摘要:

ISSN 0265-0568 NPRRDF 14(2) 83-204 (1997) Natural Product Reports A journal of current developments in bioorganic chemistry Volume 14 Number 2 CONTENTS ... 111 Hot off the press Robert A. Hill and Andrew R. Pitt Reviewing the recent literature on natural products and bioorganic chemistry 83 Recent advances in chemical ecology Jeffrey B. Harborne Reviewing the literature published between July 1992 and December 1995 99 The role of carbohydrates in biologically active natural products Alexander C .Weymou th- Wilson 111 The biosynthesis of C5-C25 terpenoid compounds Paul M. Dewick Reviewing the literature published between 1993 and 1995 145 Natural sesquiterpenoids Braulio M. Fraga Reviewing the literature published in 1995 163 Fatty acids fatty acid analogues and their derivatives Marcel S.F. Lie Ken Jie Mohammed Khysar Pasha and M. S. K. Syed-Rahmatullah Reviewing the literature published between 1988 and 1995 191 Diterpenoid and steroidal alkaloids Atta-ur-Rahman and M. Iqbal Choudhary Reviewing the literature between mid-1994 and the beginning of 1996 Cumulative Contents of Volume 14 Number 1 1 Brassinosteroids Shozo Fujioka and Akira Sakurai 11 Quinoline quinazoline and acridone alkaloids (July 1994 to June 1995) Joseph P. Michael 21 Indolizidine and quinolizidine alkaloids (July 1994 to June 1995) Joseph P. Michael 43 Lignans neolignans and related compounds (January 1994 to December 1995) Robert S. Ward 75 Cyclopeptide alkaloids (January 1985 to December 1995) Dimitris C.Gournelis Gregory G. Laskaris and Robert Verpoorte Number 2 83 Recent advances in chemical ecology (July 1992 to December 1995) Jeffrey B. Harborne 99 The role of carbohydrates in biologically active natural products Alexander C. Weymouth-Wilson 111 The biosynthesis of C,-C2 terpenoid compounds (1993 to 1995) Paul M. Dewick 145 Natural sesquiterpenoids (1995) Braulio M. Fraga 163 Fatty acids fatty acid analogues and their derivatives (1988 to 1995) Marcel S. F. Lie Ken Jie Mohammed Khysar Pasha and M. S. K. Syed-Rahmatullah 191 Diterpenoid and steroidal alkaloids (mid-2994 to the beginning of 1996) Atta-ur-Rahman and M. Iqbal Choudhary Articles that will appear in forthcoming issues include Diterpenoids (1995) James R. Hanson Marine natural products (1995) D.John Faulkner Amaryllidaceae alkaloids (2995) John R. Lewis The biosynthesis of the gibberellin plant hormones (up to September 1996) John MacMillan Synthesis of amino acids incorporating stable isotopes (1990 to mid 2996) Nicholas M. Kelly Anc.ew Sutherland an1 Christ,.ie Willis Biosynthesis of fatty acids and related metabolites (up to end 1994) Bernard J. Rawlings Biosynthesis of plant alkaloids and nitrogenous microbial metabolites (1995) Richard B. Herbert Phenethylamine and isoquinoline alkaloids (July 1995 to June 1996) Kenneth Bentley Recent progress in chemistry of non-monoterpenoid indole alkaloids (July 1995 to June 1996) Masataka Ihara and Keiichiro Fukumoto Chemistry and biosynthesis of clavulanic acid and other clavams Allan Brown Steroids reactions and partial synthesis (1995) James R. Hanson Monoterpenoids (part 1993 all 1994 part 1995) David H. Grayson COPIES OF CITED ARTICLES The Library and Information Centre (LIC) of the RSC offers a first class Document Delivery Service for items in Chemistry and related subjects. Contact the LIC The Royal Society of Chemistry Burlington House Piccadilly London WlV OBN. Tel +44 (0)171 437 8656 -Fax +44 (0)171 287 9798 -Email library@rsc.org This service is only available from the LIC in London and not the RSC in Cambridge.

ISSN:0265-0568    

DOI:10.1039/NP99714FP003

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 Uniiversity 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 Edit0 r 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 ica I Editor Telephone +44 (0) 1223 420066 Mrs Dawn Webb Facsimile +44 (0) 1223 420247 Miss Karen L. White E-mail rscl @ rsc.org Editorial Secretaries RSC Server htt p ://ch em ist ry.rsc. org/rsc/ Natural Product Reports is a bimonthly journal of critical reviews. It aims to foster progress in the study of bioorganic chemistry by providing regular and comprehensive reviews of the relevant literature published during well-defined periods. Topics include the isolation structure biosynthesis biological activity and chemistry of the major groups of natural products-alkaloids terpenoids and steroids aliphatic aromatic and 0-heterocyclic compounds. This is augmented by frequent reviews of the wider context of bioorganic chemistry including developments in enzymology nucleic acids genetics chemical ecology primary and secondary metabolism and isolation and analytical techniques which will be of general interest to all workers in the area.Articles in Natural Product Reports are commissioned by members of the Editorial Board or accepted by the Chairman for consideration at meetings of the Board. Natural Product Reports (ISSN 0265-0568) is published bimonthly by The Royal Society of Chemistry Thomas Graham House Science Park Milton Road Cambridge UK CB4 4WF. 1997 Annual subscription rate f355.00; US$640.00. Customers in Canada will be charged the sterling price plus a surcharge to cover GST. Change of address and orders with payment in advance to The Royal Society of Chemistry The Distribution Centre Blackhorse Road Letchworth Herts. UK SG6 1HN. Air freight and mailing in the USA by Publications Expediting Service Inc. 200 Meacham Avenue Elmont NY 11 003.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/NP99714FX009

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

FOURTH INTERNATIONAL SYMPOSIUM IUPAC ON BlOORGANlC CHEMISTRY Sponsored (IVlh ISBOC) BlARRlTX FRANCE 1 -6 JUNE 1997 The symposium intends to highlight the latest ideas and advances at the frontier between chemistry and biology to inspire and stimulate the development of research in this area. The scientific programme will include plenary lectures oral communications poster presentations and a vendor exhibition. The organizin Committee has chosen five interdisciplinary themes that will provide a framework for 9ectures and oral communications which will be selected from submitted abstracts. The following topics will be covered Enzymes Biotechnology Recepiors Nucleic acids The following scientists have agreed to present a lecture J.C.Chottad B. lrnperiali 0.Moras T. Costa I. lvanov Y Nagai J. FontecillaCamps G,G.Jung P. E. Nielsen J. Freyssinet J.A. Killian H Paulus E. Giralt P. Leodlay J.C. Promi R Haener J,M Lehn F. Schuber I,Huang H.W Liu B. Stoddard A workshop session will be organized by J.4.le Pecq and A. Kahn on "Gene Therapy". The Conference will be held in the Casino Bellevue Biarritz France which overlooks the Atlantic Ocean. Biarritz located in the south west of France can be reached by plane or TGV (high speed train) Biarritz was born last century as sea bathing became fashionable. High society has followed Emperor Napoleon Ill and Empress Eugenie to ensure !he continuing PO ularity of Biarritz's beaches Biarritz has become an international centre for competition (go1 P surf tennis rugby pelota games).From fiestas 10festivals there is always a season to celebrate in Biarritz. The Municipal Casino fully restored in its 1930ssplendour has been re-opened in 1994. Registration fees ; Before After March 14 1997 March 15 1997 Private organizations 3500 FF 4000 FF Academic Loboratories 2500 FF 3000 FF Students 1250 FF 1750 FF Accompanying persons 1000 FF 1500 FF IUPAC affiliate members are entitled to a 10 % reduction. The registration fees cover all the scientific evenls (sessions and book of abstracts) welcome party lunches and breaks between sessions. For the accompanying persons only the welcome party and lunches are included Inquiries should be addressed to Prof. Andrio Marqud ISBOC IV Laboratoire de Chimie Organiqut Biologique Universite Pierre et Marie Curie BoSte Courrier no 182 -4 place Jussieu 75252 PARIS CEDEX 05 -FRANCE Tel. 33 01 44 2755 35 Fax ; 33 01 44 27 71 50 e.maiI ; marquet @ccr .jus sieu.fr

