2 isolated from Strepto-myces si~yaensis.~ The most outstanding examples in the other nitrogen-bearing products are salinamide A 3 and salinamide B 4 unusual antiinflammatory cyclodepsipeptides of L-amino acids isolated from Streptomyces sp.taken from a jellyfish from the Florida Keys.' Unusual quinones have also been isolated. Thus SS-228Y 5 is an angular pentacarbocyclic example with both antibacterial and antitumoural activity though photochemically and ther- mally very labile,'" isolated from another species in the Streptomycetaceae Chainia purpurogena from Japanese sea mud." Sesquiterpene naphthoquinones marinone 6 and de- bromomarinone 7 were isolated from an unidentified actino- mycete from south Californian marine sediments.lo 453 0 N-Me-Val /f r NHCOCH2S02NH2 OAc-Thr 0 M e N s OHH IH H2NOC 2 N-Me-Phe OH Thr MeN 0 Ile 3 7,40-epoxide 4 R20 0 OH 0 5 2.2 Cyanobacteria In contrast to land-thriving cyanobacteria which always had to be grown in culture to get sufficient biomass for the isolation of metabolites several wild marine strains of cyanobacteria have been collected in abundance in tropical areas either following blooms in surface or shallow waters or as tufts fixed to a substrate. Unless otherwise indicated these are wild tropical species in the genus Lyngbya. While the alkaloids are unexceptional peptides are unusual either of the linear type and made of L-amino acids like the immunosuppressive microcolin A 8 and microcolin B 9,11or cyclic and formed by both L-and D-amino acids like the antimicrobial hormo- thamnin A 10 of Hormothamnion enteromorphoides from the Caribbean.l2 Cyclodepsipeptides of L-amino acids are represented by the strongly ichthyotoxic antillatoxin 11.l3 Curacin A 12 is an unusual acetogenin entailing a thiazoline ring. It shows strong cytotoxicity towards L1210 leukaemic cell lines and inhibits tubulin polymerization by binding at the colchicine (or overlapping) site. l4 Macrolides are represented by the tumour promoters aplysiatoxin 13 and debromo- aplysiatoxin 14,15as well as by the unusual laingolide 15 which also embodies an enamide functionality.l6 454 Natural Product Reports 1997 8 R=OH 9 R=H L-Hy-Pro OH L-H-Ser DHHA .iJoH HOVhq( L-H-Ser 0 0 0 NH D-Phe /NH I L-Leu D-aIle 10 N-Me-Val 11 OMe 12 ’H 0 \ OH OH 13 R=Br 14 R=H 0 15 2.3 Other bacteria Alteromonas is a genus of productive Gram-negative bacteria of wide occurrence in the sea either in bulk waters or in the mud or even associated with other organisms. Character- istic metabolites include rubrenoic acid C 16 which is an Br Br 16 17 18 19 H 20 aromatized acetogenin that shows bronchodilator and muscle relaxant activities. l7 In the alkaloids the shikimate penta- bromopseudilin 17 is a potent antibacterial agent,18 while isatin 18 has an ecologically relevant antifungal activity.l9 Biopolymers are represented by protease inhibitor polypep- tides called marinostatins,20 patented proteases,*' potentially antifungal chitinolitic enzymes expressed in Escherichia coli,22 and adhesive polysaccharide-melanin polymers that may find use for aq~aculture.~~ Among the products of bacteria of other genera emerges oncorhyncolide 19 isolated from cultures of an undetermined bacillus taken from surface waters near a salmon farm; 19 is a rare example of a metabolite with methyl branching derived from a~etate.'~ Anguibactin 20 stands out as an alkaloidal siderophore of the widespread fish pathogen Vibrio anguil- l~rurn.~~ Cereulide 21 and homocereulide 22 obtained from Bacillus cereus taken from the surface of a snail in Japan are cyclodepsipeptides of both L-and D-amino acids endowed with potent cytotoxic activity against P388 leukaemic cell lines though also emetic toxins.26 Carbohydrates are represented by an antibacterial aminoglucose of Bacillus sp.from deep waters 7 D-Ala I HN D-Ala D-Ala 0 OVNH L-Val 21 R=H 0 22 R=Me in the western Pacific,27 and an antitumoural heteroglycan called marinactam from Flavobacterium uliginosum taken from a seaweed in Japan.28 The isoprenoids are best represented by astaxanthin 23 a very valuable antioxidant in use both as a food additive and as a pigment for aquaculture; it is produced in cultures of Agrobacterium aurantiacum selected in an extensive screening project of bacteria from Okinawan waters.29 3 Archaebacteria The archaebacteria bear unique isoprenoid glycerol ethers as cell wall stabilizers3' but the most valuable products are thermostable enzymes-which have found use in the polymer- ase chain reaction (PCR)-isolated from thermophilic archae- ba~teria.~ No examples of biologically-active small molecules have emerged from the archaebacteria perhaps reflecting a scarce competition in the harsh conditions of the biotopes where these organisms have been collected so far.The archae- bacteria living in free temperate oceanic waters have not yet been studied by natural products chemists; if culturable these microorganisms might open a new area. 4 Euglenoids (Euglenoidea Euglenozoa Protozoa) A clear-cut distinction between marine and freshwater eugle- noids is generally difficult to draw since these organisms show wide tolerance to salinity.In this perspective the most start- ling findings about the euglenoids concern 4a-methylsterols in Euglena gracili~~~ and particularly PGE2 and PGFZ, detected in trace amounts in Euglena gracilis var. ba~illaris.~~ These compounds may have evolutionary significance the first ones as to the transition from triterpene-based to steroid-based membranes and the second ones as to the evolution of the functions from defensive to hormone-like agents.34 5 Dinoflagellates (Dinoflagellatea Dinozoa Protozoa) Marine dinoflagellates have yielded metabolites that have been shown to be valuable health foods and laboratory tools and also show promise for drugs.The laboratory tools are also potent toxins and/or tumour promoters. Moreover some metabolites of dinoflagellates are involved in their characteris- tic phenomena of biolumine~cence~~ and circadian cycles,36 or may be taken as evolutionary markers for them as is the case of 4a-methylsterols which differ from those of the euglenoids for methylation at C-23. 0 I I 23 Pietra Secondary metabolites from marine microorganisms 455 Docosahexaenoic acid an important food additive required for brain development is produced in abundance by several dinoflagellates. The non-photosynthetic cosmopolitan species Crypthecodinium cohnii in particular has been exploited for a very successful commercial production of this acid both in Japan and the United States.' C.cohnii lacking thylakoid membranes must synthesize docosahexaenoic acid through other pathways than the desaturation processes of photo- synthetic organisms. One of the earliest compounds to be studied from the dinoflagellates is okadaic acid 24 a tumour-promoter poly- ether of a new type which is biosynthesized by widespread dinoflagellates of either the planktonic genus Dinophysis or the It is produced from benthic epiphytic genus Pr~rocentrum.~~~ cultures of the latter and used for studies of cellular regulation.376 Biosynthesized from acetate and glycolate as building blocks okadaic acid is the archetypal member of a growing family of DSP (diarrhoeic shellfish poisoning) toxins that accumulate in molluscs and sponges.38 Zooxanthellatoxin A 25 isolated from cultures of the symbiontic dinoflagellate OH OH HO1 Na03SO OH OH OH I OH 0 456 Natural Product Reports 1997 Symbiodinium sp.taken from a very small flatworm of Okinawan waters is structurally related to okadaic acid like the amphidinols but it is a much larger molecule containing a 62-membered macrolide ring where the relative stereo- chemistry among the ring portions is not known.39 It is a potent vasoconstrictive agent that acts by favouring the permeability of Ca' + across cell membranes. Brevetoxin A 26 a member of the family of NSP (neurotoxic shellfish poisoning) finds use in the laboratory for the study of ion channels. First isolated from cultures of Gymnodinium breve it is also produced by other red tide dinoflagellates.Although it is not known to accumulate in other organisms it is the cause together with other polyethers of the same family of extensive marine mortalities. Evidence has been adduced that the biosynthesis of brevetoxin A follows unusual acetogenin lines although G. breve only accepts acetate directly dicarboxylic acids are required as intermediates to explain the pattern of label incorporation. Brevetoxin A was the first member of a structurally varied series of poly- ethers characterized by a continuous array of trans-fused 24 OH YOH OH OH OH OH OH HO OvNH OH OH 25 CHO 26 0-heterocyclic rings where the medium-sized saturated mem- bers may take a variety of conformations that are reflected in the NMR spectra of these compounds and may be imagined to play a role in the binding to receptor^.^' The second member to be isolated from the same organism was brevetoxin B for which the absolute configuration was directly established; this served as a reference from which the absolute configuration of the more complex members of the family discussed below could be proposed.41 Structurally related to the brevetoxins are the ciguatera toxins.Ciguatoxin was obtained in extremely low amounts from the viscera of the moray eel from both the Tuamotu Archipelago and Tahiti in French Polynesia while structurally close analogues were obtained from a strain of the benthic epiphytic dinoflagellate Garnbierdiscus toxicus taken in suf- ficient amounts for direct extraction from the calcareous red seaweed Jania sp.in the Gambier Islands French Polynesia.42 The other main causative agent of ciguatera maitotoxin the largest and most toxic of all non-peptidic secondary metabo- lite~,~~ was obtained from very large cultures of a strain of G. toxicus taken from seaweeds in the Gambier Islands. Another strain from this location gave in culture related substances with antifungal activity the gambieric acids,43" while a strain of G. toxicus from Rangiroa Atoll in French Polynesia gave in culture another ciguatoxin analogue gambier01.~~~ Palytoxin analogues were obtained in cultures of another benthic epiphytic dinoflagellate Ostreopsis siamensis from Okinawan waters.44 Both the brevet ox in^^^ and the palyto~ins,~~ together with C, acetogenins produced by red seaweeds in the genus Laurencia4' and Taxol-type diterpen~ids,~' have recently been at the forefront of organic synthesis for challenging the problems of construction of medium rings.The PSP (paralytic shellfish poisoning) alkaloid saxitoxin 27 is also used for studies on ion channels. Although ob- tained from cultures of red tide dinoflagellates in the genus Alexandrium saxitoxin is thought to have bacterial origin.49 ,OCONH2 27 However Mendelian inheritance of parental saxitoxin in toxic dinoflagellate strains proves that the latter are capable of de novo synthesis of this toxin.,' The biosynthesis of saxitoxin involves arginine as precursor of the guanidinio groups while the perhydropurine skeleton unexpectedly derives from Claisen condensation of acetate with arginine.The carbon atom in the side chain is derived from S-adeno~ylmethionine.~~ Unlike Gyrnnodznium red tides which cause extensive marine mortali- ties Alexandrium red tides only intoxicate shellfish although ultimately this can prove to be quite harmful to man through the food chain. Dinoflagellates also produce macrolides of potential phar- macological value. Both symbiotic and free-swimming Amphidinium spp. afford cytotoxic amphidinolides of which the most potent in assays against leukaemic L 12 10 epidermoid carcinoma KB and colon HCT cell lines is amphidinolide N 28,52 whose structure has not been fully elucidated.The biosynthesis of the amphidinolides goes through non-successive mixed polyketide~.~' Goniodoma pseudogonyaulax in culture gave macrolides called goniodomins which show in vitro antifungal activity.53 No obvious chemotaxonomic correlation has emerged at the level of order in the Dinozoa. Polyether toxins are produced Ho'-fi OH 28 by members of the Dinophysiales Gymnodiniales and Gonyaulacales while in the family Gonyaulacaceae toxic alkaloids are also found. Macrolide polyols are found in both the Gymnodiniales and the Gonyaulacales the latter also affording polyunsaturated dodecanoids. 6 Ciliates (Ciliophora Protozoa) Marine ciliates have colonized a variety of habitats from the sandy shoreline to mesoabissal biotopes and play important roles in the marine food web by feeding on bacteria thus bringing forth matter recycling initiated by the latter and serving as food for the larvae of marine invertebrates.There has been much recent interest in the natural product chemistry of ciliates which except for some examples of toxic polyene alkaloid^,^^ is dominated by terpenoids. From the viewpoint of function these terpenoids may be divided into two classes those that inhibit the division or kill at higher doses most other ciliates except predacious ones and those that only repel or kill the latter. Euplotins A 29 B 30 and C 31 as well as * H' -0Ac 08 O H 29 R,R=O 30 R,R = 0; A"' l1 31 R = H; A10~l1 I CHO 32 their putative biogenetic precursor preuplotin 32,55"belong to the first class.Evidence has been adduced that these com- pounds work in cell-to-cell contacts arguably serving to the producer the morphospecies Euplotes crassus to maintain and broaden its niche; in fact the euplotins are destroyed in sea water to give inactive compounds that also served to assign the absolute onf figuration.^'^ In line with these observations euplotin C 31 at 1-5 pg cm-3 was recently found to cause by interacting at the cell membrane a strong and sustained increase in cytosolic free Ca2' in the ciliate Euplotes vann~s.~~~ The terpenoids so far isolated from all other ciliates proved to be toxic towards predacious ciliates while non-harmful to non-predacious ones. The first was raikovenal 33 isolated alongside its putative biogenetic precursor preraikovenal 35 from a strain of Euplotes raikovi collected along the beach of Casablanca in Morocco.56 Further clues as to the chemistry occurring in E.raikovi stemmed from the author's laboratory where molecular mechanics calculations indicated that Pietra Secondary metabolites from marine microorganisms 457 14 Me 33 p-14 35 34 a-14 preraikovenal preferentially takes a folded conformation suit- able for an unprecedented tele-dienone-olefin [2+21photo-cycloaddition to give ent-epiraikovenal ent-34. This actually occurred in good quantum and chemical yields. Shortly after- wards epiraikovenal was isolated from both Mediterranean and Californian strains of E.raik~vi.~~ This suggests that the Mediterranean and Californian strains use the elusive enanti- omer of preraikovenal as a late biosynthetic precursor of epiraikovenal. This photochemical reaction might have appli- cations in organic synthesis e.g. in a new route to spatane diterpene~.~~ These observations have stimulated the total synthesis of raikovenal and related compounds in racemic forms.59 The conservativeness of E. crassus which affords the same euplotins and preuplotin throughout the world albeit at differ- ent relative ratios according to the particular strain suggesting a long-date adaptation is contrasted by the variability of E. raikovi. Arguably the finely tuned euplotins leave no room for further improvement while E.raikovi is still improving on its 'chemical defences' thus existing in a highly polymorphic condition. Similar is the case of Euplotes rariseta strains from northeastern Australia southern Brazil and the Canary Islands which gave rarisetenolide 36 and 1I 12-epoxyrarisetenolide 0 12 36 p-11 37 a-11 while a strain from New Zealand gave epirarisetenolide 37.60 Rarisetenolide is the first of the bioactive terpenoids from ciliates lacking highly reactive functionalities which reinforces the view that these molecules are recognized in toto by recep- tors on the cortex of the ciliates. It should also be noticed that these structural variations although involving fundamental molecular details that imply the coding of different enzymes occur within the same molecular framework for each morpho- species.Thus these metabolites may be taken as reliable taxonomic markers at the level of supraspecies. The last examples of toxic agents towards potential pre- dators are focardin 39 and epoxyfocardin 38 diterpenoids 39 7 458 Natural Product Reports I997 isolated from Euplotes focardii from Terra Nova Bay in Antarctica which exist in solution as equilibrating epimen61 Thus like certain invertebrates,62 interstitial ciliates also need chemical defence even in these cold waters of limited biodiver- sity. That of the focardins is a case where the same defensive metabolites are used by ciliates both in Antarctic waters and warm waters of high biodiversity as quite recently found for Euplotes quinquecarenatus collected in the Red Sea.63 One is intrigued by these findings as to which species came first and as to what degree of genetic relatedness exists between the species.These observations stimulated us to undertake a genomic analysis of these species. So far it has been discovered that the conservativeness of the various strains of E. crassus for euplotin production is in accordance with a perfect degree of rDNA homology among these strains.64 On the other hand there are preliminary indications that the polymorphic con- dition described above for both E. raikovi and E. rariseta is met by a poor degree of rDNA homology among the respective 7 Chrysophytes (Chrysophyceae Phaeophyta Chromista) The chrysophyte Poteriochromonas malhamensis gave in cul- ture polychlorinated straight-chain enol sulfates that exhibited modest inhibition of tyrosine kina~e.~~ 8 Diatoms (Bacillariophyceae Diatomae Chromista) Like the euglenoids red algae and dinoflagellates the dia- toms too may contain the primitive 4a-methylsterols which contribute to marine sediments.66 The massive release of methyl bromide into the atmos-here^^ is the most dramatic ecological aspect of marine diatoms.However diatoms are not unique in this respect and the major contributing organism of methyl bromide in the sea is still unknown.68 Also relevant to leisure activities in summer is the release of polysaccharides by diatoms which make foams in closed environments such as the Adriatic Sea during calm periods.69 Much more concern arises from biologically active products from the diatom Nitzschia pungens f.multiseries which intoxi- cated mussels in culture at Prince Edward Island in south- eastern Canada. The toxins include a potent neuroexcitatory amino acid domoic acid 40 produced by a strain of Nitzschia 40 0 0 &COOH OH OH 41 R =a-H 43 42 R=P-H pungens f. multiseries via the condensation of a geranyl unit with an amino derivative of the citric acid cycle followed by cyclization to form the proline ring,70 and amnesic eico- sanoids extracted from wild cells the bacillariolides 141 I1 42 and 111 43.71The latter is the first reported strictly extracellular metabolite of the diatoms.71 H OH H?-( 44 HO HO OH OH In contrast abundant production of eicosapentaenoic acid by diatoms such as in the genera Thalassi~sira~~ and Navic~la,~~ might become biotechnologically useful.9 Eustigmatophytes (Eustigmatophyceae Phaeophyta C hromist a) This group of microalgae is only mentioned here because of the abundant production of the valuable food additive eicosapentaenoic acid by Nannochloropsis sp.74 10 Raphidophytes (Raphidophyta Chromista) The genus Heterosigma best represents the raphidophytes as far as natural products are concerned. Thus Heterosigma akashivo is responsible during periodic blooms for massive killing of fish in Japanese waters. In culture it produced new diacylglycerol galactopyranoside glycolipids possessing 0-3- polyunsaturated fatty acid residues which however are not responsible for the toxic activity.75 This microalga might also be at the origin of polyunsaturated fatty acids found in certain Another strain of this alga proved lethal to fish in the waters of Brittany but no toxin has been identified so far.76 11 Prymnesiophytes (Prymnesiophyta=Haptomonada Chromista) Prymnesium parvum is a red tide prymnesiophyte that threat- ens fish farming worldwide especially in brackish waters.Obtained from Hawaii in culture it gave a toxic L-xylose glycoside polyether prymnesin 2 44.77 In this respect this species resembles certain toxic dinoflagellates. In contrast Isochrysis galbana is a good source of polyunsaturated fatty acids.78 Hymenomonas sp. from the East China Sea gave a novel sterol sulfate hymenosulphate with Ca"-releasing activity in the sarcoplasmic reticulum.79 Finally Emiliania huxleyi pro-duces both eicosapentaenoic acid in abundance and straight chain C,,-C, alken-2- and -3-ones and esters which are also found in Quaternary marine sediments.These may be used as indicators of water-column temperatures.80 12 Cryptophytes (Cryptophyceae Cryptophyta Chromista) The cryptophyte Chrysophaeum taylori collected as a 'yellow slime' on an exposed reef in Puerto Rico gave hormotham- nione 45 and its 6-desmethoxy derivative 46 which are OH 0 OH OH 45 R=OMe 46 R=H Pie t ra Secondary metabolites from marine microo rgan isms styrylchromones endowed with strong cytotoxic activity towards leukaemic cell lines.81 This raises questions of related- ness or c~nvergence~~ since styrylchromones are typical prod- ucts of angiosperms which appeared much later than the cryptophytes.13 Prasinophytes or grass-green scaly algae (Prasinophyta Plantae) The prasinophyte Tetraselmis subcordiformis produces di-methylsulfoniopropionate in analogy to the dinoflagellate Amphidinium cartheri. As a precursor of acrylic acid and because it accumulates in T. subcordiformis in the presence of grazers a defensive role has been attributed to this compound.82 Like certain diatoms Prasinococcus capsulatus may release polysaccharides into the sea.83 Of more practical use Tet-raselmis suecica is another good source of polyunsaturated fatty 14 Green microalgae (Chlorophyta Plantae) Interest in the green microalgae is currently focused on the Dunaliella complex which provides the basis of the industrial production by culture in the open air of translcis-p-carotene.This is more liposoluble and has better qualities as a free radical scavenger than the all-trans form84 and it is a candidate to gradually take the whole market as a food additive. 15 Red microalgae (Rhodophyta Plantae) In contrast with the rich literature about secondary metabo- lites from red seaweeds only Porphyridium cruentum has to be mentioned in the red microalgae. In common with euglenoids red algae dinoflagellates and diatoms this alga contains the primitive 4a-methyl~terols.~~ P. cruentum might also become of commercial interest for the production by of eicosapentae- noic acid as a food additive8' and other polyunsaturated fatty acids for cosmetics.78 However P.cruentum also pro- duces substantial amounts of toxic trichloroethylene and perchloroethylene.86 16 Fungi (Eumycota) It was more on ecological and physiological grounds than on taxonomical grounds that fungi as a group was established. Whenever matter is decaying the fungi are present. Land microfungi have been the source of therapeutically important classes of metabolites like the penicillins cephalosporins and cyclosporins. Now that the fungal biodiversity on land seems to be nearly exhausted the sea may offer a variety of unexplored fungi. However it is not yet clear whether the potential of marine fungi for drugs will ever equal that of the terrestrial fungi.When dealing with marine isolates the first question is are they true marine fungi? In certain cases particularly for lignicolous and parasitic species the answer is clear cut they belong to genera unknown on land and sporulate in sea water. In other cases typically for isolates from marine invertebrates the answer is difficult for fungi that belong to genera well known from land particularly when the literature fails to describe if they can sporulate in sea water. Fungi also sporu- lating in freshwater might have been recently transported from land into the sea surviving there as dormant spores. However organisms are defined at the species level i.e. fungi in the same genus may be found both on land and in the sea each species being exclusive to one or the other environment.In conse- quence for this review the fungi isolated from the sea have been divided into three commodious groups. In fact with fungi more frequently than with other organisms except perhaps the cyanobacteria the products from marine isolates are often similar if not identical to those from land isolates and this occurs for ‘terrestrial genera’ at the same frequency as for ‘marine genera’. Fungal products were always obtained from cultures. Basidiomycetes and protistan fungi are omitted from this discussion the first ones being very scarcely represented in the sea and the latter being not known to produce secondary metabolites although a thraustochytrid is a useful source of docosahexaenoic acid87 possibly competing commercially with production from dinoflagellates.16.1 True marine ascomycetes Representative of this group are naphthoquinone polyketides like leptospherodione 47 from a Mediterranean strain of 0 OH 47 48 49 a-OH 50 P-OH Leptosphaeria oraemaris,88 and the closely similar obionin A which has potential for CNS activity from Leptosphaeria obi one^.^^ A different class of acetogenins is represented by zopfinol 48 a chlorinated benzenoid with a long linear chain isolated from Zopfiella marina.90 New terpenoids are limited to isoculmorin from Kallichroma tethys” and the C helicascol-ides A 49 and B 50 isolated from Helicascus canal~anus.~~ Structurally complex diketopiperazines are the most charac- teristic nitrogenous compounds.Included are the melinacidins from the cosmopolitan Corollospora pul~hella,~~ and epi-polythio derivatives the leptosins from Leptosphaeria sp. of Japanese waters. Leptosin C 51 is significantly antitumoural in mice bearing Sarcoma- 180 as cite^.^^ A carbohydrate deriva- tive leptosphaerin 52 from another strain of Leptosphaeria oraemaris was one of the first secondary metabolites isolated from marine fungi.95 Metabolites carrying a carbohydrate 460 Natural Product Reports 1997 HO 51 OH / 52 53 R=CHzOH 54 R=CHO moiety linked to portions of different biogenesis have also been isolated from marine ascomycetes like a sphingosine derivative from Lignincola laevi~,~~ or an antifungal diterpene derivative zofimarin from Zojiela (likely Zopjiella) marina.97 Preussia aurantiaca a typical lignicolous land species as a mangrove isolate gave the depsidone polyketides auranticins A 53 and B 54.98 16.2 True marine deuteromycetes The most unusual metabolites from marine deuteromycetes are sesquiterpenoids like the dendryphiellins of the lignicolous Mediterranean Dendryphiella ~alina,~~ and the phomactins of Phoma sp.taken from the shell of a crab in the Sea of Japan which only sporulates at the high salinity of seawater.”’ The dendryphiellins may be subdivided into two classes the eremophilanes like dendryphiellin E 1 55 and the trinoreremo- philanes like dendryphiellin A 56; both came about as unusual classes of fungal ter~enoids.~~ All dendryphiellins bear an ester 0 55 R=CHzOH 57 R=CHO 56 I ‘CI OH 58 59 side chain that may be thought of either as a degraded terpene or a methylated fatty acid.Later an antibacterial antifungal and antifouling metabolite nearly identical to dendryphiellin El called bipolal 57 was isolated from the terrestrial deutero- mycete Bipolaris sp.'01 The phomactins in particular phomactin A 58 are potent specific PAF antagonists. loo The unusual ring-opened form of a triquinane chloriolin A 59 was isolated from an unidentified deuteromycete secured from an Indo-Pacific sponge.'02 16.3 Terrestrial-genera deuteromycetes Two fungi taken from the digestive apparatus of fishes from Japanese waters gave unusual nitrogenous compounds.Thus a strain of Penicillium fellutanum gave lipidic aldehyde tri- peptides fellutamides A 60 and B 61 which showed nerve y42 I 0ANH2 60 R=OH 61 R=H OqH H 62 R' =Me; R2= H 63 R1=H; R2=Me growth factor (NGF) stimulation and cytotoxicity towards leukaemic cell lines. lo3 Aspergillus fumigatus gave tryptoquiva- line alkaloids for example fumiquinazolines A 62 and B 63.'04 Among the acetogenins the halymecins A 64 B 65 and C 66 polyesters obtained from the deuteromycetes Acremonium sp. and Fusariurn sp. taken from seaweeds of Japanese waters are of interest. The halymecins inhibit both diatoms and dinoflag- ellate~.~~~ Two groups of unique polyketides are also pro- duced including chlorocarolides A 67 (or 68) and B 68 (or 67) isolated from Aspergillus cf.ochraceus taken from an Indo- Pacific sponge,106 and penostatins A 69 and B 70 cytotoxic agents towards leukaemic cell lines obtained from Penicillium sp. taken from seaweeds of Japanese water~.''~ Terpenoids proved uncommon. Examples are the antibac- terial and antifungal mixed-biogenesis stachybotrins A 71 and 67 (7R*,8R*) 69 R' =OH; R2= H 68 (7s 8S*) 70 R' = H; R2= OH OH HN* 0 71 R' = OH; R2= H 72 R' = R2= H B 72 secured from Stachybotrys sp. of brackish waters in Florida,lo8 a genus known from land to give trichothecenes. 17 Products of proven or alleged microbial origin isolated from marine macroorganisms The bacteria are found associated with all organisms. Out- standing examples from the sea are the 50% bacterial biomass in sponges of the order Verongida abundant cyanobacteria in many sponges and oxychlorobacteria in colonial ascidians of the family Didemnidae.In the latter Acaryochloris marina is an unusual photoautotroph based on mostly chlorophyll d with only minor amounts of chlorophyll a.1o9 Many eukaryotic microorganisms are also known as sym- bionts of marine species e.g. dinoflagellates in sponges and fungi in a variety of invertebrates algae and seagrass. It has often been surmised that certain products isolated from marine macroorganisms may have microbial origin. Much work has also been devised to find a biotechnological route to thera- peutically promising compounds isolated from rare marine macroorganisms and too complex for a practical chemical synthesis; however in only a few cases has a microbial origin been proven if 'dietary' metabolites are omitted.One of the best proven and over-reviewed cases concerns a brominated diphenyl ether isolated from both the tropical sponge Dysidea sp. and its symbiontic bacterium Vibrio sp. in culture."' In retrospect failure to isolate the desired metabolites from microbial symbionts is not surprising since it is long known from land microorganisms and now also from marine micro- organism~,~~, 43 that to be productive strict symbionts may require the growth conditions found in their host. Separating unicellular symbionts from their host cells may bypass the problem of assessing the origin of the metabolites."' Another criterion for judging whether compounds iso-lated from taxonomically well defined macroorganisms may have microbial origin is the presence of such compounds in collections of the macroorganism from only certain geographic Pietra Secondary metabolites from marine microorganisms 461 H I I locations.This is the case of the calcareous sponge Leucascan-dra caveolata from waters of New Caledonia. Thus collections from the Passe of Nakety along the eastern coast gave an unusual strongly bioactive macrolide leucascandolide A 'l2 while collections from farther north which were morphologi- cally indistinguishable did not contain such a metabolite. Against this conclusion is that differences in the two sponges may not be related to their morphology but in favour of this proposition the macrolide is an atypical product of calcareous sponges.Given the shallow-water habitat of the sponge and the compound nature dinoflagellates may be suggested as the most likely producers. The microbial-type structure of the compounds isolated from macroorganisms may in fact be another criterion for judgment. This is the case of enediyne antitumour antibiotics isolated from a didemnid a~cidian,"~ a class of compounds first isolated from terrestrial actinobacteria in the genera Micromonospwa and Actinomadura. Similar conclusions may be drawn for isoquinoline alkaloids of mimosamycin type known from the land actinobacterium Streptomyces Zaven-dulae marine sponges and tunicates.Another related case concerns marine and land cyanobacterial macrolides of scytophycin/tolytoxin type which resemble the sphinxolide- type 73 macrolides,' l4 isolated from sponges' l5 and opistho- branchs."4 '16 Although the macrolide size is variable in these compounds the general features are similar with a side chain ending in an N-methyl-N-vinylformamidegroup that under- goes slow conformational changes reflected in the NMR signals of nearly the whole molecule. Still another criterion is the isolation of structurally related products from phylogenetically distant macroorganisms like pyrroloiminoquinone 74 alkaloids,' l7 found both in ascid- ians and in taxonomically unrelated sponges as well as pyrido[4,3,2-mn]acridine 75 alkaloids,' l8 found in sponges sea 0 74 75 anemones and ascidians.However which microbial taxon may produce compounds of these classes is unknown. Conver- gen~e~~ may be an alternative explanation and therefore this last criterion should not be pushed too far particularly not when structural similarities are limited to portions of the molecules. Once any dietary origin has been excluded microbial origin for products isolated from invertebrates or seaweeds may be ruled out in the absence of suitable symbionts or epiphytes. Thus some strains of E. crassus which produce both pre- uplotin and the euplotins were accurately examined at both the optical and the electron microscope level for the presence of 462 Natural Product Reports 1997 I 73 symbiotic bacteria.None could be detected in some strains while others revealed only a few cocciform bacteria (from <5 to <lo per ciliate cell)'19 which are not known to produce either sesqui-or di-terpenoids. Production of the same terpenoids by the ciliates on either algal or bacterial diet reinforces these conclusions. In conclusion that of microbial symbionts or parasites of marine macro- and micro-organisms remains a difficult area of study in the realm of marine natural products. This work was supported by CNR Roma Progetto Singolo and Progetto Strategic0 ST/74-PS and MURST 40%. 18 References 1 L. Bongiorni and F. Pietra Chem. Ind. (London) 1996 54. 2 H. Philippe and A. 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Nomoto Tetrahedron Lett. 1996 37 655. Pietra Secondary metabolites from marine microorganisms 108 X. Xu F. S. de Guzman J. B. Gloer and C. A. Shearer J. Org. Chem. 1992 57 6700. 109 H. Miyashita H. Ikemoto N. Kurano K. Adachi M. Chihara and S. Miyachi Nature 1996 383 402. 110 G. B. Elyakov T. Kuznetsova V. V. Mikhailov I. I. Maltsev V. G. Voinov and S. A. Fedoreyev Experientia 1991 47 632. 111 C. A. Bewley N. D. Holland and D. J. Faulkner Experientia 1996 52 716. 112 M.D’Ambrosio A. Guerriero C. Debitus and F. Pietra Helv. Chim. Acta 1996 79 51. 113 L. A. McDonald T. L. Capson G. Krishnamurthy W.-D. Ding G. A. Ellestad V. S. Bernan W. M. Maiese P. Lassota C. Discafani R. A. Kramer and C. M. Ireland J. Am. Chem. Soc. 1996 118 10898. 114 G. Guella I. Mancini G. Chiasera and F. Pietra Helv. Chim. Acta 1989 72 237. 115 M. V. D’Auria L. G. Paloma L. Minale A. Zampanella J.-F. Verbist C. Roussakis C. Debitus and J. Patissou Tetrahedron 1994 50 4829. 116 K. Yamada M. Ojika T. Ishigaki Y. Yoshida H. Ekimoto and M. Arakawa J. Am. Chem. SOC.,1993 115 11 020. 117 A. J. Peat and S. L. Buchwald J,Am. Chem. Soc. 1996,118,1028. 118 M. A. Ciufolini Y.-C. Shen and M. J. Bishop J. Am. Chem. Soc. 1995 117 12460. 1 19 Courtesy of Prof. H.-D. Gortz Biologisches Institut Universitat Stuttgart. 464 Natural Product Reports I997