ISSN:0265-0568    

DOI:10.1039/NP99714BX011

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

Recent advances in chemical ecology Jeffrey B. Harborne Plant Science Laboratories University of Reading Reading UK RG6 6AS Covering July 1992 to December 1995 Previous review 1993 10 327 1 Introduction 2 Animal-animal interactions 2.1 Animal toxins of plant origin 2.2 Chemical defence in animals 2.3 Pheromones 3 Plant-animal interactions 3.1 Constitutive chemical defence 3.1.1 Nickel hyperaccumulation 3.1.2 Terpenoids 3.1.3 Alkaloids 3.1.4 Phenolics 3.1.5 Tannins 3.1.6 Bird toxins 3.2 Induced chemical defence 3.3 Hormonal interactions 3.4 Insect antifeedants 3.5 Oviposition stimulants and deterrents 3.6 Pollination interactions 4 Plant-plant interactions 4.1 Allelopa t hic agents 4.2 Plant-parasite interactions 5 Plant-microbe interactions 5.1 Phytotoxins 5.2 Constitutive antimicrobial defence 5.3 The phytoalexin response 6 References 1 Introduction Current research in chemical ecology is concentrated on the role of the wide variety of secondary metabolites in protecting plants from insect herbivory or vertebrate grazing.A notable recent discovery is that potatoes and related Solanum plants have a second line of defence to the traditional steroidal alkaloids such as solanine. They have the ability to synthesise polyhydroxytropane alkaloids such as the calystegines as well. These are potent glycosidase inhibitors and may be responsible for neurological disorders that sometimes arise in cattle feeding on potatoes.While the steroidal alkaloids of Solanum are not sequestered by larvae of Ithomiine butterflies feeding on these plants the new alkaloids can be sequestered by both butterfly and moth larvae (see Section 2.1). It is well known that certain plant species growing on particular soils have the ability to defend themselves against the toxicity of heavy metal ions by chelating them and accu- mulating the chelates in vegetative tissues. It has been assumed that such plants containing lethal concentrations of copper cadmium zinc or nickel would be toxic to herbivores but no ecological experiments have appeared to support this assump- tion. The situation has now changed regarding nickel hyper- accumulators and good evidence has been produced (Section 3.1.1) that insect herbivores such as Pieris rapae suffer mor- tality when fed on nickel-containing tissues.The accumulation of nickel chelate in the plant also incidentally protects from microbial infection. Lichens are particularly rich in secondary metabolites and they accumulate a series of phenolic metabolites many of which are specific to this algal-fungal symbiosis. They include depsides depsidones and anthraquinones which can make up Harborne Recent advances in chemical ecology some 20% of the dry weight. Here again it has been speculated for many years that these compounds are defensive agents against herbivores. At last some feeding experiments have been carried out and the results confirm that harmful effects on growth do occur in the armyworm Spodoptera littoralis.This does not mean however that herbivores cannot become adapted to the lichen substances since herbivory does occur naturally on lichen tissues. Indeed these and related studies (Section 2.1) have shown that some specialist insects and molluscs feeding on lichens actually absorb and store lichen substances from their diet for the purpose of defence. As in previous reviews,1p3 animal-animal interactions will be considered before plant-animal plant-plant and plant- microbe interactions. The fourth edition of a textbook in ecological biochemistry has appeared both in English4 and in German,' and a textbook on the plant-insect interface has been published.6 A monograph on the biochemistry of orchid scents covers the pollination biology as well.7 A symposium proceedings on terpenoid chemical ecology includes several important reviews.8 2 Animal-animal interactions 2.1 Animal toxins of plant origin Much work has concentrated on the uptake and further metabolism of alkaloids by insects sequestering and storing these dietary toxins.The Asian arctiid moth Creatonotos transiens for example is well known to sequester pyrrolizidine alkaloids (PAS) from its food plants and store them in both the larval and adult stage^.^ Von Nickisch-Rosenegk and Winkg have now found that six European species of arctiid moths are similar all converting the free bases to the respective N-oxides in vivo.Furthermore adults of three species converted 7(S)- heliotrine to 7(R)-heliotrine which is a direct precursor of the male pheromone 7(R)-hydroxydanaidal. It is thus a reasonable assumption that all these arctiid moths use the PAS both for defence and for pheromone production. Insects borrowing PAS from plants either obtain them at the larval stage from leaves or in the adult stage from nectar. The polyphagous grasshopper Zonocerus variegatus feeding on the weed Chromolaena odorata goes one better and eagerly con- sumes the PA-rich flowers. Analysis of the plant shows no alkaloid in leaf stem or nectar but high concentrations in root and flowerhead which have 1.03 and 2.44 pmol g- dry wt respectively. The grasshopper sequesters intermedine and rind- erine and transforms up to 20% into lycopsamine and echina- tine by inversion of configuration at C-3'.Pure rinderine injected into the hemolymph of Zonocerus is partly converted to intermedine indicating inversion of configuration at C-7. lo Adult leaf beetles of the genus Oreina of the Chrysomelidae are interesting in that they defend themselves by dietary sequestration of PAS by de novo synthesis of cardenolides or by a mixture of these two strategies. Pasteels et al." have surveyed 10 species to determine which strategy is the most widely used. The choice of food plant is one key since those feeding on Umbelliferae generally produce their own cardeno- lides while those feeding on Senecio borrow the PAS. One species Oreina melanocephala proved to be poorly defended.It feeds on Doronicum clusii which although it is in the Senecioneae is usually devoid of PAS and it is only able to secrete traces of cardenolide. More successful species such as Me H. .k & flMe. I ! I R 1 R=H 3 2 R=OH been found to sequester the same compound the zygaenid Pryeria sinica and the magpie moth Abraxas grossulariata. l5 Both these moths feed on Euonymus japonicus (Celastraceae) which contains sarmentosin. The high concentration (650 pg per insect) of toxin in the imago of the magpie moth suggests that the compound is defensive against predation. Aristolochic acids aromatic nitro compounds occur in the plant family Aristolochiaceae and are known to be sequestered by swallowtail butterflies feeding on Aristolochia spp.* Nishida et al.l6 have found that two Malaysian swallowtails which feed on a different genus Thottea also borrow aristolochic acids I and I1 from their food plants. The monoterpenoid iridoids have a wide distribution in plants and are sequestered by moths leaf beetles flies and grasshoppers. A new iridoid 6P-hydroxyipolamide 5 has been detected in a single specimen of the Costa Rican moth Thessalia theona but unfortunately its food plant could not be HoaMe 4 5 0 bMeOHC OH Me 6 7 OMe 0 OMe II y!% OHyOMe OMe 0 OH 8 9 0.elongata have greater flexibility in defence both synthesis- ing cardenolides and sequestering PAS. Populations of this latter species were found exclusively producing cardenolides exclusively sequestering PAS or doing both depending on the local availability of food plants.Two new classes of plant alkaloid have been found to be sequestered by insects. Thus the tropane alkaloids calystegine A 1 and calystegine B have been found in the leaves of Datura wrightii (Solanaceae) and in the pupae and adults of the Death’s Head Hawk moth (Acherantia atropus) which feeds on this plant. l2 Furthermore the ithomiine butterfly Mechanitis polymnia was also found to have the calystegines although the larvae never sequester the steroidal alkaloids which occur in their solanaceous food plants. Sequestration of steroidal alkaloid does however occur in the case of the specialist sawfly Rhadinoceraea nodicornis the larvae of which feed on the host plant Veratrum album.The major alkaloid of the haemolymph is 3-acetylzygadenine 3 probably formed in vivo in the larval gut from 3-angeloylzygadenine which is first hydrolysed and then acetylated. Protoveratrine A and B present in the host plant are degraded by the larvae and not stored. Bioassays with the ant Myrmica rubra show that the haemolymph is highly deterrent and toxic. Ants spiders and bush crickets all failed to eat the alkaloid-protected larvae. l3 Other nitrogen-containing plant substances known to be borrowed by animals for defensive purposes include cyano- glucosides and aromatic nitro compounds. The unusual cyano- glucoside sarmentosin 4 has been isolated from the Apollo butterfly Parnassius phoebus and from its food plant Sedum ~tenopetalum.’~ The body tissues contain 500 pg per insect and there is a high concentration in the wings.The eggs are also protected by this bitter-tasting toxin. Two moths have also 84 Natural Product Reports identified.l7 Traces of 8-hydroxygeraniol and its 8-glucoside have been detected in the defensive secretions of the leaf beetles Plagiodera versicolora and Gastrophysa viridula. This suggests de novo synthesis in the beetle rather than a dietary borrowing with P-glucosidase hydrolysing the 8-glucoside oxidation of 8-hydroxygeraniol to 8-oxocitral and cyclisation to the iri- doids plagiodial and chrysomolidial which are found in the beetle.’* The protective role of plant phenolics in animal-animal interactions has not been extensively explored although it is known that salicin is borrowed for defence by willow beetles and converted to salicylaldehyde.2 Hesbacher et al.l9 have found not surprisingly that lichen phenolics may be used by specialists feeding on lichen for defensive purposes.Eleven species of Arctiidae larvae have been found to have the anthraquinone parietin 6 and the depside atrenorin 7 in their tissues. By contrast generalist larvae such as Spodoptera littoralis suffer toxic effects when fed on lichens and usually avoid feeding. However two species of lichen-feeding snails Baleaperversa and Chondrina clienta also store the same two phenolics as the moth larvae.20 The possibility of metabolising commonly occurring dietary phenolics and converting them to toxins has been demon- strated in the case of the adult lubber grasshopper Romalea guttata.When fed on catnip Nepeta cataria it sequesters caffeoyltartronic acid from this food plant and stores catechol and hydroquinone in its defence gland secretions. Feeding caffeic acid-fortified diets caused excessive secretions of catechol confirming the conversion of the caffeic ester of Nepeta to free catechol in vivo.21 Since this grasshopper in its natural habitat is a generalist feeder on grasses which contain caffeoylquinic (chlorogenic) acid it is presumably able to carry out this conversion and protect itself with catechol in the field. 2.2 Chemical defences in animals Beetles are notably well protected from predation by defence secretions containing aliphatic quinonoid or terpenoid mol- ecules and more information is coming to hand on their relative effectiveness as defence agents.The defensive strategies of aleocharine and tenebrionid bark beetles have been described by Dettner22 as ‘dabbing and shooting’ at their enemies (ants flies and other arthropods) with benzoquinone and naphthoquinone secretions. Thus analysis of their secre- tions shows the presence of p-toluquinone 8 and three 6-alkylnaphthoquinones dissolved in various alkanes alkenes ethyl isopropyl and isoamyl esters. Occasionally acetophe- none benzylpropionate and methyl hydroxybenzoate were also detected. These defence secretions also appear to provide protection from fungal infection.Rove beetles of the genus Bledius secrete in their abdominal glands the toxin p-toluquinone 8 which is dissolved in the solvents y-dodecalactone and 1 -undecene or in octanoic acid and octyloctanoate. Steidle and Dettner23 have shown that the quantitative composition of the defensive secretion is adapted to the natural predators of a given beetle species so that the ratio of toxin to solvent is species-specific. For example B. spectabilis which is attacked by insects and birds has a solvent ratio y-dodecalactone 1 -undecene of 1 :2.8 whereas B. arenarius has a large surplus of octanoic acid and thus is more effective against its carabid predators. Pederin 9 a haemolymph toxin in rove beetles is highly toxic causing lesions of human skin.Its production by beetles of the species Paederus fuscipes and P. riparus has been investigated for the first time by Kellner and Dettner,24 who indicate that it is probably only synthesised by the female. Thus males always contain only small amounts (0.1-0.5 pg) whereas females may have ten times these concentrations. However females fall into two classes those with low and those with high levels. About 85% of females contain large amounts and transfer pederin into every egg they lay; the other females lay most of their eggs without pederin. The pederin content of the larvae is thus determined by the amount deposited in the egg and it is probably only in the larval stage that pederin is essential for survival. 2.3 Pheromones The various pheromones secreted by the exocrine glands of ants and their roles in recruitment recognition and alarm behaviour have been reviewed.25 Several new ant trail pherom- ones have been reported.In particular methyl anthranilate has been identified as a trail pheromone of army ants of the genus Aenictus which live in the jungles of Southern Asia. On its own however this compound is not effective it has to be ‘primed’ with traces of a second component methyl nicotinate. Why army ants should require two separate pheromones while other social insects make do with just one is still unclear.26 2,4-Dimethyl-5-hexanolide10 has been recognised as a trail 9 Me 10 11 12 0 Me\C=CHCH2CH2COMe 4;-Me/ H Me Me H 13 14 n rc! 15 16 17 18 19 20 21 22 OH 23 Harborne Recent advances in chemical ecology pheromone of the carpenter ant Camponotus herculeanus.One pl of a 0.001 pg ml-’ solution was sufficient to elicit a trail-following beha~iour.~’ N,N-Dimethyluracil 11 is the trail pheromone of the panerine ant Megaponera foetens although it is actually secreted in the poison gland. The same insect uses actinidine 12 as an alarm pheromone which is secreted in the pygidial gland.28 Ant alarm pheromones are very useful for responding to the approach of a predator but they can occasionally have disadvantages as well. Thus the alarm signal 6-methyl-5- hepten-Zone 13 is exploited by the zodariid spider Habronestes bradleyii to locate injured or alarmed workers of the meat ant Iridomyrex purp~reus.~~ Research by Pickett and his research group at Rothamsted3’ has shown that female sex pheromones of various aphids are based on (4aS,7S,7aR)-nepetalactone or (1R,4aS,7S,7aR)-nepetalactol or a blend of the two.This group have now confirmed31 that the female of the grain aphid Sitobion avenae relies entirely on the lactone to attract its mate in spite of the fact that the cells in the male antenna are responsive to the lactol as well. A study of behavioural activity using a track olfactometer confirmed the attraction of the males to the lactone and not the lactol. The first identification of an aliphatic amide as a sex pheromone has been achieved in the longhorn beetle Migdolus fry~nus,~~ a pest of sugar cane.The compound is N-(2‘S)- methylbutanoyl 2-methylbutylamine 14 and is released by the female. N-Formyl-L-isoleucine methyl ester was also obtained from the female beetle but it proved not to be as effective as a pheromone. In the red pine cone beetle Conophthorus resin- osae the female releases (@-(+)-pity0115 to attract the Curiously in the related beetle genus Pityophthorus 15 is produced by males as an aggregation pheromone. Males of Conophthorus produce a different type of signal a spiroketal 16 to warn rivals that they must contest the resident male for a female. In the scarab beetle Anomala octiescostata the males pro- duce a 4:l mixture of buibailactone and japonilure to attract female beetles for mating. When the opportunity arises they also voraciously consume flowers of the dandelion being attracted by the volatiles which contain phenylacetaldehyde phenylacetonitrile and benzyl benzoate.They then use these plant volatiles to enhance the attractiveness of their male sex pher~rnones.~~ While beetle pheromones are generally synthesised de novo by the beetle it has often been assumed in the case of the bark beetle aggregation pheromones that they are produced from dietarily derived myrcene 17. This is because of the striking structural similarity between compounds such as (4S)-(-)-ipsenol 18 and (4S)-(+)-ipsdienol 19 and the acyclic mono- terpene myrcene. In vivo radiolabelling experiments in both Ips paraconfusus and Ips pini have now shown that 18 19 and amitinol 20 can all be produced de novo in these beetles from [l-’4C]acetate.35 This of course does not prove that dietary myrcene may not also be a substrate for pheromone produc- tion or that microbial symbionts might not also be involved in oxidations along the biosynthetic pathway.Further work is still needed to establish the varying biosynthetic sources of these aggregation pheromones in the many different species of bark beetle. There was never any suggestion in the case of lepidopteran pheromones that these simple molecules are not insect-derived. There is however always the possibility that sex pheromonal signals in Lepidoptera are affected by host plant volatiles. This appears to be true in the case of both male and female cabbage looper moth Trichoplusia ni.In this moth the male releases (9-( +)-linalool p-cresol and m-cresol while the female pro-duces (Z)-7-dodecenyl acetate. Landolt et al. have shown that sexual attraction in both directions is increased by the presence of the host plant (Gossypium hirsuturn) ~dour.~~ Host plant greenleaf odour [e.g. including (2)-hex-3- enylacetate] may also synergise on occasion with female sex moth pheromones. This happens in the case of male corn earworms (Helicoverpa zea) and male codling moths (Cydia pornonella). This is because alighting females only release their sex pheromones when perched on leaves of the host plant such as maize. This result could have immediate application in insect pest strategies since the cost of green leaf volatiles is only a small fraction of that of a synthetic pheromone.37 A new class of lepidopteran female-produced pheromone has been discovered in the banded apple pigmy leaf miner Stigmella malella.Two short-chain unsaturated methylcar-binols 21 and 22 have been identified in this insect. For monitoring traps a 10:3 E:Z chiral blend at 100-1OOOpg is re~ommended.~~ The seven-carbon alcohol (2R)-(.Z,)-hept-4-en-2-01 23 has been identified as a female sex pheromone in the evolutionary archaic lepidopteran species Eriocrania ~icatricella.~~ The most striking recent discovery in the field of lepidop- teran sex pheromones has nothing to do with moths but refers to female Asian elephants. (Z)-Dodec-7-en-l-yl acetate is used by females of more than 126 species of insects as part of their pheromonal blend.Precisely the same molecule has been characterised in female Asian elephants Elephas maximus to signal to the males their readiness to mate. The natural elephant odour proved to be a 97:3 mixture of Z:E isomers.40 Bioassay with the synthetic pheromone (98:2 Z:E mixture) confirmed that bull elephants respond positively to this female pheromone. This remains a remarkable example of conver- gent evolution based on the useful properties including its volatility of this particular chemical signal. One final example of an interesting chemical signal in nature is worth mentioning before concluding this section. This is the highly noxious volatile dimethyl sulfide which now appears to be an important foraging cue to Antarctic seabirds.Dimethyl sulfide appears to be produced by phytoplankton in response to grazing by zooplankton. The release of the vapour then guides seabirds such as stormy petrels to feed on the zooplankton including Antarctic krill. The dimethyl sulfide is a byproduct of the metabolic decomposition of dimethylsulfoniopropionate which occurs in the marine phytoplankton Phaeocystis pou- ~hetii.~' Dimethyl sulfide is also a signalling molecule in polli- nation biology and will be referred to again in a later section. 3 Plant-animal interactions 3.1 Constitutive chemical defence 3.1.1 Nickel hyperaccumulation The plant Thlaspi montanum growing on serpentine soils sequesters and hyperaccumulates nickel from those soils into its tissues.Boyd and Martens42 have shown that such hyper- accumulation is defensive against herbivores. Leaves differing 167-fold in Ni content (3000 vs. 18 ppm) were obtained from plants growing on high- and low-Ni soils. Larvae of the small white butterfly Pieris rapae fed high-Ni leaves did not grow and suffered 100% mortality after 12 days whereas those fed on low-Ni leaves increased in weight and mortality was only 21%. Similarly larvae fed on an artificial diet with added Ni showed acute toxicity at 1000 ppm. Other insect larvae were similarly affected when fed high-Ni leaves including the moth Euchloe hyantis and several grasshopper^.^^ Interestingly hyperaccumulation of nickel also protects a second serpentine plant Streptanthus polygaloides from patho- gen infection.44 The toxicity of nickel to insect herbivores and plant pathogens must depend to some extent on the way that the nickel is sequestered within the plant.Recent research suggests that it is simply coordinated with the amino acid histidine since supplying histidine to a non-accumulating plant greatly increases its nickel tolerance and capacity for nickel transport to the shoot.45 There are over 400 species of metal-hyperaccumulating plants currently known so that it is likely that many of these copy Thlaspi montanum and benefit from the ability to hyperaccumulate a metal by being relatively free from herbivory. 86 Natural Product Reports 3.1.2 Terpenoids According to Hems and Matt~on,~~ perennial plants fall into two categories those with rapid growth poor chemical defence but a highly inducible system; and those with slow growth rate well defended with high levels of toxin and a poorly developed inducible resistance.Baldwin et al.47 have studied the well known insecticidal plant Chrysanthemum cinerariaefolium and shown that it belongs to the second category of plant. Thus it is rich in monoterpenoid-based pyrethrins stored in glandular trichomes in leaves and in secretory canals and oil glands in flowers. Expectedly it failed completely to respond to simu- lated herbivory and no increases in pyrethrin levels could be detected up to 13 days after mechanical damage. Another Compositae plant rich in terpenes is the rubber rabbit bush Chrysothamnus nauseasus with (a-p-farnesene P-humulene a-and y-muurolene.It is protected from her- bivory in the summer months by high levels (80 pg g -' dry wt) of terpenoids. These levels drop dramatically in the winter to 18 pg g -dry wt and the plant is then browsed by mule deer Odocoileus hemion~s.~~ Seasonal variation in terpene levels presumably provides enough protection from herbivory for the plant to survive in coexistence with this grazing animal. Since arboreal marsupials consume terpenes in quantities that are toxic to other mammals they must possess special detoxification mechanisms to deal with potential toxicity. Therefore McLean et al.49 studied the dietary fate of Eucalyp-tus terpenes in the common ringtail possum Pseudocheirus peregrinus over ten days.Six urinary terpenes detected turned out to be dicarboxylic acids hydroxy acids or lactones. Thus this possum appears to detoxify by polyoxygenating the terpenes to highly polar acidic metabolites that are readily excreted. Conjugation with glucuronic acid is notably minimal presumably in order to conserve the carbohydrate resources of the animal. One unresolved question about terpenoids is how far they can be moved around the plant during growth and differentia- tion. This clearly relates to their effectiveness as herbivore toxins. One class of terpenoid potentially capable of transport within the phloem are the glucosidic iridoids. Indeed exper- iments with antirrhinoside 24 in Asarina scandens have con- firmed phloem transport for this iridoid.Thus it was rapidly labelled when mature leaves were exposed to I4CO2 and it was then transported into the phloem along with sucrose appear- ing in the petiole within 20 min of the start of the experiment. Antirrhinoside was also isolated from the phloem by the EDTA method.50 2,3-Dihydrofarnesolic acid 25 is a unique terpene from the trichomes of the wild tomato species Lycopersicon hirsutum. It has now been shown to provide resistance to arthropod herbivores. When sprayed onto leaves of the domestic tomato it repelled feeding by a tomato pest the two-spotted spider mite Tetranychus ~rticae.~' Several studies have shown that sesquiterpene lactones characteristically present in plants of the Compositae are antifeedant and protective against herbivory.Chou and M~llin~~ have explored the interaction between the lactones of the sunflower Helianthus annuus and the western corn root- worm Diabrotica virgifera in order to understand further their defensive roles. What is interesting here is that the sunflower has seven antifeedant lactones and the concentrations of these lactones change with ontogeny. In particular a non-polar lactone such as 15-hydroxy-3-dehydrodesoxytifruticin,pre-dominant in three-week old plants is replaced by polar compounds e.g. argophyllin A 26 through later stages and at anthesis. Antifeedant activity correlated with these changes indicates that the plant is better protected in newly grown leaf and in floral tissues. The composite Encelia farinosa a plant native to North America exists as a variety of chemotypes.Some have high levels of the sesquiterpene lactone farinosin 27 and others with mainly the chromene encecalin 28. Both major toxins are detrimental to neonate larvae of the armyworm Spodoptera 3.1.4 Phenolics HO * 24 25 26 27 28 29 R=OGlc 30 R=OH H littoralis. The plant varies its defence in natural populations and hence is an obstacle to host plant recognition as well as to adaptation by herbivore^.^^ 3.1.3 Alkaloids The defensive role of pyrrolizidine alkaloids (PAS) against herbivory in Senecio jacobaea has been explored.54 Eight clones varying in their alkaloid levels were available for testing. Herbivory of the specialist flea beetle Longitarsus jacobaeae was lower in genotypes with high PA levels.Herbivory by both adults and larvae were negatively correlated with PA concen- tration. However differential attack of Senecio clones with high and low PA levels did not result in fitness and survival differences. The authors conclude that the defensive role of PAS is only weakly supported by these results. Alkaloid defence may only be important at certain stages of the life cycle of the plant. For example the young seedling may be most vulnerable and hence might accumulate high levels of protective toxin. van Dam et al.” have shown this is true for plants of Cynoglossurn oficinale. Young rosette leaves were found to contain 190 times more PA than older leaves and the absolute amount decreased with time.The defensive role of alkaloids against mammalian her- bivory is very well documented and one further example has come to light. The Australian plant the weir vine (an Ipornoea species) causes a neurological disorder when consumed by livestock. Chemical analysis showed the presence of glycosi- dase inhibitors swainsonine and calystegine B,. Histological examination of brain tissue of sheep and cattle poisoned by weir vine showed lesions similar to those observed in animals poisoned by other swainsonine-containing plants. This is the first example of a plant producing structurally distinct glycosidase inhibitors i.e. alkaloids of the indolizidine and nortropane class. 56 Harborne Recent advances in chemical ecology The simple phenols present in birch and willow are defensive to hare grazing.They are dealt with by these herbivores by detoxification and excretion of the phenols as glucuronides. The metabolic cost of phenolic detoxification has been com- pared in the mountain hare Lepus tirnidus and the European hare Lepus europaeus. The first animal is a browser on birch and willow whereas the second is a grass grazer. Excretion of glucuronide was found to be positively correlated with phenolic intake. However the European hare excreted sub- stantially more whereas the mountain hare had a greater detoxification capacity. Hence the European hare has a higher cost of detoxification and may therefore not be able to exploit forest habitats in the same way that the mountain hare The phenolic glucoside platyphylloside 29 is a defensive agent of Betula platyphylla and inhibits feeding on the leaves by hares rabbits and goats.The metabolic pathway in uivo has been studied in sheep rumen liquor. The pathway is reductive. Platyphylloside 29 is converted to 5-hydroxyplatyphyllone 30 then to 3-platyphyllone and then to the more completely reduced centrolobol 31. The reduction in digestion caused by intake of 29 is caused by the build up of centrolobol in the gut.” The birch tree Betulapubescens does not respond to simu- lated herbivory by increasing its phenolic content. Further- more feeding experiments with the vole Microtus aquestis revealed no differences in palatability between treated and untreated leaves.59 A novel phenylpropanoid 1-caffeoyl-4-deoxyquinic acid 32 has been identified in leaves of the wild groundnut Arachis paraguaensis.It causes developmental inhibition of growth in the tobacco armyworm Spodoptera litura. Together with related caffeylquinic esters and flavonol glycosides it has been shown to provide significant resistance in this plant to insect attack.60 A similar study of the effect of lichen phenolics on Spodoptera littoralis showed delays in larval growth and a high incidence of developmental malformation.6’ 3.1.5 Tannins Methods of preserving plant leaves before condensed tannin and phenolic glycoside analysis were compared using three willow taxa. Highest concentrations were measured in vacuum-dried fresh leaves.Dried leaves gave satisfactory results for phenolic glycosides but concentrations of tannins in dried leaves were down by 5-10%. Freeze-drying was not recommended for phenolic glycosides since salicin is produced as an artifact in this procedure.62 It has sometimes been assumed that all tannins act as feeding deterrent allelochemicals in a uniform manner. Mole et al.63 have now punctured this hypothesis. Feeding trials with rats showed that differences were apparent in the way that tannins affected digestion and the post-digestive assimilation of nutrients into the body. In particular while condensed tannins pass through the rat bound as PR-protein complexes hydrolysable tannins appear to undergo hydrolysis during digestion. A related experiment in which radiolabelled sorghum con- densed tannin was fed to chickens indicated that the tannins were not absorbed from the digestive tract.However the low molecular weight phenols of sorghum were absorbed and it appears that these compounds and not the tannins are responsible for toxic effects seen in chickens fed on sorghum tannin diets.64 Chronic tannic acid intake at 4% in the diet by the prairie vole Microtus ochrogaster has no direct effect on reproductive performance. Females lose weight during lactation compared to controls and this is probably due to reduced digestive efficiency caused by the tannin.65 McArthur et al. have proposed that salivary proline-rich proteins in mammals originally functioned to maintain oral homeostasis preventing excessive precipitation of hydroxy- apatite on teeth surfaces.Their ability to bind dietary tannin would then be a derived function. This hypothesis is supported by the varying levels of PR-protein complexes that were measured in a range of marsupials including kangaroos possums and the koala bear. The potential toxicity to animals of hydrolysable tannins is illustrated by the small deciduous tree yellow wood or Termi-nalia oblongata. Cattle and sheep that consume the leaves are poisoned and die. The active principles are two tannins puni- calagin and terminalin 33 which produce lesions in the liver and kidney re~pectively.~~ Terminalin has a high toxicity to mice at concentrations of 20mg kg- '. Incidentally the authors of this paper describe terminalin as a condensed tannin whereas it is clearly an ellagitannin.0 SGlc I 0 R 33 34 R=H 35 R=OMe 36 3.1.6 Bird toxins Large birds like geese consume quantities of plant material when left to graze in meadows but little is known of their feeding preferences. A study by Wink et UZ.~~ on plant palat- ability in young and adult geese belonging to three species is therefore welcome. Older birds were found to be more selective than younger ones. Detoxification systems were well devel- oped and this included geophagy in the case of alkaloid- containing plants. The only major barrier to feeding appeared to be the presence of essential oils. By contrast plants contain- ing alkaloids amines and sulfur compounds were tolerated to a remarkable degree.Birds are also frugivores and play an important role in seed dispersal by consuming fruits and carrying the seeds away from the parent plant. It is generally assumed that while the seeds may contain toxins to prevent them being eaten the fruit will be relatively free of toxic constituents. Nevertheless it is possible that some fruit species may have evolved mild toxins to prevent too many fruits from being eaten per foraging bout and hence ensuring wider seed dispersal. Barnea et al.69 have now shown that this is true for fruits of hawthorn ivy holly and yew which are fed upon by blackbirds redwings and starlings. These frugivorous birds stayed on all plant species 88 Natural Product Reports for only 1.3-5.3 min and consumed only a few fruits in each feeding bout.Phytochemical analysis revealed allelochemicals specifically in the fruits rather than the seeds. Chemical protection was provided by cyanogenic glycosides saponins and flavonoids which were variously detected in these fruits. 3.2 Induced chemical defence Hunter and Schultz7* present the first evidence that phyto- chemical induction can inhibit a pathogen of the herbivore responsible for defoliation. This refers to experiments with red oak trees which increase their concentrations of gallotannins in the leaf after attack by gypsy moth larvae. When a batch of larvae inoculated with nuclear polyhedrosis virus are put onto the foliage they are less susceptible to viral infection than the controls.This may be simply because the increased gallotannin in the larvae inhibited the virus. The authors conclude that induction of gallotannin in red oak is a generalised response to tissue damage rather than an adaptive response in the tree to herbivore attack. One of the best examples of the induction of secondary metabolite synthesis by mechanical or insect damage is the increase in furanocoumarins observed in leaves of wild parsnip Pastinaca ~ativa.~' This system has been explored in more detail and it has been shown that induction of xantho-toxin one of the principal furanocoumarins is localised to the immediate vicinity of the damage.72 Furthermore increases were still observable in damaged leaflets following their detachment from the parent plant.In attached leaves the inductive response reached a maximum within 24 h and con- centrations then declined to preinduction levels after seven days. Finally stressing the plant by nutrient limitation or light limitation had little effect on the induction response. These results with furanocoumarins in an umbellifer contrast sig- nificantly with earlier data obtained for nicotine alkaloid induction in Nicotiana ~ylvestris.~~ It now appears that jasmonic acid or its methyl ester are effective messenger molecules in several systems for setting off the induction process. Thus jasmonic acid induced increases in nicotine synthesis in Nicotiana sylvestris leaves.74 Jasmonic acid or the ester at nanomolar concentrations induced up to 20-fold increases of indole glucosinolates in cotyledons and leaves of Brassica napus (3-indolylmethyl),B.rapa (4-hydroxy-3-indolylmethyl) and B. juncea (both). Other glucosinolates in these plants were unaffected. The increases paralleled those caused by mechanical damage or by insect feeding. Many other signalling molecules were tested but all failed to give any response.75 Doughty et a1.76 obtained similar results by spraying the ester onto leaves of B. napus except that induction produced both 3-indolylmethyl-34 and 1-methoxy-3-indolylmethyl-glucosinolates 35. The same substituted salicylic acid for jasmonate and obtained the selective induction of 2-phenylethylglucosinolate,instead of indole derivatives. Yet another signalling molecule iodoacetic acid has been applied by Burden and Norris7* to induced defence in Glycine max.The major compound induced after 2-4 days in this plant is the isoflavone daidzein which has weak antifeedant activity. The classical system for studying the production of protein- ase inhibitors after mechanical or insect-feeding damage is the tomato plant Lycopersicon esculentum. Stout et al.79 have observed that proteinase inhibitors (PIS) are accompanied by increases in phenolase peroxidase and lipoxygenase activities. The tomato plants however respond differently according to which insect is feeding. Thus caterpillar feeding induced pro- duction of phenolase and lipoxygenase as well of PIS. Leaf miners by contrast only produce an increase in peroxidase activity.Increasing phenolase activity might make the leaves unpalatable because increases in oxidised phenolics could be toxic to the insect. Appelso has drawn attention to the impor- tance of enzymic oxidations in activating the toxicity of many plant phenolics. Changes in protein quality have also been observed in other plants demonstrating induced resistance to herbivory. Fourth instar larvae of Helicoverpa zea suffered 39% reduction in growth rate after 24 h and 27% reduction after 48 h feeding on wounded foliage compared with larvae on undamaged leaves. At the same time there were 53% increases in lipoxygenase activity and 34% increases in trypsin inhibitors.” Increases in oxidases were also observed in response to feeding on soya- bean leaves by a bean leaf beetle and the three cornered alfalfa hopper.82 In the tomato insect feeding induces two main proteinase inhibitors with different molecular weights.In tobacco Nicotiana tabacum six proteinase inhibitors have recently been induced by wounding. Sequencing indicates that they are closely similar to peptides reported in the stigmas of Nico tiana ala ta .83 In response to herbivory some plants have developed the means to release volatile chemicals which are particularly attractive to parasitoids which then visit the plant and destroy the herbivore. Some details of the supporting experiments were given in the last rep~rt.~ Since then several further exper- iments have been described. Brussel sprouts fed upon by Pieris brassicae larvae release volatiles that are attractive to the parasitoid Cotesia glomerata.Mechanically damaged leaves are expectedly less attractive than herbivore damaged leaves.84 Sprout plants respond to any damage by increasing volatiles such as sabinene and limonene. The key to parasitoid attrac- tion seems to be green leaf odour volatiles since they make up only 2% of the total volatiles in mechanically damaged leaves but make up 13% in caterpillar damaged leaves. In cotton seedlings twenty-two volatiles are emitted by freshly damaged plants and nine further compounds can be detected several hours after damage. These data suggest that there is a constitutive defence of odour compounds augmented by an induced mechanism mobilised in response to attack by the corn earworm Helicoverpa ~ea.~~ Twenty-three volatiles were elicited by armyworm feeding on cotton.No pronounced differences were noted from the volatiles of leaves of undam- aged leaves. However volatile emission from damaged leaves of a naturalised variety was seven-fold higher than that of damaged leaves of commercial cultivars. In spite of this army- worm larvae preferred feeding on leaves of the commercial cul tivars. 86 Other sources of volatile emission when insect larvae are feeding on plants are the insect faeces and such odour com- pounds might also be attractive to parasitoids. Agelopoulos et a1.87 have considered this possibility in the cabbage -Pieris rapae -Cotesia rubescula tritrophic interaction and concluded that plant damage is the most important source of ‘infochemi- cals’.Nevertheless 85 different chemicals could be identified as emanating from the faeces and their release from the faeces was enhanced by moisture. One final question has been raised by these tritrophic interactions involving volatiles. Do plants tap SOS signals from their infested neighbours? Present evi- dence for and against this form of interplant communication is reviewed by Bruin et a1..88 3.3 Hormonal interactions A bizarre form of defence based on ecdysteroids has been recognised in the marine arthropod Pycnogonum litorale. It discharges a mixture of eight moulting hormones at a concen- tration of 1.0 x 10-mol 1-at any enemy. The major constituent is 20-hydroxyecdysone 22-acetate.The main pred- ator is the common crab Carcinus maenas and the discharge effectively puts the crab off its In the plant kingdom the first pyrrole-containing ecdysone has been described. It is pinnatasterone 24-0-(pyrrole-2-carboxylate) 36 and it occurs in the bark of Vitex canescens along with 20-hydroxyecdysone and turkesterone.” It exhibits high hormone activity in the housefly bioassay. The plant Leuzea carthamoides is like Vitex a rich source of ecdy- steroids. Three new structures closely related to known phytoecdysones have been isolated from its roots.91 Harborne Recent advances in chemical ecology 3.4 Insect antifeedants A review has appeared on the classic insect antifeedant a~adirachtin.~~ Azedarachin C 37 a limonoid from Melia azedarach root bark inhibits feeding of Spodoptera exigua at 400 ~pm.~~ The limonoid hirtin 38 from Trichilia hirta inhibited larval growth of Peridroma sansia in a dose-dependent manner with an EC, of 13 ppm.It displayed behavioural effects on this lepidopteran as well as post-ingestive A series of limonoids including limonin were tested for antifeedant activity against the armyworm Spodoptera litura but all proved to be less active than the azadirachtins which were included in the tests as ’OH H 37 38 OBz AcO /OBz AcO.. 0--Me Me AcO Me 39 40 41 42 43 44 45 A sesquiterpenoid 2-norbornanol derivative 39 isolated from fruits of Dysoxylum spectabile proved to be an active ant repellent at a concentration of 0.l%.96 Trans-a-neerodol 40 previously reported in the defensive secretion of the rectal gland of the silphid beetle Necrodes surinamensis has now been found for the first time in a plant in the essential oil of Lavandula luisieri.It would appear to be an excellent insect repellent.97 A novel sesquiterpene of the eudesmane series mutangin 41 from Elaeodendron buchananii reduced feeding by 55% in the lepidopteran Chilopartellus when applied at a concentration of 50 pg per leaf The isobenzofuranone sedanenolide 42 an aroma component of Apium graveolens provides host plant resistance to both Spodoptera exigua and Trichoplusia ni.99 2,2,4,4-Tetramethyl -N,N-bis(2,6 -dimet hylpheny1)cyclo -butane- 1,3-diimine 43 newly reported from Arundo donax shows significant antifeedant activity against the boll weevil Anthonomus grandis the most destructive pest on cotton plants.Triacontanol and tricin also present in Arundo donax were also antifeedants.Im Termite antifeedant activity has been detected in Afromum melegneta Detarium microcarpum and Xylopia aethiopia. The well known flavour compounds 6-ginger01 and 6-shogaol occurring in A. melegneta provided the strongest activity at 1000 ppm equivalent to 8 pg cm -2 against worker termites of Reticulitermes operatus. The active antifeedants in the other two plants turned out to be known clerodane or ent-kaurane diterpenoids. lo' Cadina-4,1O( 15)-dien-3-one 44,from leaves of Hyptis verti- cillata proved to be an effective insecticide to the weevil Cylas formicarius a destructive pest on the sweet potato crop.At a concentration of 3.6 mg g-' of insect over 98 h it produced 90% mortality. '02 The fungal sesquiterpenoid lactarufin A 45 from the hot-tasting Lactarius mushroom was shown to be a particularly potent antifeedant against the storage pest Tribo-lium confusum. In a series of tests substances with lactarane (as in 45) and marasmane skeletons proved more active than isolactaranes; at the same time increasing hydroxylation of the sesquiterpene skeletons decreased antifeedance. '03 While antifeedants from one plant might be used empirically to reduce insect feeding on another the question may be asked as to whether secondary compounds present in an insect's host plant might not occasionally have antifeedant activity.Renwick and Huang'04 have evidence that a host plant may be rejected by larvae of the butterfly Pieris rapae as a conse- quence of diet-dependent sensitivity to the common plant phenolic chlorogenic acid. The garden nasturtium Tropaeolum majus is an acceptable host plant to P. rapae and the insect will go through its normal life cycle on the plant. However if second to fifth instar larvae are transferred from cabbage to nasturtium they refuse to eat and starve to death. This is because larvae reared on nasturtium have only limited sensi- tivity to chlorogenic acid and are able to avoid its antifeedant effects. Yet larvae reared on cabbage develop a sensitivity to this phenolic acid and hence are unable to transfer successfully onto nasturtium.Thus dietary experience can dramatically affect the response of an insect to a potential antifeedant. The brown plant hopper Nilaparvata lugens is a serious pest on rice and it is important to determine why some rice varieties are resistant to attack while others are susceptible. A search for a sucking deterrent in the phloem sap where they feed has revealed high levels of three glycosylflavones based on schaftoside in resistant cultivars with ten-fold lower amounts in susceptible forms. Curiously enough the same compounds are also probing stimulants so that it is only their high concentration which gives them antifeedant effects.''' Similar studies have been carried out on antifeedants to plant hoppers in the millet plant.'06 One final note on antifeedants this time to birds refers to the well established antifeedant properties of hydroxycinnamic acids in flower buds of fruit trees.The synthetic model compound cinnamamide has been successfully tested as an antifeedant to a variety of common birds and has considerable potential for protecting trees in bud from bird herbi~0ry.l'~ 3.5 Oviposition stimulants and deterrents Much interest continues to be generated by those chemicals in plant tissues which determine the choice of plant for oviposi- tion by the female. After ovipositioning females of some herbivorous insect species are known to deposit a marking substance on or near the eggs to discourage visits by other conspecifics.Such marking substances or oviposition-deterring pheromones have rarely been characterised. It is therefore of great interest to chemical ecologists that three such host markers have been characterised by extracting 150 000 eggs of the large white butterfly Pieris brassicae. The compounds have novel structures; they are the cinnamic acid substituted hydroxybenzoic acids miriamide 46,miriamide 5-glucoside 47 and 5-dehydroxymiriamide 48. These substances have been synthesised are relatively stable in the field and clearly have a possible role in protecting cabbage crops from colonisation by cabbage white butterflies. lo* 90 Natural Product Reports W C0NybOH HO 46 R =OH 47 R = OGk 48 R=H CHzOCO(CH&(CH= CH-CH2)3Me Me(CHz- CH= CH)3-(CH2)7CO0 CH CHzOGal 49 Flavonoids are well known to be oviposition stimulants to several swallowtail butterflies.Nishida'" reports another example in the case of the zeryntiine swallowtail Luehdor-a japonica which feeds on Heterotropa aspira. The major stimu- lant in the plant leaves is isorhamnetin 3-(glucosyl-~l+6-galactoside)-7-glucoside but it is only active when mixed with unidentified water-soluble leaf components. The danaid butterfly Danaus plexippus also depends on flavonol glycosides for oviposition stimulation to lay on Asclepias curassavica leaves. A mixture of four quercetin glycosides-two 3-dirham-nosylglycosides the 3-rutinoside and the 3-rhamnosyl-galactoside-have been identified.' '' Notably in this case the flavonoids are active on their own without admixture with other water soluble compounds in acting as contact stimulants.The first flavonoid oviposition deterrent a quercetin trigly- coside from Orixa japonica which deterred Papilio xuthus was mentioned in the last rep~rt.~ A second flavonoid repellent has now been described from leaves of Phellodendron amurense (Rutaceae). It is phellamurin (8-prenyldihydrokaempferol-7-glucoside) which occurs in significant amount (1.8% fresh wt). It deters females of two Papilio butterflies P. protenor and P. xuthus. Curiously in spite of this deterrence larvae of both species perform well if artificially fed on the leaves. Honda and Hayashi' " suggest that this plant has co-evolved high concen- trations of the dihydroflavonol to deter herbivory and that lower concentrations would make the plant acceptable for oviposition.The first report of a diacylglycerol galactoside 49 as an oviposition stimulant has appeared in the literature. It encour- ages oviposition by the piperine swallowtail Battus philenor on the leaves of Aristolochia macrophylla. The active constituents include D-( +)-pinitol sequoyitol and aristolochic acids I and 11 which form a synergistic mixture with 49.'12 Two phenanthroindolizidine alkaloids (+)-isotylocrebine and ( -)-7-demethyltylopharine are oviposition stimulants in the host plant Tylophora tanakae (Asclepiadaceae) to the danaid butterfly Ideopsis similis.' l3 The phenolic 2'-0-acetylsalicortin with other phenolic glycosides is the main attractant in shoots of Salix pentandra to females of the shoot galling sawfly Enura amarinae.'l4 Tropaeolin one of two major glucosinolates in the garlic mustard plant Alliaria petiolata is an oviposition stimulant to females of the cabbage butterfly Pieris rapae. By contrast sinigrin the other major glucosinolate stimulates egg laying by another cabbage butterfly Pieris napi oleracea. In this second case however the stimulation is a mistake because the plant A. petiolata is not suitable for larval survival."5 The positive cues of glucosinolates in crucifer plants for oviposition by Pieris butterflies can be counteracted by the co-occurrence in the same leaves of cardenolides. This happens in the plant Erysinum cheiranthoides and if larvae of Pieris rapae are raised on this plant they simply refuse to eat.The active antifeedants are the cardenolides digitoxigenin 3-glucoside glucodigigulo- methyloside glucodigifucoside and cheirotoxin. 'l6 While the crucial role of non-volatile chemicals at the leaf surface is well established for guiding oviposition less is known of the leaf volatiles that might determine host plant selection. Baur and Feenyl l7 therefore examined the sensitivi- ties of females of three Papilio species to odour compounds of host plants (wild carrot) and a non-host plant (Artemisia dracunculus). The results indicated a high sensitivity to com- pounds widely present in leaf odours such as linalool 4-terpineol and green leaf volatiles but a weak response to sabinene a carrot leaf constituent.The butterflies appeared to be highly conservative in their responses to odours generally. In similar experiments with the Monarch butterfly Danaus plexippus Bergstrom et al. 'I8 found that ovipositing females clearly distinguish by odour between young and old leaves of a host plant Asclepias curassavica always laying on the young leaves. But head space analysis failed to reveal any one volatile which distinguishes the young from the old. There are how- ever quantitative differences. Trans-a-farnesene and linalool accumulate in the volatiles of young leaves and there is the suggestion of a 'dual effect' in which together they function as a young-leaf signal. Additionally there are some odours which accumulate in older leaves (a-thujene methyl salicylate) so that these compounds might function as mild deterrents steering the ovipositing female towards the younger leaves.The question of young vs. old leaves has been raised in the case of oviposition by the buckeye butterfly Junonia ceonia which lays only on young leaves of Plantago lanceolata. The non-volatile iridoids present appear to be the main guide since young leaves have a high content of catalpol whereas old leaves have lesser amounts of mainly aucubin.' l9 One other class of chemical has recently been implicated in affecting oviposition that of the sugar esters. Such acyl esters have been identified in the leaf of a variety of Solanaceous plants and they appear to have a defensive role against insects.Lied1 et al. have reported that in the wild tomato Lycoper-sicon pennellii the acyl esters deter oviposition by the homop- teran Bemisia argentifolii while Puterka and Severson describe similar effects for the esters of Nicotiana gossei against pear psylla. 