ISSN:0265-0568    

DOI:10.1039/NP9971400453

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

Coumarins Ana Estbvez-Braun and Antonio G. Gonzalez Instituto Universitario de Bio- Organica Universidad de La Laguna E-38206 Tenerife Canary Islands Spain Covering January 1995 to December 1996 Previous review 1995 12 477 1 Introduction 2 Separation and identification 3 7-Oxygenated coumarins 4 C-Substituted 7-oxygenated coumarins 5 Disubstituted 7-oxygenated coumarins 6 5,7-Dioxygenated coumarins 7 6,7-Dioxygenated coumarins 8 7,s-Dioxygenated coumarins 9 5,6,7-Trioxygenated coumarins 10 5,7,8-Trioxygenated coumarins 11 6,7,8-Trioxygenated coumarins 12 5,6,7,8-Tetraoxygenated coumarins 13 4-Oxygenated coumarins 14 Coumestans 15 Miscellaneous coumarins 16 Biscoumarins 17 References 1 Introduction This review covers the literature published or abstracted between January 1995 and December 1996 (Chemical Abstracts vols.122-125 inclusive) and follows the pattern of its predecessor.' The plants of the Rutaceae and Umbelliferae continue to be the richest sources of coumarins (see Table 1). The majority of the phytochemical studies were conducted on Angelica and Peucedanum species. Some of the isolated coumarins showed interesting biological activities for example decursin from Angelica gigas and psoralidin from Psoralea coryfolia both present toxic activity against various human cancer cell lines. Another example is soulattrolide from Calophylum teysmanii which is a potent inhibitor of HIV-1 reverse transcriptase. A great deal of work on the synthesis of bioactive coumarins of the type calanolide has been published during this period.The structures of some coumarins like calanolide C and D were revised. A review on 13C NMR spectroscopy of coumarins and their derivatives has been published.2 The structure-activity relationship of a number of coumarins in xanthine oxidase inhibition has been rep~rted.~ There has been work carried out on the within-plant distri- bution of coumarins in Coronilla and Securigera specie^.^ Single-step synthesis of eight natural coumarins (ayapin limmettin scoparone leptodactylone fraxinol isoscopoletin 5,6,7-trimethoxycoumarinand 7-methoxy-8-hydroxycoumarin) has been de~cribed.~ Finally numerous synthetic coumarins have been prepared for practical applications such as herbi- cides or for treating allergies and inflammations.2 Separation and identification The literature for these two years contains several systematic applications of known techniques for the separation and identification of coumarins along with descriptions of novel methods. These papers are summarized here. The coumarin content in the crude drug of Angelicae tuhou was easily determined by micellar electrokinetic capillary chromatography with 4-hydroxybenzoic acid as an internal standard.6 A reversed phase HPLC method for separation and Estgvez-Braun and Gonzalez Coumarins determination of six coumarins in Murraya paniculata was establi~hed.~ The separation of seven closely related coumarins from Chrysanthemum segetum by capillary electrophoresis has been reported.8 Solich et al.developed a differential-fluorimetric flow-injection determination of scopoletin or umbelliferone in a binary mixture with herniarine. This method is based on the pH-dependence of the fluorescence spectra of the analytes.' Qin et a1." have described a method of separation and quantitative determination by HPLC of cichoriin esculetin and prionanthoside three coumarins found in the Chinese traditional drug Zihuadiding (Herba uiolae). A new method for specific and accurate determination of several coumarins in biological matrices has been described.' ' This method is based on a modified particle beam liquid chromatography-mass spectrometry interface. An application regarding the analysis of 18 coumarins in extracts of Smyrnium perfoliatum was reported." Tosi et ~1.'~ have evaluated by HPLC the contents of herniarin and umbelliferone in herbal preparations of chamomile sold in Italy.Sixteen planar cou- marin type substrates and inhibitors of cytochrome P 4502A5 (CYP2A5) were analysed using the comparative molecular field analysis method (CoMFA).I3 The results of the extrac- tion of furanocoumarins by ultrasound-assisted techniques were evaluated and compared with those of conventional methods. l4 Antonious and Sabry Is discussed the effect of different solvent polarity parameters on the extraction process of several coumarins. A TLC-spectrophotometric method was proposed to assay furocoumarin constituents in the roots of Heracleum candicans.16 Based on the results of GC-MS analy- sis an HPLC procedure was developed for the determination of coumarins of tonka bean extract^.'^ NMR spectroscopy and MS studies on 6-or 8-alkyl-7-oxycoumarins and dihydrofurano- coumarins have been published.' Supercritical fluid extraction of furanocoumarins with carbon dioxide was used for their isolation from the fruits of Archangelica oficinalis. '' 3 7-Oxygenated coumarins Two new monoterpenoid coumarins ( -)-(9-trans-marmin 1 and pituranthoside 2 were isolated from the shoots of Pituran-thos triradiatus.20 The S configuration at C-6' was proposed by comparing the value of the optical rotation of 1 with natural marmin which is known to be dextrorotatory with R configu-ration at C-6'. The structure of farnesiferol C 3 was confirmed by its synthesis.21 Two new 7-geranyloxycoumarins chloro- marmin 4 and aeglin 5 were isolated from the bark of Aegle marmelos.22 The roots of this plant also yielded marminal 6 and 7'-O-methylmarmin 7.23Treatment of 4 with Py-DMAP afforded epoxyaurapten 8 which furnished marmin 9 on treatment with 5% aq.H,SO,-THF. Consequently the absolute configuration of the C-6' hydroxy function in 4 was assigned as R i.e. the same in 8 and 9. The absolute config- uration of 5 was assigned as 6'R,4'R following its synthesis from 3-deoxy- 1,2:5,6-di-O-isopropylidene-~-glucofuranose. The chloroform-soluble part of the bark of Zanthoxylum schinifo- afforded acetoxy aurapten 10. This compound showed inhibitory activity on platelet aggregation in uitro.Episamar-candin 11 a new sesquiterpene ~oumarin,~~ is a constituent of Ferula assafoetida. The aerial parts of Chorilaena quercifolia26 yielded 12. Isoapiosylskimmine 13 was obtained from the roots of Angelica 465 Table 1 Sources of coumarins Species Structures Ref. Species Structures Ref. Aegle marmelos Amni visnaga 4-7 40 22,23 49 Lonicera gracilipes Metrodera fravida 88 113 77 93 Angelica edulis 31 41 Monascus anka 127-132 102 Angelica gigas Angelica pubescens Angelica shikokiana Asterolasia trymalioides Atrichum undulatum Boronia algida Bupleurum fruticosum Calophyllum lanigerum Calophyllum teysmanii Chorilaena quercifolia Citrus hassaku Citrus limonia Clematis chinensis Derris scandens Diospyros kakis Diplolophium buchananii 13,27-30 17-20 41 112 108,109,118-121 43 102 83 84-86 12,93 16,66,137,138 22 87 124 140 26 27,37 3 1,32 50 92 91 52 86 70,7 1 70,73 26 30,65,117 33 76 99 119 36 Murraya exotica Murraya paniculata Paramignya monophylla Pelargonium sidoides Peucedanum guangxiense Peucedanum japonicum Peucedanum ostruthium Peucedanum praeruptorum Pituranthos triradiatus Polytrichum formosum Psoralea coryfolia Pterocaulon alopecuroides Pterocaulon purpurascens Pterocaulon virgatum Ruta graveolens Seseli sibiricum 44 23-25 56-62 75,761150.h.c 38 32-35 15,65 34,36 1,2 108-111 118 125 103 117 104 96,97 54 53 34,35,60,61 67 88 47 42 29 44,45 20 91 100 87 95 89 85 58 Ducrosia ismaelis 64 64 Setaria italica d 101 Emericella desertorum 142-144 121 Sider it is massoniana 122 96 Eriostemon cymbiformis Eriostemon myoporoides Esenbeckia grandijora Ferula assa foetida Jatropha cilliata Lingusticum elatum 89,90 67-74 47 11 114 39 78 66 55 25 94 48 Stauranthus perforatus Stellera chamaejasmoside Tetraphis pellucida Viola prionantha Yalaha (citrus paradisi+ c.tangerine) Zanthoxylum schinifolium 42 139 106,107,116 91 66,141 10,98-101,126 51 118 90 79 65 24 RO 0 HO 0 1 R = C H 2 w H 7 R = 14 R=CH2& H OH HO' H 15 R = CH'&cOOH 16 R= 4 R=CH2 10 R=CH2& steps with a yield of ca. 27%. Four new 6-alkylcoumarins HO' H angelitriol 17 angelol J 18 K 19 and L 20 were obtained from 5 R=CH2wH 11 R= the roots of Angelica pubes~ens~~ 32 and their absolute config- HO H ,'H HO urations were determined by optical rotatory dispersion (ORD) 0-R-D-GluC spectra.Compounds 17 18 and 19 were tested on human 6 R =CH,+H 12 R=CH2& platelet aggregation and showed significant activity. 6-Acyl-7- methoxycoumarins were prepared by ammonium cerium(1v) 0 nitrate oxidation of compounds such as 21 which were ob- 13 R = 2-OP-apiofuranosyl-(1+2)-Op-~-glucosyl tained by Pechman reaction of 4-alkylresorcinols with malic 4 C-Substituted 7-oxygenated coumarins 6-Allyl-7-hydroxycoumarin and the naturally occurring linear coumarin demethylsuberosin 14 have been prepared in good yields via regioselective boron halide catalysed ortho-Claisen Me0 0 rearrangements.28 This procedure provides an efficient alterna- OH OAng tive approach to the synthesis of linear substituted coumarins 17 R=C1T O H 19 R= without using a halogen to block the C-8 (angular) position.21 R = Me,Et Pr Bu Bui H 22 R=COH The new coumarin 15 was isolated as the active principle of the H OEt ,OH roots of Peucedanum o~truthium.~~ Hasakol 16 an antispas- modic coumarin isolated from Citrus hassaku was synthesized 18 R= FroH H from 7-geranyloxy~oumarin.~~ This synthesis involves three 466 Natural Product Reports 1997 acid and etherification of the resulting 7-hydroxy~oumarins.~~ Crenulatin 22 from Citrus limonia has been synthesized follow- ing The this new procedure.coumarins 23-25 were extracted from Murraya k N 2 -i bR0 -ii H% 0 R panic~lata.~~? (2R)-2’-hydroxymarmesin-2’O-/?-~-gluco- 0 pyranoside 26 was isolated from aerial parts of Diplolophium 0 25 RO 0 26 R = p-D-glUCOSyl Ro+-mo 0 27 R = P-D-gIUCOSyI 28 R = 6-acetyl-P-D-glucosyl hu~hunanii.~~ The stereochemistry at C-2 was determined by CD spectroscopy.Marmesinine 27 6”-acetylnodakenine 28 and columbianetin-P-D-glucoside 29 were obtained from the roots of Angelica Decursin 30 was also isolated from the roots of A. gigus and identified as the cytotoxic active component;37 30 displayed toxic activity against various hu- man cancer cell lines with ED, about 5-16 pg ml- ’ and it showed low cytotoxicity against normal fibroblasts. It seems that this cytotoxicity is related to the protein kinase C acti- vation. A new short synthetic pathway to furocoumarins based on the Fries rearrangement of hydroxycoumarin chloro-acetates has been published.38 The structure of bakuchicin from Psoralea corylifolia was reassigned.The spectral data previously reported are identical with those of angelicin and not with those of the isomeric furano co~marin.~~ Oroselone and oroselol have been synthesized4’ following the procedure shown in Scheme 1. Angelicin was also synthe- sized by this methodology but using vinyl acetate instead of acetylene. The fruits of Angelica edulis afforded edulisin VI 31 .41 Four new khellactone coumarins 32-35 were isolated from Peucedunum jap~nicum;~’ 34 and 35 were obtained as a racemic mixture. ( -)-cis-Khellactone and ( -)-trans-khellactone have been synthesized from umbelliferone (Scheme 2) as part of a study on the applicability of catalytic enantioselective cis- dihydr~xylation.~~ 45 The root of Peucedanum praer~ptorum~~.yielded two new angular dihydropyranocoumarins named qui- anhucoumarin H 36 and I 34. Their absolute configurations were established as 3’S,4’R for 36 and 3‘S,4‘S for 34 respect-ively by chemical correlations with known compounds. The EstPvez-Braun and Gonzdez Coumarins Oroselone R = C(Me)=CH2 Oroselol R = C(OH)(Me) Scheme 1 Reagents i HC =C-R Rh,(OAc), PhF; ii HCOOEt NaH THF; iii DDQ PhH; iv Ph,P=CHCO,Et xylene 0 0 OR’ OR’ 0 0 0 32 R1=<; Rz=l 34 R’=< ; R2=‘r 37 chemical structure of 37 was studied in detail by COLOC (correlation spectroscopy via long-range coupling) and ’H-‘HCOSY spectra which allowed the revision of literature assignments for some of the carbon and proton signals.46 Peguangxienin 38 is a constituent of Peucedanum guang- xien~e.~~ The absolute configuration of anomalin 39 from the roots of Lingusticum elatum was established as 9R,10R by X-ray cis-Khellactone-3’-~-~-glucopyranoside 40 was isolated from the fruits of Ammi vi~naga.~~ The structure was established on the basis of acid hydrolysis and spectral data.(3’R,4‘R)-3‘-Epoxyangeloyloxy-4’-acetoxy-3’,4‘-dihydroseselin 41 was extracted from the leaves of Angelica shikokiana5’ and purified for treating allergies and inflamma- tions especially bronchitis and skin allergies. The crystal struc- ture of xanthyletin 42 from the roots of Stauranthus perforatus was reported. 51 3‘-Hydroxyxanthyletin 43 was isolated from the aerial parts of Boronia algid~.,~ The flavonoid coumarin 44 was established as a constituent of Murraya 467 iv HO 0 HO 0 Meoy&OMe / OMe I ‘O / II Me0 \ OMe 0I 0 BzO 0 OH 0 -A I -44 ii (93.5%) 1 1vi 5 Disubstituted 7-oxygenated coumarins Balsamiferone 45 and gravelliferone 46 have been synthesized BzO 0 by tandem thermal Claisen-Cope rearrangements of couma- (38%) rate derivative^.^^ 3-( 1‘,1‘-Dimethylallyl)columbianetin 47 iii previously known as a synthethic compound has been isolated P J /t\ from the roots of Esenbeckia grandiJE~ra.~~ Convenient OH two-step approaches have been described for the synthesis of (58.5%) seselin and angelicin derivatives (48-50 and 51-53) from I vii 2,4-dihydroxybenzaldehyde and 2,4-dihydroxyacetophenone respectively.These syntheses use tandem Claisen rearrange- ment and Wittig reactions.56 4-Substituted-6-acyl-7-meth-OH oxycoumarins were prepared by reaction of 4-alkylresorcinols (62%) BzO with RCOCH,CO,Et and Me,SO, and subsequent oxidation HO. of the resulting compounds with ammonium cerium(1v) ‘OH nitrate.57 OH (29.2%) iviii HO. O F O H I ‘OH 0Po 0p RiO 0 -W‘-OH OH (15%) OH 45 R’ = Bz; R2 =CH2& 47 (92.7%) \/ Scheme 2 Reagents i 2-methylbut-3-yn-2-01 THF; ii DMF 130 “C; 46 R’ = H; R2 = C 4/ iii K,[OsO,(OH),] hydroquinidine 4-methyl-2-quinolyl ether K,[Fe- (CN),]; iv I, KI EtOH; v benzyl bromide K,CO, acetone; vi ?’ 2-methylbut-3-en-2-01 Pd(OAc), DMF; vii K,[OsO,(OH),] hydro-quinidine 9-phenanthryl ether K,[Fe(CN),]; viii H, Pd/C EtOH; ix,DEAD PPh, THF / 0 pR2 0 0 PR2 -48 R’ = H; R2 = Me 49 R’ = Me; R2 = H 50 Ri = R2 = Me 0a 0 *OR2 6 5,7-Dioxygenated coumarins OR’ The crystal structure of coumarrayin 54 from Seseli sibiricum has been reported.58 The coumarin 55 has been synthesized from 3,5-dimethoxy phenol.59 Kinoshita and co-workers35,60 61 elucidated the structures of four new cou-marins (56-59) from Murraya paniculata var.omphalocarpa. They briefly discuss the chemosystematic status of M. panicu-lata var. omphalocarpa with respect to the intraspecific differ- entiation of M. paniculata.60 The absolute configuration of ( -)-omphamurin 60 at C-2‘ was determined as S by Horeau’s method.35 The absolute stereochemistry of ( -)-mexoticin 61 R-0 and (-)-sibiricin 62 has also been establi~hed~~ by their 42 R=H chemical correlation with omphamurin 60.Toddacoumaqui-43 R=OH none 63 was synthesized through a Diels-Alder reaction62 468 Natural Product Reports 1997 OMe I Me0 0 56 R= ,C=( H H 61 R= OH 57 R= 62 R= 63 R= 0 between 8-(1-acetoxy-3-methylbuta-1,3-dieny1)-5,7-dimethoxy-coumarin and 2-methoxy- 1,6benzoquinone. Compound 63 showed a weak activity against HSV-1 and HSV-2 but no activity was observed against HIV-1. Danheiser and Tr~va~~ reported an efficient synthetic route to the linear furocoumarin bergapten (Scheme 3) based on a iiii OH 60% iii I iv OMe OMe I I o\ OH OH 81% 65% Scheme 3 Reagents hv (vycor) ClCH,CH,Cl room temp.3.5 h; ii heat 2 h; iii K2C03 CH31 acetone heat 18 h; iv BuZNF THF 0 'C-+room temp. 1.75 h; v cat. H,RU(PP~~)~, acetone-toluene 185 'C 22 h; vi DDQ benzene cat. p-nitrophenol heat 32 h photochemical aromatic annulation strategy. This approach avoids the regiochemical problems that arise in some alterna- tive routes. Oxypeucedanin ethanolate 64 was isolated from the aerial parts of Ducrosia isrnaelis.64 The roots of Peuceda-num ostr~thium~~ afforded the coumarin 65. The roots of Yalaha65 (a cross of Citrus paradisi and C. tangerine) provided the new coumarin trans-clausarinol 66. Seven novel coumarins with bruceol system (67-73) and one geranyl coumarin 74 were obtained from the aerial parts of Eriostemon myoporoides.66 COSY NOESY and HMBC experiments on all these coumarins lead to unambiguous Estivez-Braun and Gonzalez Coumarins &00 HOW 0' 64 R=CH2q0.66 OH 65 $b to 0 67 R = CHp 71 R= CH2&? 72 R= CHp4 OOH 68 R = CHp OH OH I 74 75 R=H 76 R=Me assignments of all the carbons and protons present in these compounds. Two new pyranocoumarins 75 and 76 were isolated from stem bark of Paramignya m~nophylla.~~ The position of the side chain was confirmed by the alkaline induced cyclization of 76. The structures of the natural calanolides C and D have been reassigned6* to pseudocalanolides C 77 and D 78 where the orientation of the two pyran rings is opposite to that initially reported.The absolute stereochemistry of pseudocalanolide C 77 was established as (6S,7S,8R) using the modified Mosher’s method. Synthetic routes leading to the natural 4-phenyl- 4-propyl- and 5-methyl-coumarins isolated from Calophyllum sp. have been reported.69 This paper reassigns the structures of oblongulide 79 and apetatolide 80 as well as those of calanolides C and D whose reassignment by other means is reported above. The same synthetic study establishes as trans the previously unknown stereochemistry about the 2,3-dimethylchromanone ring of tomentolides A 81 and B 82. Pseudocordatolide C 83 was isolated from Calophyllum laniger~rn,~’~ 71 and calanolide F 84 from the leaves and twigs of Calophyllum teysmanii.” The 0.34 PM.Finally enantioselective total syntheses of (+)-calanolide A and (+)-calanolide B and their ( -)-enantiomers have been reported.74 75 7 6,7-Dioxygenated coumarins A new and unusual pyranocoumarin named clematichinenol 87 was isolated from the aerial parts of Clematis ~hinensis.~~ From I3C-lH COSY and COLOC experiments all carbons and protons were unambiguously assigned. Coumarin 88 is a constituent of Lonicera gracilipe~.~~ The aerial parts of Eriostemon ~ymbiforrnis~~ afforded isobaisseoside 89 and isobaisseoside-4’-p-coumarate 90. Their structures were un- ambiguously determined by NMR bidimensional experiments. Compound 89 is isomeric with baisseoside in which the Me0 0 0 HO 0 O 79 Y O 80 Y 87 R’O 0 88 R’ = H; R2= 6~-D-apiofuranosyl-(1~6)-Op-gl~~0~~l 89 R’ = 6a-rhamnosyl-P-glucosyl; R2 = H 01:: / 0 Hop ~2 = H / 0 90 R’ = 6a-rhamnosyl-~-(4’-pcoumaroyl)-g1~~0~~1; 81 R=Ph 83 82 R=Pr Me0 0 I 0 OH 0 Yo 85 ’ 84 g / 00 0 ***?OH 86 relative stereochemistry of 84 was determined by coupling constant analysis and comparison of calanolide F with known compounds and the modified Mosher’s method established its absolute stereochemistry at C-12 as S.The same authors7’ reported the isolation of a new coumarin 85 from the latex of Calophyllum teysmanii. The orientation of the trisubstituted olefin was established as E based on the observed NOE enhancements. The newly identified compounds were screened for anti-HIV activity.Kucherenko et al. 72 established anti- HIV activity for ( f )-calanolide A and also described a novel approach for its synthesis. Soulattrolide 86 isolated from Calophylum tey~rnannii~~ latex was found to be a potent and specific inhibitor of HIV-1 reverse transcriptase with an IC, of 470 Natural Product Reports 1997 91 R1= 6’-Oacetyl-P-D-glucosyl; R2 = H yoPo 0 0 0 RoP HO-7 93 rutinose moiety is attached to C-6. Prionanthoside 91 was obtained from Viola prion~ntha.~~ New sfondine derivatives 92 were synthesized as potential phototherapeutic agents.80 Improved syntheses of esculetin (6,7-dihydroxycoumarin) and 3-hydroxy-6,7-dimethoxycoumarinwere reported.81 Scopar- one and scopoletin exhibited a potent inhibitory effect on rabbit platelet aggregation.82 The aerial parts of Chorilunea quercifolia26 yielded 93.The production and excretion of the coumarins scopoletin scopolin and ayapin were studied in whole sunflower plants and isolated organs using a pre-viously reported abiotic elicitation system.83 Aesculetin scopoletin and scopolin showed marked activity as inhibitors of eicosanoid-release from ionophore-stimulated mouse peritoneal macro phage^.^^ 8 7,8-Dioxygenated coumarins ( f)-R~taretin~~ 94 was synthesized from daphnetine 95. Two new coumarin glycosides 96 and 97 have been isolated from Ruta graveolen~.’~ Their structures were confirmed by acid hydrolysis. Four new coumarins epoxycollinin 98 shinanally-lo1 99 schinilenol 100 and schinindiol 101 were obtained from Zanthoxylum s~hinifolium.~~ Shinanallylol 99 showed inhibitory activity on platelet aggregation in vitro.OR 94 R=H 96 R = a-L-rhamnosyl-(l+6)-P-D-glucosyl HO 0 OH OR 95 97 R = a-L-arabinosyl ROJp 0 OMe 98 R = CHz / 100 R= C H z U H I OH OH 9 5,4,7-Trioxygenated coumarins The roots of Bupleururnfruticosurn86 yielded the new coumarin 102. The position of the substituents on the aromatic ring was determined by NOESY and COLOC experiments. The aerial parts of Pterocaulon alope~uroides~~ afforded 7-(2,3- dihydrox y -3-methylbutyloxy)-5-hydroxy-6-methoxycoumarin 103. 13C NMR data assignments were carried out through OH 0 HO A OH 102 1 03 isolated from Pterocaulon balansae but its structure was erroneously assigned as 105.The structure of 104 was confirmed by X-ray crystallographic analysis. 10 5,7,8-Trioxygenated coumarins Two new coumarins 106 and 107 have been isolated from gametophytes of Tetraphis pell~cida.’~ The phenolic pattern of the Tetraphis pellucida is like that of the Polytrichaceae i.e. characterized by tri- and tetra-hydroxycoumarin derivatives. This is the first property of the gametophytes that demon- strates a taxonomic relationship between Polytrichaceae and Tetraphidaceae. Four glucoside coumarins 108-1 11 were obtained from the moss Polytrichum formo~um;~~ 108 and 109 were also isolated from the moss Atrichurn ~ndulaturn.~’ R20 0 OH 106 R‘ = 6-malonyl-P-D-glucosyl; R2 = H 107 R’ = Me; R2 = P-sophorosyl 108 R’ = P-D-glucosyl; R2 = H 109 R’ = P-D-gluCOSyl; R2 = Me 110 R’ = P-D-~~uCOS~~-(~~~)-P-~IUCOS~~; R2 = H 11 1 R’ = P-D-glucosyl-(Gtl)-P-glucosyl; R2 = Me 11 6,7,8-Trioxygenated coumarins The aerial parts of Asterolasia tryrnalioidesg2 yielded 112.The position of the methoxy and methylenedioxy groups was supported by NOE experiments. 6,7,8-Trihydroxy coumarin and 6,8-dihydroxy-7-methoxycoumarinwere isolated from Pelargonium sidoides.88 This paper also discusses the differen- tiation between oxygenation patterns on the basis of chemical 0 HO 0 OR OH 112 R=Me 114 OH 113 R=CHz+oH f-0 0 I AA ?dbo 104 105 NOESY DEPT and 2D ‘H-13C COSY experiments. 7-Acetoxy-5,6-dimethoxycoumarin has been isolated from Pelargoniurn sidoides.88 This coumarin was also obtained by acetylation of 7-hydroxy-5,6-dimethoxycoumarin,however standard procedures (acetic anhydride-pyridine) did not work so that somewhat drastic conditions were required (acetyl chloride-perchloric acid).This novel coumarin represents the first natural compound within this group possessing an acetoxy function. The coumarin 104 has been isolated from Pterocaulon virgat~rn.~~ This compound had been previously Estkvez-Braun and Gonzalez Cournarins shift data. The coumarin 113 of unknown stereochemistry is a constituent of Metrodoreafl~vida.~~ Fraxetin 114 was obtained as the analgesic principle of the Peruvian medicinal plant Jatropha ~illiata.~~ 12 5,6,7,8-Tetraoxygenated coumarins The coumarin 115 was isolated from Pelargoniurn sidoides.88 The conspicuous absence of any bathocromic shift in the UV spectra of 115 using standard shift reagents and of NOE association of H-4 with a methoxy group strongly supported the 6,8-dihydroxy-5,7-dimethoxyplacing of functionalities in 115.This is the first natural coumarin of this type where such a placing has been detected and it has been transformed into 5,6,7,8-tetramethoxy coumarin under treatment with Me,SO,- K,CO,. The gametophytes of Tetraphis pellucidago yielded compound 114. Purpurasolol 117 has been isolated from Pterocaulon purpuras~ens.~~ Four new tetraoxygenated 471 OH OR Meo*0 0 Me0 0 YH 117 R20v0‘0 OH 118 R’ = P-D-glucosyl; R2 = H 119 Ri = 6-aCetyl-~-D-glUCOSyl;R2 = H 120 R1 = 6-malonyl-p-D-glucosyl; R2 = H 121 R1 = 6’-malonyl-p-D-glucosyl; R2 = Me coumarins (118-121) were isolated from the moss Atrichum undulatum;” 118 was also obtained from the moss Polytrichum formo~um.’~ 13 4-Oxygenated coumarins 7-Demethylsiderin acetate 122 was isolated from Sideritis ma~soniana.’~3-Acyl-4-hydroxycoumarins were reduced in good yields to 3-alkyl-4-hydroxycoumarins using zinc powder in acetic acid/hydrochloric acid.97 Pyrano[3,2-~]coumarinic AcO 122 123 OMe 124 adducts can be obtained in synthetically useful yields by hetero-Diels-Alder reactions between 3-methylenechroman-2,4-dione and gem-di- tri- and tetra-substituted ole fin^.^* The prenylated coumarin ( f)-isoferprenin 123 has been synthe- sized following this method.” 4,4’-Di-U-methylscandenin124 although known synthetically has been reported for the first time as a natural product from stems of Derris scan den^.'^ 14 Coumestans Psoralidin 125 has been isolated by cytotoxicity-guided purifi- cation from the seeds of Psoralea corylifalia,’oo as it turned out to be the active principle.It has been shown to exhibit 472 Natural Product Reports 1997 cytotoxic activity against stomach cancer cell lines with IC, values of 53 pg ml -in SNU-1 and 203 pg ml -in SNU-16. 125 15 Miscellaneous coumarins Schinicoumarin 126 was obtained from the bark of Zanthoxy-lum ~chinifolium~~ and showed antiplatelet aggregation activity. The isolation of 5,8-dimethoxycoumarin from the leaves of Setaria italics'" was described.This compound presents antimicrobial activity against Gram positive and Gram negative bacteria. Hossain et al. lo2 elucidated the struc- tures of monankarins A-F (127-132) a new series of pigments OMe R2 126 127 and 128 R’ = R3 = Me; R2 = H 129 and 130 Ri = R2 = R3 = Me 131 R1 = R3 = H; R2 = Me 132 R1 = R2 = Me; R3 = H having a conjugated pyranosoumarin skeleton which were obtained from the fungus Monascus anka. Monankarins A-D exhibited monoamine oxidase inhibitory activity. The absolute stereochemistry at C-18 of 127 was estimated by Mosher’s method. Monankarins 128 and 130 showed spectral data very similar to those of 127 and 129 respectively except for the circular dichroism (CD) spectra.These facts suggested that 128 and 130 were diastereomers of 127 and 129 respectively. The stereochemistry at C-17 and C-18 of 127 was also analysed independently by the DADAS90 implemented MolSkop sys- tem. The analysis suggested that the stereochemistry at C-17 and C-18 position was R,R.Some new 4-substituted-3-phenyl- 6-methylstyrilcoumarins have been synthesized as antibacterial agents.lo3 The coumarin analogue of retinal 133 was synthesized by the Wittig olefination of a p01yenal.I’~ A psychopharmacologi- cal study of 3-arylsulfonyl-4-hydroxycoumarins has been reportedIo5 4-Phenoxycoumarins and their thio-derivatives have been prepared as herbicideslo6 for the control of monocotyledenous weeds in cereals. Synthesis- and structure- antiallergic activity relations of 4-substituted-3-cinnamoyl-coumarin derivatives have been published.lo7 The 6-and 7-hydroxy 7,8-dihydroxy and 5-methyl-7-hydroxy derivatives of 4-methylcoumarin have been prepared in 40-96% yields. lo’ NH2NHC02CHRNHSO WPI UOAO 134 R = alkyl benzyl .... H fN\ 135 136 The synthesis and chemotherapeutic evaluation of some diazocoumarins has been described.lo9 7-(P-tert-Alkyl-aminoalkoxy)-8-benzoyl-4-methylcoumarinswere prepared and screened for antimicrobial activity.'lo 7-(3-Piperidino-propoxy)-8-benzoyl-4-methylcoumarinshowed marked anti- fungal and antibacterial activity. 3-Chlorocoumarins resulted from the cyclization of 2-hydroxybenzaldehydes with ClCH,COCl. ' ' The synthesis of some coumarin-6-sulfono-N-amino acids 134 and the evaluation of their antimicrobial activity has been reported.' '* Xanthotoxin-5-sulfonamides135 have been pre- pared by reaction of xanthotoxin-5-sulfonyl chloride with an amino or phenolic compound.' l3 Microwave irradation accel- erates the bromination at the a,P-double bond in coumarins using bromine adsorbed on neutral alumina or iodine mono- bromide in acetic acid. 'l4 4-(1-Piperaziny1)coumarins were synthesized and tested in vitro for their inhibitory activity on human platelet aggregation. '' 7-Ethoxy-4-(1-piperazinyl) OMe I -Me0 I Ph OMe coumarin 136 showed the highest activity which was even higher than the reference compounds ASA trifluoroperazine propanolol and dipyridamole.16 Biscoumarins Optically pure (+)-and ( -)-isokotanin A have been syn- thesized.' l6 The key steps involve the asymmetric Ullmann coupling and selective demethylation (Scheme 4). The absolute configuration of the natural (+)-isokatanin A was assigned as R. The optically inactive claudimerin A 137 and B 138 were isolated from the roots of Citrus hassaku.' l7 The complete structure and relative stereochemistry of 137 were obtained from a single-crystal X-ray analysis. The relative configur- ations of four protons on the pyranopyran ring of 138 could not be determined. The authors point out that the lack of optical activity of 137 and 138 suggests that either they are artifacts or they are formed in the plant cells without the participation of enzymes.Claudimerin A 137 and B 138 are the first natural coumarins containing a pyranopyran ring. The aerial parts of Stellera charnaejasmoside' ' afforded chamaejasmoside 139. 11-Methylgerberinol 140 has been isolated from the roots of Diospyros kakis.'" The structure of 140 was subsequently confirmed by a synthesis with 59% yield by refluxing an ethanolic solution of 4-hydroxy-5-methylcoumarin and acetaldehyde for 5 min. Biqmimetic syn- thesis of some novel coumarin dimers based on oxidative coupling with Mn(OAc),-HC10 has been reported. 120 The roots of Yalaha65 afforded the new biscoumarin khelmarin D 141. The R configuration of desertorin A 142 B 143 and C 144 from Ernericelfa desertorum121 was confirmed by X-ray crystallographic analysis of 145 the di-p-bromobenzoate derivative of desertorin A.OMe OMe I 0 NHAc Me0 *OH Me0 iv Ph MeovoH OMe OMe 51yo 80% 1. OAc 0 OMe I II I Me0 viii Me0 vii Me0 vi Me0 -Meog( -Meov Meoq-( Meo*- OAc 90%Iix 0 OH 96% 0 OMe 78% 0 OMe 54% ovowoM:OH OMe Me0 Me0 00 89% 64% Scheme 4 Reagents i activated Cu DMF reflux 72 h; ii TFA H20,THF room temp.; iii Ac,O Py room temp.; iv LAH THF room temp.; v 10% Pd/C cat. TFA EtOH; vi (CH,CO),O TiCl, CH2C12 room temp.; vii TiCl, C,H, reflux; viii ClCOOMe Py 55 "C; ix Bu'OK Bu'OH 60 "C; x NaH HMPA room temp.; xi (CH,),SO, HMPA room temp. Estkvez-Braun and Gonzcilez Coumarins 473 9 137 v- 138 140 141 0 142 R’ = R2 = H 143 R1 = H; R2 = Me 144 R1 = R2 = Me 145 R’ = R2 = pBrC6H4C0 17 References 1 R.D. H. Murray Nut. Prod. Rep. 1995 12 477. 2 B. Mikhova and H. Duddeck Stud. Nut. Prod. Chem. 1996 18 971 (Chem. 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ISSN:0265-0568    