12' 3.6 Pollination interactions The evolution of plant volatile production in pollination ecology and in insect-plant relationships has been reviewed by Harrewijn et An automated system for collecting volatiles released from plants has been de~cribed.',~ It was employed to demonstrate that MirabiZis jalaba when in flower releases ocimene mainly at night between 18.00 and 20.00 hours. The influence of climatic factors on emission of floral volatiles in situ from Trifolium repens has been examined.Phenethyl acetate is the dominant compound and this is emitted in a rhythmic manner. Increasing light and tempera- ture increased volatile emi~si0n.l~~ Head space and GC-MS analysis of the volatiles of Brassica napus showed that six compounds come from the flowers 12 from the flower and leaf and 11 from the leaf alone. The average emission of volatiles is 45 ng per flower per hour. Rhythmic emission was noted for sabinene and limonene released from both leaf and flower but no rhythm was detected in the emission of a-farnesene linalool and 3-carene.125 The floral scent of eight bat-pollinated plant species has been found to be based primarily on sulfur compounds. Besides dimethyl sulfide dimethyl disulfide and dimethyl tri- sulfide irregular sulfides are present such as 2,4-dithiapentane and 2,3,5-trithiahexane.Three species contained mushroom- smelling fatty acid derivatives with a C skeleton. The presence of closely similar sulfur compounds in seven out of the eight species which all belong to different families suggest that this is a case of convergent evolution in scent composition.126 A further bat-pollinated species Dactylanthus taylorii a member of the Balanophoraceae has unexpectedly yielded squalene as Hurborne Recent advances in chemical ecology a major floral constituent. Also present are C, to C31 hydro-carbons fatty acid esters and typical fragrance chemicals such as nerol and geraniol. Although the plant is bat pollinated the nectar is attractive to possums and ship rats who browse on the flowers without pollinating them.As a result this species is threatened with extinction.'27 Two of the sulfur compounds reported in bat-pollinated flowers have been found in the odour of the voodoo lily flower Sauromatum guttatum dimethyldisulfide and dimethyltri-sulfide.128 The same two compounds are incidentally respon- sible for the foul odour released by the mushroom Phallus impudicus.'28 A reinvestigation of the well known aroid Arum maculatum failed to reveal any of the simple amines that were earlier recorded in the volatiles. Instead the analysis showed the presence of heptan-2-one (8-23% of total volatiles) indole (8-23%) germacrene B (10-14%) and p-cresol (O.2-6%).The major pollinators were confirmed as females of the dung fly Psychoda phalaenoides and a comparison of floral odours with those produced by cow dung identified p-cresol as the main common ~omponent.'~~ The faecal-smelling indole of Arum maculatum is also pro- duced surprisingly in the blossoms of Cucurbita (Cucurbita-ceae) where the pollinators are diabroticite beetles. Indole acts synergistically with 1,2,4-trimethoxybenzene and (E)-cinnamaldehyde to attract the beetles to the flowers for polli- nation. Beetle species vary in their responses to indole and it appears that the indole receptor in the beetles has been con- served over 30 million years during diabroticite evolution. '30 In the hawkmoth-pollinated Brugmansia x candida (syn. Datura candida) it was suggested that tropane alkaloids were present in the volatiles which intoxicated the moths during pollinating visits.A reinvestigation by Kite and Leon,13' however failed to uncover any alkaloids in the floral odours. The dominant components are trans-ocimene (3842%) and 1,8-cineole (5-19% of total). The leaf odour also lacked any alkaloid but contained perillene (1425%) and dendrolosin. Three forms of the orchid Ophrys insectifera are pollinated by male bees who carry out pseudo-copulation with the flowers which resemble the female bees in morphology colour and scent. Different species of male bees were involved suggesting that there might be differences in floral odours. Indeed analysis showed both quantitative differences in aliphatic hydrocarbons methyl esters and aliphatic alcohols and qualitative differences in the nine terpenoids that were present.132 In moth-pollinated orchids of the genus Platan-thera similar differences were observed in the scent volatiles in individuals and in different populations.' 33 Again in the bee-pollinated plant Pyrola grandzyora populational variation in scent production was apparent with either benzaldehyde or methoxybenzenes dominating. 134 Analysis of the floral fragrance of the early flowering shrub Daphne mezereun revealed a remarkably simple pat- tern with (S)-( +)-linalool as 95% of the total. Linalool oxide isomers made up the remainder of the By contrast the floral odours of Jacquinia macrocarpa in the Theophrastaceae have a range of sesquiterpenes aliphatic alcohols and esters pyrazines and benzenoids.Additionally there are several cyclohexane derivatives (e.g. P-cyclocitral) which could be formed by degradation of the carotenoids present in the corolla. 136 The visually-distinctive umbell-shaped flowers of the Umbelliferae are unusual in attracting a great assemblage of diverse insect species to feed on and pollinate the flowers. This would suggest the absence of species-specific cues in the volatile fraction but that the odours would attract groups of different insects. Chemical analysis has borne this out in that there are many components present. Monoterpene hydrocarbons dominate in several species but nitrogenous compounds aliphatic short-chain esters and linalool are also present.'37 138 In visiting flowers to collect their food bees may come into contact with toxic constituents.Alkaloids for example are known to be present in certain nectars. Detzel and Wink'39 therefore tested 63 allelochemicals on the feeding behaviour of Apis mellifera and found that they were not especially adapted to plant defence chemicals. Some 17 of the chemicals proved to be fatal to the bees at concentrations between 0.003 and 0.6%. Bees are thus at risk from visiting Atropa belladonna flowers where the tropane alkaloid levels in the nectar reach 273 yg g -' fresh wt. By contrast they are safer on tobacco flowers where the nicotine levels are only 0.17 pg g -' fresh wt. 4 Plant-plant interactions 4.1 Allelopathy The proceedings of a conference on allelopathy held in Ames Iowa has been p~blished.'~' One question that has often been raised regarding the allelopathic effects of plant monoterpenes is their presumed low solubilities in water and hence their inability to interact in the soil with seeds of other plant species.Weidenhamer et al. 14' have now found that this low solubility is a myth and that oxygenated monoterpenes in particular have solubilities sufficient for them to produce phytotoxic effects. Thus monoterpene ketones (e.g. menthone) are soluble in the range 155-6990 ppm while alcohols (e.g. geraniol) are soluble in the range 183-1360 ppm. When tested on seed germination these compounds show phytotoxic damage at concentrations under 100 ppm.A second question often asked about the monoterpenoids is the length of their survival in leaf litter. Wood et a1.142 have measured monoterpene quantities in leaf litter of the California Bay tree Umbellularia californica with some inter- esting results. Fresh leaves from the litter had equal amounts (ca. 16% dry wt) of terpene irrespective of whether they were green yellow or brown in colour. Decomposition soon occurred with oxygenated terpenes (80% of total) disappear- ing first. The total monoterpene content decreased by between 63 and 86% about one year after leaf fall. As expected loss of monoterpenes was correlated with water solubility (see above) and was greatest in moist sites after rainfall. Two menthofuran monoterpenes 50 and 51 in Calamintha ashei have been found to be major constituents of leaf soaks and leaf washings and concentrations of 0.66 and 0.74 mM are leached out.Bioassays showed that 0.05 mM concentrations of 50 were inhibitory to seed germination of competitor species.143 In the plant Chrysocoma pauciJlosculosa which grows like Calamintha ashei in the Florida sandhills the diterpene 17-oxogrindelic acid 52 was identified as the allelo- pathic agent. At a concentration of 12-48ppm 52 reduced germination and radicle growth of the two competing grass species Schizachrium scoparium and Leptochloa dubia. No-tably 52 had no effect on germination of lettuce seed so often used as a test organism.'4 Several lupane triterpenes includ- ing messagenin 53 have been implicated as allelopathic agents.They were isolated from Melilotus messanensis and tested against several other dicotylenous species. The indole protoalkaloid gramine which occurs in barley leaves is a potential allelopathic agent toxic to weeds but its release into the environment has been a puzzle. Yoshida et a/. have now demonstrated that it actually partly occurs on the leaf surface and can be eluted from the leaf by artificial rain experiments. Gramine content on the leaf surface varies from 0.04 to 0.18%of the total gramine in the leaf. The ability of seeds to survive in the soil for long periods is often raised in allelopathic experiments. One means of survival might lie in the presence of chemical defence agents. Indeed Hendry et a1.'47 have found that seeds in British soils often contain large amounts of o-dihydroxyphenols and additionally regularly release hydrogen cyanide on hydrolysis.Another factor in seed survival could be the presence of an allelopathic agent capable of exuding out when necessary. Such a com- pound lepidimoide 54 has been reported in seed exudates of a number of common plants.I4* 92 Natural Product Reports 50 R=H 52 51 R=OH 0 I/ -,. HpvH HO 9 How ONa I OH 53 54 Me Me 55 56 Me 57 4.2 Plant-parasite interactions The chemistry of the stimulants exuded from host plant roots to trigger off the germination of seeds of the parasitic witch- weed Striga asiatica has now been resolved. There are three closely related lactones involved.The first is strigol 55 orig-inally characterised as a stimulant from the non-host plant ~otton.~ This substance has been characterised from two genuine host plants namely maize Zea mays and millet Panicum milia~eum.'~~ A small amount is also produced by root exudates of sorghum. However the major stimulant from sorghum is a second structure sorgolactone 56.15' Finally a third compound alectrol 57 has been reported from cowpea roots Vigna unguiculata which stimulates germination of the closely related Striga gesneroides. These three substances are incredibly active in that concentrations in the range 10 -'' to 10-l5 M have been reported to set off Striga seed germination. For this group of sesquiterpene lactones active as Striga germination stimulants the collective name 'strigolactones' has been proposed.152 5 Plant-microbe interactions 5.1 Phytotoxins A new 10-membered macrolide putaminoxin 58 has been characterised as the main phytotoxin in Phoma putaminum the causal agent of leaf necrosis in Erigeron annuus.It produces necrotic symptoms at 20 yg per droplet on both E. annuus and Mercurialis annua but is relatively ineffective on most non- host plants. 153 A second 10-membered macrolide pinolidoxin 59 has been found as a phytotoxin of Ascochyta pinodes the causal agent of pea anthracnose disease. When assayed on host (pea) and non-host (bean) plants it was found to be highly Me 58 59 H Me Me CH2Me Me0 60 61 HO*o\ CO2H OH 0 OH OH 62 63 C-n 5H2 CH2CH20H OCOtCH2CH20H CH2 HO C02H 64 65 ~ EH2 CH2CH20H OCOECH2CH20CO CCH2CH20H CH2 CH2 HO OH 66 toxic.The trisubstituted salicylic aldehyde ascosalitoxin 60 is the major phytotoxin of Ascochyta pisi a phytopathogenic fungus also capable of producing anthracnose in peas. This substance like 59 is toxic to both host and non-host plants.'55 The known phytotoxin monocerin 61 has been found in a new source the fungus Exserohalium turcicurn which causes the Northern Corn Leaf Blight on the maize crop. It produces brown necrotic lesions on punctured leaves the size of which is in direct relation to the dose ap~1ied.I~~ Elicitins are toxic signalling proteins secreted by different Phytophthora species responsible for incompatible reactions and systemic leaf necroses.One such elicitin secreted by Phytophthora cactorum has been sequenced and it has 98 amino acid residues. Changes in the amino acid sequence at point mutations in the middle of the molecule can be corre- lated with phytotoxicity.157 Another elicitin from the same fungus has been named cactorein and shown to be an acidic protein. When applied to tobacco plantlets at concentrations as low as 100 mM it caused wilting desiccation. When applied to cultured tobacco cells at concentrations of 40 mM it caused the accumulation of the sesquiterpenoid phytoalexins rishitin phytuberol and capsidiol. 15* 5.2 Constitutive antimicrobial defence Antifungal compounds reported from higher plants during the period 1982 to 1993 have been surveyed.'59 Defence responses of plants to pathogens have been reviewed.16' A phytoalexin survey of the family Rosaceae in leaves of 130 representative species using both biotic and abiotic elicitation showed that there was very rarely a response.The family is very unusual in this respect compared to the Leguminosae Compositae or Solanaceae and appears to depend chiefly on constitutive Harborne Recent advances in chemical ecology antifungal agents in the leaves. Indeed about a third of the species surveyed showed the presence of catechin-like con- stituents probably in glycosidic form which are the main antifungal agents.I6' In Pyrus leaf arbutin is present and hydroquinone is released by enzymic hydrolysis on fungal infection.Chemical analysis of other tissues especially sap- wood of woody members of the family showed the presence of six other constitutive antifungal constituents p-hydroxyaceto- phenone acetovanillone chrysin sakuranetin naringenin and 5,7-dihydroxychromone 62.Whether the latter is formed by breakdown of constitutive flavanones or occurs bound to glucose has yet to be determined.'62 In independent exper- iments 62 has been shown to inhibit the radial growth of the soil pathogenic fungi Rhizoctonia solani and Sclerotium rolfsii with ED, values of 18 and 26 pM.'63 Several unusual antimicrobial constituents have been obtained from the plant Aeollanthus buchnerianus including the diunsaturated hydroxy fatty acid 63 and the novel abietane diterpenoid 64.164 More expectable bibenzyls such as batatasin I11 and 3'-methoxybatatasin I11 have been recorded as anti-microbial agents in branches of Empetrum nigrum.165 Tuliposides A-C are aliphatic glucose esters which undergo hydrolysis and rearrangement when tissue is damaged to form tulipalins A-C which are active antifungal agents. They occur in leaves stems and flowers of Tulipa and Alstroemeria species and besides being antifungal are responsible for contact dermatitis in humans handling the bulbs. Two new tuliposides D 65 and E 66 have been obtained from Alstroemeria flowers and have similar properties to the other tuliposides.166 A major advance in our understanding of disease resistance mechanisms in plants has been the recognition that cysteine- rich antimicrobial peptides have an important role in the protection of higher plants.An increasing number of such peptides have been characterised from seeds. Some are ident- ified via sequencing of cDNA clones in both monocots and dicots. They show similarities to insect defensins and the name 'plant defensin' has been proposed for them.'67 In a recent paper five new plant defensins were isolated from seeds of horse chestnut Clitoria ternatea Dahlia merckii and Heuchera sanguinea.168 All produced 50% inhibition of fungal growth at concentrations between 1 and 25 pg ml -In radish seed two defensins have been found.169 They are located in outer cell layers of the seed coat and are preferen- tially released on gemination to create a microenvironment around the seed in which fungal growth is suppressed.In addition similar antifungal peptides can be induced in radish leaf after localised fungal infection. '69 Cysteine-rich peptides have also been characterised in sugar beet 1ea~es.l~' 5.3 The phytoalexin response The best evidence to date that phytoalexins are effective in providing plants with disease resistance has been obtained by foreign phytoalexin expression in a novel plant. Stilbene phytoalexins such as resveratrol require only a single con- densing enzyme stilbene synthase to link the precursors malonyl-CoA and p-coumaryl-CoA for their synthesis. Patho- gen resistance has thus been engineered into tobacco plants by the transfer of stilbene synthase genes from grapevine a resveratrol-producer.The regenerated tobacco plant proved to have improved resistance to infection by Botrytis cinerea. This could be traced immediately to the rapid synthesis of up to 40 pg resveratrol g -' fresh wt. This is thus the first report of increased disease resistance based on an additional foreign phytoalexin and is a technique which may have important practical benefit when applied to crop ~1ants.l~' The family Cruciferae produce a series of unique sulfur- containing indole phytoalexins. '72 Two further structures cyclobrassinone 67 and 1-methoxyspirobrassinin 68 have been reported among the phytoalexins formed by kohlrabi Brassica oleracea var. gongylodes. Labelling experiments with ~-[a-'~C]tryptophanand ~-['~CH,]methionine infiltrated into I H OMe 67 68 OMe R4wRl R3 OR2 R1 R2 R3 R4 69 OH Me H H 70 OH Me OH H 71 OH Me OMe H 72 OH Me H OMe 73 H Me OH H 97 9' I A4 OMe R' R2 R3 R4 R5 R6 R7 74 H H Me OMe OH H H 75 H Me Me OMe OH H H 76H HMeHOHH H 77 H H Me H OMe H H 78 H H Me OH OMe H H 79HMeH H H H H 80 H Me H H OMe H H 81HMeH H H H OH 82 OMe H Me OH H H H 83 OMe H Me OMe H H H 84 OMe H Me OMe OH H H 85 OMe Me H H H H H 86 OMe H Me H OH H H 87 H Me Me H OMe OH H stem tuber tissue of kohlrabi indicated that these two amino acids are precursors of the indole phytoalexins.173 Whether tryptophan is a precursor throughout the family is uncertain since Tsuji et al.174 question whether it is a direct precursor of the phytoalexin camalexin of Arabidopsis thaliana. As already mentioned in Section 5.2 species in the Rosaceae rarely respond to phytoalexin induction when leaf tissue is examined.161 However sapwood of woody members of the family do respond to fungal inoculation by producing phyto- alexins and a series of five biphenyls 69-73 and 14 dibenzo- furans 74-87 have been variously identified (Table 1).'62 Most of these are new structures established by NMR measure-ments. '757 76 One surprising feature considering their obvious biosynthetic relationship is that biphenyls and dibenzofurans are almost never produced together in the same plant. Thus genera are either biphenyl-producers (Aronia Chaenomeles etc.) or dibenzofuran producers (e.g.Cotoneaster Crateagus etc.). While most woody Rosaceae produce between two and three phytoalexins in the sapwood some (e.g. the rowan tree Sorbus aucuparia) produce as many as five. The antifungal activities of the dibenzofurans are marginally greater than those of the biphenyls. Thus when tested against Fusarium culmorum dibenzofurans exhibited ED,,s of 14 to 53 pg ml -I while biphenyls had ED,,s of 16 to 84 pg ml-The dibenzo- furans 7487 are uniformly heavily 0-methylated as well as hydroxylated and presumably have sufficient lipophilicity to be able to interfere with fungal growth and development. While most phenolic phytoalexins have two or more Table 1 Biphenyl and dibenzofuran phytoalexins formed in sapwood of plants of the Rosaceae Compound Generic source Biphenyls Aucuparin 69 Malus Sorbus 2'-Hydroxyaucuparin 70 Sorbus 2'-Methoxyaucuparin 71 Malus Sorbus 4'-Methoxyaucuparin 72 Chaenomeles Sorbus Isoaucuparin 73 Sorbus Dibenzo furans a-Cotonefuran 74 0-Cotonefuran 75 Cotoneaster y-Cotonefuran 76 6-Cotonefuran 77 &-Cotonefuran78 Cydonia Eriobofuran 79 Pyracantha 7-Methoxyeriobofuran 80 Photinia 9-Hydroxyeriobofuran 81 Pyracantha 6-Hydroxy-a-pyrufuran 82 6-Hydroxy-a-pyrufuran 83 Mespilus 7-H ydroxy-6-methoxy-a-pyrufuran j3-Pyrufuran 85 y-Pyrufuran 86 Pyrus 2,8-Dihydroxy-3,4,7-trimethoxy- dibenzofuran reported as phytoalexins.The first is 2,6-dihydroxy-4-methoxyacetophenone 89 which was obtained in the root of Sanguisorba minor after fungal inoculation with Botrytis cinerea.The ED50 of 89 is 45 p~ on spore germination in eO. A ,OH OMe 89 90 R 91 R=H 93 92 R=OH 94 95 &CHO 0 96 ?H oQJrJoH OFOH aromatic rings in their structures some simple monoaro- \/ matic systems are occasionally encountered as antifungal \ agents. In particular a number of acetophenones have been 97 98 94 Natural Product Reports Botrytis.'77 The second is danielone 90,a phytoalexin from the fruit of papaya Caricapapaya. The ED50 is 50-75 ppm against Colletotrichum gloesporioides. 17' A third and fourth appear to be p-hydroxyacetophenone 91 and 3,4-dihydroxyacetophenone 92 from the cocoa plant Theobroma More important antifungal compounds elicited in cocoa however are the triterpenoid arjunolic acid and elemental sulfur.The sulfur which accumulates de novo in xylem cells is a remarkably simple molecule as a phytoalexin. Three further acetophenone phytoalexins 93-95 one of which has isopentenyl substitution have been elicited in tubers of yacon Polymnia sonchifolia (Compositae) inoculated with Pseudomonas cichorii. '8o This type of phytoalexin is new within plants of the Compositae. The presence of a more usual phytoalexin the sesquiterpene lactone lettucenin A 96 has been confirmed in challenged lettuce tissues. This is active against lettuce pathogens at a concentration of 10 -M. The success or failure of lettuce mildew Botrytis cinerea to invade lettuce is based on the balance between lettucenin A accumu- lation and degradation.'" Accumulation of 96 in lettuce is preceded by increased activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase one of the early enzymes of biosyn- thesis.'82 A sesquiterpene phytoalexin 97 has been isolated from leaves of Hypochoeris radicata stressed with copper ~hloriile."~ Curiously its structure is closer to that of the phytoalexins of the Solanaceae than to compounds like lettucenin A.In the sunflower Helianthus annuus two simple coumarin phytoalexins scopoletin and ayapin have been described in the literature. Gutierrez et al. 184 record that these coumarins are readily induced by a variety of elicitors in mature leaves but that seedlings and immature plants fail to respond.Another unrelated plant which produces simple coumarins as phytoalexins is the plane tree Platanus acerifolia. These are umbelliferone and scopoletin '85 which although showing antifungal activity in bioassays tend to accumulate too late in the stem of plane seedlings to totally inhibit conidial germination. A new plant species from a new plant family to be examined for phytoalexins is banana Musa acuminata of the Musaceae. A series of novel phenalenone-type structures are induced after infection with fungi such as Colletotrichum musae and Fusarium oxysporum. The first to be described was irenolone 97 from leaves and fruit peels186 and the second the naphthalic anhydride 98.'87 Later four related structures musanolones C-F 99-102 were obtained from infected banana rhizomes.''' Plant phenalenones are rare in nature although several similar compounds occur constitutively in members of the Haemodaraceae. The stilbene resveratrol 103 is known as a phytoalexin from several unrelated plants including the vine Vitis vinifera and the groundnut Arachis hypogaea.'59 It has been reported for the first time from the grass family Gramineae in seeds of Festuca Lolium Hordeum Stipa and Poa species. 189 It appears to occur particularly in grass species which are infected with fungal endophytes and which contain inter alia ergot-type alkaloids. The rice plant also a member of the grass family is known to produce a series of diterpenoid phytoalexins so that the discovery that it can also synthesise the flavanone sakuranetin 104 in response to infection or UV treatment is something of a surprise.'903 * ' It is accompanied by small amounts of naringenin the immediate biosynthetic precursor but this has low antifungal activity.By contrast sakuranetin has an ED50 value against spore germination of Pyricularia oryzae of 15 ppm. The amount produced in infected rice leaves is signifi- cant and 104 may well be the most important phytoalexin of the rice plant. Meanwhile further diterpenoid phytoalexins have been recorded in rice. One is oryzalene F 105 formed both on UV treatment and on blast infection. It has a relatively poor ED50 OH R' 99 R=H 101 R' = H; R2 =OH 100 R=OW 102 R1 = OMe; R2 = H OH 0 104 105 106 0 R' 107 R=H 110 R=Xyl 108 R= dH109 R=H 111 R=Gk labdadiene type oryzalexin S has a stemmarane based struc- ture.lg3 One-could argue then that the rice plant is capable of producing three biosynthetically distinct types of phytoalexin.Rice is also well protected from fungal disease by constitu- tive defence agents including high silicate leaf content and the presence of antifungal fatty acids. '59 Yet another constitutive defence is the production in the young rice seedling of 5-(8'2-heptadeceny1)resorcinol and several homologues. '94 Finally it appears that systemic acquired resistance can be induced in rice plants by treating them with probenazole as a soil drench. Fungal growth is then severely restricted by the rapid lignifi- cation of the plant tissues.In fact the enzymes of lignification increase on the first day of infection in an incompatible reaction. 195 It is unusual to find phytoalexins occurring as glycosides and even more unusual to find that tropolones can by synthesised as phytoalexins. The discovery of two tropolone glucosides 107 and 108 in the bark of Cupressus sempervirens following infection by Diplodia pinea is a notable event. In vitro 107 and 108 inhibited spore germination in several fungi.'96 In this connection it is interesting that two glycosides of the known phytoalexin betavulgarin 109 have recently been isolated from the roots of sugar beet Beta vulgaris infected by Rhizoctonia solani. They are the 2'-xyloside 110 and the 2'-glucoside 111.The authors speculate about their possible role in providing resistance away from the actual necrotic zone of infection. 197 In the celery plant it has been assumed for some time that linear furanocoumarins based on psoralen are the characteris- tic phytoalexins protecting the plant from fungal attack. However in storage it appears that two furanocoumarin precursors accumulate and provide more efficient antifungal protection. These are marmesin 112 which has 100-fold more value of 103~prn.'~~ Another new phytoalexin is oryzalexin S 19'antifungal properties than psoralen and columbianetin 113 106. Unlike all the other rice diterpenoids which are of the which is 80 times more antif~nga1.l~~ The concentration of Harborne Recent advances in chemical ecology Ho4..112 113 114 115 116 1 117 columbianetin in vivo is 38 pg g -' fresh wt and this is close to the concentration required for growth inhibition of celery pathogens in vitro. In Citrus plants the phytoalexins reported are mainly simple coumarins. An antifungal compound produced on wound- ing the peel of grapefruit and Valencia orange is the p-hydroxycinnamaldehyde derivative 114.200Is this a precur- sor of an isopentenylcoumarin also produced as a Citrus phytoalexin? In interactions between virulent strains of plant pathogens and their host plants phytoalexins formed are often tolerated because they are metabolised to inactive products by the fungus. This happens in dry rot on potato tubers caused by Gibberella pulicaris.The major phytoalexin rishitin 115 is converted to 13-hydroxyrishitin 116 and 11,12-epoxyrishitin 117 and neither of these derivatives affect the growth of the fungus.201 Many other lines of phytoalexin research have been pursued during the period under review. 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ISSN:0265-0568    