DOI:10.1039/NP9971400465

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

Monoterpenoids /+ David H. Grayson University Chemical Laboratory Trinity College Dublin 2 Ireland X Covering Part of 1993 all of 1994 and part of 1995 Previous review 1996 13 195 1 Introduction 2 2,6-Dimethyloctanes 3 Artemisyl santolinyl and chrysanthemyl systems 4 Cineol derivatives 5 Menthanes 6 Pinanes 7 Camphanes and isocamphanes 8 Caranes 9 Fenchanes 10 Thujanes 11 Ionone Derivatives 12 Iridanes 13 Cannabinoids 14 References 1 Introduction This review covers developments in monoterpenoid chemistry which were published between mid-1993 and mid-1995 and its format closely follows that of the previous article in the series.’ The chemistry of the acyclic and monocyclic monoter- penoids has been reviewed2 in an updated edition of ‘Rodd’ as has that of the bicyclic mon~terpenoids.~ Other aspects of monoterpenoid chemistry have attracted the attention of reviewers including topics such as their general chemi~try,~ catalytic asymmetric synthesis5 and the utilisation of bio- organic methods for the enantioselective synthesis of mono- terpen~ids.~ The use of monoterpenoid-derived compounds which may replace chlorocarbons as environmentally-friendly solvents for the industrial-scale degreasing of metals has been surveyed,’ and a guide to the safe use of essential oils for aromatherapy has been published.’ Two new syntheses of rosefuran 1 have been described,” l1 one of which” makes use of a new general route to furans.HO/ I’@ OAc 1 2 3 Planococcyl acetate 2,12 rac-fragranol 3,13 and rac-,14 ( -)-15 and (+)-grandis01 4,I5-l7the pheromone of the male cotton boll weevil Anthonornus grandis are cyclobutane derivatives for which further syntheses have been reported.New routes to rac-I8 and (-)-lineatin17 5 have been described and an existing synthesis of rac-5 has been improved.” Grayson Monoterpenoids Terpenyl glucosides have attracted much attention (vide infra) and a method for their synthesis has been reported2’ which employs a modified Koenigs-Knorr reaction between a terpenyl alcohol and tetra-0-acetyl-a-D-glucopyranosyl bro-mide in the presence of silver carbonate supported on silica gel followed by deacetylation. The synthesis of several mono- terpenyl 6-O-~-~-apiofuranosy~-~-~-glucopyranosides has also been reported.21 The occurrence of glycosidically-bound monoterpenoids and their role in plants has been reviewed.22 The application of modern analytical techniques to mono- terpenoids has been reviewed,23 as has the analysis of essential oils by tandem GC-FTIR and mass spectroscopic methods.24 Standard ‘fingerprint’ GC traces for five essential oils have been published,25 and the use of multidimensional GC coupled with 1R and mass spectrometry is now26 well-established as a method for detecting the adulteration of plant oils.Multi- dimensional GC techniques which utilise chiral cyclodextrin- based liquid phases have been and the use of PEG-heptakis-(2,3-di- 0-acetyl-6- 0-tert-butyldimethylsily1)-p-cyclodextrin in OV-1701-vi as a liquid phase for the enantio- selective GC separation of the various monoterpenoids present in geranium oils has been rep~rted.~’ The widely-varying enantiomeric compositions of various monoterpene hydrocar- bons which are present in different tissues of Picea abies have been studied by multidimensional GC methods,30 as have the enantiomeric compositions of the monoterpenoids of buchu leaf Stereoisomeric monoterpenoid alcohols and esters are more readily identified by mass spectrometry when CI rather than EI conditions are employed.32 The relative selectivities which P-cyclodextrin exhibits for the inclusion by it in aqueous media of a variety of monoterpenoid hydrocarbons alcohols and ketones have been measured.33 The novel spiro-ethuliacoumarin 6 has been obtained34 from Ethulia conyzoides and its structure confirmed by X-ray analy- sis.Enantioenriched asarinol-C 7 and (+)-asarinol-D 8 have been obtained35 from the roots of Asiasarurn sieboldi. HO HO HO 6 7 8 The volatile monoterpene hydrocarbons which are emitted by the flowers of members of the Apiaceae have been exam- ined.36 Variations in the composition of the volatile mono- terpenoids present in cultures of Mentha arvensis have been demonstrated to be a function of the conditions under which the cells are grown.37 The composition of the monoterpenoids of Pinusflexibilis has been studied in relation to geographical ~ariation,~’ as have the seasonal and morphological relation- ships which affect the composition of the essential oil from Salvia oficinalis L.cv. kraj~va.~’The essential oil-bearing plants of Ethiopia and their uses have been re~iewed.~’ 477 Aspects of monoterpenoid biosynthesis in higher plants have been re~iewed,~",~ as has the genetic control of monoter- penoid biosynthesis in Mentha ~pp.~~ and the biochemistry and molecular biology of isoprenoid metabolism.44 Another review45 discusses the metabolism of monoterpenoids with specific reference to metabolic turnover in intact plants and in detached tissues. Articles on the biosynthesis of monoterpe- noids which are concerned with localisation and ~ecretion,,~ isopentenyl diphosphate bio~ynthesis~~ and monoterpene syn- thetases4' have also appeared. The monoterpene cyclases which occur in grand fir callus cultures have been ~tudied.~' The various biotransformation reactions which plant cell cultures can carry out when monoterpenoids are substrates have been re~iewed,~' as have the process conditions which are required for the successful operation of such systems on a larger scale.51 The production of aroma compounds by plant culture systems has been surveyed,52 and the production of monoterpenoids by tissue cultures of Thymus vulgari~~~ and Melissa ofi~inalis~~ has been described.The stereospecificity of the hydroxylation of rac-dihydro-P-campholenolactone9 9 which is effected by Fusarium culmorum has been investi- gated,55 and shown to take place mainly at C-6 and to a lesser extent at C-5.The effects of environmental factors on the biosynthesis of i~oprenoids~~ and on the emission of isoprene from plants57 have been reviewed. Factors which lead to variability in the emission of unsaturated monoterpene hydrocarbons from ponderosa pines have been studied,58 as has the diurnal variation in the quantity and rate of emission of monoterpenes from Pinus tabulaeformis which peaks at both noon and midnight.59 Radiolabelling experiments involving emitted monoterpenes have demonstrated a close correlation between photosynthesis and monoterpenoid formation in the needles of Pinus abies.60 Seasonal variations in the composition of the monoterpenoids which are emitted from coniferous trees have been noted and quantified.61 The bioactive volatile monoterpenoids which are emitted by higher plants have been reviewed.62 Coniferous trees emit monoterpenoids which can act as protective agents against herbivores and pathogens.Thus certain monoterpenes may behave as attractive agents for bark beetles but also for their predators and parasite^.^^ Low concentrations of various essential oils and especially that obtained from Majorana syriaca have been shown to inhibit the growth of several soil-borne and foliar fungal pathogens,64 and the essential oils from Origanum marjorana and Thymus serpyllum are toxic to the insect Acanthoscelides obtectus which is a pest of kidney bean plants.65 The monoterpenoids present in the foliage of Picea sitchensis exert a negative influence on browsing by red deer.66 The development of 'natural' methods for the control of unwanted plant species in the field continues to attract atten- tion.A series of 18 volatile monoterpenoids have been screened for their phytotoxicity towards crop and weed species. All exhibited relatively high germination and growth inhibititory activity towards corn and wheat. Several oxygen- ated monoterpenoids severely inhibited the germination of four different annual weeds but had little effect on soybean germination. Geraniol 10 and a-terpineol 11 were especially selective in this respect.67 A number of monoterpenoid alde- hydes and ketones have been converted into a series of ureas amides and amines which have been surveyed for their 478 Natural Product Reports 1997 10 R=OH (S)-11 23 R=OPP antimicrobial and plant growth regulatory activities.68 Many monoterpenoids are phytotoxic at concentrations <100 ppm and must often be distributed at ground level via rainwater.A survey of typical solubilities in water has revealed6' the follow- ing patterns hydrocarbons <35 ppm ketones 155-6990 ppm and alcohols 183-1 360 ppm. Turpentine oil totally inhibits the penetration of human hair by the fungi Microsporum gypseum and Trichophyton ~anbreuseghemii.~'Myrcene 12 has been shown to prolong the 12 sleep time of rats which were treated with pentobarbital. The mechanism appears to involve interference with in vivo bar-biturate metaboli~m.~~ The inhalation of certain fragrant essential oils (which contain monoterpenoids) inhibited the motility of mice even when they were dosed with the stimulant caffeine.Monoterpenoids were found at nanogram levels in the mouse serum supporting the perceived benefits of aroma- therapy via inhalati~n.~~ Rate constants for the reaction of various monoterpene dienes with ozone have been determined.73 The reactions which monoterpene dienes undergo with C,-C aliphatic al- cohols via catalysis by synthetic zeolites have been reported,74 as have the reactions of various monoterpene epoxides with methanol in the presence of K-10 m~ntmorillonite.~~ Various unsaturated monoterpenoids have been reacted with H,S in the presence of aluminium halides at 0 "C to give mixtures of sulfur compounds which includes thiols and episulfides some of which have good organoleptic proper tie^.^^ The reactions of acyclic monoterpenoid 1,5-dienes and of their terminal epox- ides in S0,ClF-HS03F superacid media have been studied77 and re~iewed.~' The photochemically-induced homolytic fis- sion reactions of a number of chiral monoterpenoid-derived amine-boryl radicals have been examined.79 The investigation of plant essential oils continues apace and Table 1 lists species which have been examined during the period under review and for which monoterpenoid constitu- ents have been identified and quantified.A comparative study of the leaf oils of several south Australian Eucalyptus spp. has been carried out.262 2 2,6-Dimethyloctanes The linalyl glucoside 13 and its 6'-O-malonate derivative have been isolated263 from Jasminium sambac and are aroma precursors of linalool 14 in that plant.(R)-Linalyl O-~-L-arabinopyranosyl-P-D-glucopyranosideis a precursor of ha- loo1 14 in Gardenia ja~minoides,~~ and a number of substituted glycosides of linalool and of the dihydroxylinalool 15 have been obtained from Ligustrum pend~nculare.~~~ Geranyl O-~-D-xylopyranosyl-P-D-glucopyranosidea precursor of is Table 1 Sources of monoterpenoids ~____ Species Principal constituents Reference Abies balsamea P-Pinene car-3-ene bornyl acetate terpineols 80 Achillea crithmifolia a-Terpineol camphor 81 Achillea fragrantissima (Forssk.) Sch. Bip. a-Thujone artemisia ketone santolina alcohol 82 A c t in odium cunn ingham ii Schaue r .a-Pinene 83 Adenosma bracteosum Bonati Thymol linalool 84 Aesculus hippocastanum Camphene limonene cineol a-and P-pinene 85 Aframomum pruinosum Gagnep. P-Pinene 86 Aloysia triphylla (L’Herit.) Britton Cineol geranial neral 87 Alpinia breviligulata Gagnep. leaf a-Pinene 88 root Cineol borneol fenchyl acetate 89 rhizome P-Pinene 89 seed Geranyl acetate 90 fruit P-Pinene a-terpineol 90 Ambrosia maritima a-Terpineol 91 Angasomyrtus salina a-Pinene 92 Angelica decursiva (Miq.) Fr. et Sav. a-Pinene 93 Aniba canelilla (H.B.K.) Mez. a-and P-Pinene 94 Annona squamosa leaf a-and P-Pinene sabinene 95 fruit Borneol bornyl acetate verbenone 95 Arillastrum gumm ferum (-)-Limonene a-and P-pinene 96 Aristolochia asclepiadifolia Brandg.Linalool borneol 97 Aristolochia ovalvolia Duchr. y-Terpinene geraniol camphor borneol a-cyclocitral 98 Artemisia absinthum Myrcene bornyl acetate a-fenchene P-pinene 99 Artemisia annua Artemisia ketone artemisia alcohol 100 Artemisia argenta L’Her. a-Phellandrene isopinocamphone 101 Artemisia gmelinii Web. ex Stechm. Artemisia ketone cine01 102 Artemisia maritima a-and P-Thujone 102 Artemisia roxburghiana Wall. ex Bies var. hypoleuca a-and P-Thujone 102 Artemisia sieberi Bess. Artemisia thuscula Cav. Artemisia cv. ‘Powis Castle’ Camphor camphene cineol Camphor davanone (2)-Epoxyocimene p-thujone 103 104 105 Austrabaileya scandens C. White Austromyrtus spp. Baccharis dracunculifolia a-and P-Pinene (4-p-Ocimene P-Pinene 106 107 108 Baccharis latfolia a-Pinene a-thujene 108 Baccharis salic folia a-Phellandrene 108 Blepharocalyx cruckshanksii Limonene a-pinene 109 Blepharocalyx salicifolius Limonene linalool cineol 109 Blumea membranacea (DC.) Hook.f. - 110 Boswellia carterri Birdwood Caesulia axillaris Roxb. Limonene a-pinene Limonene 111 112 Calamintha grandij7ora Callistemon linearis (Schrader et Wendl.) Isopinocamphone a-terpineol Cineol 113 114 Canaga odorata Catimbium latilabre (Ridl.) Holtt. Camphene 115 rhizome Linalool cineol 116 root Citronellol cineol 116 seed fruit skin a-Phellandrene camphor a-and P-Pinene cineol 117 117 Cestrum nocturnum Linalyl acetate linalool citronellyl propionate cineol 118 borneol Chaenomeles speciosa Nakai Linalool linalool oxides 119 Chaerophyllum macrospermum - 120 Choricarpia leptopetala (F.Muell.) Domin a-Pinene 121 Choricarpia subargentea (C. T. White) L. A. S. Johnson a-Pinene 121 Chromolaena odorata a-Pinene 122 Chrysactinia mexicana Chrysanthemum indicum Cinnamomum albi$orum Nees Cinnamomum cambodianum H. Lec. Cinnamomum camphora Nees et Eberm Cinnamomum camphora Sieb. var. linaloolfera Piperi tone derivatives Camphor trans-caran-trans-2-01,bornyl acetate sabinene Geraniol terpinen-4-01 a-terpineol cineol a-Terpineol terpinen-4-01 linalool Camphor Linalool 123 124 125 126 127 128 Cinnamomum sulphuratum Nees Linalool 129 Cinnamomum tamala Citrus aurantium Linalool limonene p-cymene a-and P-pinene Linalool 130 131 Citrus paradisi MacFad. Citrus sinensis Osbeck. Clausena excavata Burm.f. Linalool cis-Piperitol P-Phellandrene 131 131 132 Cleistocalyx fullageri Cleistocalyx operculatus Roxb. Merr. et Perry a-Pin e n e (E)-and (Z)-p-Ocimene myrcene 133 134 Grayson Monoterpenoids 479 Table 1 Sources of monoterpenoids continued Species Principal cons ti tuen ts Reference Cot in us coggygr ia a-and P-Pinene limonene 135 Crithmum maritinum Cupressus sempervirens Cyclotrichium niveum (Boiss.) Manden. et Scheng. Cymbopogon Jlexuosus (Steud) Wats. Cymbopogon jwarancusa Cymbopogon jwarancusa Olivieri y-Terpinene p-cymene P-pinene methyl thymol Myrcene limonene a-terpinyl acetate a-pinene car-3-ene Pulegone isomenthone Geraniol geranial neral citronellol Piperitone Citral 136 137 138 139 140 140 Cymbopogon schoenanthus Limonene 140 D iploloph ium a fricanum Doryphora sassafras Endl.Dracaena refIexa Lam. var. angustifolia Baker Echinophora tenuifolia subsp. sibthorpiana (Guss.) Tutin Elsholtzia blanda p-Mentha-l,3,8-triene a-and 0-pinene Camphor Citronellyl acetate a-Phellandrene p-cymene a-Phellandrene cineol 141 142 143 144 145 Elsholtzia incisa Erodium cicutarium Eucalyptus calmadulensis Pet ford y-Terpinene cineol p-cymene P-pinene Citronellol geraniol isomenthone Cineol a-and P-pinene a-terpineol borneol 146 147 148 Eucalyptus denticulata p-Cymene y-terpinene 149 Eucalyptus goniocalyx F. Muell. p-Cymene limonene cineol 150 Eucalyptus nitens Cineol a-pinene 149 Eucalyptus patens Benth. Cineol limonene a-pinene 150 Eugenia brasilensis Lam. Limonene a-and P-pinene cineol linalool 151 Eugenia plicato-costata a-Pinene 152 Eugenia schuechiana a-Pinene 152 Fluorensia cernua - 153 Foeniculum vulgare Miller subsp.piperitum (Ucria) Coutinho Fenchone 154 Hedera helix var. hibernica Limonene sabinene a-and P-pinene 155 Hedychium coronarium Koenig Linalool 156 Hyptis mutabilis (Rich.) Briq. Camphor myrcenone cis- and trans-dihydrocarvone 157 Hyptis spicigera Lam. a-Pinene sabinene 158 Hyptis suaveolens Cineol 159 Hyssopus oficinalis Pinocamphone isopinocamphone pinocarvone 160 Hyssopus oficinalis subsp. aristatus (Godr.) Briq. Cineol isopinocamphone P-pinene 161 Iuicium micranthum Dunn. Cineol 162 Jasminium sambac Aiton Linalool 163 Juglans nigra a-and P-Pinene sabinene 1 64 Juniperus communis a-Pinene sabinene 165 Juniperus convallium Rehd.et Wils. a-Pinene myrcene limonene 166 Juniperus oxycedrus - 167 Juniperus przewalskii Kom. Juniperus przewalskii Kom. forma pendula (Cheng and L. K. a-Pinene limonene sabinene piperitone a-Pinene sabinene 168 168 Fu) R. P. Adams & Chu Ge-Lin Juniperus rigida Mig. a-Pinene bornyl acetate 169 Juniperus saltuaria Rehd. et Wils. Sabinene cis- and trans-sabinol 170 Lagoecia cuminoides Thymol 171 Ledum groenlandicum Sabinene 172 Ledum palustre Sabinene a-terpinene 173 Ledum palustre angustum - 174 Lepechina Jloribunda Benth. Borneo1 175 Lindera benzoin Blume. var. benzoin Cineol a-and P-phellandrene 176 Lippia adoensis Hochst. ex Walp. Limonene perillaldehyde piperitenone ipsdienone 177 Lippia alba (Mill.) N. E. Br. Lippia integrifolia Lippia junelliana y-Terpinene p-cymene Camphor Myrcenone myrcene limonene camphor tagetone 178 179 179 dihydrocarvone Lippia rnicromera Schauer Lippia polystacha Carvacrol y-terpinene p-cymene a-Thujone carvone 180 179 Lippia turbinata a-Thujone carvone 179 Litsea cubeba Citral 181 Lysicarpus angustifolius (Hook.) Druce a-and P-Pinene limonene a-terpineol 182 Magnolia biondii Pamp.a-Pinene sabinene limonene cineol a-terpineol 183 Magnolia obovata Marjorana hortensis Bornyl acetate camphene a-and y-Terpinene sabinene hydrates terpinen-4-01 linalyl 184 185 acetate Melaleuca ho weana Cineol 133 Melaleuca leucadendron Cineol 186 Melaleuca parviJlora Link. Micromeria fruticosa Druce subsp. serpyllifolia (Bieb.) P. H. Terpinen-4-ol a-and y-terpinene p-cymene Pulegone piperitenone 187 188 Davis Mintostachys andina Murraya koenigii Spreng.Nepeta caesarea Boiss. Nepeta discolor Benth. Pulegone menthone P-Phellandrene a-pinene terpinen-4-01 Nepetalactone Sabinene cineol 189 190 191 192 480 Natural Product Reports 1997 Table 1 Sources of monoterpenoids continued Species Principal constituents Reference Nepeta racemosa Lam. Nepeta troodi Holmboe Nepeta tuberosu tuberosu Ocimum keniense Ocimum tenugorum Origanum hypericijolium before flowering after flowering Origanum rotundfulium Bioss. Origanum solymicum P. H. Davis Origanum syriacum var. bevanii Ostericum grosseserratum (Maxim.) Kitag. Pelargonium citronellum Pelargonium grossularioides Persea tolimanensis Peucedanum zenkeri Engl.leaf rhizome Phagnaton sinaicum Bornm. et Kneuck Phallus impudicus Pinus heldreichii Christ. Pinus peuce Pinus purnila Regl. Pinus tabulaeformis Carr. Piper gaudichaudianum Kunth Piper mikanianum (Kunth) Steud. Pittosporum balfourii Plectranthus coleoides Porophyllum ruderale Cass. Psidium guyanensis Pers. Psidium incanum Psidium luridum Psidium pohlianum Berg. Pycnanthemum Joridanum E. Grant and Epling clone 1 clone 2 Rhus coriaria Robinia pseudoacacia Rosa centifoh Salvia cryptantha Montbret. et Aucher ex Benth. Salvia dorisiana Standley. Salvia pomifera Salvia sclarea Satureja holivianu Briq. Satureja cilicia P. H. Davis Satureja odora Sutureja parvijora Satureja wiedemanniana (Lallem.) Velen.Schistostepium heptulobum Oliver et Hiern. Sideritis athoa Papanikolaou et Kokkini Sideritis hispida P. H. Davis Sideritis romana Solenostemon munostachys (P. Beauv.) Briq. Solidago canadrnsis Solidago gram in folia Salis b. Spilanthes acmella Tagetes argentina Tagetes erecta Tagetes laxa Tagetes laxa cubrera Tagetes ternijora Tanacetum dolichophyllum Tanacetum gracile Tunacetum longfolium Wall. aerial parts roots Tarchonanthus camphoratus Telosma cordutu Merrill Terminaliu bentzoe f. subsp. rodriguesensis Wickens Teucrium jlavum Thuja occidentalis Thymus bornmuelleri Velen. Grayson Monoterpenoids 193 Nepetalactone cineol 194 Camphor linalool cineol 195 5,9-Dehydronepetalactone,geranyl acetate Cineol 196 Camphor 197 Carvacrol p-Cymene 198 199 cis-Sabinene hydrate p-Cymene 200 Carvacrol 201 202 a-and P-Pinene p-cymene 203 Neral geranial Citronellol geraniol isomenthone 147 Sa binene 204 Limonene myrcene Car-3-ene 205 Thymol 206 Linalool (a-ocimene 207 a-Pinene limonene 208 Hydrocarbons 209 Hydrocarbons 210 -21 1 P-Pinene linalool 212 Limonene 212 Myrcene citronellyl acetate a-and P-pinene 213 Fenchene bornyl acetate isobornyl acetate 214 Limonene 215 Cineol a-and P-pinene 216 Cineol linalool 217 Cineol linalool 217 Cineol a-pinene a-terpinyl acetate p-cymene 216 Menthone pulegone piperitone 218 Neomenthol pulegone 218 Limonene 219 Car-3-ene linalool 220 Geraniol citronellol 22 1 Cineol camphor a-pinene camphene 222 Perillyl acetate methyl perillate 223 a- and P-Thujone cineol 224 Linalyl acetate 225 Menthone isomenthone carvacrol isopulegone 226 Carvacrol 221 Pulegone piperitone 228 Piperitone oxide menthol piperitenone oxide 228 Borneol limonene 229 Cineol (z>-P-ocimene 230 M yrcene 23 1 Carvacrol 232 Limonene carvacrol 233 P-Pinene 234 a-Pinene limonene myrcene 235 Sabinene P-pinene P-phellandrene 236 Limonene (z>-P-ocimene myrcene 237 (a-and (2)-Tagetenone 238 Terpinolene (E>-p-ocimene piperitone limonene 239 238 (a-(a-and (a-p-ocimene and (2)-Tagetenone (a-and (3-tagetenone tagetone 240 (2)-Tagetone 238 P-Thujone 24 1 P-Thuj one 24 1 trans-Sabinyl acetate trans-sabinol 242 Terpinen-4-01 sabinene p-cymene 242 a-Fenchyl alcohol cineol a-terpineol 243 Geraniol p-ionone dihydro-P-ionone 244 Citronellyl acetate 245 a-and P-Pinene 246 a-and P-Thujone fenchone 247 Thymol 248 48 1 Table 1 Sources of monoterpenoids continued Species Principal constituents Reference Thymus cilicius Boiss.et Bal. a-Pinene 249 Thymus decassatus Thymol 250 Thymus longicaulus chaubardii var. chaubardii Thymol 251 Thymus serpylloides subsp. serpylloides Carvacrol c-terpinene p-cymene 252 Thymus syriacus Boiss. Thymol carvacrol p-cymene borneol 253 Torreya taxifolia Arnott Limonene a-pinene 254 Vitex agnus-castus Cineol limonene sabinene a-pinene 255 Wedelia paludosa a-and P-Pinene limonene 256 Wedelia trilobata a-Pinene limonene a-phellandrene 256 Xylopia nitida p-Cymene 257 Zanthoxylum armatum Linalool limonene 258 Zanthoxylum bungeanum Piperitone 4-terpineol linalool 259 Zan thoxylum bungeanum Maxim.P-Phellandrene piperitone P-pinene 260 Zingiber ojicinale Citral cineol 261 been isolated269 from Artemisiu sulsoloides Willd. and the so-called 'acacia lactam' has been reformulated as 21 following its synthesis from (S)-(+)-halo01 14.270The major phytotoxic metabolite of the fungus Phomopsis foeniculi which causes fennel stem necrosis has been isolated and identified as the acetylenic derivative 22.271 A careful study utilising 2H and I3C NMR and mass 13 R = P-D-GIc 15 spectrometric techniques which has been made of the enzyme- 14 R=H catalysed cyclisation of doubly-labelled geranyl diphosphate 30 R=Ac 23 to give monocyclic and bicyclic monoterpenoids has pro- vided convincing evidence for the correctness of currently accepted mechanisms.272 The conformation of the geranyl diphosphate-Mn2' complex has been probed by NMR spec-troscopy and the results obtained indicate that chelation of the metal to the diphosphate group causes folding of the hydro- carbon chain due to interaction between its unsaturated centres and the bound Mn2+ ion.273 A study of the localisation O-P-D-GIC 0-p-D-Glc of linalool synthase in Clurkiu breweri has revealed that 16 17 linalool 14 is mainly produced in the flower petals and that this is the precursor for the linalool oxides which are found in the pistils.274 The levels of geraniol dehydrogenase found in various Cymbopogon cultivars correlate well with the qualities of the essential oils which are obtained from them.275 The '+OH metabolism of citronellol 24 in Pelurgonium roseum has been in~estigated.~~" OH HO OH roH 18 R=OH 19 R=H 24 The effect that various essential oil constituents have on the growth of Aspergillus Juvus and on the production of ,J OH aflatoxin by this fungus has been examined.277 Geraniol 10 citronellol 24 and nerol 25 all exert inhibitory effects on both OH processes at concentrations of 500 ppm.The stereoselectivity O=@' 0 and regio-selectivity of hydroxylation of various monoter-20 21 22 penoid derivatives by Aspergillus niger has been and the same fungus effects enantioselective ring-opening of the epoxygeranyl N-phenylcarbamate rac-26 to yield the 6s form 26 together with the 6s diol 27 which is obtained in 60% yield geraniol 10 in266Camellia sinensis var.sinensis cv. 'Shuizian' and 96% ee.279 Various basidiomycetes including Gunoderma and the bis-glycoside 16 has been isolated267 from Linariu uppluntum Pleurotus jlubellutus and P. sajorcuju have been juponicu together with the citronellyl derivative 17. The novel screened for their ability to produce volatile oxygenated compounds penproside-A 18 and penproside-B 19 have been metabolites from myrcene 12.280Cultures of the brown-rot obtained268 from Penstemon procerus.Photooxonerol 20 has fungus Gloephyllum odorutum can produce mainly linalool 14 482 Natural Product Reports 1997 34 35 36 R=Me 40 R=P-Me 28 R=CO 25R=Hr( lo 29 R=CO- 26 geraniol 10 or citronellol 24 depending upon the type of culture used and on the nature of added elicitors such as chitin or D-( +)-glucosamine.28'Hairy roots of in vitro grown Artemisia absinthum which has been transformed with Agro-bacterium rhizogenes LBA 9402 carrying plasmid pRI 1855 produce neryl isovalerate 28 and neryl butyrate 29.99 A series of five lipases have been screened for their ability to catalyse the esterification of terpenyl alcohols under non-aqueous conditions.282 The best combination which was found involves geraniol 10 as substrate tributyrin as acyl donor and lipase-AY from Candida rugosa.Another lipase this time from Candida antarctica is also useful for the synthesis of geranyl and a lipase from Candida antarctica SP435 which 41 catalyses the transesterification from triacetin of gerani01,~~ 10 and of citronellol 24284,285 has been employed under optimised The Mucor miehei lipase catalyses the esterification occasions. conditions284which permit its re-use on up to 17 of geraniol 10 in hexane and also without any great advantage in supercritical CO when propyl acetate is the acyl donor.286 The utilisation of Lipodex-E chiral GC columns for deter- mining the enantiomeric purity of linalyl acetate 30 in authen- tic essential oils has been as has the use of chiral P-cyclodextrin-based stationary phases for the GC analysis of linalool 14 in coriander oils.288 (3R)-Linalool ent-14 is regioselectively dihydroxylated using AD-mix-a to give the trio1 31 with the opposite configuration 37 38 39 0 42 43 rOCH2-N"" 44 n NHBz fOCH2-45 NaBH,CN-BF,.Et,O yields the diol 42 which has been con- verted into quercus lactone 43.296Asymmetric dihydroxylation of the geraniol-derived ether 44 followed by biomimetic open- ing of the oxirane ring leads ultimately to the antibiotic 31 32 33 monoterpenoid (+)-tuberine 45 whose absolute configuration is now corrected to that at C-6 being attained when AD-mix$ is employed.(3s)- A fully worked-out protocol for the conversion of geraniol 10 into (S)-( -)-citronello1 ent-24 of 98% ee via asymmetric hydrogenation which is catalysed by Ru(OAc),-[(R)-BINAP] Citronellyl acetate 32 similarly yields 33 with AD-mix-~x.~~~ has been described,298 as have routes from geraniol 10 to The regioselectivity and enantioselectivity of the osmylation of geraniol 10 and of some geranyl sulfonamides has been exam- ined,290 and the asymmetric dihydroxylation of geranyl and neryl acetates has been shown to occur in high ee with a marked preference for dihydroxylation of the 6,7-double bond.29' Myrcene 12 can be selectively ep~xidised~~~ to yield either of the mono-epoxides 34 or 35.Nerol 25 is epoxidised by the reagent combination 0,-Mn( 3)-TPP-[Rh(q5-C,Me,)(bipy) ClJ-HC0,Na-Bz,O under phase-transfer conditions to give a mixture of the regioisomeric epoxides 36 and 37.293The epoxi- dation of (z)-P-ocimene 38 by Na,W04-H,O under phase- transfer conditions in the presence of (n-Oct),PhCH,N'Cl- yields mainly the oxirane 39.294The epoxide 40 obtained from geraniol 10 via asymmetric Sharpless epoxidation reacts with CS,-NaH to give 41 which is converted into (9-linalool 14 on treatment with Bu,SnH in the presence of catalytic amounts of (R)-( +)-citronello1 24 and from nerol 25 to (5')-( -)-24 which employ chiral modified cationic Rh catalysts.299 The influences of the Ru catalyst precursor the support and the solvent on the outcome of the hydrogenation of citral (@/(a-46have been in~estigated.~" When RuC1 is the catalyst precursor and alcoholic solvents are employed the aldehyde function is first acetalised and this is then followed by regioselective reduction of the 6,7-double bond.When chloride ions are absent from kcHo ecHo A &OH Et3B.295 (R)-Linalool is likewise obtainable from the enantio- 46 (R)-47 (-)-48 49 meric epoxide. Reduction of the geranyl epoxide 40 using Grayson Monoterpenoids 483 the catalyst precursor hydrogenation occurs mainly at the 2,3-double bond to give citronella1 47 but cyclisation takes place to give isopulegol 48 when cyclohexane is the solvent. The authors conclude that incompletely reduced Ru species and acidic sites favour the cyclisation and acetal-forming pathways.300 Bimetallic Rh-Sn catalysts supported on silica have been found to effectively hydrogenate (E)-citral 46 to geraniol 10 with 96% selectivity at 100% conversion;301 (2)-46 affords nerol 25 under the same conditions.The selectivity of these reductions is related to the Sn content of the catalyst.302 Linalool 14 is reduced to dihydrolinalool 49 when it is irradiated in water in the presence of Pt-Ti02.303 The regioselective ozonolysis of (s>-( +)-dihydromyrcene 50 at its 6,7-double bond provides a route to the chiral ketone 51 50 51 52 which is an ant alarm pheromone. Alternatively the 6,7- double bond of 50 can be protected by its epoxidation to give 52. The remaining 1,2-double bond can then be ozonolysed and the protected 6,7-bond later regenerated via reaction of the ozonolysis product with 13A1.304 Myrcene 12 reacts with butenal in the presence of askanite- bentonite clay to give the ether 53.305The Diels-Alder reaction between myrcene 12 and the valuable (8-dihydroxyethylene equivalent 54 provides a route to the trans-diol 55,306and 53 54 OH 55 56 57 cycloaddition of myrcene 12 with thiobenzophenone yields the regioisomeric adducts 56 and 57.307Myrcene has been (inevitably!) reacted with c6()and the cycloadduct has been ~haracterised.~'~ Geraniol 10 has been directly converted into the derived allylic sulfone 58 by reaction with PhS0,Na-HC02H in aqueous i~opropanol.~~~ 310 Geraniol 10 has been utilised as starting material in a synthesis of the interesting macrocyclic diepoxides 59 and tetraepoxides 60.31 Six diastereoisomeric tetraepoxides 60 were isolated and their relative configurations were determined by NMR and X-ray methods.Most of them exhibited ionophoretic activity and could transport Ca2' and K' ions across human erythrocyte cell membranes. The C,-symmetric diastereoisomer of 60 which is shown was the most effective for the transport of Ca2' ions. 484 Natural Product Reports 1997 58 R=SO*Ph 59 60 61 R=OAc 64 R=Br Geranyl acetate 61 reacts in the presence of Lewis acids such as SnCl to yield rac-karahana ether 62 via the intermediate 63.312Geranyl bromide 64 undergoes Sn-induced coupling with aldehydes or ketones to give the rearranged products HO R1 62 63 65 66 67 68 65.313The homogeranyl derivative 66 reacts with TMSOTf and then Et3N to give the limonene derivative 67 via the intermediacy of the sulfonium species Linalyl acetate 30 has been converted into the cyclopenten- one 69,315and the acetoxy sulfone 70 undergoes a Pd-catalysed intramolecular metallo-ene cyclisation to give the cyclopentane 71.316 69 70 71 (R)-( +)-Citronella1 47 has been converted into the malonyl- idene derivative 72 which undergoes a low temperature dia- stereoselective ene cyclisation catalysed by FeC1,-A1,03 to give almost exclusively the p-menthyl compound 73.317The citronellyl imine 74 undergoes in situ hetero-Diels-Alder cycli- sation to give mixtures of octahydroacridine derivatives 75,318 but the related chromiumcarbonyl complex 76 obtained from 77 yields the q6-octahydroacridine derivative 78 with good trans-selectivity when the cycloaddition is catalysed by Lewis acids.319 The entire sequence can be effected in a one-pot process by reacting together the aldehyde the o-toluidine chromiumtricarbonyl complex and a Lewis acid.Other deriva- tives of the aldehyde 77 include the chiral a-sulfinyl-a,P- unsaturated ketone 79 which undergoes Lewis acid-catalysed 3 Artemisyl santolinyl and chrysanthemyl systems The glycoside 86 which is a precursor of hotrienol has been isolated326 from peelings of Solanurn vestissimum D. and the 73 74 72 H 4 75 76 tl.; 77 78 ? 1 79 80 F COR A 81 R=Me 82 R=OMe intramolecular hetero-Diels-Alder cyclisation to give the hexahydrobenzopyran derivative the sulfinyl ketone 81 which also undergoes Lewis acid-catalysed hetero-Diels-Alder cyclisation in good de,321 and the related sulfinyl ester 82 which affords ene reaction products also with good de.321 Geranial (a-46 reacts with trimethylaluminium in the pres- ence of catalytic quantities of CuBr to yield the conjugate addition product 77.322 The acid-catalysed cyclisation of (E)-46 affords323 the tetralin derivative 83.83 84 85 A synthesis of rac-ipsenol 84 which employs sulfonyl anion chemistry has been described,324 and an organozinc-based route to the same compound and to rac-ipsdienol85 has been reported.325 Grayson Monoterpenoids highly-oxygenated irregular monoterpenoid 87 has been obtained327 from Aster farreri. The two new chrysanthemyl hydroperoxides 88 and 89 have been found in Achillea nobilis.328 O-P-D-GIC '\ 1 86 87 OAc OOH OAc OOH I I I I 88 89 3-Hydroxy-3-methylbutynereacts with the prenylindium reagent 90to give yomogi alcohol 91 in 88% yield.329 Reaction of the chloro diene 92 with formaldehyde in the presence of SnCI and catalytic CuCl affords lavandulol93 in 58% yield.330 1 90 91 92 HO+ +OH \\ 93 94 95 96 A synthesis of lasiol 94 found in the mandibular gland secretion of male ants Lasius meridionah involves desym- metrisation of the epoxide 95 via initial cleavage with a chiral lithium amide.331 A synthesis of (+)-artemeseole 96 has been described.332 The synthesis of pyrethroic acids from starting materials available from the chiral pool has been reviewed,333 as has the synthesis of chrysanthemic acid derivatives from monoter- pen~ids.~~~ The dimedone derivative 97 has been converted into the homochiral cis-and trans-chrysanthemic acids 98 and 99 in a route which involves a lipase-catalysed hydrolytic resolution step.335 A lipase-mediated kinetic resolution of the rac-trans-ester 100 has been described.336 97 98 99 R=H 100 R=Me (15‘)-trans-Chrysanthemic acid has been converted into (1R)-cis-caronaldehydic acid hemiacetal 101 a key intermediate for the synthesis of the insecticide deltamethrin.337 An X-ray study has revealed that very short intramolecular hydrogen bonds exist in crystals of the potassium and ammonium salts of cis-caronic acid monohydrate 102.338 102 M = K+ or NH4+ 101 4 Cineol derivatives Cultured cells of Eucalyptus perriniana convert cineol 103 into a mixture which consists mainly of the two diastereoisomeric glycosides 104 and 105.339The mechanism and stereochemistry of the hydroxylation of 1,4-cineol 106 by Bacillus cereus has been probed using 2H-labelled substrates.340 103 R’ = R2 = H 1 06 107 R = CH20H 104 R’ = 0-P-D-Glc; R2 = H 108 R = C02H 105 R’ = H; R2 = O-P-D-GIC 109 R’ = H; R2 = OH The novel cineol derivatives 107 and 108 have been iso- lated341 from the urine of brushtail possums Trichosurus vulpecula which had been fed a cineol-enhanced diet as have the hydroxylated products 109 and 110,342and lll.343 The alcohol 109 and the diol 110 have both been ~ynthesised,~~~ as has the diol 111 and its 2B-e~imer.~~~ 110 111 5 Menthanes The three novel glycosides 112-114 have been isolated344 from PerilZa frutescens and the diol 115 and the triols 116 and 117 have been from the roots of Cyanchum hancocki- anum.Acetylsaturejol 118 (known) and isoacetylsaturejol 119 have both been in Satureja gilliesii. Novel labile compounds which have been obtained from the root cortex of ~O-P-D-GIC C02-P-D-GlC I 0 0 A A a-112 114 P-113 115 116 117 118 119 120 Paeonia suflruticosa Andrews include paeonisothujone 120 which is the first o-menthane monoterpenoid incorporating a cyclopropane ring to be The novel dihydrochal- cones named adunctins A-E 121-125 have been in 0 OH Ph OMe PhwoMe / 0 OH 121 122 a-1 23 125 p-124 the leaves of Piper aduncum.New p-menthane derivatives in which the ring has been aromatised include 126 from Bahia schaflneri var. ari~tata,~~~ the five compounds 127-131 from hula crithm~ides,~~’ and the epoxy ester 132 from Eupatorium stoe~hadosmurn.~~~ A crystalline alcohol obtained from Zan- thoxylum rhetsa which was originally named ‘mullilam diol’ and given the 1,4-cineol structure 133 is not a diol at all but is352the rac-trio1 134. A dimer of a-phellandrene has been obtained from elemi The species-specific biosyntheses of (+)-limonene 135 by Citrus unshiu and of ( -)-limonene ent-135 by Mentha spicata which occur via enantiomeric endo-spatial rearrangements of intermediate linalyl cations have been Shoot cul- tures from Mentha arvensis produce pulegone 136 when they are grown in liquid media in darkness.The same cultures begin to produce menthol 137 and menthone 138 as well as pulegone when they are exposed to light.355 486 Natural Product Reports 1997 QOH +OH 126 CI 127 R' = H; R2=Ac 128 R'=R2=H (-)-141 142 a-143 p-144 MeoQ OH RO&OTigloyl OH 129 R=H 130 R =Ac +ova'eroy' 131 The biotransformations that suspended cell cultures of Bupleurum falcatum effect on monoterpenoids such as (+)-limonene 135 and ( -)-carvone 141 have been reviewed.360 Cell cultures of Nicotiana tabacum convert (3-(-)-limonene ent-135 into oxygenated products but have little effect on the (R)-i~omer.