DOI:10.1039/NP9971400083

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

The role of carbohydrates in biologically active natural products Alexander C. Weymouth-Wilson Dextra Laboratories Ltd The Innovation Centre University of Reading Reading Berkshire UK RG6 6BX 1 2 2.1 2.2 2.3 3 4 5 5.1 5.2 5.3 6 7 7.1 7.1.1 7.1.2 8 8.1 9 9.1 10 10.1 10.2 10.3 10.4 11 Introduction An thracyclines Molecular biology Sugar derivatives Prodrugs Aureolic acids Avermectins Enediyne antibiotics Calicheamicins Esperamicins Neocarzinostatin Glycopeptides Macrolide antibiotics The side effects of macrolides Gastrointestinal Cytochrome P450 complexes Pol yenes Mode of action Phosphoglycolipids Structure-activity relationship Miscellaneous Angucycline antibiotics Recently discovered enediynes Cholestane glycosides Marine natural products References X is also a tumour-associated antigen and it is present to an abnormal extent in certain tumours.8 Tumour cells which contain sialyl Lewis X bind to areas of the vascular endo- thelium which express E-selectin.This adhesion may be an explanation for tumour metastasis and could lead to a new therapeutic approach. Areas of interest in the field of glyco-biology include cellular adhesion and cell-cell recognition; fertilisation; glycosylation of proteins; neurobiology; vaccines; and xenotransplantation. Fig. 1 shows some of the more common monosaccharides which are found in nature. ,OH OH OH OH p-D-glucose P-D-galaCtOSe a-D-mannOSe HOa OH HO OH OH NHAc HO N-acetyl-p-D-galactosamine Kacetyl-a-D-neuraminic acid Fig.1 Common natural monosaccharides There is increasing interest in carbohydrates and this review will try to illustrate the role that carbohydrates have on important biologically active natural products and the inter- esting carbohydrate derivatives of these products that are formed. This review will not include steroid glycosides some of which have been used as cardiac drugs for many years but will concentrate on newer biologically active natural products. 2 Anthracyclines Anthracycline antitumour antibiotics for example 24 are among the most potent and widely used anticancer agents. The anthracycline structure is based on a tetracyclic aglycone which is attached to at least one sugar residue.Daunomycin 2 is used for the treatment of acute leukaemia and adriamycin 3 is used for the treatment of various solid tumours. These two anthracyclines have been studied in great detail.' As a group the anthracyclines exhibit good antibacterial activity against 1 Introduction Carbohydrates are often thought of as food sources including the much talked about complex carbohydrates that are import- ant for maintaining a healthy diet. The biological roles of oligosaccharides have in many ways been overlooked in the past compared with the roles of proteins and nucleic acids. Although for many years it has been known that the antigenic determinants of the ABO(H) blood group and the related Lewis blood groups are carbohydrate structures,' the import- ance of carbohydrates in other areas had not been fully recognised.A great deal has been learnt in recent years about the biological role of oligosaccharides and this has led to a new field named glycobiology. For example one area of interest is the carbohydrate binding ligands for L E and P selectin of which sialyl Lewis X 1 has received the most attention.2 This molecule plays an important role in the process of recruiting neutrophils and monocytes from the blood into damaged tissue. It has been hoped that sialyl Lewis X may be of therapeutic use as an antiinflammatory drug but due to the complexity of chemical synthesis a mimic of the natural product appears to be the way for~ard.~-~ Sialyl Lewis HO' OH AcNH HO NHAc OH Gram-positive bacteria mycoplasms and some yeasts but in general are not active against Gram-negative bacteria.Due OH to their toxicity they are not used clinically as antibacterial 1 sialyl Lewis X agents. Weymouth- Wilson The role of carbohydrates in biologically active natural products 0 OH OMe 0 OH 0 OR2 sugar = L-daunosamine 2 daunomycin R1 =COMe; R2=H 3 adriamycin R1 = COCH20H; R2 = H 4 rubomycin R' = COMe; R2 = Me+ N(M42 OH L-rhodosamine 2.1 Molecular biology The antitumour activity of daunomycin which can be extended to anthracycline as a whole is attributed to the incorporation of the drug into DNA." X-Ray crystal struc- ture analysis of a complex of daunomycin" with self-complementary DNA hexamer d(CGTACG) has shown that two daunomycin molecules intercalate at each of the two C:G sites at either end of the duplex.The intercalative binding results in an increase in base-pair separation from 3.4 to 6.8 8 as the molecule associates which in turn leads to an unwinding of the helix and the formation of a non-covalent complex with the DNA. This results in inhibition of DNA replication and RNA transcription. Rings B-D of the aglycone are intercalated into the DNA leaving ring A and the amino sugar as anchoring units. The cationic amino sugar and the hydroxy group of ring A fill the minor groove which displaces water and ions from the groove. This has led to speculation that no more than one drug molecule can be intercalated per three base pairs.The hydroxy group of ring A forms two hydrogen bonds with N3 and N2 of the adjacent guanine base stabilising the drug DNA complex. The amino sugar does not hydrogen bond to adjacent bases although it is thought that due to its snug fit in the minor groove it has a stabilising effect in the complex. The amino and hydroxy groups of the sugar face out of the minor groove and it has been suggested that they may interact with polymerases which could prevent or retard the action of these enzymes. It has been noted that anthracyclines which contain several sugars have in general less side effects than other anti- tumour antibiotics. For example marcellomycin 5 ex Actino-OH C02Me OH 0 OH 0 I R2 2-deoxy-L-fucose 5 marcellomycin R1=OH; R2 = Mei& OH 6 aclacinomycin A R' = H; R* = 0 L-cinerulose 100 Natural Product Reports sporangium species and aclacinomycin A 6 ex Streptomyces galileus MA144-M1 which are active yet show much less cardiotoxicity than daunomycin.l2 To cancer cells that have acquired a resistance to doxo- rubicin aclacinomycin A 6 has been used in combination with this drug to enhance its effectiveness.I3 A recent NMR study of aclacinomycin A and B complex to d(CGTAGCG) showed two main differences compared with daunomycin and doxo- rubicin.l4 Firstly aclacinomycin covers four base pairs and the trisaccharide which lies in the minor groove projects further into the solvent region. Secondly the 2'-deoxyfucose of the trisaccharide was found to be close to the A4 nucleotide which forces the DNA helix to kink towards the major groove.This structural distortion could be recognised in a unique way by relevant enzymes. These changes suggest that aclacino- mycins may have different modes of biological action com- pared with daunomycin and doxorubicin both of which contain charged monosaccharides. Nogalamycin 7 is somewhat unique in the anthracycline family in that it contains two sugars one attached to the D ring (a-3,6-dideoxy- 3 -dime th ylaminoglucose) and the second (nogalose) attached at C-7 of the A ring. Me ?H HI 0 OH 0 Me0 7 nogaloamycin NMR~S-I8 and studies on nogalomycin 7 have shown that the aglycone is intercalated with the drug spanning the two grooves of the DNA helix with the nogalose occupy- ing the minor groove and the aminoglucose occupying the major groove.More recently nogalomycin has been used to probe bulged DNA structures the structure is often associated with important biological functions. It has been noted that some intercalators bind to bulged structures tighter than to normal B-DNA.~~ 2.2 Sugar derivatives In general the amino sugars attached to the aglycone have to be the natural a-glycoside linkage as the p-analogues were found to be devoid of activity. A review by Ar~amone~~ describes in detail the amino sugar derivatives and their activities. The important clinical derivatives were found to be the L-arabino analogues of daunomycin and doxorubicin 4'-epidaunomycin and 4'-epidoxorubicin 8.24These analogues when tested were shown to have similar activity to their parent 0 OH 0 OMe 0 OH 0 HO M& H2N 8 4'-epidoxorubicin compounds.Epidoxorubicin was shown to have reduced cardiotoxicity compared to its natural counterpart and is used clinically.25 There has been interest in the neutral sugar derivatives of the anthracyclines e.g. SM-5887269 which shows potent anti- tumour activity with reduction in local tissue toxicity and cardiotoxicity compared to adriamycin 3. Structural NMR analysis of SM-5887 suggests that the intercalation of SM-5887 was a hybrid between adriamycin 3 and nogalamycin 7 and similar to aclacinomycin 6. There has been growing interest in the area of drug resistance and ways to combat this problem.It has been suggested that the amino group of daunosamine in doxorubicin or more generally the presence of a basic centre might be an important structural feature recognised by the P-gpt multidrug tran~porter.~~ Comparative studies of doxo- rubicin and hydroxyrubicin 10 in drug-resistant tumour cells have shown that hydroxyrubicin partially or completely cir- cumvents P-gp-mediated drug resistance due to its decreased transport by P-gp compared with doxorubicin.28 OMe 0 OH 0 OH 9 SM-5887 0 OH 0 OH 10 hydroxyrubicin(WPl59) 0 OH 0 OMe 0 OH 0 mAB-P-glucosidase 1 0 OH 0 OMe 0 OH 0 OH Scheme 1 prodrugs described of which doxorubicin attached via an oligopeptide chain to poly[N-(2-hydroxypropyl)methyl-acylamide] exhibited promising activity.35 3 Aureolic acids The aureolic acid group of compounds are highly potent antimicrobial agents which are active against gram-positive bacteria and mycobacteria but are too toxic to be used as antibiotics.However they show good activity as antitumour agents and chromomycin A (CRA,) 11 olivomycin A 12and OMe k o OR2 Ho30&O&Me OMe OH A 7' Since the glycosidic bond of 2-deoxy sugars is inherently less stable and the associated loss of the sugar moiety is thought to ' ' D-chromose D-chromose *\ (R2=COMe) '' 0 OH 9 10 be one of the causes of toxicity various C-2' fluoro analogues have been prepared. The reasoning for this strategy is that electronegative fluorine strengthens the glycosidic link and there should also be little steric effect from its introduction.D-oliose OH OH 0 (R2 = H) This work has been documented by Ts~chiya.~~ Of interest was 2'-P-fluorodaunorubicin which although slightly less active (L 1210 murine leukaemia) than daunorubicin has a higher dose range (P 1210) and was also found to be less toxic.,' 2.3 Prodrugs In certain type of cell recognition carbohydrates were found to play an important role for example L1210 leukaemia cells have receptors specific to f~cose.~' This approach has led to the synthesis of various anthracycline prodrugs with the aim of reducing side effects. One area of interest is the antibody directed enzyme-prodrug therapy (ADEPT)32 or antibody directed catalysis (ADC)., Scheme 1 shows the use of ADEPT where the P-glucoside derivative of daunomycin was used as a probe for this approach.34 There have been other anthracycline tP-glycoprotein (P-gp) is an energy-dependent transmembrane drug transporter.Weymouth- Wilson The role of carbohydrates in biologically active natural products D-olivose 1330 OH E D-olivomycose 11 chromomycinA3 R1 = Me; R2= R3 = COMe 12 olivomycinA R1= H; R2 = COMe; R3 = COCH(Me)* mithramycin 13 are used clinically. These molecules were found to be inhibitors of DNA-dependent RNA polymerase. It was discovered in degradation studies of CRA, that CRA lacking some of the sugars was less active36 and that the aglycone did not bind to DNA at all.37 The mode of action of the tricyclic aromatic antibiotics was first thought to be similar to the anthracyclines whereby the drug was intercalated into the DNA.However intercalation was not found to be important and studies have shown that the 23 HHO o ~ o ~OMe OH o ~ M e Me 22A*-Me ' D-OliVOSe D-olivose Me I 0 OH OH OH 0 '8 HO&O H O W 0 D D-olivose D-OliVOSe OH D-mycarose 13 mithramycin members of the aureolic acid family bind to GC rich regions of DNA thereby inhibiting replication and translation pro-ce~ses.,~ Recent NMR studies by Gao and Pate14' have shown that CRA forms a symmetrical dimer with self complemen- tary d[T-T-G-G-C-C-A-A] with the hydrophobic edge of the chromophore located next to the GC:CC half site.The sugars in the CDE trisaccharide extends towards the 3'-direction of the octanucleotide in the minor groove. The AB disaccharide and the hydrophilic side chain are directed to the phosphate back bone. The dimer induces conformational perturbations in the DNA helix resulting in a wider and shallower minor groove at its binding point. The NMR work of the interaction of chromomycin A with DNA duplexes by Shafer and co-~orkers~~ is in agreement with the findings of Gao and Patel.40 Magnesium was found to be necessary for binding and the NMR studies on CRA by Gao and Pate14' showed that CRA forms a 2:l drug-Mg2' complex which lies in the minor groove. Silva and Kahne4' have shown that the 2:l complex with Mg2' is due to the stabilisation effect of the sugar not of the DNA.They predicted a possibility of eight octahedral diastereomers for this complex but they also found that there was one complex that was predominant. This complex was shown to have the appropriate shape to bind to the right handed DNA duplex used in their studies and it is suggested that it is this 2:l dimer complex that then binds to the DNA. They also showed by degradation that the trisaccharide CDE is essential to stabilise the dimer whereas the removal of sugar B of the AB disaccharide had no significant effect on the complex formation. The role of sugar A was undetermined. The action of mithram~cin~~ 13 is essentially the same as CRA but two differences have been noted.First it has been shown that when chromomycin binds to form a chromomycin- DNA complex all four sugar residues in the central tetra- nucleotide adopt A-helix sugar puckers and glycosidic torsion angles while only the central cytosine in the tetranucleotide forms A-sugar pucker in the mithramycin d(T-C-G-C-G-A) duplex. This is believed to be due to the increased hydrophobic nature of the sugars of mithramycin compared with chromo- mycin A,. Secondly there is a reduction in sequence specificity of mithramycin dimer for sequences flanking the central (G-C):(G-C) step. The E-sugar of mithramycin lies on the floor of the minor groove but the interactions are more hydrophillic in nature when compared to the E of chromomycin A,. The additional hydrogen bonding potential of D-mycarose has been used to explain the reduced sequence specificity of the mithramycin dimer.4 Avermectins The avermectins were discovered in 197943 as fermentation products of Streptomyces avermitilis. This new class of macrolides showed extraordinary activity against endo- and ecto-parasites. Avermectin B, 14 and ivermectin 15 are primarily employed as animal health care agents although 102 Natural Product Reports 0 Hwof..o&Me Me OH 14 avermectin B1 Me O Mg.y+o-J Mh0 OMe I I /I H bH 15 ivermectin ivermectins the semi-synthetic derivative are used for human treatment of onchocer~iasis.~~ The mode of action of the avermectins has been explored but as yet the exact mechanism of action remains unclear.45 The action of avermectin is believed to stimulate specific chloride ion transport systems increasing the membrane permeability of the membranes to chloride ions via GABA (y-aminobutyric acid) receptors46 and non-GABA receptors.47 Although the 13-bis-oleandroside substituent is not obliga- tory for anti-parasitic activity it does contribute desirable properties to the macrocyclic ring and this has led to a number of modifications of the oleandrose sugars.However the agly- cones of avermectins have poor anthelmintic activity.48 The 4"-amino-4"-deoxyoleandrose derivatives of avermectin B and ivermectin were synthesised following the observation that most macrolide antibiotics contained a basic amino Of interest in the amino analogues is 4"-epi-acetylamino-4"- deoxyavermectin B which showed the most promising activity and is currently under development as a novel avermectin ende~toside.~~ The 4"- and the 4'-position have been modified in a number avermectinhvermectin derivatives primarily due to the ready accessibility of these position^.^' The synthesis of C-2"-a-fluoro- and C-2"-P-fluoroavermectinswere pre-pared the rationale being to strengthen the glycosidic bond.The derivatives showed interesting biological properties with potencies in some screens comparable to the parent cornpo~nd.~' The synthesis of 13-epi-avermectins showed them to be substantially less toxic than the natural product but the anthelmintic activity was not reduced. This increase in thera- peutic index may allow their use in broader application^.^^ 5 Enediyne antibiotics The discovery of the enediyne antibiotic^,^^ which include calicheamicin y1I 16 esperamicin 18 and neocarzinostatin 19 has led to great interest because of their complex structure biological activity and mode of action.53 The enediynes are remarkable DNA cleaving compounds the mechanism of the DNA cleaving is believed to involve a Bergman-type cyclo- ar~matization~~ of the aglycone which generates a diradical species.The diradical in the presence of oxygen cleaves two strands of DNA in a concerted manner55 (Scheme 2). Nu 3 S-S-SMe I\ HOm -s u g a r HO 0-Sugar Bergman cyclization I 0 Cleaved DNA mom R = C02Me Scheme 2 5.1 Calicheamicins The calicheamicin family was isolated from Micromonospora echinospora ssp.calichensis; calicheamicin y ,I 16 was discov- ered when sodium iodide was added to the broth.56 The family of calicheamicins possess activity against Gram-positive and Gram-negative bacteria but they also show unprecedented activity against murine tumours and solid neoplasms. The biological activity of the enediynes has been attributed to the DNA cleaving properties these compound exhibit. Calichea- micin binds to the minor groove of DNA showing a high degree of base pair sequence-specificity and a high degree of double strand to single strand cleavage of DNA. This occurs primarily at homo pyrimidine-homopurine sites such as 5'-TCCT/AGGA and 5'-TCTCIGAGA.The double stranded scissions suggest that calicheamicin binds in the minor groove with the diradical positioned in such a way as to allow hydrogen abstraction from each strand.57 The degree of specificity has been attributed to the carbohydrate moiety of the molecule with the tail of the oligosaccharide aligned towards the 3'-end of the DNA. Studies have shown that the aglycone calicheamicin binds to DNA with low affinity and in a non-sequence-specific manner.5s There has been voluminous amounts of work in this area which have revealed many clues as to how the carbohydrate affects binding selectivity. The sugar functionality of calicheamicin y ,I has some un- usual structural features these being the iodine substituted aromatic ring C the thio sugar B and the hydroxylamine glycosidic link between sugars A and B.The carbohydrates in calicheamicin are liphophilic in nature which makes them favourable to interact with DNA which along with the other features gives the molecule the required chemical architecture to bind in a sequence selective manner to DNA. Recent NMR studies of calicheamicin DNA complex suggest that the DNA distorts to accommodate the drug and that the drug does not change much upon binding.59 This Me 0 0 OMe OH I Me-N inflexibility of calicheamicin may lead to the sequence specifi- city as certain sequences of DNA may be easier to distort to fit the drug. There has been growing evidence that the iodine of aromatic ring C interacts with the C-2 NH groups of one or both guanines in TCCT:AGGA duplex DNA.60 The binding en- ergy of the oligosaccharide domain of calicheamicin60 to TCCT:AGGA sites within duplex DNA was shown to be 7.6 kcal mol-'.Substitution of the iodine for a hydrogen resulted in a loss of 2.3 kcal mol-' in binding energy and a series of analogues showed that I>Br>Cl>F>CH,>H for binding affinities.61 This has led to the suggestion that the polarizable iodine interacts with the C-2 NH of both guanine residues at the target site and that the interaction is not simply van der Waals contacts. However the difference in binding energy of 2.3 kcal mol -cannot be due to hydrogen bonding alone and it has been proposed that there are other inter- actions as of yet undetermined which led to this difference.The sulfur atom of the sugar group B was shown to form hydrogen bonds with an exposed amino proton from 3'-guanine in a drug DNA complex.62 This sugar was shown to be positioned edgewise in the minor groove allowing the aromatic ring C to be placed between the minor groove with its iodine and methyl group positioned deep in the minor groove. A hydrolysis product of esperamicin A, esperamicin C 17 has been viewed as an analogue of calicheamicin which has a hydroxy group at C-12 and isopropylamino and methyl- sulfanyl groups in place of the ethylamino group and the rhamnose aromatic moieties re~pectively.