~~' Cells of N. tabacum contain cytochrome P,,,-dependent monooxygenases which mediate the allylic hydroxylation of (9-a-terpineol 11 to the diols 142-144.362 The epoxide 145 derived from (+)-limonene 135 is converted into a mixture of the diol 146 and the diastereoisomerically 132 133 134 (+)-135 (+)-136 (-)-137 (-)-138 The enantiomers of terpinen-4-01 139 and of a-terpineol 11 which both occur in the essential oil from Melaleuca alterni- folia have been successfully separated by enantioselective GC $H go 139 (+)-140 using a j3-cyclodextrin-based chiral column.3s6 Similar meth- odology has been applied357 in a determination of the enan- tiomeric purity of (R)-( +)-pulegone 136.The enantiomers of menthone 138 and of isomenthone 140 can be resolved by GC using an octakis-(3-O-butyryl-2,6-di-O-pentyl)-y-cyclodextrin stationary phase.358 Under these conditions optically pure ( -)-menthone 138 and (+)-isomenthone 140 have been detected in some oils e.g.those obtained from Micromeria fruticosa and Calamintha incana. The extraction of menthol 137 and of menthone 138 from peppermint leaves using supercritical CO has been investi- gated and the rates of extraction at different bioconcentrations have been measured.3s9 Grayson Monoterpenoids 145 146 147 (-)-148 pure epoxide 147 by Aspergillus nige~.~~~ The latter could be (lconverted into the sesquiterpenoid (+)-a-bisabolol via reaction ;:i 9.bOH with prenylmagnesium chloride in the presence of CuI. A cell A suspension culture of Catharanthus roseus converts (R)-(-)-piperitone 148 into a mixture of the hydroxylated products 149-151 .363 Racemic piperitone rac-148 is transformed into the PoHo$o P o OH 149 150 a-OH; P-Prl 151 p-0~;P-Prl (-)-153 (-)-154 R = H R=OH 152 p-OH; a-Pri 6S,7S ketol 152 by Rhizoctonia ~olani.~~~ Cultured cells of Mentha piperita convert ( -)-isopiperitenone 153 into the ( -)-7-hydroxy derivative 154.365 The brown alga Dictoya dichotoma var.implexa produces piperitone 148 as its major metabolite.366 The biological effects exerted by some common monoterpe- noids which may find application as environmentally-friendly pest-control agents have attracted attention. (R)-( +)-Limonene 135 which is present in the leaves of the yellow squash Cucurbita pep0 cv. 'Early Prolific Straightneck' is weakly repellent to the female pickleworm moth Diuphunia nitidalis (Stoll.) and the (3-(-)-limonene ent-135 simul-taneously present in the same plant causes a slight but signifi- cant reduction in oviposition by the moth.367 The essential oil of Citrus sinensis which is 94.8% limonene 135 is strongly toxic to various fungal pathogens when it possesses an activity profile better than that of the synthetic substances Carbendazim@ or Maneb@.368 Although limonene 135 has been identified as being the active constituent in this instance limonene on its own does not have as powerful an effect as the complete essential oil does.Limonene 135 which is liberated from the exocarp of wounded orange fruits has been found to stimulate the germination of adventitious conidia of PeniciZ- Zium digitat~m.~~~ (8-Carvone 141 has been shown to inhibit the healing of wounds which have been made on potato tuber tissue.370 Piperitone 148 is more active as an insect repellent than is the commercial product DEET@ (N,N-diethyl-rn- toluamide).259 The I3C NMR spectra of the various stereoisomers of dihydrocarveol and of dihydrocarvyl acetate have been sub- jected to a full analysis.371 Esters of stereoisomeric p-menthyl alcohols with the enantiomeric 0-p-fluoroaryl lactic acids 155 155 a-or p-Me 156 157 158 159 exhibit 'H NMR spectra which permit assignment of the absolute configurations of the Theoretical calcu- lations of the rotatory power of a-phellandrene 156 have been carried The phase behaviour of binary mixtures of limonene 135 or of cineol 103 with supercritical ethene has been Both monoterpenoids behave in a similar manner.Equilibrium constants and thermodynamic par-ameters for the mutual interconversion of the o-menthenes 156159 have been determined.375 The dimetallation of limonene 135 yields a linearly-conjugated dianion which is identical to that obtained from terpinolene 160.376A synthesis of (4S)-( -)-[9-3H]limonene 160 161 162 161 from the aldehyde 162 has been The reactions which limonene 135 undergoes with various C,-C alcohols in the presence of synthetic zeolites to give ethers some of which possess fragrant properties have been described.378 (+)-Limonene 135 reacts with aqueous acid in the presence of synthetic zeolites to give mixtures containing a-terpineol 11 and mainly carvone 141.379The Ritter reaction of (+)-limonene 135 with various nitriles yields isolable iminium perchlorates 163 which are diastereoselectively reduced by sodium borohydride to give cyclic amines Limonene 135 reacts with MeCOSH to yield a mixture of the three thioesters 165-167,381and the 8,9-double bond of limonene can be selectively protected via the [1,3]-dipolar cycloaddition of benzyl azide which yields 168.382Limonene 135 reacts with 488 Natural Product Reports 1997 NHCOR2 NHCOR~ 163 164 SCOMe is bA ,J,,SCOMe 165 166 ASCOMe Ph 1 67 16 8 styrene under catalysis by askanite-bentonite clays to give amongst other products the bicyclic compound 169.383Under the same conditions y-terpinene 170 affords 171 together with the disproportionation product p-cymene 172.(+)-Limonene 135 has been converted via the corresponding alcohol into the useful allylic chloride 173.384 169 170 171 172 1 73 Oxidation of limonene 135 by 0 in the presence of sodium disulfite yields the diol 174 which is forrned via the intermedi- ate epoxide 175.385An improved method for conversion of the diastereoisomeric epoxide 176 into trans-l)-terpineol 177 has been described.386 The oxidation of a-terpinene 178 by 174 175 a-epoxide 177 176 P-epoxide molecular oxygen has been demonstrated to proceed via a I radical-chain mechanism. The primary products are hydrogen peroxide and p-cymene 172.387( -)-Limonene ent-135 has C02Me C02Me been converted into a separable mixture of the diastereoiso- 9;$4:; meric cyclic carbonates 179 and 180 which have been further processed to give the optically pure diols 181 and 182.388 0 C02Me 0-$0 3 fl O-4 0 OH 179 p-bond 181 P-OH 180 a-bond 182 a-OH The selective hydrogenation of the conjugated double bond of some a,P-unsaturated ketones has been achieved using limonene 135 as hydrogen donor in the presence of Pd/C catalysts.389 The isolimonylborolane 183 has been prepared from (+)-isoterpinolene 184.390 183 184 185 186 187 188 Terpinolene oxide 185 reacts with CuSO which has been calcined at 350 "C to give mainly the dienol 186 but reaction with CuSO supported on bentonite clay yields mainly kara- hanenone 187.391Epoxylimonene 175 is converted into a mixture consisting mainly of ketones together with some diols when it is treated with the mixed oxides A1203-y203.392 The product ratios are reversed when less Y203is employed.The structures of the rac-l,2:4,8-diepoxy-p-menthanes arising from terpinolene 160 have been revised and the major product has been shown to be the trans-isomer 188.393 Terpinolene 160 reacts photochemically with methyl aceto- acetate to give the adducts 189-192 which afford the retro- benzylic acid rearrangement products 193 and 194 on heating followed by methylati~n.~~~ The aroma properties of menthol 137 and of menthone 138 and of menthyl esters have been reviewed.395 The kinetics of the hydrogenation of thymoll95 over Ni-Cr,O catalysts have been studied.396 The products which are formed initially are the less thermodynamically stable cis-configured neo-and neoiso-menthols.Hydrogenation of thymol 195 over Pt or Rh catalysts also favours the formation of neo-and neoiso- menthol in a process which proceeds via the intermediacy of the ketones.397 However hydrogenation over Ir catalysts affords these alcohols directly as the primary reduction prod- ucts. Manufacturing processes for the production of rac-and ( -)-menthol 137 have been reviewed.398 Grayson Monoterpenoids 189 190 191 0 192 193 194 A A 201 195 4 196 R=CO 197 R = CH2SnBu3 198 R=CH2NC 199 R = CH2CN 200 R=C=CH Some chiral 'two-layer' phosphines have been prepared from menthol 137,399and conditions for the one-step palladium- catalysed conversion of menthol into its isovalerate ester 196 via reaction with CO-H,-isobutene have been ~ptimised.~" Anodic oxidation of the stannyl ether 197 in the presence of cyanotrimethylsilane in Bu,NBF,-THF yields401 the iso- cyanide 198.When the electrolyte-solvent combination is changed to Bu,NC10,-CH2C1 the product formed is the nitrile 199. The acetylenic ether 200 reacts photochemically with methanol to give the acetic ester 201 via a [1,3] O-+C migration involving an intermediate ketene.,02 The synthesis and properties of hexa-( -)-menthylditin tri-( -)-menthyltin bromide and allyltri-( -)-menthyltin have been described.403 The menthyl-and neomenthyl-tetraphenylcyclopentadienes 202 and 203 have been prepared Ph Ph 202 p-bond 203 a-bond together with their Rh-cod complexes both of which have been the subject of X-ray crystal structure determination^.^'^ The thioether 204 reacts with (-)-menthol 137 in the presence of Br,-pyridine-AgBF to give the menthoxysulfo- nium salt 205 whose structure has been confirmed by X-ray analysis.40s Treatment of the disulfide 206 with bromine leads to the diastereoisomeric menthoxyphenylthioxophosphor-anesulfinyl bromides 207 (67%) and 208 (33Y0).~'~(-)-Menthol 137 has been converted into the chiral neo-configured (-)-Meno.+,Me MeS SMe 'S SMe I I I I 204 205 (-)-Men0 S S ,0-(-)-Men (-)-Meno ./s (-)-Meno ,SBr // \\ I P-s-s-P P Ph' / p\ SBr Ph" "S Ph' 'Ph 206 207 208 r r1 For 205208 (-)-Men = o-,1 sulfinic acid salt 209 via a route which has also been applied to other monoterpenoid alcohols.407 Reaction of ( -)-menthol 137 with PCl .yields menthyl phosphorodichloridite 210 and this can be utilised on a large scale as a resolving agent for ~~c-BINAP.~'~ I I A A 209 210 An extraordinary number of enantio- and diastereo-selective reactions have been effected with the aid of menthyl-based chiral auxiliaries and reagents.The multifarious cycloaddition reactions which the buteno- lide 211 undergoes with diazo compounds nitrile oxides nitrones and azomethine ylids have been examined and A A 211 R' =R2=H 214 R=Me 212 R1 = R2 = Br 215 R = Ph 213 R1 =Nu; R2 = Br product de values have been determined.409 A synthesis of the dibromobutenolide 212 has been reported and its reactions with S 0 and N nucleophiles to give products 213 via addition-elimination sequences have been de~cribed.~" Menthyl propionate 214 is deprotonated by LDA in THF to give a 3 1 mixture of 2 and E enolates which have been reacted with electrophiles such as ally1 bromide benzaldehyde and MoOPh.The highest de values (ca. 60%) were obtained using MOOP~.~~' The related phenylacetate ester 215 could also412 be deprotonated using lithium bases but aggregation of the derived lithium enolates led to poor de values being obtained in subsequent alkylation reactions. However deprotonation of 215 using the strong phosphazene base Bu'P afforded a 'naked anion' which could be alkylated in high de.The homochiral alkoxy allene 216 is a synthon for a-hydroxy allenic ethers which are formed in good de via deprotonation and then reaction with aldehydes. Hydrolysis 490 Natural Product Reports 1997 of these ethers leads to hydroxy ketones 217 with little loss of stereochemical integrity.,l The R and S diastereoisomers of 218 have been synthesised from ( -)-menthy1 phenylglyoxylate 219 and conjugate addition of RMgBr-CuI to the R isomer affords products where the introduced alkyl group is syn to the phenyl substituent.,14 Menthyl phenylglyoxylate 220 undergoes diastereoselective Lewis acid-catalysed ene reactions with homoallylsilyl 216 217 I I A0 Ph 0-0 A 218 219 R=H 220 R=Ph The clay-catalysed Diels-Alder reactions which take place between menthyl acrylate 221 and cyclopentadiene have been studied.416 The a,P-unsaturated menthyl ester 222 reacts with I 221 222 I @ HO j 0 Cop-(-)-Menthy1 223 a-bond 225 224 P-bond furan under catalysis by BF,-Et,O to give a mixture of the diastereoisomeric products 223 and 224.,17 Only the P-configured diastereomer 224 then reacts with rn-CPBA to yield the decalin derivative 225.The stereochemical outcome of the reactions with chiral aldehydes of B-enolates derived from the bromoborane 226 has been examined and thioester-derived enolates have been shown to give superior results in these aldol reactions.418 Three of the four possible diastereoisomeric 1-ferrocenylalkylamines 227 have been synthesised from ( -)-menthol 137 and exam- ined for their usefulness as chiral templates in Ugi four- component condensation reactions.,19 The cyclopentadiene derivatives 228 react with [(q5-CSMes)Co(acac)] to give chiral cobalticinium PF -6 complexes which are anion receptors and which can distinguish between the R and S enantiomers of camphor-10-sulfonic 226 227 228 R=MeorPh The phosphonate anion derived from the 8-phenylmenthyl ester 229 reacts with rac-2-phenylpropanal to give a mixture of the 2 and E products 230 of S configuration but the corre- sponding phosphine oxide anion derived from 231 leads to R I 0-(-)-8-PhenylmenthyI 229 R=OMe 230 231 R=Ph b0%R 0 I'Ph I'Ph 232 R=MeorPh 233 The silyl enol ethers or ketene silyl acetals derived from the esters 232 react under Lewis acid-catalysed conditions to give either syn-or anti-aldol products the outcome of the reaction depending upon the geometry of the starting The formation and stereochemistries of the ketene silyl acetals 233 derived from a series of 8-phenylmenthyl arylacetates has been studied and the 2 isomers have been shown to be the thermodynamic Radical-initiated ring-closure reactions of the dienoic esters 234 proceed best when Lewis acids are present.424 The cyclopentene 235 is then formed in 90% yield and 88% de and the cyclohexene 236 is produced in 72% yield and 84% de.The a,P-unsaturated bromoesters 237 and 238 undergo Michael- induced ring closure reactions when treated with suitable nucleophiles.Three- five- and six-membered ring systems have been produced with de values of up to 95%.425 The acrylate ester 239 can be reacted with chloral in the presence of base to 234 n=l or2 235 n=l 236 n=2 I 237 R=H; n=1,3or4 238 R=Ph; n=1,3or4 give the expected Baylis-Hillman adduct in 70 de.426 Diels- Alder reactions of menthyl acrylate 221 or of 8-phenylmenthyl acrylate 239 with cyclopentadiene which are performed in I 239 240 Me 241 1 242 R = H Ph 2-Naphthyl 243 CF,CH,OH or in CF,CH(CF,)OH proceed with increased rates and endolexo selectivities phenomena which are explained terms of the conformational preferences of the dienophiles in these non-hydrogen bonding donor solvents.427 The 8-phenylmenthyl derivative 240 undergoes ZnC1,-mediated Diels-Alder cycloaddition reactions to give tricyclic products 241 in high de.428 The thioglyoxylates 242 undergo hetero-Diels-Alder reactions with cyclopentadiene to yield mainly 243 which are the first chiral 2-thiabicyclo[2.2.llhept- 5-ene systems to have been prepared.429 Michael addition of MeCH(CN) to the activated imine 244 in the presence of catalytic amounts of La(Pr'O) affords mainly the 2s diastereomer of the adduct 245 whose relative configuration was determined by X-ray analysis.430 Radical addition of the selenide 246 to the vinyl ether 247 leads in good de to the adduct 248 which was also analysed by X-ray I I I 244 245 246 I Ph/ 'OMe 247 248 249 R= C02R C02R PhA Ph 250 Grayson Monoterpenoids 491 methods.431 The photochemical addition of methanol to 1,l- diphenylpropene which can take place in the presence of chiral sensitisers has been The best result which led to the formation of the (S)-( -)-249 was obtained when the sensitiser 250 was used.The oxazinyl 2-fury1 ketone 251 has been synthesised and can be reduced in good de in accord with the Felkin-Anh 1 I Me0 251 252 prediction.433 An X-ray crystallographic analysis has revealed the structure and absolute configuration at phosphorus of the oxide 252.,, Menthone 138 is reduced by aqueous dithionite in the presence of heptakis-2,6-di-O-methyl-~-cyclodextrin give to a 1:2.5 mixture of menthol 137 and of neomenthol 253 in 68% yield.435 The yield is only 10% in the absence of the cyclodextrin.CI A A A A 253 254 255 257 256 Ar = 4-CI-C6H4 ( -)-Menthone 138 is irreversibly deprotonated by strong sterically hindered bases to give an a-anion which reacts with aromatic aldehydes to afford the menthyl arylidene com- pounds 254. Under equilibrium conditions the major products of these condensations are the isomenthyl compounds 255.436 When (+)-menthone ent-138 is condensed with aromatic aldehydes mixtures of the E and 2 forms of ent-254 are said to be formed (cJ:ref. 436) of which only the E isomer undergoes Michael reaction with secondary amines. Reduction of the adducts using LiAlH leads to amino alcohols which have been examined for their analgesic activities.437 The p-chlorobenzylidene compound 256 reacts with thiophenate ion to give the adduct 257 of high de.438 ( -)-Menthone toluene-p-sulfonylhydrazone 258 has been obtained in pure form for the first time and has been found to epimerise to the isomenthyl derivative which by contrast with the parent ketone is the more thermodynamically stable form under acidic conditions 150 times more rapidly than menthone does.439 Shapiro reaction of 258 in the presence of excess BuLi A A 258 259 R=Li 260 R=I leads to the valuable menthenyllithium 259 without epimerisa- tion at C-4.Reaction of 259 with iodine affords the vinyl iodide 260. ( -)-Menthone 138 has been utilised as a chiral auxiliary for the optical resolution of 3-hydroxycarboxylic acids via the derived 1,3-dioxan-4-0nes 261.,,' The chiral pyrazole deriva- tive 262 has been synthesised from (-)-menthone 138,,,l as 261 262 263 a-Pri 264 p-Pri 265 266 Ph Ph 0 267 have the isomeric pyrazole ligands 263 and 264.442The latter have been subjected to X-ray crystallographic analysis.Reac- tion of the pyrazole 264 with CH,Br leads to the chiral bis-pyrazolyl ligand 265.443Acylation of 262 leads to 266 which reacts with phenylmagnesium bromide to give the R ketone 267 of 35% ee together with recovered au~iliary.,~ ( -)-Isopulegol 48 has been converted into both (-)-mintlactone 268 and (+)-isomintlactone 269.445A palladium- catalysed carbonylation reaction has been used to convert @ & tLH& --t-/ 0 O A HO 268 a-H 270 271 272 269 P-H isopulegol into the fused 6-lactone 270.446Both isopulegol 48 and neoisopulegol271 react with Tl(OAc) or with Tl(NO,) in AcOH-H,O to yield the ethers 272.447a-Terpineol 11 and cis-carvol 273 react under the same conditions to give 4a-hydroxypinol 274 or 275 respectively.The reaction of a-terpineol 11 with Tl(OCOCF,) also leads to 274.48 The pulegol derivative 276 is epoxidised in regio-and stereo-specific fashion to give 277.49 Various standard transformations of 6-terpineol 278 have been described.450 The unsaturated diol 279 can be effectively451 oxidised to the ketone 280 by H,O,-(NH,),Mo,O,,. 4H,O-NaOAc-Bu4NC1. (R)-( +)-Pulegone 136 has been utilised in a synthesis of the spiroacetal 281 which is a major component of the abdominal gland secretion of the shield bug Cantao parentum (White).452 Pulegone 136 has been converted into the chloroisopulegone 282 and thence into the synthetically useful sulfone 283.453The four possible cis- and trans-isomers of the odiferous thio ketone 284 have been synthesised from each of (+)-pulegone 492 Natural Product Reports 1997 Hc 290 291 273 274 275 276 An experiment suitable for teaching purposes wherein (+)-pulegone 136 is converted into the chiral carboxylic acid 291 has been described.459 The Diels-Alder adduct 292 has been converted into opti- cally pure ( -)-carvone 141.460The carvone hydrohalides 293 277 278 279 R=H,OH 280 R=O & yx 0;ATcN 294 0 292 293 X=BrorI 0 $0 Po9 S02Ph SH react under radical conditions with electron-deficient olefins 281 282 283 284 such as propenenitrile in the presence of Bu,SnH-AIBN to give the diastereoisomeric products 294.461Carvone 141 reacts iith Me,Al-TMSBr-CuBr to yield the conjugate addition 136 and its enantiomere Isomers were separated by liquid product 295 Conjugate addition Of the lithiated chromatography and their stereochemistries were determined via X-ray analysis of derived 3,5-dinitroben~oylthiolates.~~~ (+)-Pulegone 136 has been converted into the thio alcohol 285 OH eSH 'C02Me 285 286 which has been utilised for the optical resolution of the ketone vac-286 through formation of the mixed a~etal.~~~ Reduction of (+)-pulegone 136 with sodium borohydride in alcohol solutions followed by exposure to strong acids leads directly to the allylic ethers 287.456A fully worked out 1 1 287 288 procedure for the conjugate addition of BuMgCl to pulegone 136 in the presence of MnC1,CuCl which leads to the Michael product 288 as a mixture of diastereoisomers has been des~ribed.~~' (+)-Pulegone 136 has been converted into the keto sulfone 289 which is a precursor for the chiral cyclohexenone 290.458 Grayson Monoterpenoids I On Ph SMe 295 296 297 298 Ry ,,SiMe2Ph HOX::"' 299 R=O 301 300 R=a-H; P-OH thioacetal 296 to (-)-carvone 141 yields the product 297 together with an isomer.Simultaneous desulfurisation and debenzylation using RaNi leads to the pyridone 298.463The keto silane 299 is reduced by Li-NH to give the hydroxy silane 300 which reacts with Bu4NF followed by H202to give the diol 301 with retention of stereochemistry at the C-Si centre.464 The latter steps of the sequence provide a method for the conversion of a silane into an alcohol in the presence of an olefinic bond.The photochemistry of the hydrazone 302 and of some related molecules has been explored and reaction rates have been measured.465 493 302 303 304 I I OQ 0 Of& 0 305 306 R=CI 307 R=H (-)-Carvone 141 reacts with dichloroketene to give a mixture of adducts from which the diastereoisomer 303 can be isolated by simple recrystallisation. Bu,SnH-induced radical formation leads to 304 which rearranges to the ring-expanded radical 305.The latter captures a hydrogen atom to give 306 whose structure was determined by X-ray methods. Excess Bu,SnH reduces 306 to form 307.466 The use of carvone and its derivatives as chiral pool syn- thons for the synthesis of higher terpenoids and other bio- logically active molecules continues in popularity. Thus (+)-dihydrocarvone 308 has been utilised as precursor in a 308 (-)-309 a-Me; R = H 31 1 (+)-310 P-Me; R = H 312 P-Me; R= Me synthesis of the isomeric octalones 309 and 310,467and the carvone-derived 0-diketone 311 has been used in a synthesis of a-cyperone 312 which was obtained optically pure via a phenylalanine-induced intramolecular asymmetric aldol reac- ti~n.~~~ (-)-Carvone 141 itself has been employed as a precursor in syntheses of prostaglandin intermediate^,^^^ of the biomarker eudesmane 313,470of the useful synthon 314,471 of ( -)-3-hydro~yhirsutene,~~~ and of (+)-paeoniflorigenone 315 and (+)-paeonilactone-C 316.473 Syntheses of (+)-paeonilactone-B 317 and of ( -)-paeonisuffrone 318 have also been 0 31 3 31 4 315 0 0 o< 0 -Y0 316 31 7 31 8 494 Natural Product Reports 1997 (+)-Carvone ent-141 has been exploited for the synthesis of some drimane sesq~iterpenoids,~~~~ a synthesis477 476 in of (-)-Ambrox@ and in routes to (-)-coriolin and to ( -)-epi~oriolin.~~~ The double Michael reaction of carvone 141 with methyl methacrylate yields the useful bicyclo[2.2.2]octanone 319.479 Anodic methoxylation of thymol 195 in methanol solution leads mainly to the keto acetal 320.480 I A 31 9 320 321 322 R=H,H 324 323 R=O A synthesis of the leishmanicidal and trypanocidal com- pound espintanol321 has been reported.481 Both menthofuran 322 and evodone 323 have been synthesised via a novel route to fused 3-methylfurans which involves the reaction of allenic A sulfonium salts with ketone en01ates.~~~route to rac-andirolactone 324 has been described.483 6 Pinanes The trans-pinocarveyl hydroperoxide 325 has been isolated from Achillea ptarmi~a,~~~ and the novel dimeric peroxide 326 assumed to have the absolute configuration shown and which &,.OR 0-0 fl6.LH 0 'OH 325 R=OH 326 328 329 327 R=H possesses strong antimalarial activity has been obtained from Amomum krevanh which also contains trans-pinocarveol 327 and the new 4-hydroxymyrtenal 328.485Myrtenol 329 has been found in the alga Cladophora vagab~nda.~~~ The new glycosides 330-332 occur in Artemisia ~ieberi.~~~ Paeonisuf-frone 318 and paeonisuffral 333 have been in the roots of Paeonia sufruticosa as have deoxypaeonisuffrone 334 and isopaeonisuffral 335.347 Isotope effects have been used to reveal details of the reactions catalysed by pinene synthases which are present in Salvia ofi~inalis,~~~ and labelled geranyl diphosphates 23 have been used to study the stereochemistry of the proton elimina- tion step in the S.oficinalis monoterpene cyclase-catalysed reactions en route to a-336 and 0-pinene 337.490a-Pinene 336 is oxidised to verbenol 338 by cells of Penicillium citrinum NFU-901.491 The stereochemistry of reduction of the double bond of verbenone 339 by cultured cells of Nicotiana tabacum has been examined.492 The product cis-verbanone 340 is I 0' I OR ~-D-GIC-~-OAC-GIC 330 R = P-D-GIc 332 331 R = ~-D-GIC-~'-OAC-GIC OH 334 335 333 (+)-336 (+)-337 338 (-)-339 340 formed via addition of the pro-4S hydrogen atom of NADPH to C-2 of 339 with the new 3-H coming from the medium.A molecular modelling exercise together with a definitive 'H NMR study using the Ag(fod)-Yb(fod) shift reagent combi- nation has led to a final (?) assignment of the allylic proton resonances of P-pinene 337.493The 13CNMR assignments for a series of bicyclo[3.1.llheptane derivatives have been revised on the basis of 2D-INADEQUATE experiments.494 The cis- and trans-pinocamphones 341 and 342 exhibit different CI I 341 p-Me 343 344 345 342 a-Me mass spectra which are explicable on the basis of the preferred conformation of each molecule.495 CI-MS employing chiral reagent gases has been used to differentiate the (+)-and ( -)-isopinocampheols 343 and the enantiomers of menthol 137 via diastereomeric ion-molecule reactions in the gas phase.496 a-Pinene 336 trans-isolimonene 344 and a-terpinene 178 all give the same EPR spectrum (of the radical cation of a-terpinene) when they are examined on H-m~rdenite.,~~ Rate constants for the reaction of a-pinene 336 with atomic hydrogen at 295 K which gives mainly pinane 345 have been measured using a fast-flow reactor coupled to a mass spec- tr~meter.,~*( -)-a-Pinene 336 is reduced with very high selectivity by hydrogen over an electrolessly-deposited Ni-P catalyst supported on y-alumina to give cis-pinane 345.499-501 The oxidation of the cis- and trans-pinanes 345 by in situ generated RuO has been studied,502 as has the oxidation of a-pinene 336 using molecular oxygen as oxidant in the presence of Fe3+ or Mn3' phthalo~yanines.~~~ The oxida- tion of neat a-pinene 336 by oxygen in the presence of (4-MePy),CoBr2 yields verbenone (339).504 Ozone can act as an initiating agent for the liquid phase oxidation of a-pinene 336,505and the effect of the partial pressure of ozone upon this process has been examined.506 The addition of NaOH to the system retards the reaction but increases the selectivity for the production of verbenol 338 and verbenone 339.507P-Pinene Grayson Monoterpenoids 337 affords pinocarvone 346 when it is oxidised by either of the systems Fe3+-Bu'OOH or Fe3'-Bu~OOH-picolinic A procedure for the oxidation of (+)-a-pinene 336 by Pb(OAc) coH 346 347 to give ultimately verbenone 339 of high ee without isolating either of the intermediate acetate or alcohol has been described,509 as have conditions for the conversion of P-pinene 337 into perillyl alcohol 347 via its reaction with P~(OAC),."~ The results of further studies on the isomerisation reactions of a-pinene 336 which take place in the presence of acidic zeolites have been p~blished.~" The formation and reactivity of the radical cation 348 generated from ( -)-a-pinene ent-336 by single electron transfer has been reported.512 A 348 A systematic study of the reaction which occurs between a-pinene 336 and acetic acid to give bornyl acetate 349 has been carried Best results were obtained using liquid- phase boric acid-derived catalysts.a-Pinene 336 reacts with ,CHO 349 R=Ac 351 352 353 350 R = COCH=CHz acrylic acid to yield mixtures of bornyl acrylate 350 and fenchyl acrylate 351.514Both a-pinene 336 and P-pinene 337 react with CO-H in the presence of [Rh6(CO),,] to give the hydro- formylation product 352 regiospecifically but when phosphines are added to the Rh catalyst or when [Co,(CO),] is used both alkenes yield the internally-substituted aldehyde 353 instead.51 354 355 356 357 P-Pinene 337 reacts with the enone 354 generated in situ to give the hetero-Diels-Alder adduct 355.516Treatment of P-pinene 337 with PCl leads to a mixture of both 356 and 357.'17 Ritter reaction of ( -)-p-pinene ent-337 with various nitriles in the presence of perchloric acid affords iminium salts which can be reduced to give the bicyclic piperidine derivatives 358.380 The solubilities of a-pinene 336 and of verbenol 338 in supercritical CO have been -P NHCOR 3 358 359 R=C02Me 360 R=CHO ( -)-p-Pinene 337 has been utilised as the starting material in syntheses of robustadial-A and robustadial-B.'I9 The keto ester 359 is obtained from ( -)-S-pinene ent-337 and an exten-sive study has been made of its face-selective Diels-Alder reactions and of the chemistry of the derived add~cts.~~' The regiochemistry and stereochemistry of the cycloadducts ob-tained from the related keto aldehyde 360 via Lewis acid-catalysed reactions with dienes at low temperatures has also been in~estigated.'~'The keto ester 359 has been converted into the antimalarial (+)-qingha~su.'~*, 523 The epoxidation of a-pinene 336 by hypochlorite which is catalysed by manganese complexes of tetraphenylporphine (best) phthalocyanines or certain Schiff bases (worst) has been examined.524 The mechanism of the acid-catalysed ring-opening of a-pinene epoxide 361 does not involve participation 361 362 363 364 365 366 by the cyclobutyl ring in the rate-determining step.525Evidence has been adduced which suggests that the reaction may proceed via initial fission of the internal C-C bond of 361 to give the intermediate 362.The best of a series of alumina-rare-earth catalyst combinations tried for the isomerisation of the epoxide 361 to the aldehyde 363 were Al,O,-Eu,O and Al,O,-Nd,03.526 Aminium salts also catalyse the conversion of the epoxide 361 into 363.',' The peroxide 364 reacts with radical species to give 365 which then forms the epoxides 366.528 Application of a combination of spectroscopic and X-ray methods has revealed the structural details of the novel hetero-Diels-Alder dimer 367 of pinocarvone 346.529Pinocarvone V 367 368 369 ent-346 reacts with cycloheptatriene in the presence of catalytic amounts of its own molybdenumcarbonyl complex 368 to give the adduct 369 which was obtained as a single diastereoisomer whose structure was assigned by X-ray analysis.530 (+)-Nopinone 370 has been converted into the tertiary allylic alcohols 371 which suffer oxidative rearrangement to yield ( -)-verbenone 339 and its derivatives 372.531An X-ray and CD study of the ketene thioacetal 373 derived from (+)-370 371 372 373 R = Et Bu or ally1 Me 374 375 (+)-nopinone 370 has been reported.532The methyl ester 374 has been converted into the q5-complex 375 which has been characterised by X-ray and NMR The myrtenyl derivative 376 undergoes base-catalysed re-arrangement to yield exclusively the allylic alcohol 377.534 376 377 378 379 380 381 382 Nopyl toluene-p-sulfonate 378 has previously been reported to give the cyclobutanol 379 on acetolysis but a second look at this transformation has revealed that the correct structure for the solvolysis product is in fact 380.535This tertiary alcohol which has been named fortesol can be prepared on a large scale is very resistant to rearrangement under strongly acidic conditions and has found applications in the optical resol-ution of carboxylic and phosphinic acids.The synthesis of (+)-apopinene 381 via the decarbonylation of (+)-myrtenal 382 has been reviewed.536 The diol 383 cyclises to a mixture of the tetrahydrofurans 384 and 385 when it is treated with Reaction of the a-pinene-derived cyclopropane 386 with Pt" in the form of Zeise's dimer leads to the methylenebornane 387.538 Applications of chiral organoboranes based upon the pinane skeleton continue to be developed.The apopinene derivatives 388 and 389 which are potentially useful chiral auxiliaries have been synthesised from a-pinene 336 and their hydro-boronation has been examined.539A method for obtaining the bis(2-organylapoisopinocampheylborane)-TMEDA com-plexes 390 in optically pure form has been de~cribed.'~'The apopinenes 381 388 and 389 and a-pinene 336 have been upgraded to optically pure materials via reaction of their 496 Natural Product Reports 1997 383 384 385 387 388 R=Bd 386 389 R=Pri ,BH2 .TMEDA l2 390 R = Et Pri Bui etc 391 392 393 boranes with for example cyclooctadiene followed by a dis- placement reaction with a~etaldehyde.~,~ The new chiral rea- gent 391 derived from 389 is more effective for the asymmetric hydroboronation of prochiral olefins than are the correspond- ing methyl ethyl or phenyl derivatives previously The asymmetric reduction of prochiral ketones has been reviewed in the context of applications of the chloro- borane 392,54’ and the uses of DIP-chloride 393 for this purpose have also been summari~ed.~~~ A systematic study of the asymmetric reduction of a-fluoromethyl ketones with DIP-chloride 393 and with B-isopinocampheyl-9-borabicyclo[3.3.llnonane has been carried The di- methyl sulfide complexes of the isopinocampheylhaloboranes 394 effect clean mono-hydroboronation of both internal and CI A 394 X = CI Br or I 395 terminal alkynes with high regio~electivity.~~~ These reagents especially the iodoborane also exhibit very good regioselec- tivity in the hydroboronation of terminal alkene~.~,~ The stere- oselectivities which they exhibit in the reduction of cyclic ketones follow the sequence I >Br>Cl.B-Enolates derived from the chloroborane 395 react with aldehydes to yield anti- aldol products which have much lower ee values than those predicted by transition state modelling The chiral thioethers 396 form sulfonium perchlorates 397 which react with aldehydes in the presence of base to yield optically-active epoxides 398 together with recovered 396.549 The best ee value (43% for the trans-epoxide) was obtained using the dibenzyl salt with benzaldehyde as the substrate.The use of the amino alcohol 399 as a catalyst for the asymmetric addition of diethylzinc to benzaldehyde has been Grayson Monoterpenoids 396 R = Me P$ or PhCH2 397 Me ‘N-Ph PhAPh @ @OH 398 399 400 reviewed,550 and the pyridine derivative 400 and some related compounds have been synthesised and surveyed for the same purpose.55’ The novel pinanone ketimine chiral auxiliaries 401 have been synthesised and the asymmetric alkylation of the derived 401 R = CO-(-)-menthyl Ph CH2-(2-pyridyl) or CH2-(2’-furyl) 402 anions has been Those that embody an aryl or heteroaryl ring afforded the best de values in these reactions and a transition state model has been proposed to account for the outcome.Diastereoselective alkylation of the p-alanine- derived imine 402 affords after hydrolysis enantiomerically pure a-substituted p-amino acids.553 II 403 R=OMe 405 406 404 R=Ph 407 408 R=H 409 R = 2-norpinanyl The diol 403 reacts with lithium tetrahydridoaluminate (LiAlH,) to give a reagent which reduces prochiral ketones to secondary alcohols of 8-72% ee.554 The diol 404 reacts with allyl(dich1oro)methylsilaneto yield the stable silylene 405 as a single diastere~isomer.~~~ This reacts with the aldehyde 406 in the presence of SnC1 to give the alcohol 407 of 100% de. Syntheses of the chiral terpyridines 408 and 409 have been 7 Camphanes and isocamphanes Bornyl 6-~-~-~-xylopyranosy~-~-~-glucopyranoside, which is a precursor of borneol 410 has been found in Gardenia ja~minoides.~~, The novel trio1 monoglycoside 411 occurs in '&OH 26 OH OH 0-P-D-GIc 410 411 41 2 (+)-413R= H 41 4 415 R = 0-P-D-Glc Diplolophium b~chananii,~~~ and the new camphanol derivative shionoside-C 412 has been isolated from the roots of Aster tatari~us.~~' Enantiomerically pure ( -)-camphor en?-413 is present in Chrysanthemum parthenium Bernh.559 ( -)-Borneo1 ent-410 is glycosylated by cultured cells of Eucalyptus perriniana to give mainly 414,560and the same system yields mainly the glycoside 415 when (+)-camphor 413 is the substrate.561 The behaviour of a cytochrome P450-dependent (+)-camphor hydroxylase which is present in tissue cultures of Salvia oficinalis has been Complete 'H NMR assignments for a broad range of 3-halocamphor derivatives have been The I7O NMR spectra of a series of 27 hydroxylated bicyclo[2.2.1]- heptanes and -heptenes have been recorded and assigned and the observed shielding effects have been correlated with relevant 13C chemical shifts.564 A multinuclear (13C I5N and 170) NMR study of various camphorsulfonyloxaziridines such as 416 has been carried The EI mass spectra of 14 41 6 41 7 41a 419 420 4-substituted camphor derivatives have been measured and analysed.Methane was the only reagent gas which was found to be effective for obtaining the corresponding CI spectra.566 The CI mass spectra of the enantiomers of isoborneol417 and borneol 410 are distinguishable when optically active amino- propanol derivatives such as (5')-(+)-1-aminopropan-2-01 are utilised as reagent gases.567 The He-1 photoelectron spectra of a number of camphor derivatives have been recorded and assigned.568,569 The elastic and inelastic neutron-scattering behaviours of (+)-camphor 413 and of rac-borneol rac-410 have been and a similar study has been carried out on rac-camphene ra~-418.~~' Ab initio calculations of the vibrational CD spectra of camphor 413 and some related molecules have been made.572 Details of the transformations of camphene 418 which occur in the gas phase over alumina silica-alumina and zeolite catalysts have been reported.573 Camphene 418 reacts with methacrolein over p-zeolite to yield the aldehyde 419 which is formed via a Wagner-Meerwein ~earrangement.