~~ Esperamicin C causes double strand DNA cleavage but with considerably less sequence ~electivity~~ than calicheamicin and the pattern of cleavage indicated a bidirectional mode of binding.These results suggest that the aromatic and rhamnose fragments of calicheamicin determine the sequence specificity and the mono- directional mode of binding but they have little or no effect on orientating the aglycone within the DNA. The removal of the rhamnose sugar D of calicheamicin reduces the cutting efficiency but the sequence specificity is not altered. This sugar in NMR studies65 of the drug-DNA complex was shown to lie less deeply in the minor groove and to interact with hydroxy groups of the phosphate backbone. In the same studies the sugar group E was found to straddle the DNA sugar-phosphate backbone with the amino group interacting with the backbone phosphates.Molecular modelling work of the hydroxylamine glycosidic bond of calicheamicin yI1 has shown that the A and B sugars adopt an unusual eclipsed conformation due to this unusual linkage.66 This conformational induced shape of the drug was shown to be complementary to the shape of the minor groove. It is thought that the hydroxylamine glycosidic linkage gives the oligosaccharides the correct shape and rigidity to allow selective binding in the minor groove of DNA. Nicolaou et a1.60 have synthesised the unnatural stereochemistry at the hydroxylamine nitrogen-bearing C-4 position. In footprinting studies the natural oligosaccharide provided concentration- dependent protection whereas the unnatural stereochemical derivative did not give the same protection at the same concentration.The monomer and the head to head dimer of the oligosac- charide segment of calicheamicin have been shown to inhibit MeSSS L HO HO NHC02Me IV' dMe OMe 16 calicheamicin yl Weymouth- Wilson The role of carbohydrates in biologically active natural products MeSSS Me L HQ MeS I OMe Me 17 esperamicin C DNA transcription factors.67*68 The DNA targets chosen contained TCCT sequences (sites which the sugar targets) which were within or adjacent to the binding sites of tran- scriptions factors. The inhibitory concentration was found to be in the micromolar range with the dimeric carbohydrate being much more effective compared with the monomer. The inhibitory effect has tentatively been attributed to the confor- mational change in DNA as the sugar interacts with it this then interferes with the transcription factors DNA recognition.The interference with the transcription factor by the carbo- hydrates also display itself with inhibition of transcriptional stimulation. This carbohydrate-induced repression of tran-scription was observed in live cells by Liu et aL6' which was not observed by Ho et al.67 The base sequence selectivity of the calicheamicin carbohydrate to DNA may open up a new arena for the chemical control of genetic information. 5.2 Esperamicin The family of esperamicins were isolated from cultures of Actinomadura verrucosospora these compounds show incred- ible antitumour activity due to DNA cleavage caused by the agly~one.~~ Esperamicin A 18 causes mostly single strand DNA cleavage and is far less sequence specific than calichea- micin y,*.Both compounds have a very similar aglycone. Structure analysis of esperamicin A d(CGGATCCG) duplex showed that the drug bound in the minor groove with the methoxyacrylanthranilate segment intercalating at the (G2-G3):(6'-C7') step.70 The isolated deoxyfucoseanthranilate group was shown not to interact with DNA however this group was shown to contribute 1-2 kcal mol -to the binding energy of esperamicin A,. When esperamicin A [double strandedxingle stranded (ss:ds) ca. 3.4 11 is compared to espe- ramicin C (ss:ds 100% ds) which lacks the deoxyfucoseanthra- nilate a change in where deoxyribose hydrogen abstraction occurs.Esperamicin C causes ds lesions involving deoxyribose hydrogen abstraction from the 4'-and 5'-positions whereas esperamicin A causes lesions from the 1'- and 5'-positions. This change to the 1'-position for esperamicin A is believed to be due the deoxyfucoseanthranilate which intercalates into the DNA. The A-B sugars of the A-B-C trisaccharide is positioned deep in the minor groove of the drug DNA complex with the C sugar directed up over the edge of the minor groove. The hydroxy groups of the trisaccharide A-B-C are positioned close to potential hydrogen bond acceptors and favourable van der Waals interactions thus help to stabilise the c~mplex.~' MeSSS Me Meyh&c he OMe 5.3 Neocarzinostatin Neocarzinostatin 19 was isolated from Streptomyces carzino- staticus Var.F-41 as a 1:l mixture of chromophore and protein.72 The chromophore exhibits potent antibacterial and antitumour activities whereas the protein component stabilises the drug. Neocarzinostatin 19 has been shown to induce single and double strand DNA scission with a ratio of 5:l ss:ds with a preference for T and A residues (T>A$C>G).73 The sequence specificity of neocarzinostatin differs from calichea- micin and esperamicin which may be partly due to the reduction in glycosylation compared to the latter compounds. There are certain similarities between the way esperamicin A and neocarzinostatin cleave whereby lesions occur from the 1'-position of the deoxyribose. Recent NMR studies74> 75 on the post-activated 20 neocarzinostatin-DNA complex (neocar- zinostatin is too reactive to be used in a DNA complex study) has shed light on the way the molecule interacts with DNA.The 2'-N-methyl-~-fucosamine naphthoate and tetra-hydroindacene of the postactivated-neocarzinostatin 20 were shown to have the correct structure to bind to specific sequences of DNA such as AGC sites. Specific sites are suggested for chemical modification which may alter selectivity of bindingcleavage one of these sites being the N-2 of the 2'-N-methyl-~-fucosamine. 6 Glycopeptides This group of antibiotics is made up of complex polypeptide aglycones to which a variety of mono- di-and tetra-saccharides are attached.76 Bleomycin A 21 is produced by Streptomyces verticillus and shows activity against Gram- positive and Gram-negative bacteria but is also a clinically useful antitumour agent.The mode of action of bleomycin is DNA strand scission which occurs in the presence of Fe2' and molecular oxygen.76 It has been shown that the sugars do not contribute to the binding affinity or the DNA cleavage selec- tivity although the presence of the sugars enhances the DNA cleavage efficiency by 2-5 times.77 There has been speculation on whether the terminal 2-O-(3-O-carbamoyl)-~-mannose moieties play any role as a ligand for the It has been shown that demannosylbleomycin A and bleomycin A are indistinguishable suggesting that the terminal sugar is not acting as a ligand. The sugars are suggested to contribute in the binding of 0 and activation and protection of the reactive iron-oxo or perferryl intermediate to activated ble~mycin.~~ 7 Macrolide antibiotics The macrolides are an important group of compounds which have seen much success due to their excellent activity against Gram-positive bacteria.Many are used clinically these include erythromycin 22 oleandomycin spiramycin josamycin and midecamycin. They are classified according to the size of the aglycone lactone ring which is either 12 14 or 16 members in size. The 12-membered ring macrolides have not been used clinically. The 14-membered ring macrolides have been studied greatly of which erythromycin A 22 has been used clinically for years. The mode of action of erythromycin antibiotics involves HO ,OMe IJ Me I OH 'OMe 09OMe 18 esperamicinA 104 Natural Product Reports ye Me OMe I OH 19 neocarzinostatin 2'-N-methyl-D-fucosamine OH Me /Me OH 0 naphthoate HO 0 R = H02CCH(NH2)CH2CH2CONHCHCONHCH2C02H I CH2(S) 20 post-activated neocarzinostatin IY I Me 0 L-S AMeVs' WIG HO I H I OH L-gulose OH -3-Ocarbomoyl-D-manose OANH2 21 bleomycin A2 the inhibition of protein synthesis of specific binding to the 50s ribosomal subunit but with a specific target at the 23s ribosomal subunit and various proteins." The binding is competitive which appears to be a 1:1 stoichiometry between the drug and the ribosomal subunit.80 However the exact interaction of the macrolide and the ribosome subunit is still not fully understood.The current theory states that the macrolides stimulate dissociation of peptidyl-tRNA from the ribosome during the elongation phase,81 which in turn would inhibit protein synthesis. In principle the macrolide antibiotic should inhibit mammalian mitochondrial protein synthesis but they are unable to penetrate the mitochondrial membrane and so the agent is not cytotoxic. Early work established that the sugar moieties of the macro- lides are essential for the microbial activity of the com-po~nd.~*-~~ It is difficult to consider the activities of the sugar 0 0 OR1 L-cladinose R' = Me; R2 = H OR2 L-mycarose ~1 = ~2 = H 22 erythromycin A R1= Me; R2 = R3 = H; R4 = OH 23 erythromycin B R1= Me; R2 = R3 = R4 = H 24 erythromycin C R1 = R* = R3 = H; R4 = OH 25 erythromycin D R1 = R2= R3 = R4 = H without the aglycone as the exact mode of action is unclear.However the sugar moieties have been linked with many pharmacological properties displayed by the macrolides anti- biotic~.*~ The presence of the basic nitrogen has been linked with the possibility of active transport of the drug into the cell although whether the amino sugar is protonated or not does not affect cellular uptake. Studies have shown that the more basic macrolides are the more potent ones.86 The 2'-esters of erythromycin are preferred due to their favourable pharmaco- logical and pharmacokinetic properties. These esters of erythromycin show lower incidences of gastrointestinal side effects and it is known that these unhydrolysed esters have biological activity.87 Erythromycin is acid labile and undergoes decomposition to give the 6,9,9,12-spiroketal 27 via the 8,9-anhydro-6,9- hemiketal 26 (Scheme 3) with these products having low Erythromycin Me Me IH+ Ho.:B e ! M N(Me)2 Me'. Me Me 00 Me 26 ye Me Me0 e M e ' 0 0 OH Me 27 Scheme 3 antimicrobial activity. Much of the recent research has been aimed at the synthesis of acid stable macrolides.88 The 16-member macrolides e.g. miocamycin 28 are found to bind to 23s rRNA. These compounds are found to inhibit Weymouth- Wilson The role of carbohydrates in biologically active natural products 105 OAC A.-Me Me 28 miocamycin peptidyltransferase reactions whereas the 14-membered macrolides inhibit the translocation of peptidyl-tRNA.’l 7.1 The side effects of macrolides 7.1.1 Gastrointestinal It is well known that erythromycin readily decomposes due to the action of stomach acid to give firstly the 8,9-anhydro-6,9- hemiketal 26 and then the 6,9,9,12-spiroketal 27 (Scheme 3) these compounds tend not to have antibacterial activity.Furthermore the 8,9-anhydro-6,9-hemiketalwas found to bind to the same receptors as motilin the peptide hormone responsible for intestinal smooth muscle contraction thus causing gastrointestinal side effects.89 Structure-activity studies on the hemiketal has revealed that the dimethylamino group on desosamine and the neutral sugar cladinose are essential for activity.89 Modifications on the 3‘-amino group increased smooth muscle contraction whereas acylation of the 4”-OH led to compounds with greatly reduced activities.” A recent publication describes the 4”-deoxy derivative of L-cladinose has an activity of an order of magnitude greater than the natural compound.91 These new compounds are now under investigation as potential agents for the treatment of gastrointestinal motility disorders such as diabetic gastro- paresis nonulcerative dyspepsia irritable bowel syndrome and paralytic ileus.It is also noted that the sugar moieties attached in parallel to the aglycone rather than series was important for activity.92 7.1.2 Cytochrome P450 complexes The formation of inactive cytochrome P450 complexes with macrolides can interfere with the metabolism of other The N(CH,) group is oxidised to the corresponding NO group which then binds strongly to the haem moiety with this binding being dependent on the accessibility of the amine.94 It has been shown that esterification at the P-position of the dimethylamino group of desosamine leads to a lowering in pK leading to a better drug cytochrome P450 complex.95 This has been shown not to be the case for all esters as the 2’-propionate ester of erythromycin forms very little of the complex whereas the introduction of a benzoate ester at the same position prevents complex formation occurring.This has led to NMR studies of various 2’-esters of erythromycin and erythrosamine with the aim of correlating their confor- mations as to the abilities of the compound to complex with cytochrome P450.95 This work shows that the different aglycone conformations were observed which in turn induced five orientations of the desosamine sugar.It has been found that certain conformations correlated well with the formation of cytochrome P450-nitroso-metabolite complexes. 8 Polyenes The polyene macrolides are characterised by having a macro- cyclic lactone a polyene chromophore and a polyhydroxylated chain which often processes a glycosidic residue. Most of the polyenes are produced by soil actinomycetes of the genus Streptomyces. These compounds show excellent activity against a variety of dermatophytes and fungi but little or no 106 Natural Product Reports antibacterial activity.Amphotericin B 29 and the closely related structure nystatin A 30 are used clinically how- ever they cause serious side effects such as nephrotoxicity haemolytic anaemia and electrolyte abn~rmalities.~~ OH OH ,OH c02w Me‘ D-mycosamine 29 amphotericin B OH OH .OH OH 0.. HOVlelco2w 6 Me OH 8.1 Mode of action The mode of action of the polyenes is not fully ~nderstood,~~ although it is known that they interact with sterol containing plasma membranes with a preference for prokaryotic cells (ergosterol containing) over mammalian cells (cholesterol con- taining). De Kruijff and Deme198 have proposed that ampho- tericin B 29 (A) and cholesterol (C) in the plasma membrane forms an aggregate (-A-C-)’ cylindrical complex with the hydroxy groups of amphotericin facing each other while the lypophilic backbone interacts with the sterol.This half pore which is deemed quite mobile could then align with another half pore from the other side of the plasma membrane allowing a channel to form in which ions can be leached.99 Structure-activity work on amphotericin B 29 has shown the importance of the basic nitrogen of the sugar D-mycoFsmine for activity. loo The polyene is orientated with the polar head at the membrane-water interface and it has been suggested that the basic amino group and the carbonyl group forms a ‘cage’ like hydrogen bonded structure with sterol containifig a 34-(4 ,H-O<-i) H ste roi -0; amphotericin B H‘ -O\ C d’ (b) H-O(-i] Y sterol-0; amphotericin B RO\ C d’ R=Me Fig.2 Schematic view of hydrogen bonds formed between the polyene derivative’s polar head and the sterol hydroxy group hydroxy group and water. It was found that when both the carbonyl function and the amino function was present the polyene did not show less selectivity for ergosterol over cholesterol. This result was explained because the hydrogen bonds in the system to the 3-P-hydroxy sterol were strong and that the hydrophobic interactions with the alkyl chain of the sterol became secondary [Fig. 2(a)] When the acid group is esterified the amino group is free to rotate which leads to a system that is relatively weak so the hydrophobic interactions become an important feature [Fig.2(b)]. This led to the polyene becoming more selective for ergosterol than cholesterol as the polyene has greater affinity for sterols containing a double bond at the C-22 position. A number of derivatives of amphotericin B have been reported"' in which the N-glucosyl derivative shows reduced toxicity and similar activity to the parent compound but increased water- solubility. 9 Phosphoglycolipids The phosphoglycolipid antibiotics are a group of related com- pounds that contain an oligosaccharide moiety phosphoric 8' lo-. '0' 0-P=O .NHAc OH AcHN NH I OYYO acid and a lipid unit which is either moenocinol or diumyci- nol. lo2 These substances were isolated from Streptomyces cultures and show good activity mainly against Gram-positive bacteria.The most studiedlo3 of the phosphoglycolipid antibiotics is moenomycin A 31 which has been shown to inhibit the appearance of the linear glycan of peptidoglycan from 33. The N-acetylglucoaminyl-N-acetylmuramyl-(pentapeptide)-pyrophotyl-undecaprenol 33 is the final precursor before polymerisation a two step process which consists of trans- glycosylation and transpeptidation. Moenomycin A 31 appears to be a competitive inhibitor of the peptidoglycan polymerase of E. coli and the penicillin-binding protein which is the enzyme responsible for transglycosylation. lo4 9.1 Structure-activity relationship Structure-activity relationships have been carried out on moenomycins A 31 and C 32 which has established the minimum active structure^.'^^ These results show that for moenomycin A the disaccharide 35 is the smallest unit required for activity whereas studies on moenomycin C H&Oo 31 moenomycin A R1 = Me; R2 = OH; R3= HO HO 32 moenomycin C R1 = OH; R2 = R3 = H NHAc ,OH m-DAP I D-Ala I D-Ala 33 Kacetylglucoaminyl-l\l-acetylmuramyl(pentapeptide)prophotylundecaprenol 34 = Me; R2= OH; R3 = HO H&Oo HO 35 R' = OH R2= R3 = H Weymouth- Wilson The role of carbohydrates in biologically active natural products have shown that the trisaccharide 34 was necessary for similar biological activity.lo6 The carbamoyl group in the moenuronic acid moiety was found to be essential for activity along with the phosphate group C acid group D and the alkyl chain E which can be saturated.The simplified products of moemycin A and C possess closer structural analogy to the bacterial cell wall precursor 33 and allow for an easier synthetic approach which could prove important in the synthesis of analogues.'o7 10 Miscellaneous This review has covered compounds for which structure-activity has been established and in which the mode of action has been or is being elucidated. There are however many interesting carbohydrate natural products in which the mode of action of the carbohydrate moiety is not fully understood some of which are mentioned below. 10.1 Angucycline antibiotics The angucyclines,'os most of which are glycosylated belong to an expanding family and show interesting biological activities.The angucycline PI-080 36 shows inhibition of platelet aggre- gation with the aglycone found to be inactive thus showing that the sugars play an important role in activity. MeF ? OH oMe$ 0 Me+0 36 PI-080 10.2 Recently discovered enediynes The enediyne antibiotics are an expanding family of com-pounds with the precise role of the sugar of the newer compounds such as kedarcidin"' 37 having not yet been fully evaluated. \/ 0 WOH OMe h070 HO M*o Me 37 kedarcidin 10.3 Cholestane glycosides The cholestane glycosidel10 named OSW-1 38 which is extracted from the bulbs of Ornthogalum sandersiae was shown to be more potent in vivo against lung cancer and certain types of leukaemia compared to established drugs such as adriamycin cisplatin and taxol.The compound was shown to be less toxic to normal cells when compared with other drugs in vivo against leukaemia in mice. 0 -...icy HO HHoWo&H HO 0 Me0H0 38 OSW-1 10.4 Marine natural products The isolation of natural products from marine microorgan- isms"' is still in its infancy when compared to its terrestrial counterparts. 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ISSN:0265-0568    