~~ Optimised conditions for the synthesis of a-acetylcamphene 420 have been described.575 Camphene 418 reacts with aqueous potas- sium permanganate to give the diol 421 together with the I I \OH C02H NHAc 421 R=OH 422 423 424 R=H 425 426 R = Me Ph or PhCH2 427 hydroxy acid 422.576The former undergoes a Ritter reaction with MeCN-H,SO to afford the unsaturated amide 423.The alcohol 424 derived from camphene yields unrearranged amides 425 under the same conditions. The tricyclanols 426 undergo a Bertram-Walbaum reaction with AcOH-H,SO to give the camphene derivatives 427.577 Borneo1 410 and isoborneol 417 are oxidised to give cam- phor 413 when they are treated with aqueous Ca(OCl) in the presence of P-cyclodextrin.The yields obtained are propor- tional to the alcohol-P-cyclodextrin ratio.578 The reduction by sodium borohydride of camphor 413 as its P-cyclodextrin complex has been Camphor 413 reacts with TMSCN-ZnI to give the cyano- hydrins 428 which yield rearranged unsaturated nitriles when they are treated with Tf20-pyridine.580 Camphor undergoes quantitative nucleophilic addition reactions with organo-lithium reagents provided that the carbonyl group is first complexed with CeC13.58' (+)-Camphor 413 has been converted into the dienol 429 which reacts with acids to give two diastereoisomeric 'dibor- nacyclopentadienes'.Reaction with BuLi then leads to the diastereoisomerically pure lithium salt 430 which reacts with for example ZrC1 to yield 431 whose structure was deter- mined by X-ray crystallographic analysis.582 The enolate anion of camphor 413 reacts sequentially with selenium and then oxygen to give a mixture of the epimeric diselenides 432.583 The selenocyanide 433 undergoes base-catalysed elimination to give presumably the selenoketone 434 which dimerises to a mixture of the diastereoisomeric metallocycles 435. The related thiocyanate 436 similarly yields the 1,3-dithiacyclobutane derivative 437. The diastereomerically pure P-chiral mixed anhydrides 438 and 439 have been synthesised from (+)-camphorsulfonic acid M0.584 498 Natural Product Reports 1997 by Pseudomonas putida cytochrome P,50-CAM.589 A series of 1,3-diols 449 which may find application as chiral auxiliaries have been synthesised from (+)-camphor 413.590 Some minor products arising from the bromination of 3-bromocamphor 450 have been identified,591 and further bromination of 3,3-dibromocamphor 451 leads inter alia to the unusual tertiary alcohol 452.592 3-Bromocamphor 450 R' Br ,Br Br 429 430 M = Li(OEt2)" 428 431 M=ZrCI3 432 433 X=Se 434 435 x=s +k 40 SO3P II"Ph 40 S03H 0 435 X=Se 438 a-Bu'; P-Ph 440 437 x=s 439 P-Bu'; a-Ph 441 442 443 The sterically hindered imines 441 and 442 have been synthesised from (+)-camphor 413 by reaction with the amine in the presence of Si(OEt) and catalytic amounts of H,S0,.585 Quantum yields for the EIZ photoisomerisation of the UV-blocking benzylidene derivative 443 (R =H) have been measured.586 A series of benzylidene derivatives 443 have been converted into their arylsulfonyl chlorides using ClS0,H- SOCl, and thence to sulfonamides etc.which have been screened as candidate pesticides and pharmaceutical agents.587 (+)-Camphor 413 has been converted into the olefin 444 which reacts with HBr-AcOH to give the rearranged bromide 444 445 R=Br (+)-446 R = OH Y (-)-447 448 449 445. This affords the (+)-alcohol 446 which can be oxidised to give ( -)-4-methylcamphor 447.588 The (1R)-5-methylenecamphor 448 and its (15')-enantiomer have been synthesised and both are oxidised to the derived exo-epoxides Grayson Mono terpeno ids Br 450 R1 = R3= H; R2= Br 452 451 R1 = H; R2= R3= Br 454 R1= R2 = R3= Br \ 453 455 456 reacts with DMSO-NaI-0 to give camphorquinone 453 in quantitative yield.593 Reaction of 3,3,8-tribromocamphor 454 with KOH leads to the cyclobutane 455 which undergoes a Favorskii-like transformation to the amide 456 when it is treated with PhNH,-KH.594 Thiocamphor 457 is converted into a mixture of thiols 458 and 459 when it is irradiated at 254 nm in the gas phase and 457 R= H,H 458 459 460 R=S=O 462 R=C-OH 461 R=NMe 463 this represents the first example of a Norrish type-2 reaction of an aliphatic thi~ketone.~~~ The thioketene 460 derived from thiocamphor 457 reacts with methylamine via attack at C rather than at S to give the imine 461.596The thiocamphor derivative 462 has been converted into the cobalt complex 463 which is a catalyst for the asymmetric cyclopropanation of for example styrene with ethyl dia~oacetate.~~~ (+)-Camphor 413 continues to find application as a chiral pool-derived starting material for the synthesis of complex molecules.Perhaps the most spectacular exploitation of this ketone has been its use as a starting material in Holton and co-workers first synthesis of Tax01.~~~ Paquette and co-workers have described599 full details of the anionic oxy- Cope rearrangement of the camphor-derived alcohol 464 which leads to the Taxol intermediate 465 and have also published the results of a study on the exo-hydroxylation reactions of the ketone 466 to give the ketol 467.600(+)-Camphor 413 has been converted into the bromoalkyne 468 499 464 465 &C=C-Br wo \\ 'n /I u \' " ' OH Br' 'I 466 R=H 468 469 467 R=OH which reacts with I in the presence of the Koser reagent PhI(0Ts)OH to give the useful synthon 469.601(+)-(1R)-9-Bromocamphor 470 has been utilised as the starting material for a synthesis of (+)-haplaindole-Q 471 which confirms the absolute configuration of that compound.602 (+)-3,9- Dibromocamphor 472 is a key intermediate in a synthesis603 of ( -)-monoterpenylmagnolol 473 which is found in Magnolia oficinalis Rehd.et Wils. II U H 470 R1= Br; R2 = H 471 472 R1= R2 = Br 473 The camphorimine 474 has been converted into the chiral quinoline derivative 475 via treatment with Bu'OK in THF.604 The asymmetric oxidative coupling of the carbanion of the hN,R N-&$ c 474 R = o-CF~-C~H~ 475 476 R = CH2-(3'-pyridyi) imine 476 provides mainly threo products of good de,605 and erythro-threo ratios for the products arising from the oxidative coupling of several related compounds have been determined by 'H NMR spectroscopy.606 The results of extensive work on the use of derivatives of camphor 413 as chiral auxiliaries has been published during the period under review.MIRC reactions of the bromoenoates 500 Natural Product Reports 1997 477 n=1,3or4 478 R=CI 480 R = CH2COMe 479 R=SR' 481 R = COCH2COMe H 482 R = COQ Me 477 with suitable nucleophiles affords 3- 5-and 6-membered ring compounds of high de.425 The bornyloxybutenolide 478 has been prepared and reacted with various thiols to yield addition-elimination products 479.607The copper(I1) chelate of ethyl acetoacetate can be transesterified with borneol 410 to give after treatment with HCl the previously unknown bornyl acetoacetate 480.608A similar sequence carried out using the copper chelate of ethyl oxaloacetate yields bornyl oxaloacetate 481 which reacts with hydrazine to give the chiral pyrazole derivative 482.(+)-Camphor 413 has been converted into the thioalcohols 483 which are potential chiral auxiliaries,609 indeed the thio- alcohol 483 (R=H) has been utilised as an auxiliary for the 483 R = H CONHPh or CH2But 484 Ph 485 486 487 R3 = :or 0 enantioselective synthesis of some P-chiral phosphines and diphosphines.610 The derived sulfoxide 484 undergoes Diels- Alder cycloaddition with cyclopentadiene to give adducts of high de.611 A series of sulfenimines 485 have been prepared and these are oxidised to the sulfinimines 486.Both 485 and 486 react with Grignard reagents to give products 487 which can be cleaved to yield chiral primary amines.612 Another synthesis of sulfenimines analogous to 485 has also been described.613 The synthesis and applications of the amino-alcohol auxil- iaries 488 and 489 have been reviewed,614 and a large-scale synthesis of 488 has been de~cribed.~'~ The scalemic pyrrole derivative 490 (60% ee) catalyses the conjugate addition of a chiral alkoxydimethylcuprate 491 (?) to (E)-cyclopentadec-2-en-1-one to give (I?)-( -)-muscone of up to 99% ee.616 The chiral amplification has been explained by assuming that the reaction is effectively catalysed by a homochiral dimeric cluster of the cuprate.A further study617has revealed that 490 also generally catalyses the asymmetric conjugate addition of dimethylcuprates to 10-1 8-membered (a-and (2)-cycloenones to give chiral 3-methylcycloalkanones. The 488 R=H 489 R=H (+)-495 R= Me (-)-492 R = Me 490 R=H 493 491 R = CuMezLiz &!Me2 -&NMez 7 1 0-Zn-Me 0-Zn-Me I I -0 Me -Zn N,N-dimethylamino alcohols 492 and 493 react individually with dimethylzinc to give complexes such as 494.618When mixtures of the diastereoisomeric amino alcohols 492 and 493 are treated with dimethylzinc heterochiral dimers are favoured.Both of the amino alcohols 492 and 495 catalyse the enantioselective conjugate addition of diethylzinc to chalcones and scalemic samples of the exo,exo-amino alcohol 492 exhibit positive non-linear chiral amplification in this reaction.619 The cyclopropanation of the cinnamic amides 496 and 497 using ’ OH 4% 497 PhLC02Me 498 exo; n = 1-3 500 499 endo; n = 1-3 Et,Zn-CH,I leads to the formation of the 1S,2S derivative (ca. 58% de) from 497 and the 1R,2R derivative (ca. 80% de) from 496.620A complete reversal of these diastereoselectivities occurs when the triisopropylsilyl ethers of 496 and 497 are cyclopropanated and both products are then obtained in up to 98% de.The polyethers 498 and 499 have been successfully prepared and shown to catalyse the low-temperature Michael addition of phenylacetic acid ester anions to methyl acrylate to give products 500 of up to 83% ee.621 The acylated oxazolidinones 501 react with cyclopentadiene in a Lewis acid-catalysed Diels-Alder reaction to yield after further processing the adducts 502.622The isomeric exo-compounds 503 conveniently afford ent-502 under the same conditions. A study of the Diels-Alder and conjugate addition reactions of the acylated regioisomeric oxazolidinones 504 has Grayson Monoterpenoids n 0 501 502 0 503 504 R=R’* 505 R= R’-506 507 been reported together with data on the diastereoselective alkylation reactions of the saturated analogues 505.623An asymmetric Michael addition reaction of the oxazoline 506 has been utilised in a synthesis of the keto acid 507 whose absolute configuration was established by CD A series of unstable but enantiomerically pure oxazoline N-oxides 508 has 508 509 R = H Me Et or Pri been generated and these have been used for in situ asym-metric [2+31-cycloaddition reactions which procede with good regio- and diastereo-~electivity.~~~ Methods for the synthesis and reduction of the diastereo- isomeric camphorimines 509 have been described,626 and an X-ray crystallographic study has been carried out on a series of imines including 509 (R=H) with a view to rationalising the outcome of asymmetric reactions of the derived anions.627 Alkylation of the phosphonoimine 510 leads after hydroly- sis to the chiral (S)-aminophosphonates 511 whose ee values were determined by 19F NMR spectroscopy of their Mosher amide derivatives.628 The acylated oxazolinone 512 reacts with N-substituted a-phenylnitrones to give the [2+31-cycloaddition products 513 of good de.629 Methylation of the anions derived from the N-acyl derivatives 514 affords products 515 in a diastereoselective fashion.630 The oxazolidinone 516 which was prepared from camphene 418 yields unsaturated N-acyl derivatives 517 which undergo Diels-Alder cycloaddition to cyclopentadiene with almost complete asymmetric induction.631 The camphor-derived pyrazole ligand 518 has been synthesised.a2 The ready availability of (+)-camphor- 10-sulfonic acid 440 has led to the exploitation of many of its derivatives for asymmetric synthesis.The derived thioether 519 has been converted via its (E)-l’,2’-dichlorovinyl ether into the cobalt- carbonyl complex 520 which loses one CO group at room temperature under a current of nitrogen to give 521. Exposure to CO reverses this transformation and 521 reacts with norbornadiene in a stereoselective Pauson-Khand reaction to yield the adduct 522 which has been converted into (a-(-)-50 1 H2N/(f P(OEt)2 510 511 R = Me Et ally1or benzyl 0-0-0 524 525 526 0T$-Rl a 0 Ph k0 \S02NCy2 0%R 527 528 Nko 0 0 512 R = OMe or OPr 51 3 514 Ri = H; R2 = Me allyl or benzyl 515 R1 = Me allyl or benzyl; R2 = H 516 R=H 518 517 R =COWR' 519 520 521 522 (-)-523 4-methylcyclopent-2-enone 523 with high ee.632 The diene 524 undergoes cycloaddition with methyl acrylate in the presence of LiClO or ZnC1 to give the adduct 525 with 100% regioselectivity and with high stereo~electivity.~~~ The related sulfinylmaleimide 526 undergoes ZnC1,-catalysed Diels-Alder cycloaddition reactions with furan and with cyclopentadiene to give adducts of good de.634 The chiral cyanoisobornylate ester 527 can be diastereoselec- tively alkylated in an efficient manner to give products which after further transformations yield the p-lactams 528.635An X-ray study has revealed that the (a-sulfonamidocrotonate 529 and the (2)-sulfonamidocrotonate 530 each exist as s-cis conformers but that the sulfonamido- and sulfonyl-acrylates 531 and 532 prefer to exist as s-trans forms.636 502 Natural Product Reports 1997 &OR1 +Ol?S02Ph 532 The sulfonylimine 533 generated in situ via radcal bromi- nation of the corresponding glycine derivative reacts with Danishefsky's diene to yield the adduct 534.637The best product de values were obtained in the presence of 25 mol% of Ti(OPr'),.The sulfonylimines 535 can be deprotonated and then alkylated using alkyl iodides or ethylene Hydrolysis using aqueous acids then yields sulfonamides 536 of 6695% ee. 533 534 N/SOP-R S02NRJ6 R' RASO*NH2 535 R1 = Pri or Cy 536 The N-acryloylsulfonamide 537 undergoes EtAlC1,-catalysed Diels-Alder reactions with dienes which tend to procede in poor yields due to competing self-polymerisation.This can be suppressed by the addition of galvinoxyl and a large-scale synthesis of the cyclohexenylcarboxylic acid 538 has been achieved by this means.639 The same acrylamide 537 has been converted into the aziridine-2-carboxylic acid derivative 539.640A comparison of calculated and experimental results obtained in respect of the cycloaddition reactions of 537 with nitrile oxides has suggested that their diastereoselectivities are controlled by Coulombic repulsion factors.641 The methacrylic derivative 540 reacts with for example ethanethiolate ion to give mainly the diastereoisomeric addition product 541.642 This 537 R=CH=CH2 538 539 540 R = C(Me)=CH2 542 R=H 543 R = CH=CH-C(Me2Br 546 R = CH=CH-(o-MeO)CsH4 541 544 Yo 545 547 can be cleaved with retention of stereochemical integrity to yield the derived a-thio acid together with recovered sultam 542.Effective methodology for the N-acylation of the sultam 542 with a,P-unsaturated acids has been reported.M3 The asymmetric 1,4-addition reactions of higher-order silyl cu-prates with a,P-unsaturated acyl derivatives of 542 leads to products of better de than those obtained when lower order silyl cuprates or monocuprates are used.644 The S,i reaction which takes place between the acylated sultam 543 and R,Cu(CN)Li,.BF leads to 544 which can be reductively cleaved to yield the alcohols 545 of good ee.M5 Factors affect- ing the stereoselectivity of the palladium-catalysed cyclopropa- nation of acylated sultams such as 546 have been investi- gated.646 The compound 546 yields the 1R,2R derivative 547 the structure of which has been verified by X-ray analysis.The stereochemical outcomes of radical allylation reactions of the iodo-compound 548 and of radical-induced intramol- ecular cyclisation reactions of 549 have been determined.a7 548 R =Me 550 549 R = (CH2)3CH=CH2 551 552 The N-fluorosulfonamide 550 reacts with the enolate anion of 3-methyl-2-tetralone to give the (R)-a-fluoroketone 551 of 75% ee together with recovered sulfonylimine 552.648 The imine 552 catalyses the asymmetric oxidation by hydrogen peroxide of sulfides to sulfoxide~.~~ A synthesis of ( -)-pinidine 553 which commences with the acylated sultam 554 has been reported.650 The reagent 555 has been utilised in the optical resolution of a chiral ironcarbonyl complex.651 The first optically pure Grayson Monoterpenoids 553 554 555 556 X = CI Br 4-TolS03-or 3,5-DNBC02- halo- arylsulfonoxy- and carboxylato-selenuranes 556 have been reported.652 These react with sodium hydrogencarbonate to give the selenoxides 557 which can be reconverted into the haloselenuranes 556 with complete retention of configuration via treatment with HX in MeOH.Camphanoyl iodide 558 can be utilised for the asymmetric acylation of meso diols. Thus the diol559 has been converted v A o$0 +OH COI OR 558 559 R = H 560 R = Camphanoyl I 561 562 into the camphanoate ester 560 which was obtained in 98% de after one recry~tallisation.~~~ The keto ester 561 has been synthesised from campholic acid chloride and tert-butyl ac- etate.654 Its Eu3+ complex behaves as a chiral lanthanide shift reagent.The chemistry of some epoxides 562 which were derived from a-campholenic aldehyde has been studied in the context of their development as novel odiferous compounds. 655 8 Caranes A (+)-car-3-ene 563 synthase from Pinus contorta has been partially purified and this converts [1-3H]geranyl diphosphate into the [5-3H]carene 564.656Further experiments with doubly- labelled [3H,'4C]geranyl diphosphate have shed further light on the mechanism of this reaction and (3S)-linalyl diphos- phate has shown to be an important intermediate en route to 563.(+)-563 R = H (+)-565 566 567 568 564 R=3H 503 rac-Car-2-ene rac-565 has been synthesised from both geo- metric isomers of citral 46 by reaction with an organozinc carbenoid generated from Zn-Me2SiC1(CH2)2SiMe2C1.657 (+)-Car-3-ene 563 has been converted into the dienes 566-568 and the Diels-Alder reactions of these dienes with maleic an- hydride and with tetracyanoethylene have been studied.658 A number of known enones ketones and diols have been pre- pared from (+)-car-3-ene 563 and subjected to 'H 13Cand 170 NMR investigation .659 (+)-Car-2-ene 565 is converted into a mixture of derived carenols and carenones via its biotransfomation by callus tissue cultures from Myrtillocactus geornetrizans or Nicotiana tabacurn.660 The acylation reactions which (+)-car-3-ene 563 and (+)-car-2-ene 565 undergo with acetic and propanoic anhydrides proceed normally when they are catalysed by synthetic type Y zeolites.661 The unstable dienol 569 is obtained when (+)-3a,4a-epoxycarane 570 is treated with formic acid in the 569 570 571 572 c): KOPh 575 573 R=CI 0 574 R=CH2=( OH 576 577 578 presence of synthetic zeolites.662 The same epoxide 570 reacts with TMSOTf to yield optically active carvenone 571.663 The caran-2-01 572 obtained via the hydroboronation of (+)-car-2-ene 565 reacts with BuLi-TiCpC1 to yield 573 which is then converted into the enol complex 574 when it is treated with LiOAc.This reacts with benzaldehyde to give (R)-3-hydroxy-3-phenylpropanoic acid of 64% ee.664 The borane 575 which is derived from (+)-car-3-ene 563 reacts with aldehydes to give products 576 whose oxy anions undergo palladium-catalysed cyclisation to the methylenetetrahydro- furan derivatives 577.665 The photolysis of (+)-car-3-ene 563 to give synthetically useful chiral bicyclo[3.2.0]heptenes has been reviewed,666 and (+)-car-2-ene 565 has been utilised as the starting material in a synthesis of (+)-a-elemene 578.667 9 Fenchanes The biotransformations of ( -)-fenchone 579 and of its (+)-enantiomer 580 by cultured cells of Eucalyptus perriniana have been studied. ( -)-Fenchone 579 is hydroxylated to the endo- ketol 581 whereas the (+)-ketone 580 is converted into the regioisomeric exo-ketol 582.668 504 Natural Product Reports 1997 579 R=H 580 R=H 581 R=OH 582 R=OH ( -)-Fenchone 579 undergoes quantitative 1,2-addition reactions with organolithium reagents when it is precomplexed with CeC1,.581 ( -)-Fenchone 579 has been converted into the diol 583; (+)-camphor 413 likewise yields669 the diol 584.A series of optically active 1,3-diols 585 has been prepared from ( -)-fenchone 579.590 HO& HO OH &R OH 583 584 585 586 587 Reaction of (+)-fenchone 580 with SO yields the fenchone- 10-sulfonic acid 586 which has been converted into the oxaziri- dine 587.670This like its camphor-derived relative oxidises sulfides to sulfoxides of reasonable ee but the reaction rates are very low.Fenchyl hydrazone 588 reacts with tert-butyl methyl ketone to yield 589 which can be lithiated and then treated with 588 589 R=H 590 R = PPh2 PhANAN OA Ph 591 592 chlorodiphenylphosphine to give the ligand 590 whose struc- ture was determined by X-ray methods,671 and which has been converted into the molybdenumcarbonyl complex 591. The more complex imine 592 has been synthesised and its configuration and conformation have been established.672 3-Bromoisocamphanone 593 reacts with MeLi to give a multicomponent mixture which consists mainly of alicyclic +f0 Br 6R 0 604 R' = H; R2 = P-D-GIc 607 593 594 605 R' = OH; R2 = P-D-GIc 606 R'=OH; R2=H alcohols and ketones.673 The hindered olefin anti-(2)-bis(fenchy1idene) 594 has been synthesised and its crystal structure has been determined.674 10 Thujanes The novel umbellulone derivative 595 has been isolated from Umbellularia ~alifornica,~~~ and the new phloroglucinol- based euglobal-B1- 1 596 has been obtained from Eucalyptus A I 595 596 bl~kelyi.~~~ The biosynthesis of the thujane class of mono- terpenoids has been reviewed with special emphasis on the application of recombinant DNA techniques to the exploi- tation and study of the monoterpene cy~lases.~~~ The electron-transfer photochemistry of (+)-sabinene 597 has been examined.678 Optically-active ring-opened products are obtained indicating that there must be retention of con- figurational integrity in the intermediate radical cations.Both 598 a-Me 600 p-Cl 602 603 599 p-Me 601 a-CI thujone 598 and isothujone 599 react with sulfuryl chloride to give a mixture of the cyclopentanone derivatives 600-602.679 The structure of 602 has been confirmed by X-ray analysis. Applications of a-thujene 603 sabinene 597 and thujone 598 as intermediates for organic synthesis have been reviewed,680 as have the uses to which thujone can be put in natural product synthesis.681 11 Ionone derivatives The novel 3-oxo-a-ionol-~-~-g~ucoside 604 has been obtained from Oreocnide rubescens,682 and the more fully hydroxylated compound 605 has been isolated from Cunila ~picata.~~~ The known compound blumenol-A 606 has been obtained from Perrottetia muhiflora together with the novel 4,5-dihydro- derivative 607.684Another novel dihydro-a-ionol derivative 608 has been found in Averrhoa ~arambola.~~~ The hydroxy- lated dihydro-(3-ionol glycoside 609 has been extracted from Grayson Monoterpenoids O-P-D-GIC ("y--R10p2 HO" 608 609 R' = P-D-Glc; R2 = H 610 R' = H; R2 = P-D-GIc 611 R' = R2 = P-D-GIc 61 2 61 3 Prunus prostrata,686 and it also occurs in Linaria japonica together with the glycosides 610 and 611.267The dihydroxy- ketone 612 occurs in Taxus ~allichiana.~~~ The epoxide 613 has been isolated from Malus domestica.688 The known compound loliolide 614 found in Eucommia ulmoides has been shown to exhibit immunosuppressive activity against T- and B-lymphocyte~.~~~ 61 4 61 5 61 6 61 7 618 61 9 620 Immobilised cells of Nicotiana tabacum reduce the a$-unsaturated ketone system of a-ionone 615 to give a mixture of the ketone 616 and the alcohol 617.690p-Ionone 618 behaves similarly yielding 619 and 620 under the same conditions.The enzyme xanthine oxidase converts p-carotene into p-ionone 618 via a free-radical mechanism.691 Rhizomes of Iris pallida which have been stored for three years accumulate ca. 400 mg kg -of irones which are valu- able perfumery compounds. If the rhizomes are incubated with the bacteria Serratia liquefaciens and Pseudomonas maltophilia both of which can be isolated from the rhizomes then the irone content rises to 1000 mg kg-after only eight days.692 Syn- theses of a-621 p-622 and y-irone 623 in optically active form have been described,693 and the racemates of all of these irone isomers have been resolved by chiral capillary GC and their organoleptic properties assessed.694 505 621 622 623 a-Pyronene 624 has been selectively epoxidised using MCPBA to yield 625 which can be converted into the three cyclocitral isomers 626-628.695The cyclogeranyl derivatives 629 can be deprotonated by BuLi to give enolates.The enolate 624 625 626 627 628 629 R = OMe OPh or SPh (-)-630 632 631 R=Me derived from the thioester 629 (R =SPh) affords (S)-(-)-629 when it is reprotonated using ( -)-N-isopropylephedrine and this has been converted into (9-cyclocitral and into (3-a- damascone 630.696The lithium enolate of the cyclogeranyl ketone 631 undergoes tandem Michael and ring-closure reac- tions with methyl acrylate to give the decalone 632.697 P-Cyclogeraniol 633 has been converted into (8-(-)-camphonanic acid 634 via a sequence which involves an 633 R=OH (-)-634 635 R = OTS C02Et 640 R=S02Ph 636 = Nu 637 R = OH or &H C02Et asymmetric epoxidation The cyclogeranyl tosylate 635 reacts with nucleophiles to give substitution products 636 but hydroxide ion and diethyl malonate ion also yield the cyclopropane derivatives 637 via the intervention of homoallylic participation.699 The cyclogeranylketene 638 reacts with the lithium salt of 4-fluorothiophenol and then with ( -)-N-isopropylephedrine to give the thioester 639 of high ee.700 The cyclogeranyl sulfone 640 has been converted into tran~-retinal,~~' and the allylic silane 641 reacts with butynone in the presence of ZnI and 4 8 molecular sieves to give after desilylation y-ionone 642.702 The (3-(-)-ketol 643 has been converted into (+)-(6R)-a-ionone (+)-615 of 85% ee,703 and the alcohol 644 obtained via 638 639 641 OH OH 642 643 644 chemoenzymatic resolution of the racemic acetate or by the asymmetric reduction of the corresponding ketone has been converted into (9-a-damascone 630.704 Syntheses of the Riesling acetal 645 and of its enantiomer have been described.705 Preparative GC using a modified cyclodextrin column was utilised to purify the enantiomeri- cally enriched products which were intially obtained.The hydroxyionone derivative 646 reacts with Cs,CO to yield 647 0 0LCO2Me '0 %646 0d0OY\\ 645 647 648 649 650 651 652 653 which is a precursor in a synthesis of f~rskolin.~'~ p-Ionone 618 has been converted into the tetralin 64tL707The p-ionone ironcarbonyl complex 649 has been stereoselectively converted into the E,E and E,Z isomers of P-ionylideneacetaldehyde 650.708The E,Z isomer of 650 was then used to synthesise (92)-retinoic acid.p-Ionol 651 affords the allylic cation 652 which can be trapped with for example (E)-BrCH=CHOSiMe to give 653. Elimination of HBr from 653 affords P-ionylidene- acetaldehyde 650.709 a-Ionone 615 undergoes ene reactions with singlet oxygen and MMP2 and MNDO calculations have been carried out with a view to deciphering the effects of secondary orbital interactions be tween the react ants.71 Reaction of the epoxy-P-ionone 654 with catalytic quantities of aminium salts provoke its rearrangement to the diketone 655.527 New methods for the zeolite-mediated selective cyclisation of Y-ionone 656 to give a-ionone 615 have been rep~rted,~" and the methylated Y-ionone derivatives 657 are converted into 658 and 659 when their P-cyclodextrin complexes are irradiated in methanol.712 506 Natural Product Reports I997 654 655 I 0 656 R= H 658 659 657 R=Me 12 Iridanes Recent developments in the structure elucidation synthesis and acid-catalysed reactions of iridoids have been re~iewed.~' The distribution of iridoids within the subgroup Psyllium of the genus Plantago has been reviewed,714 as has the localis- The ation of iridoids in Castilleja ir~tegra.~~~ structure of nishiandaside which is found in Vitex negundo has been revised to 660 and the novel isonishiandaside 661 has been obtained HO 660 R1 = OMe; R2 = H 661 R1 = H; R2 = OMe \ U[CHO \ 1 H CHO 662 663 664 from Vitex ~annabifolia.~'~ Five new iridoids which are struc- turally analogous to compounds found in many Viburnum spp.have been isolated from Viburnum ti nu^.^^^ The biosynthesis of chrysomelidial 662 and of plagiodial 663 by the larvae of chrysomelid beetles proceeds via the dialdehyde 664.7'* The biosynthesis of iridoids in the Forsythia spp. E viridissima and F. europaea has been studied,719 as has that of catalpol from aucubin in Scutellaria albida and in Paulownia tomento~a.~~~ The biosynthesis of iridoid glycosides in Thunbergia data has also been investigated.721 Known iridoids extracted from Paederia scandens Merrill using hot water have been assessed for their individual physiological effects in respect of plant growth and antimicrobial activities.722 The production of secoiridoid glycosides by plant tissue cultures of Gentiana spp.has been reviewed,723 as has the production of iridoid glycosides by tissue cultures of Genipa ameri~ana.~'~ The in vitro culture of Penstemon serrulatus Menz. for the production of penstemide and of serrulatoside iridoids has also been reviewed.725 The FAB and field-desorption mass spectra of a series of iridoid glycosides have been measured and the negative ion FAB technique has been shown to be the most advantageous for structure elucidation.726 The CD spectra of gardenone and of gardendiol have been obtained and have been used to assign absolute configurations to these compounds.727 Grayson Monoterpenoids 665 666 667 668 669 670 Approaches to the synthesis of iridoids which utilise cy- clopentadiene as the starting material have been reviewed.728 (+)-Pulegone 136 has been converted into the iridoid syn- thons 665 and 666.729Racemic forms of iridomyrmecin 667 isoiridomyrmecin 668 isodehydroiridomyrmecin 669 and al- lodolicholactone 670 have been ~ynthesised.~~'(+)-Isoiridomyrmecin 668 has been synthesised via an aza-Claisen 7 732 rearrangement strateg~,~~ and (+)-arabinose has been converted into (+)-668 and into (+)-boschnialactone 665.733 (+)-Carvone ent-141 has been converted into the chloroketone 671 which undergoes Favorskii rearrangement to the THPOYY4 'C02Me A 671 672 673 R=Me 675 674 R=H OMe 676 677 678 679 ooMe @oMe Ho ' OMe OMe HO OH 680 681 682 683 R = H or alkyl 684 685 Table 2 Sources of iridoids Species Compound( s) Reference Alangium platanlfolium var.trilobum 7-0-Benzoylloganic acid 686 743 Arbutus unedo Asperuloside and gardenoside 744 Astroloma humifisum 10-Benzoylgalioside 687 745 Chelonanthus chelonoides Chelonanthoside 688 746 Dihydrochelonanthoside 689 Chironia krebsii Chironoside 690 747 Cistanche deserticola 6-Deoxycatalpol and 8-epiloganic acid 748 Citronella gongonia Kingisidic acid 691 749 Clerodendrum inerme 692 and 693 750 Clerodendrum inerme Inermosides 694 695 and 696 751 Escallon ia myr toidea 6‘-0-~-~-Glucosylasperuloside 752 Fon tanesia ph illy re0 ides 697 and 698 753 Fraxinus angustifolia Ligstral 699 754 Angustifolioside-A 700 Angustifolioside-B 701 Fraxinus insular is Insularoside 702 755 Fraxinus insularis Oleayunnanoside 703 (revised structure) 756 Fraxinus malacophylla Butyl isoligustrosidate 704 757 Fraxinalacoside 705 Fraxinus ornus Ornoside 706 758 Fraxinus uhdei Fraxuhdoside =Oleayunnanoside 703 759 Fraxinus uhdei Uhdoside-A =Ornoside 706 760 Uhdoside-B 707 Gardenia jasm inoides Gardenone 708 761 Gardendiol709 Gelsemium elegans Benth.7-Deoxygelsemide and 9-deoxygelsemide 762 Gentiana depressa 710 763 Gentiana formosana 3'-Acet ylsweroside 764 Gentiana macrophylla Qinjiaoside-A 711 765 Hydrangea macrophylla Seringe var. Hunbergii Makino Hydromacroside-A 712 766 Hydromacroside-B 713 Kigelia pinnata Known iridoids 767 Lathraea squamaria 714 768 Linaria dalmatica 7,8-epi-Antirrinoside 715 769 Linaria genistifolia 6P-Hydroxyantirrinide 716 769 L inaria japon ica 6P-Hydroxyantirrinide 716 770 Linarioloside 717 5-Deoxyteuhircoside 718 Secolinarioside 719 Linaria japonica Iridolinarin-A 720 771 Iridolinarin-B 721 Iridolinarin-C 722 Linaria peloponnesiaca Linaria sp. Lonicera morrowii 6P-Hydroxyantirrinide 716 5-0-Allosylantirrinoside 723 Kinginoside 724 769 772 773 Mentzelia cordifolia Mentzefoliol 725 774 Glucosylmentzefoliol 726 Osmanthus asia t icus 727 775 Paederia scandens Paederinin 728 776 Patrinia scabra Patriscabrol 729 777 Isopatriscabrol 730 Paulownia lomentosa Tomentoside 731 778 7-Hydroxytomentoside 732 Pedicularis alaschania 1 0-0-Acetylaucubin 779 Pedicularis plicata Plicatoside-A 733 780 Plicatoside-B 734 Penstemon acuminatus Acuminatuside 735 78 1 Penstemon barrettiae 736 782 Penstemon ovatus Ovatuside 737 783 Penstemon parryi Phlomis younghusbandii Plumeria obtusa 738 739 and 740 741 and 742 743 782 784 785 Premna corymbosa var.obtusifolia RauwolJia grandijlora Retzia capensis Rhinanthus angustifolius subsp. grandijlora Schismocarpus matudai Scrophularia korainensis Premcoryoside 744 Isoboonein 745 Holmioside 746 6’-0-Benzoylshanziside methyl ester 747 Schismoside 748 Scropheanoside-1 749 786 787 788 789 790 79 1 Scropheanoside-2 750 Scropheanoside-3 751 508 Natural Product Reports 1997 Table 2 Sources of iridoids Continued ~~ ~~ Species Compound( s) Reference Scrophularia scorodonia Scrophularia spicata Swertia panicea Swertia panicea Thevetia peruviana Thunbergia data Known iridoids 751 and 752 Swertiapunimarin 753 Amarogentin-B 754 755 and 756 Alatoside-1 757 792 793 794 795 796 797 Thunaloside 758 Timonius timon Utricularia vulgaris Valeriana oficinalis sambucifolia 10-Deoxysecogalioside 759 Aucubin derivatives Valdiate 760 798 799 800 Verbascum nigrum Veronica anagallis-uquatica 761 762 and 763 7-0-p-Hydroxybenzoyl-8-epi-loganicacid 764 801 802 7-0-p-Hydroxybenzoylgardoside 765 Viburnum lantana Lantanoside 766 803 Viburnum suspensum 767 and 768 804 Vitex agnus-castus Agnuside and aucubin 805 Zaluzianskya cupensis Zaluzioside 769 806 \ C02Me I Bzo*oH BzO 0-P-D-GIc 0-P-D-GIc HO 686 687 688 689 690 692 R= +OH 693 R= F O H HO@oH O qI O0-P-D-GIc OH OH 694 R=H 0 OR 691 695 R=HOa> R = glucosylatesas for 692696 OH OR' C02Me I 0-P-D-GIc 697 R=HOm 699 700 R=OH 701 R=H 698 R= H Grayson Monoterpenoids BuO2C C02 HO OEt -.-I."i;o 0-P-D-Glc 0-P-D-GIc 704 705 708 HOG O M e Rw HO' 0-P-D-GIc 709 710 711 HO HO HO HO HO*o R0 o*o OH OH O-P-D-GIC O-P-D-GIC Me 0-P-D-Glc 71 4 715 716 71 7 718 71 9 cyclopentyl ester 672 a precursor for (+)-iridomyrmecin 667 been converted into gastrolactone 675 which is found in the and (+)-dihydronepetalactone 673.734An eight-step synthesis defensive secretion of larvae of the chrysomelid beetle Gastro-of enantiomerically pure (+)-mitsugashiwalactone 674 from phy~acyanea.~~~ Racemic forms of the hydroxyloganin and (S)-( -)-citronello1 ent-24 and another synthesis of (+)-674 in deoxygeniposide aglycons have been synthesised from the four steps from hexaacetyl aucubin have been bicyclic acetal 676.737, 738 The related ester 677 rearranges These syntheses confirm the absolute configuration which photochemically to give 678 which can be isomerised to the was earlier assigned to the lactone (+)-674.Citral 46 has conjugated ester 679.739The latter is a formal synthon for the 720 R = ?Me 721 R= 0-P-D-GIc 0-p-D-GIC OH OH OH 722 R= 723 724 0 0-P-D-GlC $Q OMe HO' 0-P-D-GIc 725 R= H 727 728 729 R=P-Me 731 R=H 726 R = P-D-GlC 730 R=a-Me 732 R=OH 510 Natural Product Reports I997 HOq HO q yp OH R* O-0-D-GlC OH OMe 733 R' = CHO; R2 = CH20H 735 736 R' = 2'-0foliamethoylglucosyl; R2 = H 734 R' = C02Me; R2 = H 739 R1 = 2'-QcoumaroylglucosyI; R2 = OH 738 R' = P-D-GIc; R2 = OH 737 R = 740 R1 = 2'-QcoumaroylglucosyI; R2 = H synthesis of racemic mussaenoside 680 and racemic 8-epi-loganin 681. (+)-Genipin 682 has been converted into the marine diterpenoid ~doteatrial.'~' The stereoselectivity of hydrogenation of nepetalactol 683 and of some of its derivatives has been determined.741 Oxymer- curation of 684 leads to 685 and a study has been made of its reductive demercuration reactions.742 New iridoids which have been isolated from plant sources during the period under review and plant species which have provided some known iridoids are listed in Table 2 and structural formulae are provided where appropriate (68G769).The two new monoterpene alkaloids 770 and 771 have been isolated from Tecoma st an^,''^ and the tetrahydroisoquino- line-monoterpene glucosides 772-775 have been obtained from Cephaelis ipecacuanha.808 ( -)-7-Geranylindolactam-5 776 is produced by Streptoverticillum blastmyceticum.'09 13 Cannabinoids The novel bibenzyl cannabinoid perrottetinen 777 has been isolated from Radula perrottetii.'" (R)-Citronella147 condenses with resorcinol in the presence of dichlorophenylborane to yield the isomeric compounds 778 and 779.'' ' When these are heated the hexahydrocannabinol analogues 780 and 781 are produced.Citral46 reacts similarly with 3-methoxyphenol to yield after heating of the initial products a mixture of stereoisomers of the 3-methoxy-A9-tetrahydrocannabinol analogue 782. The A'-tetra-hydrocannabinol 783 has been converted via its methyl ether into both C-9 epimers of the A7-tetrahydrocannabinol 784."* A number of fluorinated and iodinated cannabinol ana-logues have been synthe~ised.~'~ The heterocycle 785 obtained via condensation of citronella1 47 with barbituric acid co C02Me I D O HOH C02Me O-P-D-GIC 741 R = CI 742 R = OH HO-H@o 0 743 C02MeHr~o OH OH 744 745 Ho O-P-D-GIc O-P-D-GIC-~-O-BZ 746 747 748 749 Grayson Monoterpenoids 51 1 C02Me Po HO 0-P-D-GIc 7-‘ OH 0-P-D-Fructopyranosyl OH 753 754 755 C02Me HO C02Me I &o YYo HO 0-P-D-GIc 757 758 759 760 OR’ .OR2 H 0 q 3 -c 0 2 C02HI ~ 0 C02H IHo+02flo- HO 764 0-P-D-GIc 765 0-P-D-Glc /HO fOH HOp::;0-P-D-GIc Ho* 0-P-D-GIc 766 769 OH .J R1O R20 OR2 ,O’I OMe Me02C 770 771 772-(E) R’ = R2 = H 776 773-(E) R’ = Me; R2 = H 774-(2) R’ = R2 = H 775-(2) R’ = Me; R2 = H 5 12 Natural Product Reports 1997 777 778 R1 = OH; R2 = Me 779 R1= Me; R2 =OH 780 R1=OH; R2 = Me 781 R1 = Me; R2 =OH I I I 782 783 784 I Me Me 785 786 undergoes intramolecular hetero-Diels-Alder ring-closure to give 786 and the diastereoselectivity observed for this reaction agrees well with MM2 calculations on the likely transition state structure^."^ 14 References 1 D.H. 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ISSN:0265-0568    

DOI:10.1039/NP9971400477

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

Biosynthesis of polyketides Bernard J. Rawlings Department of Chemistry University of Leicester University Road Leicester UK LEI 7RH Email bjr2Ble. ac. uk Covering mid-1993 to the end of 1994 Previous review 1995 12 1 1 Introduction 2 Eubacteria 2.1 Gram negative bacteria 2.2 Gram positive bacteria 2.2.1 Aromatic polyketides 2.2.2 Non-aromatic polyketides 3 Archaea 4 Protists 5 Fungi 6 Plants 7 Animals 8 References 1 Introduction This review summarises the literature up to the end of 1994 following on from a recent review on fatty acid biosynthesis and related reviews in the series.’ * Polyketide metabolites have been loosely classified as aromatic and non-aromatic including the macrolides polyenes and polyethers.It is now established that the mode of assembly of the two classes is quite different. The ‘aromatic polyketides’ are assembled using a series of discrete enzymes in a repetitive manner reminiscent of Type 11 FAS whilst the non-aromatic are probably all assembled using multifunctional proteins with a separate catalytic activity for each step in a manner more reminiscent of Type I FAS except that each activity is only used once. The manner in which aromatic Type I1 polyketide synthases assemble their products has until now been a very black ‘black box’. Working at the appropriately named Department of Chemical Engineering Stanford Khosla and co-workers have engineered the biosynthesis of novel polyketide~.~ Examining the products produced by various combinations of genes from the actinorhodin polyketide gene cluster Khosla and co-workers have been able to suggest the molecular mode of assembly of aromatic polyketides and the role of each protein.There are two genes adjacent to each other both with very similar ‘KAS like’ sequences one with the highly conserved Cys but the other without. This second gene has been called the ‘chain length determining factor’ (CLF). There is now evidence to suggest that ring A is the first to aromatise and that the single reduction so often seen in ring A occurs after complete poly-P-keto chain assembly in a ‘non-processive’ manner but before aromatisation. Questions such as the nature of the binding of the reactive polyoxo intermediates or their transfer from e.g.KAS to KR and how the enzymes control and regulate these reactions may now be seriously addressed. Now that the Type I multienzyme complexes that assemble macrolides such as erythromycin are available work is concentrating on understanding the molecular basis of the assembly and examining the extent to which genetic engineer- ing can enable the formation of new products. Quite un-expectedly Leadlay and co-workers found that erythromycin synthase only utilises (2S)-methylmalonyl CoA as an extender unit.’ Presumably there is an epimerase activity associated with three of the 3-oxoacyl synthase activities. Katz Cane Ra~vl ings Biosy nthesis of poly ke t ides and Khosla and co-workers have overexpressed all three modules in a heterologous streptomyces and produced 6-deoxyerythronolide B whilst overexpression of just the first module produces the corresponding triketide la~tone.~ The recent progress in understanding the molecular basis of aro- matic and non-aromatic polyketide assembly brings the elusive dream of rationally engineering antibiotic production by mix- ing and matching genes one step closer but the limited success in altering the macrolide nucleus by targeted gene deletion is a salutary reminder that genetics cannot achieve the chemically unreasonable.Progress has been rapid on understanding the molecular assembly of 6-methylsalicylic acid in the fungus Penicillium patulum. Schweizer and co-workers have suggested that the single 3-OX0 reduction occurs at the expected triketide stage but that the corresponding dehydration is postponed until the tetraketide stage when it now dehydrates a 5-hydroxy group and that it may be this postponed dehydration that triggers cyclisation and aromatisation.’ It is commonly assumed that polyketide building blocks are primarily glucose derived acetate and malonate and succinate or propionate derived methylmalonate.O’Hagan and co-workers have reported that the methyl group from thiamine was efficiently incorporated into the seven C units in the polyether monensin suggesting that redundant DNA may be a source of nutrients during monensin production.8 The rapid advances in this area are dependent upon research by a wide range of scientists from synthetic chemists to enzymologists microbiologists and molecular biologists and just as importantly upon communication between them.The interdisciplinary nature of the topic results in an extensive variety of nomenclature so a list of abbreviations used in this review follows A,Aspergillus; ACC acetyl CoA carboxylase; ACP acyl carrier protein; Act actinorhodin; AT acyl CoA ACP transacylase; ARO aromatase; Avm avermectin; BCCP biotin carboxy carrier protein; CLF chain length determin- ing factor; CYC cyclase; DEBS deoxyerythronolide B syn- thase; DH 3-hydroxyacyl ACP dehydratase; E. Escherichia; ER enoylacyl ACP reductase; Ery erythromycin; FAO fatty acid oxidation; FAS fatty acid synthase; Fren frenolicin; glycol ethane-l,2-diol; KAS 3-oxoacyl ACP synthase (3-ketoacyl ACP synthase); kb kilobase pairs; kDa kilodalton; KR 3-oxoacyl ACP reductase (3-ketoacyl ACP reductase); lactate (25‘)-hydroxypropanoate; MMCoA methylmalonyl CoA; MSAS 6-methylsalicylic acid synthase; MT malonyl CoA ACP transacylase; MeT methyl transferase; NAC N-acetyl cysteamine; Otc oxytetracycline; P.,Penicillium; PKS polyketide synthase; pyruvate 2-oxopropanoate; S.Streptomyces; SAM S-adenosyl methionine; SU synthase unit; succinate butane- 1,4-dioate; Tcm tetracenomycin; TE acyl ACP thioesterase; TAL triacetic acid lactone 4-hydroxy- 6-methyl-(2H)-pyran-2-one; Tyl tylosin. A unified set of symbols for precursor incorporation has been used as outlined in Fig. 1. Occasionally reasonable assumptions are used based upon available experimental evi- dence for instance an intact acetate bar when only a single label was used but it is clear which would be the expected intact acetate units.The taxonomic system used in the previous fatty acid review is used.’ Isolated methyl carbon from acetate (not thought to be now part of an intact acetate unit) Isolated carbonyl carbon from acetate (not thought to be now part of an intact acetate unit) / Acetate unit intact Butanoate unit intact Propionate unit intact 3-Methylbutanoate unit intact Intact acetate unit and intact C-0 bond from [I-l3C 180]acetate A. Intact propionate unit and intact C-O bond n 6U Intact malonate unit Intact succinate unit /-\ A Glycerol A Carbon from [13CH3]methionine via SAM *O From dioxygen in atmosphere as 1802 *H Hydride from NAD(P)H Fig.1 Biosynthetic symbols used throughout this paper 2 Eubacteria the acyl chain and with methionine derived chain methylation 2.1 Gram negative bacteria in contrast to most actinomycete derived polyketides. Hofle Phylogenetic analysis (based upon ribosomal RNA sequences) Jansen and co-workers have examined the biosynthesis of of the eubacteria suggests at least 12 different groups or phyla. disorazole A 1 by Sorangium cellulosum So ce12 using For the purposes of these reviews one of these the Gram [1-13C]acetate and ['3CH,]methionine showing that all positive bacteria (containing an outer cell-wall that reacts with four gem-dimethyl groups originate from methionine. lo The the 'Gram' stain) will be considered separately and all the oxazole moieties presumably originate from serine.others loosely aggregated as Gram negative bacteria. These Moore and co-workers have examined the biosynthesis of 11 remaining phyla include the purple bacteria (also called borophycin 2 obtained from lipophilic extracts of the blue- proteobacteria) (e.g. Escherichia Pseudomonas Salmonella green alga (cyanobacteria) Nostoc linckia and found that the and Myxococcus-the gliding bacteria) the purple sulfur bac- teria (e.g. Thiocystis) and the cyanobacteria (e.g. Anabaena) that are also called the blue-green algae-not to be confused with the eukaryotic algae. The cyanobacteria contain chloro- plasts and are thought to be the first oxygen-evolving photo- trophic organisms probably responsible for oxygenating the Earth's atmosphere.Phylogenetic studies also suggest a very close relationship between Gram positive bacteria the mito- chondria purple bacteria chloroplasts and cyanobacteria. The gliding bacteria (myxobacteria) are a rich source of metabolites derived via the polyketide pathway but frequently using amino acids such as serine or threonine as components of Borophycin 2 A' / 20'121' v Disorazole Al 1 ,A 19 0 chain is assembled from acetate units with four methylations -Serine 7 on the starter acetate unit (C-19) the gem-dimethyl (C-21/22) and C-20 all derived from methionine. Labelled propanoate did not enrich C-19 or C-20. It is interesting to compare this biosynthesis with that of the structurally closely related 524 Natural Product Reports 1997 0 NH2 O+-Y ijI Boromycin 3 c3 ? maionate)} Valine metabolites boromycin 312 and aplasmomycin 4,13,14 l5 which are however both derived from the Gram positive strepto- mycetes.All three metabolites have unusual primers the Me$=q Me ‘.%,.OH d Me‘ A Aplasmom ycin 4 Glycerol \ GIyceroI Rawlings Biosynthesis of polyketides cyanobacteria derived borophycin uses methionine methylated acetate the streptomycete derived aplasmomycin and boromy- cin use a glycerol derived starter unit. Floss and co-workers suggest that the glycerol is converted via a pyruvyl intermedi- ate to an activated lactate (as the glutathione thioester?) without the intermediacy of free acids as these are not incor- porated.After transthioesterification lactoyl CoA could then serve as the actual starter unit. In contrast to most other actinomycete derived metabolites methionine is also used to methylate a partly reduced acetate derived polyketide chain including the assembly of the unusual gem-dimethyl groups. It is hard to accept that the Streptomycetes evolved a new pathway for convergent evolution to produce just these metabolites presumably this pathway is perhaps an example of a ‘fossilised’ streptomycete pathway that existed in early streptomyctes in common with cyanobacteria before the strep- tomycetes evolved the more sophisticated macrolide assembly with the utilisation of propanoate.2.2 Gram positive bacteria The Gram positive bacteria can be divided into those whose DNA has a low G+C content (clostridium subdivision) and those with high G+ C content (actinomycetes subdivision). The clostridium subdivision include the lactic acid bacteria (e.g. Lactobacillus) the Gram positive cocci (e.g. Staphylococcus and Streptococcus) and ‘endospore’ formers (e.g. Bacillus and Clostridrium). The actinomycetes subdivision which is of great interest to the natural product chemist can be split into major groups including the rod shaped coryneforms (e.g. Corynebacterium Arthrobacter and Brevibacterium); the anaerobic propionic acid bacteria (e.g. Propionibacterium and Eubacterium);and the filamentous actinomycetes. The filamen- tous actinomycetes group can be further split into several genera the actinomycetes (e.g.Actinomyces); the mycobacteria (e.g. Mycobacterium); the maduromycetes (e.g. Actinomadura); the actinoplanes (e.g. Actinoplanes); the norcardias (e.g. Nor- cardia Saccharopolyspora and Rhodococcus); the streptomy- cetes (e.g. Streptomyces Streptoverticillium and Chainia); and the micromonosporas (e.g. Micromonospora and Thermo-actinomyces). The filamentous actinomycetes are notable for their complex lifestyle along with the fact that they produce most of the five thousand antibiotics so far isolated from microorganisms. The streptomycetes are filamentous bacteria that usually exist in the soil. When nutrients are readily available their vegetative mycelium continues to grow.However when this local supply of nutrients run out an aerial mycelium forms which develops into spores that can be readily dispersed by e.g. the wind. This aerial mycelium develops by cannibalising the nutrients which are released by the proteolysis of the vegetative mycelium. In order to prevent opportunistic neighbouring microorganisms from exploiting this source of nutrients many streptomycetes produce antibiotics at this time. Thus the triggering of anti- biotic production is frequently intimately involved with nutri- ent starvation and sporulation. Several genes have been located notably the bld genes that regulate both antibiotic production and aerial mycelium formation. There are several known chemical ‘triggers’ that cause sporulation and antibiotic production in various streptomycetes the most well-known being A-factor (autoregulatory factor) 5.’ Once part of a colony ‘senses’ that conditions require sporulation it can communicate this to the rest of the colony by producing a diffusable extracellular molecule such as A-factor that can exert its effect at concentrations as low as 10-M.A-Factor binds very strongly to a receptor protein comparable to eukaryotic hormone receptors. Mutant strains unable to pro- duce this protein sporulated early suggesting that the receptor protein is a repressor type regulator of antibiotic production and sporulation and addition of A-factor causes derepression. A recent paper examines the role of A-factor in S. griseus that produces streptomycin.l6 There then follows a cascade 0 m (3aR 4q-Differolide (3aS 4s)-Differolide 6 7 sequence ending in an A-factor responsive proteinI7 that binds a region of DNA upstream of the transcriptional part of a regulatory gene for streptomycin biosynthesis. 18-*’ Differolide (mixture of 6 and 7) is a microbial regulator for the formation of aerial mycelium in Streptomyces glaucescens and occurs naturally as a racemate. A synthesis of both enantiomers of differolide has been reported22 which will allow a determination of which enantiomer 6 or 7 is the active compound. It is now clear that there are two distinct classes of actino- mycete polyketides the aromatic polyketides with a high oxidation level produced by discrete Type I1 enzymes in a repetitive manner and the non-aromatic polyketides including the macrolides polyethers and polyenes with much lower oxidation levels and all probably assembled using large multi- functional Type I enzymes with a separate activity for each step required.An excellent review by Katz and Donadio discussing the prospects for producing hybrid antibiotics has appeared,23 and Cane has presented an overall perspective on polyketide biosyn thesis .24 2.2.1 Aromatic polyketides A gene cluster in Streptomyces coelicolor is responsible for the early steps in the assembly of actinorhodin (Act) 8 (Fig. 2). -act1 act111 * actVII actIV (Streptomyces coelicolor) ‘MinimalPKS’ Fig. 2 ‘Act’ gene cluster from S. coelicolor This cluster codes for a series of discrete Type I1 like proteins some of whose sequences have homology with known ACP KR KASlAT and cyclases/dehydratases.Khosla Hopwood and co-workers have developed a Streptomyces coelicolor host-vector system (CH999) for the efficient construction and expression of recombinant polyketide synthase components. The hosts’ entire act gene cluster was first deleted by homolo- gous recombination. Various combinations of PKS compo- nents from a variety of sources can now be inserted using plasmids and are expressed at the appropriate stage of the growth cycle.25 This has allowed the function and importance of components of the PKS gene clusters to be determined by overexpressing various combinations of proteins and analys- ing the effect on the product formed.A series of papers tests the role of PKS subunit^,^'^^-^^ for example the ACP from some related PKSs can be interchanged with the Act ACP without affecting product structure in agreement with earlier The Act KR normally catalyses reduction at C-9 and one of the proteins previously referred to as a second KAS has been characterised as the chain length determining factor (CLF). Interestingly this protein has a high homology with the KAS but is missing the highly conserved Cys residue characteristic of KASs. Presumably the protein arose through gene duplication of the KAS followed by divergent evol- ution to serve another role perhaps binding only to the fully assembled chain preventing further chain extension by the KAS.The role of each of the act genes in the assembly of the precursor to actinorhodin was examined by insertion into CH999 with other act genes three protein subunits were found to be always required for assembly of an aromatic polyketide. This ‘minimal PKS’ consists of an Act ACP a bifunctional Act KAS/AT and an Act CLF. This minimal PKS produced SEK4 9 an unreduced octaketide in which the first cyclisation is the same as in actinorhodin but which failed to undergo the subsequent cyclisations that occur for actinorhodin. Presum- ably the ‘minimal components’ are able to control the first cyclisation and the chain length in the absence of either a KR or CYC. Addition of the Act KR (actIII) gene to this minimal set resulted in reduction at C-9 and resulted in aberrant second and third cyclisations to give mutactin 10.Addition of actVII gave SEK34 11 and SEK34b 12 suggesting that actVII was an aromatase (ARO) catalysing two dehydrations in the first carbocyclic ring to give 13. Presumably this aromatisation causes one of the two side chains to fold in a different manner to that for mutactin 10. Addition of actIV gave 3,8-dihydroxy- 1-methylanthraquinone-2-carboxylicacid (DMAC) 14 sug-gesting that actIV is a cyclase (CYC) catalysing the intra- molecular aldol condensation to form the second ring. However addition of the KR and the putative CYC (actIV) with or without the ARO was necessary for correct cyclisation to give DMAC the shunt product of actinorhodin in the absence of ‘post PKS’ proteins.Presumably in the absence of ARO (actVII) the dehydration to give the aromatic ring can now occur chemically or through the action of a dehydratase. Addition of only CYC to the minimal PKS resulted in aberrant products; presumably the oxidation level at C-9 affects the second intramolecular aldol condensation allowing the for- mation of the desired second aromatic ring.4 The probable order of assembly of actinorhodin is thus as in Scheme 1. Another series of experiments by Khosla Hopwood and co-workers examined the products from various combinations of genes derived from the octaketide actinorhodin PKS cluster (S. coelicolor) and the decaketide tetracenomycin gene cluster (S. glaucescens) expressed in the above host-vector system (CH999).27 The Tcm minimal PKS produced the decaketide SEKl5 15 again through CLF controlled cyclisation of the first ring followed by aberrant subsequent cyclisations as for actinorhodin.Addition of Act KR to the Tcm minimal PKS gave RM20 16 showing that the Act KR can reduce the C-9 (relative to the original carboxy carbon) of a decaketide as well as an octaketide and affect the second cyclisation but not the first (Scheme 2). Decarboxylation has occurred in the formation of RM20 16. Streptomyces roseofulvus produces both the nonaketide fre- nolicin 17 and the octaketide nanaomycin 18. Genes from a PKS (the fren fragment probably responsible for the biosyn- thesis of both frenolicin 17 and nanaomycin 18 containing a putative KAS/AT CLF ACP KR and CYC)30 were function- ally expressed in the above host-vector system (CH999).26 The major product was the novel tricyclic molecule RM18 19 derived from a nonaketide with two octaketides DMAC 14 (along with its decarboxylated cometabolite aloesaponarin 20) and RM18b 21 suggesting that the Fren CLF has a slightly relaxed specificity (or that a butanoate is the starter unit in frenolicin biosynthesis).The single 0x0 reduction occurred at C-9 in RM18 19 and DMAC 14 but at C-7 in RM18b 21 supporting earlier proposals that in aromatic polyketides 0x0 reduction occurs after full chain assembly (Scheme 3).26 A model is described in which some ‘slippage’ in the PKS allows reduction both at C-9 and C-7 after chain assembly.All 526 Natural Product Reports 1997 Acetate + 7 x malonate --EnzSH 0 SEnz HO OH OH SEK 4 0 9 Me &SEnz 0 0 0 OH ARO acNll Mutactin I/ I \ 10 kYC Me OH Me O=(Me HO SEnz 13 CYC acflV GSEnz i 0 OH 0 OH SEK34 SEK34b 11 12 \ OH 6SEnz< OH 0 acNl acNA acNB -EnzSH mOOH \ OH 0 3,8-Di hydroxy-1 -methylanthraquinone-2-carboxylicacid (DMAC) '1 14 OH 0 COOH Actinorhodin 0 Scheme 1 Minimal Tcm PKS 0 SEnz HO OH SEK15 15 0000 Minimal Tcm PKS -SEnz Act KR HO O& // RM20 16 Scheme 2 Rawlings Biosynthesis of polyketides / 0 I COOH Nanaomycin A 17 18 S.roseofulvus S.roseofulvus t T 00 0 0 0 0 0 JKU SEnz 0 JKR SEnz 0 SEnz lKR 0 0 0 000 HO -SEnz HO HOe OH l SEnz6 OH I -co* Jco2 f OH 0 Me OH 0 DMAC RM18 14 RM18b 19 (and minus COOH = Aloesaponarin 20) 21 Scheme 3 possible combinations of Act or Fren derived KAS/AT CLF gene clusters also showed this feature.Dendrogram analysis and ACP genes were inserted into CH999 along with Act KR suggested that this duplication event occurred early on in the and Act CYC; most of the transformants were able to express evolution of PKS gene sets. This duplication suggests that this polyketides (Table 1). The effect Act KR has is illustrated in protein may contain two domains that may be in contact with the two ends of the polyoxo chain to direct and control the second ring cyclisation.The corresponding Tcm and whiE Table 1 Effect on polyketide production of swapping cyclases do not possess this duplication but it is suggested that genes from different sources within the 'minimal they may act by forming h~modimers.~' PKS' The oxytetracycline 22 (Otc) PKS has been sequenced from S. rimo~us.~~ The Otc minimal PKS (Otc KAS/AT CLF and ORFl ORF2 ORF3 ACP) along with Act KR were cloned into the above host- (KAS/AT) (CLF) (ACP) (with act KR and act CYC) vector system (CH999) to give two epimeric polyketides act act act DMAC RM20b 23 and RM20c 24.32These are the same molecules fren act act DMAC produced when Tcm minimal PKS and Act KR were inserted act fren act No product into the host-vector system and showed that the Otc minimal act act fren DMAC RM18 and RM18b PKS system is capable of efficiently assembling an acetate fren fren act DMAC RM18 and RMl8b initiated backbone.Thomas and Williams33. 34 had suggested fren act fren No product that the assembly started with a malonate primer 25 with act fren fren No product subsequent insertion of nitrogen to give the amide. However fren fren fren DMAC RM18 and RM18b neither RM20b or RM20c starts with malonate despite its ready availability in this system suggesting an acetate starter unit 26 with subsequent conversion into the corresponding amide (Scheme 5). Scheme 4. Minimal Act PKS plus Act KR gives the octaketide The entire gene cluster responsible for the biosynthesis of mutactin 10. Minimal Tcm PKS plus Act KR gives RM20 tetracenomycin C 27 (Tcm C) from S.glaucescens (Fig. 3) has 16. However minimal Fren PKS plus Act KR gives three been sequenced and the products of each Tcm gene are products mutactin 10 RM18 19 and RM18b 21. currently being overexpressed characterised and their roles Analysis of the sequence of the Fren fragment showed that examined (Scheme 6). The gene cluster has been transcription- A cell-free system containing the gene cluster the ORF4 (a bifunctional cyclase/dehydrase with homology ally analy~ed.~~ to actVII) arose from an internal gene duplication that gave can assemble Tcm F2 28 from acetate and mal~nate.~~ rise to the 3' and 5' ends. Analysis of homologues in the Hutchinson has been examining the biosynthesis of the tetra- Act Gra (granaticin) Gris (griseusin) and Mon (monensin) cenomycins by overexpressing various components of the Tcm 528 Natural Product Reports I997 Minimal Tcm PKS Minimal Act PKS and Act KR and Act KR u20 0000 0 SEnz 0 SEnz rr HO 000 SEnz SEnz HO‘ ’ Me 0 OH Mutactin RM20 Minimal Fren PKS 10 16 and Act KR 1 0uSEnz 0 SEnz I U O HOW S OH E II-““ n z HO OH I-.SEnz 0 OH RM18 RM18b 19 21 Scheme 4 fcmlV tcmll -tcmlA- -tcmVI-S.glaucescens ‘-YPJ fcmM = Acyl carrier protein Minimal Tcm PKS tcmL and fcmK = 3-Oxoacyl ACP synthase Fig. 3 ‘Tcrn’ gene cluster from S. glaucescens PKS and post PKS enzymes. The Tcm PKS was identified by tcmKLMN allowed the cell-free conversion of acetyl CoA and overexpression of genes in a strain of S.glaucescens in which malonyl CoA into Tcm F2 28 production of which was An earlier tcmGHIJKLMNO genes are not expressed. Overexpression of enchanced four-fold by the inclusion of t~mJ.~~ Rawlings Biosynthesis of polyketides CCOOH \ 0 SEnz ?\ 25 or S.rimosus Oxytetracycline 22 0 SEnz 26 Minimal Otc PKS and Act KR OH OH I I $Me and &Me 9 $Me gMeand 9 HO H I HOW' HO O YHO HO '1 HO 0&OH 0QOH RM20b RM20c 23 24 Scheme 5 Acetyl CoA + tcrnKLM * " O/ h 0 o 9 x Malonyl CoA OH 0 0 Me SEnz r 1 OH OH 0 Me Tcm F2 28 tcrnl tcmH -HozoH OH 0 OH Me 0 Tcm F1 Tcm D3 29 30 0 0 Tcm 83 Tcm E Tcm A2 Tcm C 31 27 530 Natural Product Reports I997 Scheme 6 report describes the isolation and identification of Tcm F2 28 and Tcm Fl 29.37 The Tcm ACP is encoded for by tcmM.Overexpression of this gene in E. coli yields apo-ACP but overexpression in S. gluucescens yields solely the function- ally active h01o-AcP.~~ Mutation studies in the tcmVI region of the S. gluucescens suggested the presence of three genes (tcmH I and J) that encode a C-5 monooxygenase that converts Tcm F1 29 to Tcm D3 30; a D ring cyclase that converts Tcm F2 28 to Tcm F1 29; and a B ring cyclase that acts in the biosynthesis of Tcm F2 28 re~pectively.~~ The tcmI gene product the Tcm F2 cyclase has been purified to homogeneity and exists in solution as a functional homo- trimer converting Tcm F2 to Tcm F1 when the pH>8 requiring no cofactor.Under acidic conditions it catalyses the formation of 9-decarboxy Tcm F1. Neither reaction occurred in the absence of enzyme.40 This pH dependence suggests a two step mechanism in which an enzyme bound enolate attacks the starter unit carbonyl in an intramolecular aldol reaction. Under basic conditions dehydration would be initiated by deprotonation at C-9 whilst under acidic conditions the hydroxy group would now be a better leaving group. The authors suggest that this facile dehydration is coincident with decarboxylation though presumably on stereochemical grounds the dehydration is driven by phenolic lone pairs and the decarboxylation is non-concerted or the acid conditions decarboxylate Tcm F1.The Tcm F1 monooxygenase encoded by the tcmH gene which converts Tcm Fl 29 to Tcm D3 30 has been purified to homogeneity characterised and is a homotrimer in sol~tion.~' It may be an 'internal monooxy- genase' requiring only dioxygen for enzymatic oxidation not flavin or heme groups or metal ions but thiol groups and histidine residues appear essential with a radical mechanism. The Tcm A2 oxygenase encoded for by the tcmG gene that triply hydroxylates Tcm A2 31 to Tcm C 27 has been purified and found to be monomeric requiring FAD reduced nicotin- amides and dio~ygen.~*> Hutchinson and co-workers have 43 also found that introducing Tcm PKS genes into S. gfuuces-cens such as the tcmM ACP gene on high copy vectors under the control of strong promoters caused an increase in levels of production of up to thirty-f~ld,~~ which may have commercial implications and suggests that ACP levels may regulate the levels and activities of other components of the Tcm synthase.Carminomycin 4-O-methyltransferase which catalyses the final step in the biosynthesis of daunorubicin has been over- expressed in E. coli in its active form and has a binding site for SAM.45 The uctVI region from S. coelicolor has been sequenced and found to contain six ORFs. One ORF resembles known 3-hydroxyacyl dehydrogenases and two of the ORFs resemble enoyl reductases suggesting that the actVI region is involved in the reductive processes leading to the pyran ring.46 47 The act1 gene has been used to identify and clone a DNA fragment from the nodusmicin producer Succharopolyspora hirsutu 367 that contains homology with KAS KR ACP and BCCP.48 A PKS gene cluster from S.coelicolor called whiE (pro-nounced whiteE) is believed to encode for the production of a grey pigment that occurs in the spores. A similar cluster has been found in about half of streptomyces species so far examined. A segment of such a cluster from S. halstedii (the sch cluster) has been sequenced.49 50 Sequence comparisons with spore pigment genes and those responsible for the production of antibiotics suggest divergence of spore pigment genes from antibiotic genes early on in the evolution of the streptomyces genus. Extensive studies using labelled precursors have been pub- lished and the expected decaketide origin confirmed for the assembly of the angucycline PD 1 16740 32.However 80-labelled sodium acetate does not label C-6 which requires a bis-reduced decaketide intermediate 33. The successful incor- poration of deuterated tetrangulol34 into PDll6740 32 shows that this deoxygenation-reduction occurs as part of the PKS assembly. The corresponding hydroxy analogue deuterated Rawlings Biosynthesis of polyketides OYEnz 20 Me HO& 15 0 0 OH 33 34 Dehydroabelomycin Tetrangulol 35 \ 2 x [O]IHz0 SAM &cH*bH &/fcH20H / *OH / OH OMe 0 OH OMe 0 OH A PD 116740 32 Scheme 7 dehydroabelomycin 35 was not incorporated. Epoxidation of the C-5-C-6 double bond of tetrangulol 34 by dioxygen followed by enzyme catalysed hydrolysis generates the trans diol in a manner reminiscent of mammalian oxidation of procarcinogenic polycyclic aromatic hydrocarbons (Scheme 7).51 However the biosynthesis of an apparently related metabolite PD 1 16 198 36 from S.phaeochromogenes probably involves a rearrangement. The final oxidation levels would correspond to a decaketide 37 reduced at both C-9 and C-15. Rearrangement of a linear tetracyclic intermediate with subse- quent dehydration gives the double bond. However a retro- aldol type cleavage of the C-C bond would work if the C-I5 of 38 was still at the carbonyl oxidation level (Scheme 8).s2,53 OH OYEnz ""my"" 'OH hr-PYfY 0 0 [o] 0 OH 0 PD 116198 37 36 CjH 38 D from CD3 acetate \ retro-Aldol f -co;! Scheme 8 53 1 Retention of a deuterium at C-5 of 36 from [l-'3C,2H,]acetate presumably rules out any dehydration of a C-15 hydroxy group of 38 prior to the rearrangement.The biosynthesis of the angucycline landanomycin A 39 from S. cyanogenus has been examined using labelled acetate and dioxygen. Under the conditions of limited oxygen supply during the '*02feeding experiment 5,6-anhydrolandanomycin 40 was isolated instead of landanomycin A 39 and label from dioxygen was found in the C-7 carbonyl. This is supportive of prearomatic reduction occurring as in the biosynthesis of angucycline PD116740 32 (vide supra).54 A likely pathway is illustrated in Scheme 9 starting with a decaketide that has been reduced to the corresponding hydroxy groups at the C-9 C-13 and C-15 positions (41) with these reductions occurring prior to aromatisation.I OH 3 x [O] Hexasaccharide 1 OH OHowMe Hexasaccharide 5,6-Anhydrolandanomycin 40 #Me wMe / / 6 0\ *O OH 0 \ 0 OH Hexasaccharide Hexasaccharide Landanomycin A 39 Scheme 9 The biosynthesis of aquamycin and urdamycin has been investigated by locating mutants of S. fradiae which produced five new intermediates. 55 The structure of the kinamycins from S. murayamaensis has been reported for many years as benzo[b]carbazole cyan- amides 42 based upon chemical 'H NMR IR (2150 cm -') and X-ray data. However on examining the 13C NMR data Gould and co-workers could not locate the cyanamide reso- nance expected at ca.6 110-120 but instead located an un- assigned resonance at 6 78. Reasoning that the original X-ray data set might not easily distinguish between R,NCN and R,CN2 they considered an alternative structure based upon that of 5-diazobenzo[b]fluorenesrather than benzo[b]carbazole cyanamides and has obtained a new X-ray structure of a kinamycin D derivative agreeing with the diazo structure 43.563 57 The kinamycins are derived from the benzo[a]anthra- quinone 44 via a decaketide 45 reduced at C-9. Oxidation to 46 followed by ring closure can give prekinamycin whose struc- ture has also been revised to the diazo structure 47 that is produced by a blocked mutant. A benzo[b]phenanthridine 532 Natural Product Reports I997 48 has recently been isolated from a UV mutant which along with the minor cometabolite 49 supports the proposed mechanism (Scheme 0 SEnz HO& 000 45 -coa I2x[O] 44 46 Prekinamycin 47 Blocked mutant + OAc A HO.xMe Minor metabolite 49 WN2+ OH 0 MMe Kinamycin D UN+J 43 6H 6 '. Minor metabolite 0 HO.RMe 48 v o CN -OH 0 42 Previous structural assignment for Kinamycin D Scheme 10 The biosynthesis of the dodecaketide xanthone antibiotic lysolipin X 50 from S. violaceoniger has been examined by Rohr Floss and co-workers using labelled acetate malonate SAM and dioxygen. Several surprises were found. Firstly an intact malonate unit was used as a starter unit with the activated thioester carbon (C-25) being attached to the ring nitrogen and the carboxy carbon (C-23) initiating the carbon chain.This would be consistent with the intermediacy of malonamoyl CoA (H,NCOCH,COSCoA) but the order of attachment of carbon atoms is the reverse of that observed for the tetracyclines and cycloheximide the only other two examples of a (putative) malonate starter. Secondly the labelling of so many oxygens by dioxygen again implicates prearomatic deoxygenation the original dodecaketide prob- ably being reduced at the C-1 1 C-17 and C-19 positions (Scheme 1l).59 This raises the possibility of a non-processive Me0ANAO Me Lysolipinx 50 2’ maionate OH OH 0 0 A *o-o* A ”3C3]Malo~ iic acid HO Me6 A Me A Scheme 11 assembly of a fully oxygenated dodecaketide followed by three reductions either by a single KR or by three different KRs all as part of the PKS complex.It is interesting and fun to contemplate the molecular mode of assembly of these large aromatic polyketides. The putative dodecaketide before reduction or aromatisation would be an extremely reactive intermediate that would need ‘taming’ by being constantly bound to and chaperoned by the protein presumably as an arginine-lysine bound polyenolate. Each enolate may remain constantly attached to a specific part of an ACP or slide along a groove to the ‘next’ arginine as the polyketide chain is extended. Other proteins in the complex (e.g.KAS) may have enolate binding residues or the chain may remain bound onto the surface of the ACP whilst being chain extended subse- quently reduced cyclised and even aromatised. Elegance would require the moving of such active sites to these molecules rather than vice versa. A series of papers have appeared on the biosynthesis of the dodecaketide benzo[a]naphthacenequinone antibiotics the pradimycins from Actinomadura verrucosospora delineating later steps in the pathway and using directed biosynthesis to form A series of intermediates between 51 and pradimycin A 52 have been identified. The biosynthesis of cytogenin 53 from Streptoverticillium eurocidium M143-37F11 has been investigated by Ishizuka and co-worker~.~~ 2.2.2 Non-aromatic polyketides The previous review2 reported on the publication by Leadlay and Staunton in Cambridge and Katz of Abbott laboratories of the gene cluster coding for the erythromycin PKS from Saccharopolyspora erythraea (formerly Streptomyces eryth- reus) and that it consisted of three Type I multifunctional proteins (DEBS1 2 and 3) each transcribed in the same Rawlings Biosynthesis of polyketides COOH HO &Me / \ ’OH OH 0 OH OH 51 J 4 I Me0 OH 0 0NHMe OH Pradimicin A 52 Me0 A T O H Me/oPOH OH 0 53 OH 0 direction with sufficient enzymic activities to complete two of the six condensation cycles (synthase units; SU).Each of these proteins can be overexpressed in E. coli (apo 68 69 and they have been isolated from wild type Saccharopolyspora er~thraea.~’A ‘Ford production line’ can be envisaged for the assembly of the growing polyketide chain with the function- ality adjusted in the processive manner with one activity for each of the required reactions until release as the lactone 6- deoxyerythronolide B 54 by the last component a thioesterase prior to ‘tailoring’ by discrete oxidases and glycosidases to give erythromycin A.2 Attention has now been focused on deter- mining the molecular basis of chain assembly the activity and mechanism of the various components the topology of the overall domain structure and how this knowledge could be exploited to produce novel (designer?) metabolites through gene deletion or even mixing and matching components from different sources.An attractive idea is to disrupt in turn the KR/DH/ER genes and examine the resulting metabolite; unfortunately only limited success has been achieved with this approach probably due to ‘chemical constraints’. The first example was the deletion by Katz and co-workers of the KR in Ery SU5 that resulted in the expected 0x0 compound 5,6-dideoxy-5- oxoerythronolide B 55.71The Abbott laboratories have also produced 56 (isolated as its corresponding disaccharide anhydroerythromycin C) by changing two amino acid residues in the NAD(P)H binding motif of the single ER in the Ery PKS cluster (Scheme 12).72 Other genes have surely been deleted but in the absence of the reporting of other such examples presumably succeeding steps in particular lactonis- ation can no longer occur due to chemical unfeasibility preventing formation of other Ery analogues.The stereochemistry of the methyl groups in erythromycin would suggest that chain extension is from three (2s)-and three (2R)-methylmalonyl CoA (MMCoA) building blocks. In classic studies Cane and co-workers showed that C-2 SU = Synthase unit c=o su resulting in a carbonyl porate any label from deuterated succinate via (2R)-MMCoA = Predicted non-functional activity OH SU resulting in a hydroxy group into erythromycin! -Direction of translation of DNA DB SU resulting in a double bond DEBS1 behaves as a dimer of RMM 660 kDa on gel 0= ORF (open reading frame) CH2 SU resulting in a methylene filtration though it is not yet known whether it is a head-to- tail dimer as with mammalian FAS a head-to-head dimer or a ‘twisted dimer’ somewhere between the two.Limited proteolysis by Leadlay Staunton and co-workers of DEBSl has allowed identification of the linker regions the cleavage pattern following expected domain boundaries as predicted by sequence analysis linker regions are rich in alanine proline ACP and charged residues.74 Three main fragments are produced the N-terminal AT/ACP the central section containing a KAS/AT and the C-terminal containing KR/ACP/KAS/AT/ KR/ACP domains. Interestingly though the C-terminal half of DEBS 1 remained extremely resistant to further proteolysis and like DEBSl itself behaved as a dimer on gel filtration the N-terminal and central fragments behaved as monomers.The N-terminal fragment was acylated by radiolabelled propanoyl CoA and the other two fragments were acylated by radio- labelled MMCoA as expected thus the acyl transferases remain active after limited proteolysis. A system for overexpressing recombinant modular PKSs in a streptomyces host S. coelicolor has been developed by Khosla and co-workers. Expression of the Ery PKS (DEBS1 + 2 +3) resulted in the biosynthesis of 6-deoxyerythronolide B 54 and 8,8a-deoxyoleandolide (nor-6-deoxyerythronolide B) the latter product arising from relaxed specificity of acetate versus propanoate on selection of the starter unit. Presumably the ACPs in this system have been phosphopantetheinylated unlike in the E.coli overexpression system and these products proved that all the necessary precursors and activities for macrolide assembly were present.75 DEBS1 on its own has been overexpressed in S. coelicolor resulting in the biosynthesis of heptano-5-lactone 57 from non-TE mediated lactonisation of (2R,3S,4S,5R)-3,5-dihydroxy-2,4-dimet hylheptanoic acid (Scheme 13).6 The triketide lactone 57 had previously been Me 54 * Me* 0 Me ERM[ACP SUl SU2 SU3 SU4 SU5 SU6 Me 55 * M 0 e 6 ,Me sU1 SU2 SU3 SU4 SU5 SUE ACP Me 56 Scheme 12 deuterated propanoyl CoA (which would be carboxylated to (2S)-[2-2H]methylmalonyl CoA) labelled the three sites in erythromycin that would be expected to arise from (29-MMCoA. However attempts to perform the complementary experiment addition of tetradeuterated succinate the immedi- ate precursor of deuterated (2R)-MMCoA failed to incorpor- su1 su2 -1 ERM Me ate deuterium at the other three sites.This was interpreted as being due to an extensive loss of label due to adventitious exchange processes.73 Leadlay Staunton and co-workers have (2R,3S,4S,5 R)-3,5-Di h yd roxy- 2,4-dimethyl heptanod-lactone 57 reexamined this problem using overexpressed DEBSl DEBS2 and DEBS3 proteins (protein not ph~sphopantetheinylated).~~ Scheme 13 Incubation of radiolabelled racMMCoA with either DEBSl 2 or 3 gave labelled protein. The protein bound radioactivity isolated from a mutant strain of Saccharopolyspora er~threa.~~ decreased with extended incubation time falling to zero after two hours suggesting that the acyl transferase domains Thus a TE domain and the other two DEBS polypeptides are not essential to the activity of DEBSl.were active and that a slow hydrolytic step was active in the The eryF gene has been overexpressed in E. coli and the absence of an active ACP. Addition of additional radiolabelled resulting soluble cytochrome P450 dependent enzyme respon- sible for the conversion of 6-deoxyerythronolide B to erythronolide B has been purified ~haracterised,~~ the substrate specificity examined78 and a preliminary X-ray The report suggests that previous isolations of this protein contained a mixture of two P450 enzyme^.'^ The sequence contained an alanine residue at a position that normally contains threonine in all other P450 enzymes and that has been thought essential for oxygen binding and cleavage suggesting an unusual active site.79 The gene encoding the cytochrome P450 responsible for the final hydroxylation step the conversion of Ery B to Ery A or Ery D to Ery C has been located 50 kbp downstream of the resistance gene ermE and called eryK.Gene disruption resulted in the accumulation of Ery B and Ery D.80 Merson-Davis and Cundliffe have examined the tylIBA region of genome from S. fradiae that is responsible for the racMMCoA again temporarily labelled the protein suggesting that the acyl transferase was still active after two hours. More interestingly addition of purified MMCoA epimerase from Propionibacterium shermanii restored about half of the original level of radiolabelled protein demonstrating that only one half of the original radiolabelled (14C) racMMCoA had reacted with the enzyme and that the DEBS protein itself could not epimerise MMCoA.Again surprisingly all three DEBS pro-teins gave similar results and when mixed together they all competed for the same enantiomer of racMMCoA.’ Each radiolabelled enantiomer of MMCoA was then prepared in situ and it was soon clear that all six acyl transferase sites on the Ery PKS react with the same 2S enantiomer of MMCoA. Thus additional enzymatic steps on the PKS complex must be necessary to epimerise three of the methyl branched centres to the configuration found in the final metabolite. In addition this explains why Cane and co-workers were unable to incor- 534 Natural Product Reports 1997 biosynthesis of tylosin and found it to contain five open reading frames.Sequence analogy suggests they are a cyto- chrome P450 a glycosidase two sugar synthases and a fifth protein that shows homology with mammalian medium chain length thioesterases such as the mallard duck uropygial gland thioesterase. It has been suggested that this thioesterase may release any aberrant polyketide synthase products preventing blockage of the PKS complex.8’ Mycinamycin I11 0-methyltransferase from Micromono-spora griseorubida that 0-methylates the mycinose sugar has been cloned and functionally overexpressed in E. coli.82 A large ORF has been located in Bacillus subtilis that has extensive similarities to eryA has been called pksX presum- ably responsible for the biosynthesis of an as yet unknown macrolide.Sequence analysis suggests the presence of a KAS DH KR and ACP domain.83 A cell-free extract from S. antibioticus can glycosylate ole- andomycin 58 on the 2‘-hydroxy group of its desosamine Mfi,ugTd site for glycosylation Me. Me &?H Me Oleandomycin 58 moiety thus deactivating the antibi~tic.’~ A gene cluster has been identified that may code for this glycosyltransferase and for integral membrane proteins responsible for translocation Me Me OMe concanamycin ’ 59 Me OH n un wit Okadaic acid 61 H Rawlings Biosynthesis of polyketides of this inactivated glycosylated antibiotic across the cellular membrane.85 A highly substrate specific enzyme has been purified from the extracellular culture supernatant that can remove this glucose molecule reactivating the antibiotic.86 The biosynthesis of the ATPase inhibitor concanomycin 59 by a Streptomyces sp.(strain GO 22/15) has been investigated by Bindseil and Zee~k.