DOI:10.1039/NP9971400099

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

The biosynthesis of C&, terpenoid compounds Paul M. Dewick Department oj Pharmaceutical Sciences University of Nottingham Nottingham UK NG7 2RD Covering 1993 to 1995 Previous review 1995 12 507 1 Introduction 2 Mevalonic acid 3 Hemiterpenoids 4 The dihydroxyacetone phosphate pathway 5 Prenyltransferases 6 Monoterpenoids 7 Sesquiterpenoids 8 Diterpenoids 9 Sesterterpenoids 10 References 1 Introduction This report reviews the literature that was published during the period 1993-1995 on the biosynthesis of terpenoids in the range C5-& and continues the coverage described in Volume 12 of Natural Product Reports.' This three-year report brings coverage effectively up to date and hopefully future reports will now be produced on a more regular basis.This report describes the biosynthetic pathways leading to those terpe- noids smaller than C30 (terpenoids larger than C30 are covered separately under triterpenoids and steroids and carotenoids) the enzymes and enzyme mechanisms involved and informa- tion about genes encoding for these enzymes. A number of specific aspects more appropriate to other journals are not included. Such topics as the regulation of terpenoid biosyn- thesis particularly where the emphasis relates predominantly to steroidal compounds and higher terpenoids the genetic control of biosynthesis and biotransformations are not covered. The biosynthesis of meroterpenoids is also omitted. These compounds contain a terpenoid unit as part of a more complex structure and are adequately treated in other reports according to the major substructure e.g.alkaloids polyketides and shikimate metabolites. Some recent review articles describe general aspects of terpenoid biosynthesis and metabolism. These include biosyn- thesis of mono- sesqui- and di-terpenes,2 new aspects of isoprenoid biosynthe~is,~,~ biochemistry and molecular biology of terpenoid metabolism,5p7 sesquiterpene cyclases,8 and environmental effects on isoprenoid biosynthesis.' Volume 9 in the series Methods in Plant Biochemistry" is devoted to 'Enzymes of Secondary Metabolism' and contains several articles of interest; prenyltransferases and cyclases,' 'cyto-chrome P-450terpene hydroxylases,I2 enzymes of gibberellin biosynthesis,l3 and abscisic acid metabolism.l4 A special issue of Plant Growth Regulation is devoted to gibberellins. ' 2 Mevalonic acid The condensation of acetyl-CoA and acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) 1 is catalysed by the enzyme HMG-CoA synthase (Scheme 1). Two forms of the enzyme are known in mammals a cytosolic enzyme which is the starting point for the mevalonic acid (MVA) pathway and a mitochondria1 enzyme which together with HMG-CoA lyase is involved in ketone body synthesis. The latter mito- chondrial enzyme is not considered further in this report. An avian recombinant HMG-CoA synthase has been obtained by cDNA cloning and expression in E. coli the soluble enzyme Dewick The biosynthesis of CS-C, terpenoid compounds YSCOA ii n ysc-A n iii INADPH 4 MVA 5-diphosphate 3 MVA 5-phosphate 2 MVA vi ATP 1 @Boy 2*@@OT 5 IPP 6 DMAPP Scheme 1 Enzymes i acetoacetyl-CoA thiolase (AACT); ii HMG- CoA synthase; iii HMG-CoA reductase (HMGR); iv mevalonate kinase; v phosphomevalonate kinase; vi mevalonate 5-diphosphate decarboxylase; vi IPP isomerase accounting for more than 20% of the total cellular protein.I6 The recombinant enzyme was identical to the normal wild-type enzyme.By directed mutagenesis it was demonstrated that a cysteine residue Cys-129 was essential for formation of a covalent acetyl-enzyme reaction intermediate. A second reactive cysteine has also been implicated but none of those at five other possible sites was crucial for the reaction.l7 Human enzyme has also been obtained by gene cloning and expression in E. coli." Again mutation of Cys-129 to Ser or Ala was shown to destroy the activity by disrupting the first catalytic step enzyme acetylation by acetyl-CoA. Two HMG-CoA synthase genes have been identified in the insect Blattellu gerrnani~a.",~~ Both genes have been cloned and sequenced and shown to have similar amino acid sequences to vertebrate enzymes. The two insect proteins had 69% identity at the amino acid level but were expressed to different extents as the insect developed. Studies of plant-derived HMG-CoA synthases have sug-gested that in radish (Raphanus sativus) both this activity and that responsible for ace t oace tyl-CoA synthesis (ace toacetyl- CoA thiolase) are localized on a single polypeptide.2'.22 Thus the soluble monomeric protein from radish membranes cata- lysed the two-step conversion of acetyl-CoA into HMG-CoA and unlike yeast or animal systems could not be resolved into separate activities.Activity was lost on purification but could be reconstituted by the addition of Fe2' chelates and quinone cofactors especially pyrroloquinoline quinone. It is suggested that the reaction mechanism may involve radical formation by the redox couple Fe2'/quinone mimicking a natural cofactor system which has yet to be identified and facilitating the unfavourable Claisen reaction. In Catharanthus roseus a simi- lar situation probably exists and although the two activities acetoacetyl-CoA thiolase and HMG-CoA synthase were sep- arately proved to be operating they could not be separated and a single protein may be involved.23 A cytosolic HMG- CoA synthase preparation from rubber (Hevea brasiliensis) has been isolated; this preparation also utilized acetyl-CoA 111 HL without any requirement for an exogenous supply of acet~acetyl-CoA.'~ A cDNA from Arabidopsis thaliana that has been isolated and characterized showed a strong similarity in the deduced amino acid sequence with mammalian HMG- CoA syntha~e.~~ In marked contrast to the above studies this HMG-CoA synthase was devoid of any acetoacetyl-Co thiolase activity.The next enzyme on the mevalonic acid biosynthetic path- way is HMG-CoA reductase (HMGR) which catalyses the ASP-766 K O ?J H I 1 HMG-COA '1 I 7 mevaldate &sco~ thiohemiacetal NADPH-dependent reduction of HMG-CoA to mevalonate H-(MVA) 2 (Scheme 1).This enzyme activity provides an import- (NAD P H) ant control mechanism for the flow of metabolites into meval- onate and especially into steroid biosynthesis and its study continues to stimulate much research. Recent reports on the isolation and sequencing of genes encoding HMGR include those from Syrian hamster,26 the cockroach Blattella ger- mani~a,~'fungi Parasitella parasitica Absidia glauca Mucor mucedo Blakeslea trispora2' and Ustilago maydi~,~~ the plants wheat (Triticum ae~tivum),~' rice (Oryza ~ativa),~ radish Camptotheca a~uminata,~~ (Rhaphanus ~ativus),~~ rubber (Hevea brasiliensi~),~~ Arabidopsis thaliana,35 and potato (Sola-num t~berosum).~~ Plants tend to possess small multigene families rather than a single gene as in animals.Wheat is reported to have four genes,3o radish two genes,32 rubber three genes,34Arabidopsis two genes35 and potato seven genes.36 The fungi Mucor mucedo and Parasitella parasitica also each con- tain two genes.28 The enzyme from Pseudornonas mevalonii has been ~rystallized.~~ HMGR is considered a key regulatory enzyme controlling isoprenoid metabolism in mammals and fungi but the rate- limiting nature of this enzyme in plants is less well defined. Expression of one of the rubber (Hevea brasiliensis) HMGR genes in tobacco plants resulted in an increase in HMGR activity and about a six-fold increase in total sterol levels covering both intermediates and end-prod~cts.~~ Introduction of the hamster gene into tobacco gave a three- to six-fold increase in HMGR activity a three- to ten-fold total sterol increase but a much lower (two-fold) increase in end-product sterols.38 Cycloartenol levels were up more than 100-fold but levels of other isoprenoids were relatively unaffected.These results were interpreted to indicate that compartmentation channelling or other rate-limiting enzymes must also be involved in determining relative accumulation of terpenoids. Two HMGR genes in potato are expressed at different levels by methyl jasmonate or by fungal elicitors.39 The accumula- tion of hmgl transcripts by treatment with methyl jasmonate results in production of steroid glycoalkaloids whilst hmg2 transcripts that accumulate from treatment with arachidonic acid (the fungal elicitor) are responsible for the biosynthesis of sesquiterpene phytoalexins.The mechanism of the reaction catalysed by HMGR from Syrian hamster has been probed by site-directed mutagenesis His-865 Glu-558 and Asp-766 have all been established as essential for enzyme activity. The catalytic domains of wild-type enzyme and mutants E558Q (Glu-558 replaced by Gln) H865Q and D766N were over-expressed and investigated with respect to partial reactions also catalysed by HMGR. Only the wild-type enzyme catalysed all five reactions studied reductive deacylation of HMG-CoA to MVA reduc- tive deacylation of mevaldate thiohemiacetal 7 to MVA reduction of mevaldate 8 to MVA oxidation of mevaldate thiohemiacetal to HMG-CoA and oxidative acylation of mevaldate to HMG-CoA.Mutant D766N was inactive for all reactions but E558Q and H865Q catalysed various reactions at significant rates. In particular H865Q catalysed the reduc- tion of mevaldate to MVA at about the wild-type rate. Based on these and related data it was deduced that Asp-766 functioned during conversion of HMG-CoA 1 into mevaldate thiohemiacetal 7 and mevaldate 8 into MVA with Glu-558 and His-865 playing a role during the mevaldate hemithioacetal/mevaldate transformation. The suggested mechanism is shown in Scheme 2. The catalytic domain of 112 Natural Product Reports H O"ibi Jvvv.Glu-558 1 .AAAr I 0Lo-? 6 2 MVA 0 8 mevaldate I R+HH c-R+H YCoA H-H (NADPH) (f N I H "Yo-Scheme 2 Enzyme HMG-CoA reductase HMGR from Arabidopsis thaliana has been expressed in E. coli to yield an enzyme of extremely high specific The activity was reversibly inactivated by phosphorylation at a single site (Ser-577) using the enzyme HMGR kinase from Brassica oleracea. It is concluded that the activity of higher plant HMGR may be regulated by direct phosphorylation. A common feature of all known plant HMGR isoforms is the presence of two highly conserved hydrophobic sequences in the N-terminal quarter of the protein. Study of the Arabidopsis enzyme has led to the proposal that the two hydrophobic sequences become a membrane spanning segment with just a short interconnecting loop exposed to the lumen.The N-terminal end and the C-terminal catalytic domain are then positioned on the cytosolic side of the membrane.& The role of HMGR in the biosynthesis of terpenoid phyt~alexins~~ and HMGR inhibit01-s~~ have been reviewed. 3 Hemiterpenoids Mevalonate kinase catalyses the ATP-dependent phosphoryla- tion of mevalonate to mevalonate 5-phosphate 3 the first of the reactions leading up to isopentenyl diphosphate (IPP) 5 and dimethylallyl diphosphate (DMAPP) 6 (Scheme 1). The substrate and inhibitor specificity of pig liver mevalonate kinase have been studied with a range of substrate analogues.47 Substitution of the hydroxy group and/or methyl at C-3 yielded a range of inhibitors whilst systematic chain extension of the 3-methyl gave non-competitive inhibitors.Methylation at C-2 and C-4 produced competitive inhibitors which were also quite good substrates. Reliable assays for the three enzymes mevalonate kinase phosphomevalonate kinase and mevalonate 5-diphosphate decarboxylase have been de~cribed.~' The mechanism of action of mevalonate 5-diphosphate decarboxylase has been probed by studying its reaction with some substrate analogues.49 The fluoromethyl analogue 9 was decarboxylated 2500-fold more slowly than 4 9 10 11 and was a competitive inhibitor of the enzyme. At saturating concentrations of 9 and ATP nearly one equivalent of 3-phosphorylated 9 and ADP were bound to the enzyme.The 3-demethyl derivative 10 was phosphorylated at the 3-hydroxy group and released without decarboxylation. Both these ana- logues have decreased electron density at C-3 relative to the normal substrate and the transition state for the decarboxyla- tion step therefore has considerable cationic character. In confirmation the 3-aza analogue 11was also a good inhibitor. The results strongly support a two-step mechanism for the enzyme in which phosphorylation of the 3-hydroxy group is followed by decarboxylation and the transition state has carbocationic character (Scheme 3). 4 MVA 5-diphosphate 5 IPP Scheme 3 Enzyme mevalonate 5-diphosphate decarboxylase Isopentenyl diph0sphate:dimethylallyl diphosphate isomer- ase (IPP isomerase) catalyses the 1,3-allylic rearrangement reaction converting IPP into DMAPP via a postulated two- base cationic mechanism (Scheme 4) resulting in the observed antarafacial transposition of hydrogen.Active site cysteine residues Cys-138 and Cys-139 have been identified in the yeast (Saccharomyces cerevisiae) enzyme over-expressed in E. coli. In recent studies Glu-207 has also been found to play a role.50 A mutant C139S (Cys-139 replaced by Ser) was active but was a poor catalyst. When treated with 3-(fluoromethyl)but-3-enyl diphosphate an electrophilic active-site-directed irreversible inhibitor the enzyme became inactivated by covalent modifi- cation of Glu-207. The mutants E207Q and E207V were found to be totally inactive.The nucleotide sequence of the IPP isomerase gene from Clarkia breweri has been rep~rted.~' The deduced amino acid sequence was found to be 90% identical with that from Arabidopsis but less than 50% identical with the sequence from yeast. Isoprene 12 is traditionally associated with the terpenoids as the hypothetical building block but this hemiterpene is also a natural product emitted by a number of plants. An enzyme isoprene synthase catalyses the elimination of diphosphate from DMAPP though an equivalent non-enzymic acid- catalysed reaction is feasible (Scheme 5). In velvet bean Dewick The biosynthesis of CrCzs terpenoid compounds -I I +BH* 6 B @@o+HE ' Hs HZ +BH~DMAPP I .MN. Scheme 4 Enzyme IPP isomerase +L / DMAPP 12 isoprene Scheme 5 Enzyme isoprene synthase (Mucuna sp.) changes in extractable isoprene synthase activity exactly parallel isoprene emission thus supporting the enzymic derivation of this volatile hydr~carbon.~ The enzyme from aspen (Populus tremuloides) has been purified and shown to be a heterodimer dependent on Mg2+ or Mn2' for Partial amino acid sequences for the subunits indicated they were closely related but they did not share strong homology with any other reported proteins.4 The dihydroxyacetone phosphate pathway A number of studies have provided evidence that some bac- teria employ a biosynthetic pathway to terpenoids which is different from the well-established mevalonate path~ay.~~,~~ The origins of IPP in cell-free extracts of several bacteria were studied.Extracts from Myxococcus fulvus Staphylococcus carnosus Lactobacillus plantarum and Halobacterium cutiru- brum all converted labelled acetyl-CoA or HMG-CoA into MVA. Also labelled MVA MVA 5-phosphate and MVA 5-diphosphate were all metabolized to IPP thus showing operation of the normal pathway via acetoacetyl-CoA and MVA. In contrast no intermediates of this reaction sequence could be detected using cell-free extracts of Zygomonas mobilis or E. coli. In further studies with 13C-labelled glucose acetate pyruvate and erythrose substrates the origins of the carbon atoms of ubiquinones and triterpenoid hopanes were investi- gated., In 2. mobilis E. coli Methylobacterium fujisawaense and Alicyclobacillus acidoterrestris the labelling patterns were consistent with a novel route for the early steps of isoprenoid biosynthesis and explained some earlier incompatibilities noted when the biosynthesis of hopanoids had been studied.The C framework of the isoprenoid units is most probably built up by condensation of a C unit derived from decarboxy- lation of pyruvate (i. e. a thiamine-activated acetaldehyde 13) onto the C-2 carbonyl of a triose phosphate derivative 14 (Scheme 6). The triose phosphate is probably formed from dihydroxyacetone phosphate and not from pyruvate and several alternative possibilities could be considered. Lastly there is a transposition step in which a methyl migrates from the triose C unit to the C unit. The whole sequence resembles that operating in L-valine biosynthesis but this amino acid and 113 H+ G<'% 7 pyruvate thiamine diphosphate 0 dihydroxyacetone H+ 14 triose phosphate OH X = ( C@( @@( =o phosphate derivative HTHTH 9 R IPP L O W o@@ DMAPP Scheme 6 its C precursors were excluded as intermediates.This pathway is not operative in chloroplasts from young spinach (Spinacia oleracea) plants.56 Acetate was a much better precursor of chloroplast isoprenoids than pyruvate and the normal mevalonate pathway was utilized. 5 Prenyltransferases Prenyltransferases are responsible for the alkylation steps involving DMAPP and one or more IPP residues reactions which provide the polyprenyl diphosphate precursors for the various terpenoid families.Many prenyltransferase enzymes are relatively non-specific and catalyse the condensation of DMAPP with IPP units building up polyprenyl diphosphate chains of different lengths. A geranyl diphosphate synthase purified from grape vine (Vitis vinifera cv. Muscat de Frontig- nan) cell cultures formed only geranyl diphosphate (GPP) as the product.57 Divalent metal ions Mn2+ or Mg2+ were required as cofactors. Available evidence points to the initial ionization of the chain starter unit to provide an allylic cation prior to addition of IPP extender units (Scheme 7). An allylic cation has now been detected by NMR spectrometry in the reaction of GPP 15 and IPP to farnesyl diphosphate (FPP) 17 catalysed by avian FPP synthase.,' Incubation of [1-13C]GPP and IPP with the enzyme led to detection of signals for C-1 of GPP C-1 of -0@@ 15 GPP I OW 1 OW 17 FPP Scheme 7 Enzyme farnesyl diphosphate synthase I14 Natural Product Reports geraniol C-1 of linalool and C-5 of FPP with a further signal assigned to and consistent with C-1 of the allylic cation 16.This signal was also observed in the absence of IPP and was not present when heat-treated enzyme was used. The signal disappeared on addition of the inhibitor 6,6'-dinitro-3,3'-disulfanediyldibenzoic acid but this treatment did not affect either FPP or GPP signals. The new signal reappeared on further treatment with the reactivator dithiothreitol and the FPP signal increased whilst the GPP signal decreased. A thermos ta ble FPP syn t hase from Bacillus stearo thermophilus has been obtained by molecular cloning and over-expression in E.