~~ Due to their unusual side chain folding it has been proposed that concanamycin and related metabolites be called ‘plecomacrolides’. Hydrogen bonding between the oxygenated functionalities in the centre of the molecule causes the two ‘ends’ to fold back over each other. It was expected that acetate propanoate and butanoate would label all the acyl groups in the usual manner with post-PKS oxidation accounting for any other oxygenation.However no intact incorporation from labelled acetate could be detected at C- l/C-2 or C- 15/C- 16. Incorporation from the carbohydrate pool possibly via trioses has been observed for geldana- mycin,88 le~comycin,~~ boromycin 3 aplasmomycin 4,90 tetro-nasin (ICI139603) (60 see Scheme 21)91 and more recently okadaic acid 61.92No incorporation was observed on feeding [l-’3C]hydroxyacetate (glycolate) but feeding [1(3)-13C] glycerol gave incorporation at C-1 and C-15 but not C-2 or C-16 of concanomycin 59. Uniform labelling was observed as expected at all propanoate positions and the carbons derived from the methyl group of acetate. Thus concanamycin is a tetradecaketide assembled from four acetates seven pro-panoates and one butanoate with the other two C units at C-1/C-2 and C-15/C-16 arising from a triose pool metabolite.It is suggested that glycerol is incorporated via an activated form of glycolate possibly 2-phosphoglycolate though 2-hydroxymalonate or even 2-methoxymalonate remain possible intermediates. Two pentano-5-lactones 62 and 63 were isolated from the concanamycin 59 and bafilomycin 64 producing microorganisms respectively corresponding to the cyclisation of expected tetra- and tri-ketide intermediate^.^^ Bindseil and Zeeck then go on highlight the biogenetic similarities between concanamycin 59 the viranamycins 65 the bafilomycins 64 PC-766B 66 and the hygrolidins 67 (Scheme 14). A Glycerol Me 62 63 glycolateCalcium Ho..3Me Me OMe 0 H OH oH OH 38 Me \ \ Me Meo 37- OMe Me Me Bafilornycin A 64 535 G P P P P A G P P A P OMe Me Me Et Me Me Concanamycin 0 OH OH 59 OMe Me Me Et Viranamycins 0 OH 3H 65 OMe Me Me Bafilomycins 0 3H 64 PC-766B 3H 66 Me Hygrolidins 67 Scheme 14 OH OH OH OH OH OH OH OH Me Me Oasomycin B 68 OH OH OH OH U (via4-aminobutanoyl CoA) The biosynthesis of the 'marginolactones' such as oaso- mycin B 68 has been examined by Thiericke and co-workers.This numerous group of macrolactones all possess a macro- lactone ring of more than 31 carbon atoms with a side chain derived from arginine or ornithine and give rise to the term marginolactones.The marginolactones (the desertomycins and the oasomycins) are produced by Streptoverticillium baldacii. Studies involving a detailed analysis of the fermentation time course and of pH static fermentations have revealed both the late (post PKS) stages of the biosynthesis and the relationships between the various desertomycins and oasomycins (Scheme 15).94 Desertomycin A 69a and desertomycin B 69b are the first detectable biosynthetic intermediates undergoing oxidative deamination to give lactonisation of the side chain. A second paper examines the origin of each part of desertomycin A and shows polyketide biosynthesis initiated by either arginine or ornithine probably as 4-aminobutanoyl CoA or the guanidino analogue and attack by an oxygenase at C-22 using dioxygen.Labels from aspartic acid glutamic acid arginine ornithine and 4-aminobutanoic acid were all incorporated at the polyketide starter unit of desertomycin A 69a or B 69b.95 The biosynthesis of the immunosuppressant rapamycin 70 in S. hygroscopicus has been examined .96-99 An unusual shiki- mate derived starter unit (3,4-dihydroxycyclohexanecarbonyl CoA) is used along with a lysine derived piperidine-2-carboxylic acid (pipecolic acid) (via pipecoyl CoA?) termin- ation unit. I3C-Enriched shikimic acid was obtained from an aromatic amino acid auxotrophic mutant strain of Klebsiella pneumoniae fed on labelled glucose. FK-506 71 was first re- ported'" in 1987 from S. tsukubaensis and is an immunosup- pressant currently undergoing clinical trials for the prevention of organ transplant rejection.It is assembled from acetate propanoate butanoate pipecolic acid shikimic acid and methionine. An enzyme has been isolated and purified that specifically methylates 3 1-0-desmethyl FK-506. lo' The related immunosuppressant FK520 72 (also called ascomycin or im- munomycin) also has a 3,4-dihydroxycyclohexanylmoiety that has been shown to come from ~hikimate.~~, lo2 Wallace and Reynolds have investigated the conversion of shikimate 73 to (1 R,3R,4R)-3,4-dihydroxycyclohexanecarboxylicacid 74 in S. hygroscopicus. Specifically deuterated shikimates 73 trans-4,5-dihydroxycyclohex- 1-enecarboxylic acids 75 and trans-3,4-dihydroxycyclohexa-1,5-dienecarboxylic acids 76 were successfully and specifically incorporated into FK520 72 consistent with a pathway that commences with either a syn or anti 1,4-conjugate elimination of water followed by enoyl reduction of the A'-double bond to give either 77 or 78.Isomerisation (suprafacial or antarafacial as required) of the remaining double bond from the A2 to the A' position gives intermediate 75. Enoyl reduction would now result in the product 74 (Scheme 16). Many of these reactions have a parallel in the biosynthesis of cyclohexanecarboxylic acid in S. collinus and Alicyclobacillus caldarius. ' A novel macrolide quinolidomicin 79 has been isolated by Seto and co-workers from the mycelium of a Micrornonosporu sp. containing a benzoquinone chromophore and a 60 536 Natural Product Reports 1997 e C OH OHM OH OH W g Me / / I 0 0 ro'sugar 41 0 Me OH NHR 69a Desertomycin A R = H 69b DesertomycinB R = C(NH2)=NH r 1 COOH OH OasomycinC DesertomycinD 0 Me Me COOH COOH OasomycinD Oasomycin F oj;pugar -ojo;!I;gar o!;toH -Me 0 0 OH Oasomycin E 0 0 Oasomycin B OasomycinA 68 -+Major pathway Minor pathway Scheme 15 membered lactone ring the largest ring so far in natural prod- encoded for in the cluster; nine KR domains are required but ucts and a molecular weight over 15Oo.'O4 The ever increasing eleven are encoded for; suggesting that several domains are size of such reported macrolides such as quinolidomicin some non-functional (Scheme 17).Two of the DH domains may be polyenes and marine toxins such as maitotoxin (RMM inactive due to mutations at the active site a conserved proline 4322 Da),2 will require enormous Type I gene clusters for their residue is missing.The Avm sequence is functionally assigned assembly and a remarkable investment both of genetic by comparison with the sequence information available for the material and protein by the organisms assembling them. For closely related compound nemedectin 81.lo8 This 'gene assign- example quinolidomicin would require protein weighing ment' may now allow selective gene deletions in the future to around 5 MDa. The organism concerned must surely possess produce novel avermectins. The overall size of the gene cluster a very definite if not essential reason for their assembly responsible for avermectin biosynthesis was found to be 95 kb no longer can secondary metabolism be passed off as a by gene cluster di~placement.'~' A 60 kb central region deter- mere artefact or afterthought.Progress in this area is keenly mines the assembly of the macrolide ring a 13 kb region awaited! synthesises and attaches the oleandrose disaccharide and a The avermectins (e.g. avermectin B2b 80) milbemycins and 10 kb region at the other end of the cluster has functions for nemedectins are closely related pentacyclic 16-membered positive regulation and O-methylation. The biosynthesis of macrocyclic lactones derived from a starter unit seven acetate nemedectin 81 and related compounds from S. cyaneogriseus and five propanoate units with potent antiparasitic activity.Io5 has been investigated by Ahmed and co-workers using The PKS cluster for avermectin from S.avermitilis has been radio-labelled and 3C-labelled acetate propanoate and sequenced,Io6 as outlined in the last review.2 A recent paper by 2-methylpropanoate (or valine) and other potential units. lo MacNeil and co-workers of the Merck laboratories correlates A mutant strain of S. avermitilis lacks a branched chain the 12 synthase units (SU) of the Avm PKS to the twelve 2-0x0 acid decarboxylase the normal source of the starter unit condensation cycles involved and the 56 Avm PKS activities and thus can only produce extremely low levels of avermectin. required by sequence analogy to Ery PKS.'07 There are excess Addition of a wide range of exogenous unnatural starter units domains (activities) four PKS DH are required but six are to the culture allowed the biosynthesis of a wide range of new Rawlings Biosynthesis of polyket ides 537 rfMe f:X2 OH 73 Shikimic acid I 0 -H20 syn or anti I Me 6Me Me Me Rapamycin 70 OH OH 76 \ 0 A /Isyn +2[H] anti Y+O 4 0 OH OH OH A Starter unit (3,4-dihydroxycyclohexane carbonyl CoA?) from shikimic acid Termination unit (pipecoyl CoA?) derived from pipecolic acid OH OH OH 75 74 Scheme 16 avermectins."" One of these avermectin CHBl 82 derived from the starter unit cyclohexanecarboxylic acid is under commercial development as an antiparasitic agent.Staunton and co-workers synthesised the N-acetylcysteamine (NAC) thioester of the expected diketide using cyclohexanecarboxylic acid as starter unit (Scheme 18).'13 Addition of racemic '3CH3-labelled diketide isotopomer 83 gave the expected q O Me Me M 'OH e resonance in the 13C NMR spectrum.Addition of racernic tetradeuterated diketide isotopomer 84 gave an enhanced 71 R = CHzCH=CHz; FK506 72 R = Et; FK520 (AscomycinAmmunomycin) (M+4+Na)' peak in the electrospray mass spectrum I +OH Me Me Me Quinolidomicin A OH 79 538 Natural Product Reports 1997 OMe NAC-S4 NAC-S& 0 OH 0 OH (racernic) (racernic) 83 84 Averrnectin B2b 80 Me 6H Averrnectin CHB1 82 Scheme 18 demonstrating intact incorporation without prior oxidation to the corresponding 3-0x0diketide which would have resulted in the loss of one deuterium.Nernedectin The polyenes continue to be sadly neglected due to the 81 difficulties in isolating handling and analysing many of these OH compounds. The pab gene from S. griseus has been sequenced found similar to the E. coli pab gene and is thought to be located between other genes involved in candicidin biosyn- thesis. The pab gene is responsible for the biosynthesis of p-aminobenzoic acid the presumed candicidin starter unit. l4 High phospate levels in S. acrimycini strongly inhibited 34 Me 35 OH 30 Me' Me MeO.. 29 33 Me 85 SU3 SU4 SU5 SU6 SUlO SUll SU12 SU7 SUB SU9 ____--____ # -4 \ # SU1 SU2 SU3 SU4 SU5 SU6 SU12 SU11 SUlO SU9 SU8 SU7 A"M ~~~ElEl~ Elnnnnn 6.3Kb 5.8 3.6 4.85 4.85 5.8 5.6 5.5 5.6 6.8 3.0 4.8Kb ' KAS' KAS KAS KAS KAS KAS KAS KAS KAs & :t us us & AT AT AT AT AT? AT AT AT DH DH DH AT AT MeO..# KR KR KR KR KR KR KR KR ACP ACP? ACP? ACP? ACP ACP ACP ACP ACP ACP ACP HO TE # hCOOH 485Kb48 58 56 55 64 66 45 45 \ KAS KAS KAS KAS? KAS? KAs KAS KAS KAS \ AT? AT? AT AT AT? AT AT AT? AT # # DH DH? DH DH? DH? DH? HNJfe -HooCYMe -Hoocl'Me COSCoA 0A N H2N SU = Synthase unit ACP? = Gene predicted from module size lDHl= Predicted non-functional activity 0= ORF H Thymine (2R)-3-amino-2-rnethylbutanoate --+Direction of translation of DNA -= DNA not yet fully sequenced 86 87 Scheme 17 Scheme 19 Rawlings Biosynthesis of polyketides 539 candicidin production by affecting PABA synthase activity.'l5 Streptomyces sp. FR-008 produces a heptaene antibiotic that only differs from candicidin in the sugar residue and is not only a potent antifungal agent but also displays high toxicity for mosquito larvae. A large gene cluster has been isolated and shown to contain repeated PKS modules extending over 105 kb of the genome enough for all 21 modules or syn- thase units expected by analogy with other Type I macrolide systems.' l6 The source of C units (MMCoA) for assembly into actino- mycete derived antibiotics has apart from the direct carboxyl- ation of propanoyl CoA (if available) to give (2S)-MMCoA traditionally been considered to be succinate. Methylmalonyl mutase which converts succinoyl CoA to (2R)-MMCoA via a vitamin B, mediated rearrangement has just been cloned and overexpressed by Robinson and co-workers from S.cinnamon-ensis that produces the polyether monensin A 85.Il7 An epime- rase interconverts (2R)- and (2S)-MMCoA. More recently it has been found that the branched chain amino acids valine and isoleucine are readily converted into MMCoA. For example valine is efficiently converted to the corresponding 2-0x0 acid and then decarboxylated to 2-methylpropanoyl CoA which through oxidation of a methyl group is converted into MMCoA. 2-Methylpropanoyl CoA can also be converted by cell-free extracts of streptomycetes into butanoyl CoA (the source of C acyl units) and vice versa. The above over-expressed MMCoA mutase was unable to perform this trans- formation so there must be a separate 'isobutyryl CoA mutase' (2-methylpropanoyl CoA mutase).O'Hagan and co-workers have investigated the incorpor- ation of the methyl group of thymine 86 and (2R)-3-amino- 2-methylbutanoate 87 into the polyether monensin A 85 in S. cinnamonensis (Scheme 19).8 ,C-Labelled methyl groups from either substrate 86 or 87 were efficiently incorporated into the seven propanoate 'Me' positions of monensin A 85 with a smaller but significant incorporation into the methyl position derived from butanoate (C-33) possibly via 2-methyl-propanoate. Most antibiotics are produced during the tro- phophase after all cell growth and DNA synthesis has ceased. Thus metabolism of unwanted DNA to release thymine may be a valuable source of C and C units during antibiotic production.The biosynthesis of inostamycin 88 from Streptomyces sp. MH816-AF15 has been investigated by Kawada and Umezawa using (1-''C)-labelled acetate propanoate and butanoate,"' and found to be assembled from six propanoate units and five butanoates. Acetate was incorporated intact at the butanoate positions. However C-1 of acetate was incorpor- ated at both the C-1 and C-2 positions of propanoate labelled units and C-1 of butanoate was also incorporated at C-1 lnostamycin Me 88 HO HO OH HO HO HO -COSCoA COSCoA COSCoA + MCOSCOA ACOOH Scheme 20 540 Natural Product Reports 1997 of propionate positions. This suggests that the main meta- bolic pathway was not butanoate-succinate-MMCoA but butanoate-(2-methyl)propanoate-MMCoA with oxidation occurring on a methyl group (Scheme 20).The biosynthesis of the ionophore tetronasin 60 from S. longisporoflavus which is of commercial interest as a cattle growth promoter and antiparasitic agent has been inves- tigated by the groups of Staunton Leadlay and Ley in Cambridge.' 19-'23 Preliminary studies had shown that the car- bon chain is assembled from seven acetate and six propanoate units but that the origin of C-33 and C-34 was not yet known however radioactive hydroxyacetate (glycolate) and glycol were efficiently incorporated (Scheme 21).88 124 125 These re- sults led to the postulation that a tetronasin PKS product 89 HO Tetronasin 60 HOa t glycolatelglycerate? Scheme 21 underwent ring closure to form the terminal tetrahydrofuran and tetronic acid rings with the possible simultaneous closure of the cyclohexanyl and central tetrahydropyranyl rings.' l9 The first series of experiments carried out were to confirm the postulated tetronasin PKS intermediates by feeding 2H- and 13C-labelled NAC analogues and examining the resulting tet- ronasin by 2H and I3C NMR spectroscopy. Labelled (2E)-butenoate 90 could not be incorporated intact presumably due to the high levels of fatty acid oxidation (FAO) of this diketide precursor to acetate. However the correct stereoisomers of the putative tri- and tetra-ketides 91 92 and 93 gave intact incor- poration when supplied to senescent cultures.Other (incorrect) stereoisomers 94 95 96 and 97 gave little or no specific incorporation (Scheme 22). In order to achieve specific incor- poration a delicate balance was required between feeding the precursors too early when the FA0 enzymes were active but tetronasin production was high and feeding too late when the FA0 enzymes were inactive but the microorganism had finally 'croaked' and ceased metabolite production. However it was Rawlings Biosynthesis of polyket ides 0 90 CD NAC-S./&/ NAC-S-CD~ -0 OH 93 NAC-S+-0 OH 95 NAC-S+-0 OH 96 NAC-S++-+ 0 OH 97 Scheme 22 found that the more advanced the precursor the more resistant it was to oxidation. To investigate the possible simultaneous closure of the cyclohexanyl and central tetrahydropyranyl rings a synthesis of a tetronasin analogue was performed based upon a metal-templated polyene cyclisation (Fig.4). An V \ Fig. 4 Metal template-assisted synthesis of tetronasin electron deficient diene facilitated pyran ring formation via conjugate addition and intramolecular Michael cyclisation to give the cyclohexanyl ring demonstrated the plausibility of this biosynthetic scheme. 123 The enediynes are a remarkable class of polyketides which were not discovered until the late 1980s possessing potent anticancer activity through a previously unknown mode of action involving the formation of a benzene diradical resulting in highly efficient DNA strand scission. Their chemistry and biology has been reviewed.126 The biosynthesis of the neocar- zinostatin chromophore (NCM) 98 by S.carzinostaticus 127 dynemicin A 99 by Micromonospora chersina lZ8 and esperamy- cin A 100 by Actinomadura verr~cosospora,~~~ have now been examined. Neocarzinostatin comprises an apoprotein and the methanol extractable NCM 98. The assembly of the NCM has 541 Me 25 h26 0 Me0 OH Neocarzinostatin chromophore (NCM) 98 A MeO' / II lo* / Y e -&OOH Crepenylic acid 101 been previously reviewed,2 and it was shown that a standard hexaketide assembly of the naphthoic acid ring whilst the C14 enediyne portion was shown to be assembled from six intact acetates and two terminal acetates each of which undergoes C-C cleavage.I2' The authors proposed that this C14 chain was derived from oleate via crepenylate 101 [(9Z)-octadecene- 12- ynoate] rather than de novo synthesis.Schreiber and Kiessling suggested an alternative source of the enediyne portion from 102 with a 12n electrocyclic ring closure followed by iso- merisation and transannular cyclisation. 30 Dynemicin 99 is assembled from two heptaketide chains which form the an- thraquinone nucleus and the bicyclic enediyne unit. Interest- ingly in NCM the two carbons in each of the yne units are derived from a single acetate whilst in dynemicin they are derived from adjacent acetate units.'28 Tokiwa and co-workers suggest that a common octaketide precursor 103 may be used to assemble both dynemycin and the esperamycin diyne nu- cleus 104.To assemble dynemycin the octaketide 103 loses two carbons from the carboxylate of the chain (path b) and for esperamicin it loses one carbon (path a) (Scheme 23). Intrigu- ingly the naphthoic acid portion of 99 could also be assembled from the same enediyne precursor using the benzene diradical chemistry of Bergmann.13' Glycine or SAM labels the methoxy moiety. Esperamicin A 100 produced by Actinomadura verru- cosospora has been investigated by Lam and co-workers who examined the incorporation of singly and doubly labelled acetates methionine and Na234S04.129 This showed that the C, enediyne unit was assembled from seven intact acetate units and one C-2 of acetate. The yne units are each derived from two separate acetate units.Addition of cerulenin totally inhibited esperamicin production which was not restored by the addition of oleate [(9Z)-octadecenoate]. This strongly suggests that the oleatexrepenylate pathway previously suggested for the assembly of the putative octaketide precursor of NCM is not operational at least in this case. This seems reasonable due to the higher oxidation level of the proposed intermediate 103. 542 Natural Product Reuorts. 1997 OH 0 OH Dynemycin A 99 OMe rn m 17-1-# OH OH u = LL-'-bJyIycine acetate methyl' COOH HOOC n la \ / 0 MeSSS NHCOOMe 6sug Esperamycin enediyne portion 1 04 Scheme 23 (acetate labelling of enediyne portion) " 1) Esperamicin A 100 HO Serpentene 105 Bergman cyclisation I COOH ~ Scheme 24 The biosynthesis of the bright yellow polyene serpentene 105 in a Streptomyces sp.may be related to that of the Naphthomycin A 106 enediynes.13* The benzene ring may be assembled via an aldol reaction of a C, precursor followed by dehydrations. Alter- natively an enediyne precursor could undergo a Bergmann cycli~ation'~~ either in the fermentation media or during workup (Scheme 24). The biosynthesis of the C,N metabolite naphthomycin A 106 in S. collinus has been investigated by Floss and co-workers who showed that it was assembled from a C,N derived starter unit and a polyketide chain derived chain from six acetate and seven propanoate units.'33 3-Amino-5-hydroxybenzoic acid 107 was shown to be a specific precursor of the C,N unit by feeding [7-'3C]-labelled precursor which gave product specifi- cally labelled at c-27 (Scheme 25).It is known that neither shikimate or dehydroquinate is incorporated into C,N metabo-lites. Unpublished work by Floss suggests that a whole new pathway the 'amino shikimate pathway' may be operational in which a shikimate hydroxy group is replaced by an amino group resulting in the formation of 3-amino-5-hydroxybenzoic acid 107. This would explain the inability to incorporate shikimate itself into these metabolites. The biosynthesis of delaminomycin A 108 in S. albulus has been investigated using 3C-labelled acetate propanoate methionine glycine and alanine.134 The pyrrolidine ring was 0H # O H A ['3C3]glycerol OH 107 Scheme 25 HO HL/ Glycine ' Me Me Delaminomycin A Me Me - ( C O - "0); Ho$;-T$; 0 - o/&nzJvvv. I .rvv I Jvvv. I I Delaminomycin C Delaminomycin A Delaminomycin B 109 108 110 Scheme 26 Rawlings Biosynthesis of polyketides Me Aldecalmycin 111 I assembled from the chain terminating acetate and glycine and a pathway is proposed based upon the isolation of cometabolites delaminomycin C 109 and delaminomycin B 110 (Scheme 26). The biosynthesis of aldecalmycin 111 in a Strep-tomyces sp. has been investigated and found to involve a decaketide with a glucose attached. 35 Both delaminomycin and aldecalmycin look likely candidates for a Diels-Alder cyclisation during their biosynthesis.The biosynthesis of streptazolin 112 from a Streptomycete sp. has been investigated by Mayer and Thiericke and found to be via a pentaketide.'36 The origin of the carbamoyl carbon and nitrogen are under investigation. A possible scheme is outlined which would also explain the occurrence of cometabolites strep- tenol D 113 and the hydroxy piperidine 114 (Scheme 27). 3 Archaea No reports of polyketide biosynthesis. 4 Protists This section contains unicellular eukaryotic microorganisms other than those considered under fungi. Protozoa are uni- cellular eukaryotic microorganisms that lack chlorophyll lack cell walls and are generally motile and include the flagellates (Mastigophora includes Trypanosoma Giardia and Leish-mania) amoebas ciliates and sporozoans (e.g.Plasmodium responsible for malaria). Sponges can be considered as a colony of unicellular microorganisms. Algae are unicellular (or colonial e.g. seaweed) eukaryotic microorganisms containing chlorophyll and carrying out oxygenic photosynthesis and possess cell walls. They include the green algae (Chlorophyta) brown algae (Phaeophyta) red algae (Rhodophyta) golden brown algae or diatoms (Chrysophyta) and dinoflagellates (Pyrrophyta). A major series of reviews on marine natural product chem- istry appeared in Chemical Reviews during 1993 including microalgal metabolites the biosynthesis of marine natural products bioactive metabolites of symbiotic marine micro- organisms carbocyclic oxylipins of marine origin marine toxins marine invertebrate chemical defences and marine haloperoxidases.'37-143 The following reviews have recently appeared the biosynthesis of bioactive metabolites of marine blue-green green red and brown algae; marine sponges coelenterates and molluscs; and the structure and biosyn- thesis of marine algal oxylipins.'45 See also the review by Pietra in this issue of NPR. The dinoflagellates Prorocentrum lima Dinophysis furtii and Dinophysis acuminata produce a group of polyether toxins the dinophysistoxins which have been responsible for a red tide phenomenon known as diarrhetic shellfish poisoning. The structure of these toxins are structurally analogous to the terrestrial polyethers containing only oxane and oxolane rings however their biosynthesis appears to be more related to the large ring polyethers such as brevetoxin.2 Norte and co-workers have examined the incorporation of '3C-labelled acetate into dinophysistoxin- 1 115 from bacteria-free unialgal cultures of Prorucentrum lima.'46 All carbons except for C-37 and C-38 were enriched by acetate with the incorporation of 16 intact acetate units but not in the manner expected for eubacterial polyketides.Norte and co-workers divide the molecule into five fragments. The carbon backbone of fragments A C and E are assembled through a classical head-to-tail acetate assembly. However the source of the Me Me # -HCOOH 0 '.VQ 0 Streptazolin 112 #A Hooc~cooH 0 OH OH Streptenol D 113 114 - IH1 112 -H20 (~2) EnzS Scheme 27 544 Natural Product Reports 1997 40 I OH ' ninnnhvsistnyin1 115 7 OH HO A branching methyl groups was [2-13C]acetate.Similar alkyl- 117 and xestoquinone 118 which are both cardiotonic and ations have been observed for parts of the brevetoxin like cytotoxic pentacycIic quinones isolated from the tropical 14''47,molecules in myxovirescen A from the gliding bac- marine sponge Xestospongia exigua and X. supra respectively; terium Myxococcus virescens,'49 in virginiamyin M from Streptomyces virginiae '50 and in oncorhyncolide from a sea- water bacterium. l 51 Kingston and co-w~rkers'~~ suggest that a probable mechanism is an aldol condensation between 3-oxoacyl CoA and acetyl CoA somewhat similar to the assembly of mevalonic acid with subsequent decarboxylation.Fragments B and D are more problematical. Fragment B containing a four carbon chain derived from the C-2-C-2-C- 2-C-1 carbons of acetate may be obtained from succinate and the citric acid pathway as has been postulated for the breve- they were then able to isolate 116 from the sponge.154 5 Fungi A review paper by Schweizer and co-workers compares the hexameric yeast (Saccharomyces cerevisiae) FAS with the tetrameric 6-MSA synthase (MSAS) (RMM 750 kDa) from Penicillium patulum that assembles 6-methylsalicylic acid 119.7 Previous crosslinking experiments suggest that MSAS is a functional dimer whose active sites are shared between two subunits.15' the phosphopantetheine binding residue in yeast FAS was Targeted in vitro mutagenases indicated that toxins.'48 The authors speculate that the origin of fragment D 156 might be 3-hydroxy-3-methylpentane-1,5-dioate (glutarate) or Ser-180 in the KR containing a-subunit a region with little ~a1ine.l~~ The biosynthesis of the closely related polyether sequence homology to bacterial ACPs. In addition point mutations in the distal C-terminal region prevented attach- ment of the prosthetic group to an intact Ser-180 suggesting toxin okadaic acid 61 also a dinophysistoxin from the marine dinoflagellates has been examined by Norte and co-workers and an acetate labelling pattern obtained.'52 Little or no label that there is no distinct ACP region but that binding and was incorporated into the first two carbons the starter unit.phosphopantetheine function may depend upon interactions Wright and co-workers fed calcium [1,2-'3C2]hydroxyacetate with widely separated distal regions of the peptide sequence. (glycolate) and obtained intact incorporation at the starter unit This portion had previously been assigned as the ACP region position with only very low levels of incorporation at other due to these point mutations' prevention of pantetheine attach- positions.' 53 There remains the possibility that these distal confor- Harada and co-workers have synthesised a putative bio- ment. 15' mational interactions may be intermolecular as is believed to synthetic precursor prehalenaquinone 116 to halenaquinone occur with other interactions in yeast FAS hexamer.The KR of both yeast FAS and MSAS transfer the pro-S hydrogen from NADPH but the KRs displayed different substrate specificities. The yeast FAS KR reduced both 3-oxobutanoyl and triacetic acid ester model substrates whilst the MSAS KR is specific for the triacetyl derivative 120 to give 121 (Scheme 28). Omission of NADPH causes MSAS to produce only triacetic acid lactone (TAL) 122 at 10% the normal rate of 6-methylsalicylic acid synthesis; the unreduced enzyme bound c6 polyketide 120 does not appear to be chain extended by Halenaquinone Xestoquinone 117 118 Prehalenquinone 116 Rawlings Biosynthesis of polyketides the enzyme to the tetraketide 123.155MSAS will accept 3-oxobutanoyl CoA as a starter but with a much reduced V,,,.MSAS KR reacted specifically to reduce the 3-0x0 group in triacetic acid 120 and could not reduce the 3-OX0 group of its lower 124 or higher homologue 123. However the DH did not dehydrate the product 3-hydroxy compound 121 until it had been chain extended to the corresponding tetraketide 5-hydroxy compound 125 (Scheme 28). Thus the KAS acts at each cycle to chain extend but the KR only operates during Me HO 0 Triacetic acid lactone (TAL) 123 122 I 0 0 0 0 0 124 120 OH 0 (NADPH,KRpr&) * U S E121 n z KAS,ACPAT W S E125 n z OH 6-Methylsalicylic acid (6-MSAS) 119 Scheme 28 the triketide cycle and the DH now acting as a 5-hydroxy dehydratase only operates during the tetraketide cycle.This is contrary to the original sequence of activity proposed by Dimroth Walter and Lynen in which both the KR and DH act during the triketide cycle. 158 Presumably this ‘postponed’ dehydration triggers the subsequent aldol cyclisation and aromatisation. There is no indication of a TE activity from the sequence and MSAS is not inactivated by phenylmeth- anesulfonyl fluoride which reacts with mammalian TEs. The paper concludes that the yeast FAS and MSAS containing very similar catalytic domains produce their very different end products as a consequence of the different substrate specificities of their KR DH and terminal acyl transferase components. In unpublished work Jordan and co-workers have isolated orsellinic acid synthase (OAS) from Penicillium cyclopiurn.Spencer and Jordan then compared this enzyme with MSAS.’59 The key difference between the two enzyme systems is the presence of an NADPH dependent KR in MSAS with the RMM being correspondingly lower (MSAS 180 kDa per subunit OAS 130 kDa per subunit) presumably reflecting the absence of a KR and DH. The origin of the oxygen atoms in mevinolin 126 from Aspergillus terreus has been revised.I6’ Vederas and co-workers initially reported that four oxygen atoms (at C-8 C-1 1 C- 13 and C- 15) were derived from dioxygen on the basis of ’80,feeding studies followed by mass spectral analysis. Using improved strains they have now been able to demon- strate by NMR spectroscopy that the oxygens at C-11 C-13 and C-15 are all acetate derived.The high level of chemical exchange may be responsible for the incorporation of dioxygen at these sites and provides a cautionary warning for similar feeding experiments using labelled dioxygen. The observations are still consistent with a possible Diels-Alder cyclisation during the biosynthesis (Scheme 29). The aflatoxins are acutely toxic teratogenic and potent carcinogenic and mutagenic reagents produced by the wide- spread fungal contamination of foodstuff derived from e.g. maize and peanuts. Aflatoxin B 127 is produced by 546 Natural Product Reports 1997 HoY-Yo Me A Me’ A Mevinolin 126 t I COSEnz COSEnz Scheme 29 Aspergillus flavus and A.parasiticus by a PKS assembly of norsolorinic acid 128 followed by at least 16 enzyme catalysed steps (Scheme 30). The aJ-2 gene has been cloned from A. flavus and has been shown to be involved in the assembly of norsolorinic acid and may also regulate expression of the PKS.I6’ The apa-2 gene from A. parasiticus has been cloned and found to be functionally homologous to a$-2 and com- plemented aJ-2 mutant strains. 162 Norsolorinic acid 128 was oxidised to a single product averantin 129 the stereochemistry of which has been determined to be (1’s) by chiral HPLC. Only this isomer not the corresponding (l’R) was further oxidised but to both (l’S 5’R)-5’-hydroxyaverantin130a and the epimeric (l’S,5’59 compound 130b (Scheme 30).’63130a and 130b were both converted into averufin presumably via the ~‘-OXO intermediate.Dioxygen has been incorporated during the Baeyer-Villiger oxidation of hydroxyversicolorone to versiconal hemiacetal acetate 131.164 The stereochemistry of the conversion of 131 to versicolorin B 132 has been examined OH 0 OH 0 OH 0 OH OH afC2 or apa-2 Me Me HO 0 0 Norsolorinic acid Averantin 128 /12,\ OH 0 OH OH OH 0 OH OH OH Me Me and HO HO 130a 130b / OH 0 OH OH 0 OH 0 OH Me HO 0 0 Averufin 0 OH d + OH + HO HO / /OH HO / /OH 0 0 0 Hydroxyversicolorone Versiconal herniacetal acetate Versiconal 131 ___) / HO / OH HO / /o HO 0 0 Versicolorin B Versicolorin A Dernethylsterigrnatocystin Sterigrnatocystin O-Methylsterigrnatocystin Aflatoxin B 133 134 127 Scheme 30 and product stereochemistry found to be determined by the versicolorin B ~ynthase.'~~ The gene encoding for a SAM dependent methyltransferase that converts sterigmatocystin 133 to O-methylsterigmatocystin 134 in A.parasiticus has been cloned166 and the protein purified. 167 The oxidative cleavage and rearrangement of O-methyl-sterigmatocystin 134 to afla- toxin B 127 has been examined using the conversion of radiolabelled O-methylsterigmatocystin 134 by cell-free extracts to aflatoxin B 127 (Scheme 31).16' This complex rearrangement may be performed by a single enzyme.'69 Chatterjee and Townsend showed clean loss of C-10 from O-methylsterigmatocystin 134 its conversion into carbon dioxide and discuss possible mechanisms.'68 A red pigment isolated from a mutant strain of A.parasiticus is reported as a possible new biosynthetic intermediate. 170 Rawlings Biosynthesis of polyketides Squalestatins produced by Phoma sp. are potent inhibitors of squalene synthase and are under investigation as cholesterol lowering agents. The biosynthesis of squalestatin was covered in the previous review and is from two polyketide chains one of which has a shikimate derived benzoic acid starter unit.2 17' Around 50 analogues have now been produced by feeding benzoic acid analogues to the microorganism including fluorinated aromatics heteroaromatics cyclohexanyl- cyclopentanyl- and cyclobutanyl-carboxylic acids and some natural analogues isolated.172-174Zaragozic acid A 135 pro-duced by an unidentified fungus appears to have an identical structure to squalestatin. A biosynthetic study appeared shortly after the previous study on squalestatin and both organisms appear to use the same biosynthetic pathway involving a phenylalanine derived benzoyl CoA starter and a Me0Oo L I Me0 0 134 OMeth ylsterigmatocystin Me0 Me0-0 I L J 0 ? II -HCOOH OHC Me0 Me0 Aflatoxin B 127 Scheme 31 oh Me ?Ac Me Me COOH A A COOH Zaragozic acid A (squalestatin) 135 Succinate WH:OOH_ 0' WCOOH-COOH Scheme 32 succinate derived unit (Scheme 32).'75 A range of analogues has been produced using directed biosynthesis in which the phenyl ring is replaced by fluorinated thiophene and furan rings.76 The decarestrictines are inhibitors of cholesterol biosynthe- sis which have been isolated from Penicillium simplissimum. 0 OH Ho@. OH 'OH They are usually 10-membered lactones with an exocyclic methyl group. Mayer and Thiericke examined the biosynthesis Decarestrictine B Decarestrictine D of decarestrictine B 136 and decarestrictine D 137 using 136 137 labelled acetate and dioxygen.'77 They then reported on the later biosynthetic steps leading to various members of the decarestrictine family and found that a non-enzymatic reac- tion was a key step in the bio~ynthesis.'~~ Under acidic conditions of the fermentation the epimeric decarestrictines A and A 138 are converted into the major product decar- OH fio& HO OH *Oa.estrictine D 139 by the non-enzymatic opening of the epoxide 548 Natural Product Reports 1997 0 0 0 DecarestrIr;al~t:r PKS Product 0 -OH 0-0 OH Me 0 Decarestrictine Aland A2 Dncarnstrictine B 138 I \. J H O W 'OH r J. 1 OH Decarestricine 0 . .-?Ao] Me 0 ,LHO-Decarestrictine E Decarestrictine I ro4 / I HO -OH 7 OH Decarestrictine N HO-'*.OH OH Decarestrictine D HO' 139 Decarestricine M Scheme 33 ring followed by hydrolysis. In contrast under neutral fermen- tation conditions high yields of decarestrictine B 140 along with other decarestrictines can be obtained (Scheme 33). The fungus Cochliobolus spicifer (Nelson D-5) responsible for leaf spot disease in wheat produces the phytotoxic spici- Me Me-O W M e Me 0 Spiciferinone 144 A R' 1 Me R2*o Me 141 R' =Me; R2 = Me Spiciferone A 142 R1= CH20H; R2 = Me Spiciferone B 143 R'=Me; R2=CH20H Spiciferone C COOH COOH 010 010 Me* Me Me' COOMe Me A COOMe A Spicifernin A 1 45 ferones A 141 B 142 C 143 spiciferinone 144 and the plant growth promoter spicifernin 145 which exists as a pair of tautomers.Despite their different carbon skeleta it was pro- posed by Nakajima and co-workers that they arose from a common polyketide prec~rsor."~ Preliminary biosynthetic experiments were hindered by low yields of the metabolites.'so however addition of methionine has increased yields four-fold and has allowed incorporation studies using [1,2-' 3C2]acetate and ['3CH3]methionine (Scheme 34).The data suggested that a single hexaketide chain with two SAM derived C units undergoes a retro-aldol reaction to form spiciferones or spicifernin.''' Alternaric acid 146 is produced by the causal fungus of early blight disease on potato or tomato Alternaria solani as an antifungal metabolite. It is assembled from two polyketide chains though whether from a combination of a di- and a hepta-ketide or from a tri- and a hexa-ketide has not estab- lished.lg2 Ichihara and co-workers have obtained five metabo- lites 147-152 from A. solani that are less oxidised (some of these were obtained by addition of cyctochrome P,, inhibitors to the medium) and they postulate that they may be inter- mediates or shunt metabolites (Scheme 35).Ig33 Is4 The origin of the oxygen and hydrogen atoms was also investigated by feed- ing labelled acetate.O'Hagan and co-workers have suggested that SAM derived methyl groups in fungal polyketides are inserted by a methyltransferase (MeT) that is an integral part of the Type I PKS m0du1e.l~~ The chemically most rational time of insertion would be onto the 3-oxoacyl thioester i.e. straight after the 3-oxoacyl synthase (KAS) step as an integral parcof processive chain assembly. 0x0 reduction and dehydration would leave none of the original 'acetate' hydrogens on the C-2 position of 153 thus no deuteriums would remain from feeding [1-13C *H,]acetate at those methylated positions derived from Rawlings Biosynthesis of polyketides 0"--i";cl.-EnzS C1 Me + Me Et' CHO / "\\ J Spiciferinone 144 Spicifernin Et' CHO 145 0 Scheme 34 \ Me Me I Me /14*\ 0 OH \.= 0 OH 147 Proalternaric acid 1 149 OH 0 OH OH 0 OH Me Me.(shunt metabolite) 150 151 Me 152 Alternaric acid 146 Scheme 35 550 Natural Product Reports 1997 D from CD3-acetate D& 1 46 ~ SEnz MeT D3CxsEnZ KR Me -D3ch~~nz CD3C00H D3c% 00 00 OH 0 Me DH D3CAsEnZ 5-Final D,C%sEnz --+ -+ Metabolite 146 0 0 153 Scheme 36 C-2 of acetate (Scheme 36). No deuterium could be located at such positions in particular C- 12 supporting O'Hagan's pro- posal whilst deuterium was incorporated at the analogous non-methylated position C- 16.The stereochemistry of the methyl groups also suggest a common stereochemical outcome for the enoyl reductases. When sodium [l-'3C,180,]acetate was fed isotope shifts were observed at C-1 C-15 and C-17. No shift was observed at C-1 1 or C-3 presumably due to high levels of exchange with the medium. J-JY 0 Cercospora spp. are responsible for leaf spot disease on many crops. The structure of yellow Cercospora beticola toxin (CBT) has been determined as 154 and found to be a 2:2 complex -of two magnesiums with two identical polyketides each 154 2 assembled from two C, units an anthraquinone and a xanthone. 186 Its biosynthesis has been investigated using labelled acetate and both C, units are found to be octaketides O'H 0 H0.II I with loss of carbon dioxide. Elminthosporin 155 was occasion- ally present in the cultures and could be a common precursor to both the xanthone and anthraquinone moiety (Scheme 37) the xanthone moiety 156 being assembled by an oxidative cleavage of elminthosporin (Scheme 38) followed by free rota- tion and addition-elimination cyclisation as has been observed in other fungal xanthones."' This free rotation of ring opened elminthosporin 155 followed by ring closure accounts for the label randomisation observed in one aromatic ring (Scheme 38). The xanthone and anthraquinone portions are then joined 'End-to-end'coupling end-to-end in a chemically intriguing and as yet unexplained reaction to form the central seven membered ring of CBT 154 (Scheme 37).The xanthoquinodins are a series of anticoccidial antibiotics produced by Humicola sp. FO-888 whose structure is a heterodimer also consisting of xanthone and anthraqui- none rnoieties,lg8 with a heterodimer core similar to the betico- linsIR9 and cebetins. 19' The biosynthesis of xanthoquinodin A1 157 and xanthoquinodin B1 158 has been investigated using COOH Elminthosporin labelled acetate and methionine. As above oxidative cleavage 156 155 of elminthosporin 155 to 156 provides a reasonable route to the Scheme 37 xanthone portion and explains the randomisation of label. End-to-end coupling gives 157 or 158 (Scheme 38). A mechan- ism is proposed for the conversion of 157 to xanthoquinodin transfers the 4-pro-S hydrogen of NADPH to give the R A3 159.alcoh01,'~' probably at the triketide stage reminiscent of MSAS in P. p~tulurn.'~~ In the absence of NADPH the derailment product triacetic acid lactone 122 is formed as 6 Plants with the fungal systems (Scheme 28) suggesting that the third 6-Hydroxymellein 160 is a pentaketide produced by carrots Claisen condensation is specific for the NADPH reduced (Daucuscarota) in response to an infection or when induced by triketide. The synthase was found to be inhibited by thiol certain chemical elicitors subsequent 0-methylation resulting containing reagents but not by cerulenin the first example of in the phytoalexin 6-methoxymellein 161 (Scheme 39).'91p193 a PKS not to be inhibited by cerulenin.A later paper shows 6-Hydroxymellein synthase has been purified 240-fold from the that cerulenin forms an unstable covalently bound complex soluble fraction of elicitor-treated carrot and is a single with the enzyme which can be replaced by substrate.192 The multifunctional polypeptide chain'94 resembling Type I animal PKS was shown to have a very high affinity for acetyl and FAS and is extremely unstable. The synthase stereospecifically malonyl CoA much higher than the corresponding carrot Rawlings Biosynthesis of polyketides 55 1 0000 AAAA Xanthone 156 Me Me Me 0 L Elminthosporin 155 OH I 0 I1 OH I OH 0 OH Me 0 155 COOH 156 156 ‘End-to-body’coupling J ‘End-to-body’coupling OH 0 OH Me Me HO’ A Xanthoquinodin B1 Xanthoquinodin A1 158 157 Scheme 38 Acetyl CoA + 2 x malonyl CoA I Me Me Xanthoquinodin A3 159 FAS suggesting that when expressed it had ‘priority’ for OH 0 OH 0 substrates over FAS’93 and was strongly inhibited by the I I1 I II ultimate pathway product 6-methoxymellein.Use of NAD2H led to an isotope effect of ca. five suggesting that the NADH dependent reduction is one of the key rate determining /oTj. HOm, reactions in 6-hydroxymellein biosynthesis. Me0 6-Methoxymellein .Me 6-Hydroxymellein ‘Me Genes involved in flavanoid and stilbene assembly have been 161 1 60 isolated from grape (Vitis vinifera) using heterologous probes Scheme 39 from maize and snapdragon. The phenylalanine and stilbene synthase genes encoded large multifunctional proteins.19‘ A review has appeared that discusses the metabolic engineering of plant secondary products. The review lists the transfer of Orchid rhizomes produce bibenzyls such as 3,3’,5-stilbene and chalcone synthases from a variety of sources into trihydroxybibenzyl 162 which subsequently can be converted tobacco or petunias.’97 into dihydrophenanthrenes such as 2,4,7-trihydroxy-9,10-552 Natural Product Reports 1997 OH I Ho~CoscoA (slow) 3 x Malonyl CoA * (Bibenzyl synthase) (2€)-enoyl CoA-3-(3-Hydroxyphenyl)prop 3,3',5-Trihydroxystilbene 165 166 OH (fast) 3 x Malonyl CoA HorCoscoA (Bibenzyl synthase) 3-(3'-Hydroxyphenyl)propanoylCoA 3,3',5-Tri hydroxybibenzyl 164 162 HO 2,4,7-Tri h yd roxy-9,lO-di hyd rophenan ph rene 163 Scheme 40 dihydrophenanthrene 163 in response to fungal infection.Bibenzyl synthase has been isolated from rhizomes of Bletilla striata (Orchidaceae) as a homodimer of subunit mass 46 kDa19' that uses 3-(3'-hydroxypheny1)propanoyl CoA (dihydro-rn-coumaryl CoA) 164 as starter and three units of malonyl CoA. The enzyme did not use (2E)-3-(3'-hydroxypheny1)propenoyl CoA (m-coumaryl CoA) 165 as substrate suggesting a separate pathway to the stilbenes such as 166 (Scheme 40). 7 Animals The biosynthesis of mellein 167 and 2,4-dihydroxy-acetophenone 168 by the Australian ponerine ant (Rhytido-ponera chalbaea) has been investigated by feeding the ants aqueous sodium [1,2-'3C,]acetate (along with mealworms) confirming the expected polyketide pathway as in the fungi.199 OH 0 ?H I? Mellein 2,4-Dihydroxyacetophenone 167 168 OH 0 The siphonariid limpets (Pulmonata Mollusca) produce a wide range of polypropanoate derived metabolites such as the siphonarins.Garson and co-workers have examined their biosynthesis in Siphonaria zelandica using radiolabelled pro- panoate and succinate.200 Siphonarin A 169 is assembled from one acetate and nine propanoates with terminal decarboxyl- ation and siphonarin B 170 from ten propanoates. 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ISSN:0265-0568    

DOI:10.1039/NP9971400523

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

Book review Biochemical aspects of marine pharmacology Eds. P. Lazarovici M. E. Spira and E. Zlotkin Alaken Inc. Fort Collins Colorado 1996 pp. xi+227. Price $84 ISBN 1 880293 07 02 The seminal review of marine chemistry by John Faulkner (Tetrahedron 1977) heralded the huge upsurge of interest in this area which has continued unabated for the past 20 years. His many reviews for this journal (16 since 1984) are always chock full of mainly novel structures that bear witness to the vast diversity of natural products produced by marine organ- isms. Unlike their terrestrial counterparts there is very little folklore associated with the use of marine plants and animals and where this exists it is mainly in oral rather than written form. In consequence the search for novel compounds has been somewhat random and difficult or exciting depending upon one’s inclination towards scuba diving.None the less a growing number of metabolites with both interesting and potentially useful pharmacology has been identified. The bryo- statins cephalostatins and dolastatins are three classes of compounds which appear to show clinically useful anti-tumour activity; but it is the compounds which act upon the central nervous system that have provided much of the academic interest over the years and these are the main subject of this book. The neuropharmacological effects of marine natural prod- ucts like tetrodotoxin kainic acid and domoic acid have been well documented previously and the strength of this book lies in its coverage of the newly emerging neurochemical tools.These include the conotoxins (from marine snails) which target receptors on sodium channels or calcium channels depending upon structure; sea anemone toxins which have high affinity for sodium channels of heart muscle neuromuscular junctions or for potassium channels of several cell types; and pardaxin a toxin from a Red Sea fish that causes a massive release of neurotransmitters from several types of neurones. In fact a whole chapter is devoted to the strange metabolites produced by organisms that live in the Red Sea an environment that is unique by virtue of the great seasonal variation in sea and water temperatures. The investigations described in the 15 chapters demonstrate how marine organisms can use different cocktails of natural products to paralyse or hyperexcite their potential prey and how subtle changes of peptide sequence can change a fish toxin into a molluscicide -the trick here is to ensure that the mollusc is quickly immobilised outside of its shell rather than being paralysed within it. There is much fascinating information here especially for pharmacologists though the amount of chemistry is somewhat limited. However I would encourage natural product or synthetic chemists who have not yet been tempted to work with marine natural products to at least skim the pages of this book. They will find much of interest and their sea bathing especially in warmer waters will never be the same again! John Mann University of Reading UK

ISSN:0265-0568    

DOI:10.1039/NP9971400557

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

Corrigendum Biosynthesis of gibberellin plant hormones Jake MacMillan Nut. Prod. Rep. 1997 14 221-244. There is an error in Table 1 on p.222 in which the second entry has the correct common name of ‘Turnip rape’. The numbering of ent-kaur-16-ene in Scheme 3 on p.223 has been corrected and is shown below. There is a footnote missing from the caption of Fig. 2 on p.231 which should read Fig. 2 Natural 2P-hydroxy-C2,GAs “Tentative identification (see text). In Scheme 21 on p.240 the structure of GA has a hydroxy group missing. The correct structure is shown below. Ref. 107 should read M. Kobayashi Y. Kamiya A. Sakurai H. Saka and N. Takahashi Plant Cell Physiol. 1990 31 289. Ref. 194 should read M. Hutchinson P. Gaskin J. MacMillan and B. 0. Phinney Phytochemistry 1988 27 2695. ent-kaur-16-ene

ISSN:0265-0568    

DOI:10.1039/NP9971400558

出版商:RSC    

年代:1997

数据来源: RSC