~oli.,~ The recombinant protein was identical to the native enzyme and was still active after 70 min at 65 "C.The deduced amino acid sequence showed 42% similarity with E. coli FPP synthase. In contrast to thermolabile prenyltransferases which have 4-6 cysteine residues this thermostable enzyme carried only two but mutagenesis studies showed neither was essen- tial for catalytic function.60 Three C-terminal amino acids Arg-295 Asp-296 and His-297 were similarly shown to be non-essential for activity despite the arginine residue being completely conserved throughout FPP synthases of pro-karyotes and eukaryotes.61 A single gene encodes for FPP synthase in Arabidopsis thaliana and the deduced amino acid sequence of the enzyme showed ca.50% identity with that known for yeast rat and human enzymes.62 The Arabidopsis gene was able to functionally complement a yeast mutant deficient in the enzyme. Two cDNA clones encoding FPP synthase were isolated from white lupin (Lupinus albus) and expressed in E. c01i.63,64The deduced amino acid sequences were 90% identical ca. 80% identical to the Arabidopsis enzyme 51% to the yeast enzyme and 44% to rat and human systems. One enzyme displayed both GPP synthase and FPP synthase activities. Polyprenyl transferases from a variety of organisms contain several highly conserved amino acids including aspartic acid rich domains. Site-directed mutagenesis studies with rat FPP synthase have established that Asp- 104 Asp-107 Arg-112 Arg-113 and Asp-243 are essential for catalytic activity and are presumably involved in either the condensation or release steps.65 Similar experimentation using yeast enzyme has indicated that mutations affecting Asp- 100 Asp- 10 1 Asp- 104 Arg- 109 Arg- 1 10 Asp-240 and Asp-241 dramatically lowered catalytic activity.66 The crystal structure of avian liver FPP synthase has been reported.67 Two aspartate-rich sequences were found on opposite walls of a large central cavity suggesting these residues participate in substrate binding or catalysis.The catalytic versatility of avian liver FPP synthase has been demonstrated by incubating the enzyme with substrate analogues containing both DMAPP and IPP moieties joined by a one-carbon bridge in a single molecule.68 69 Thus 18 was transformed into 20 and 21 suggesting a sequence in which ionization of the DMAPP portion then allowed alkylation by the IPP portion but in doing so generating a cyclic cationic product 19 (Scheme 8).A 1,2-hydride shift established by labelling studies occurs in the formation of 21. The products were optically active as shown indicating strict control of the substrate conformation. Pig liver FPP synthase will accept a wide range of substrate analogues with modified hydro- carbon chains or with ether linkages.70 71 The enzyme from Bacillus stearothermophilus was more stringent in its specificity and analogues with oxygen-containing chains were poor 18 1 11,Phydride shift I I 21 Scheme 8 Geranylgeranyl diphosphate (GGPP) synthase from bovine brain has been purified and shown to be specific for the condensation step between FPP and IPP.733 74 A separate FPP synthase enzyme utilizing DMAPP and IPP was also purified indicating the synthesis of GGPP 22 from C precursors requires the action of two enzymes.GGPP synthase was a homooligomer with 4-5 subunits whereas FPP synthase was a homodimer. Two different GGPP synthase activities giving different products have been detected in rat tissues.75 A membrane associated activity synthesized (E,E,Z)-GGPP and is involved in the biosynthesis of long chain polyprenols whilst a cytosolic activity producing (E,E,E)-GGPP is concerned with protein prenylation reactions.Most eubacteria and eukaryotes seem to possess distinct FPP and GGPP synthases. In contrast bifunctional FPP/GGPP synthases from the archaebacteria Methanobacterium thermoforrni~icum~~ and M. thermoauto-trophi~um~~ have been isolated and purified. These enzymes are thermostable with optimal activity at 65 'C require Mg2+ or Mn2+ as cofactor and catalyse prenyl transfer of IPP to all three substrates DMAPP GPP and FPP. It thus appears that only one prenyltransferase enzyme is involved in the biosynthesis of squalene and other polyprenyl lipids. The gene encoding the M. thermoautotrophicum enzyme has been isolated and sequenced.78 The deduced amino acid sequence for the protein contained five conserved regions as found in eubacterial and eukaryotic FPP and GGPP synthases including aspartate-rich motifs commonly found in prenyl- tran~ferases.~~ An Erwinia uredovora enzyme produced by overexpression of the gene in E.coli would accept both FPP and GPP as substrates but not DMAPP.80 Gene sequences encoding for GGPP synthase have been characterized from a variety of sources including pepper (Capsicum annuum),81 The procedure for obtaining a thermostable GGPP synthase from Sulfolobus acidocaldarius by gene cloning has been patented .8 Activities for three key enzymes in the biosynthesis of membrane lipids namely IPP isomerase GGPP synthase and geranylgeranylglyceryl phosphate (GGGP) synthase were found in cytosolic fractions from the strict anaerobe Methano-bacterium thermoautotrophicum and the extreme halophile Halobacterium halobium.86 The membrane lipids consist of glyceryl ethers bearing saturated isoprenoid side-chains e.g.26 and in M. thermoautotrophicum the diethers are then joined covalently at the ends of the isoprene chains to form dimeric membrane spanning tetraethers e.g. 27 (Scheme 9). In both organisms (5')-glyceryl phosphate 23 was the preferred acceptor for the prenyltransferase reaction catalysed by GGGP synthase though both GGPP and phytyl PP could be utilized. Two enzyme activities were subsequently separated a cytosolic enzyme catalysing the alkylation of (8-glyceryl phos- phate by GGPP to give the monoether 24 and a microsomal enzyme catalysing a second alkylation to produce 25.87The cytosolic enzyme was purified and shown to require a divalent metal ion as the cofactor with Mg2+ preferred.88 A novel farnesylgeranyl diphosphate (FGPP) synthase from the haloalkaliphile Natronobacterium pharaonis has been sepa- rated from GGPP synthase which is the major prenyltrans- ferase in this organism.89 This enzyme had highest activity with GGPP but also accepted DMAPP GPP and FPP.The GGPP synthase was most active with GPP whilst DMAPP was preferred to FPP. The enzyme is believed to be involved in the biosynthesis of C,,-containing diether lipids such as 28 and the FGPP precursor may thus be formed by the action of just two activities GGPP synthase and FGPP synthase the former enzyme probably utilizing DMAPP.A review discussing the role of prenyltransferases in the biosynthesis of a range of isoprenoid systems has been published.90 6 Monoterpenoids The conversion of geranyl diphosphate (GPP) into simple cyclic monoterpenes involves initial isomerization to (3R)-( -)-or (3S)-(+)-linalyl diphosphate (LPP) (29 and 33). GPP is typically bound to the enzyme as a complex with a divalent metal ion and undergoes ionization to the allylic linalyl cation 29 which allows formation of LPP and the opportunity for cyclization via the stereochemically favourable cation 32 (Scheme 10). A series of cation-diphosphate ion pairs partici- pates in the sequence and both the isomerization and cycliza- tion reactions are catalysed by a single enzyme.Interestingly the biosynthesis of linalool34 in the flowers of Clarkia breweri does not proceed as far as LPP but involves quenching of the linalyl cation with water." An enzyme isolated from the freshly opened flowers converted GPP into (8-linalool but would accept neither enantiomer of LPP as substrate. The enzyme showed a preference for Mn2' over Mg2+ as cofactor. Linalool is one of the volatiles in the floral scent of C. breweri and is subsequently transformed into the other characteristic monoterpenes 6,7-epoxylinalool 35 and the linalool oxides (36 and 37).92 Linalool synthase activity is highest in the petals stigma and style and no such activity was detected in leaves which merely hydrolysed GPP to geraniol 33. Geraniol is the precursor of citral 38 in leaves of lemongrass (Cymbopogon jlexuosus) and an NADP' -dependent geraniol dehydro- genase has been ~haracterized.~~ The reaction catalysed was reversible and the enzyme activity was distinct from alcohol de h ydrogenase.Acyclic monoterpene alcohols ipsenol 39 ipsdienol 40 and amitinol 41 are components of the aggregation pheremones produced by the pine bark beetles Ips paraconfusus and Ips pini. In previous studies it had been established that these were derived from the olefine myrcene 42 and since this is a Arabidopsis thaliana82 83 and white lupin (Lupinus ~lbus).~~ component of the pine trees colonized by the beetles it was Dewick The biosynthesis of CrCZ5 terpenoid compounds \ \O 22 GGPP B@ C \ OH @ 0O/ T/ o/ H/ T 23 24 kGGPP 25 I 1 26 -1 ,I 0 "LOH 27 Scheme 9 28 GPP 29 linalyl cation 30 (3R)-LPP 31 (3s)-LPP OH 39 ipsenol 40 ipsdienol 41 amitinol 42 myrcene 33 geraniol 34 (S)-linalool 32 a 44 I I 36 linalool oxide thought the alcohols were biotransformation products.In (pyranoid) recent studies,94 feeding experiments in male beetles using labelled acetate demonstrated de nuvu biosynthesis without any requirement for plant-derived myrcene; female beetles did not 38 citral 35 6,7-epoxylinalool produce any pheremone components. The enzyme limonene synthase catalyses the isomerization- cyclization of GPP to limonene the chirality in the product HoA being determined by the enzyme and its source.Thus 37 linalool oxide limonene synthase from spearmint (Mentha spicata) yields (furanoid) (4S)-(-)-limonene 43 whilst that from unshiu orange (Citrus Scheme 10 116 Natural Product Reports unshiu) produces (4R)-( +)-limonene. How the different stereo- chemistries are generated has been studied using deuterium- labelled GPP and LPP substrates with partially-purified enzyme systems from the two sources.95 Thus (1S)-[1-2H]GPP with the Mentha enzyme gave ( -)-limonene with deuterium label cis to the 4-hydrogen. Only (3S)-( +)-LPP 31 was utilized and the [lZ-*HI-labelled substrate also gave ( -)-limonene with deuterium label cis to the 4-hydrogen. Of the various stereochemical sequences possible only one is accommodated by these findings.This requires elimination of the diphosphate group from the (2Re,3Si)-face of GPP and isomerization to a cyclizable linalyl cation having an endo-spatial arrangement (Scheme 11). Complementary studies with the (+)-limonene + \ 31 (3s)-(+)-LPP endo 43 (4s)-(-)-limonene Scheme 11 Enzyme limonene synthase synthase from Citrus established the same restrictions though in the enantiomeric series. Since the linalyl cations formed in the two limonene synthase systems are mirror image related the active sites of the enzymes must also be enantiomeric. In the later stages of the sequence to limonene a proton is lost from one of the methyl groups thus generating the propenyl side-chain. It has now been established in enzyme systems from both Citrus sinensis [(+)-limonene synthase] and Perilla frutes- cens [( -)-limonene synthase] that the proton lost is almost exclusively provided by the cis terminal methyl group (C-9).96 This contrasts with the (+)-and ( -)-pinene synthases from Salvia oficinalis where eliminations occur from both the cis (55-65%) and trans (3545%) methyls in GPP.The cyclopropyl analogue 44 of GPP has been demonstrated to be a potent inhibitor of ( -)-limonene synthase from Mentha spicata and of several other monoterpene cycla~es.~~ It formed a 1:1 stoichiometric complex with the enzyme but became irrevers- ibly bound probably by undergoing ionization and cyclization steps to an allylic cation that then alkylated the protein. Several limonene synthase genes are known to be present in the genome of Mentha spicata." Three cDNA isolates were obtained and functionally expressed in E.coli to give a catalytically active protein which elaborated a product mixture identical to that of the native enzyme. This contains predomi- nantly limonene with small amounts of a-pinene P-pinene and myrcene. Sequence comparisons showed a significant degree of similarity with the sesquiterpene cyclase epi-aristolochene syn- thase from tobacco and the diterpene cyclase casbene synthase from castor bean. Antibodies raised against the M. spicata Dewick The biosynthesis of CrCzs terpenoid compounds enzyme were highly specific for the denatured protein but did not recognize the native protein.99 However they did cross-react with the cyclase expressed in E.coli. The formation of cyclic monoterpenes is usually via the intermediacy of the a-terpinyl cation which may be produced in either 4R 45 or 4s 48 configurations depending on whether the LPP produced from GPP has the 3R or 3s configuration respectively. Biosynthesis of many other monoterpenes then requires migration of the positive charge from the side-chain into the ring and this may be achieved by hydride shifts. The precise nature of such shifts in the formation of y-terpinene 47 in thyme (Thymus vulgaris) of a-terpinene 49 in American wormseed (Chenopodium ambrosioides) and of ( -)-p-phellandrene 51 in lodgepole pine (Pinus contorta) has been investigated using cell-free enzyme systems and specifically- labelled GPP substrates.'oo It was established that a 1,2-hydride shift from C-4 to C-8 of the a-terpinyl cation thus generating the terpinen-4-yl cation 46 was occurring during y-terpinene formation and that the pro4 hydrogen from C-5 of GPP was subsequently lost (Scheme 12).The reaction rate with (3R)-( -)-LPP 30 was higher than with (3S)-( +)-LPP 31 although both enantiomers were utilized. Utilization of both enantiomers of LPP by monoterpene cyclases is not unusual but the disfavoured enantiomer often gives aberrant product mixtures as was noted here with (3S)-LPP. The stereochemical features of the transformation assuming a suprafacial isomeri- zation step and an anti-endo conformation for the cyclizing intermediate are shown in Scheme 13.a-Terpinene 49 forma-tion also involved the same 1,2-hydride shift and a similar proton loss this time from C-3 of the terpinen-4-yl cation (Scheme 12). The proton lost was originally the 1-pro-S of GPP. However for a-terpinene (3S)-LPP was the preferred substrate and therefore the isomerization and cyclization steps are antipodal to the reactions seen with y-terpinene. For ( -)-P-phellandrene 51 a 1,3-hydride shift from C-3 to C-8 of the a-terpinyl cation giving the terpin-3-yl cation 50 and the intermediacy of (3S)-LPP was established (Scheme 12). The biosynthesis of (+)-3-carene 52 a major component of turpentine has been studied previously but the results from feeding experiments with labelled MVA precursors led to a hypothesis which now seems untenable in the light of more recent enzymic studies of monoterpene cyclases.In particular the double bond appeared to have been transposed during the biosynthesis. Accordingly the formation of (+)-3-carene has been reinvestigated using cell-free extracts of Douglas fir (Pseudotsuga menziesii) and a partially purified carene synthase preparation from lodgepole pine (Pinus contortaj and these studies have produced results different from the earlier ones but consistent with what might now be expected based on the growing pool of enzymic data."' Using both plant prepara- tions it was established that label from [l-3H]GPP was retained at C-5 in carene in keeping with the intermediacy of a terpinyl cation then cyclopropane ring formation by loss of a proton from C-5 of the cation (Scheme 14).It was then shown that in lodgepole pine the 5-pro-R proton of GPP was the one eliminated during ring closure. Because (3S)-LPP 31 was found to be the preferred substrate ring closure and hydrogen loss must therefore be accompanied by inversion of configuration at C-5 of the (4q-a-terpinyl cation 48. To accommodate this it is deduced that cyclopropyl ring closure most likely occurs by an anti-l,3-elimination from the a-terpinyl cation which needs to be in a half-chair conforma- tion with the tertiary cationic centre in an axial position (Scheme 14). In this way the leaving group (5-pro-R proton) can be coplanar with the three carbons forming the cyclo- propane ring. There then needs to be a 90" rotation about the 6,7-bond (carene numbering) to allow the anti- 1,3-elimination with inversion of configuration.A 1,8-cineole synthase activity has been partially purified from glandular trichomes of sage (Salvia oficinalis) con-verting GPP into the symmetrical monoterpene ether 1,8-cineole 54 (Scheme 15).'02 This has properties of a typical 7 66; 1 ,Phydride shift 5 HS ,,a9 30 (3R)-(-)-LPP 45 (4R)-a-terpinylcation 46 terpinen-4-yl cation 47 y-terpinene a \ GPP 1,2-hydrideshiftW -HR+ QH Hs -'HR 31 (3s)-(+)-LPP 48 (4S)-a-terpinylcation 49 a-terpinene 50 terpinen-3-ylcation 51 (-)-P-phellandrene Scheme 12 I GPP 30 (3R)-LPP anti-endo / 47 y-terpinene 46 terpinen-4-ylcation 45 (4R)-a-terpinylcation Scheme 13 Enzyme y-terpinene synthase h 31 31 (3s)-LPP 48 (4S)-a-terpinylcation 52 (+)-3-carene 40 52 Scheme 14 Enzyme carene synthase monoterpene cyclase requiring a divalent metal ion (Mn2+ or Mg2+) being inhibited by cysteine-and histidine-directed reagents and being protected from inactivation by pretreat-ment with the substrate-M2+ ion complex.(3R)-LPP 45 was a much better substrate for the enzyme than (3S)-LPP indicat-ing its stereospecificity and the cyclization of LPP was faster than the coupled isomerization-cyclization of GPP showing the isomerization was the slow step as with other monoterpene 118 Natural Product Reports A OH 30 (3R)-LPP 45 (4R)-a-terpinylcation 53 (4R)-a-terpineol Q,.o 54 1,&cineole 1 HO-H GPP 45 53 I 55 54 Scheme 15 Enzyme 1,8-cineole synthase cyclases.Labelling studies showed the oxygen atom was derived from water which also supplied one hydrogen atom. The pathway is formulated as involving capture of the a-terpinyl cation 45 by water giving a-terpineol 53,followed by attack of the alcohol function onto the remaining double bond (Scheme 15). However a-terpineol was not utilized by the enzyme system and neither were the phosphate or diphosphate esters. This may be a consequence of a protein I c104-(OH 56 63 64 (-)-sabinene 65 (-)-P-pinene 66 (-)-a-pinene 67 terpinolene conformational change during the normal reaction cycle with the enzyme thus failing to recognise intermediates. The sulfo- nium analogue 56 of the linalyl cation was a good reversible inhibitor providing evidence for ion-pair intermediates.The overall stereochemistry of the reaction was also confirmed by feeding [1-3H]GPP oxidation of the cineole produced to (R,S)-3-keto-1,%cineole 55 and resolution via diastereomeric ketals. All activity resided in the R antipode (Scheme 15). (-)-cis-piperitone oxide (-)-trans-piperitone oxide (+)-piperitenone oxide (-)-cis-perillyl alcohol (-)-cis-isopiperitenone oxide 43 (-)-lirnonene (-)-trans-isopiperitenol 61 (-)-isopiperitenone The pathways to the Mentha monoterpenes and related structures are now well characterized at the enzymic level and were outlined in the last report.' The author regrets that the scheme given in that report contained errors which misplaced some of intermediates.A corrected version is now shown in Scheme 16. Some of these conversions have now been con- firmed by using cell suspension cultures of Mentha piper it^."^ The cultures retained most transformations of the menthol pathway although they were not able to incorporate the early precursor ( -)-limonene 43. Thus (+)-pulegone 58 was trans- formed into (+)-isomenthone 62 and ( -)-menthone 59 and (-)-menthone into (-)-menthol 60. The feeding of (-)-isopiperitenone 61 yielded (+)-pulegone piperitenone 57 ( -)-menthone ( -)-menthol ( -)-7-hydroxyisopiperitenone 63 plus other compounds. lo4 Monoterpene cyclases from gymnosperms appear to have slightly different characteristics to those from angiosperms.'05 Monoterpene cyclases from lodgepole pine (Pinus contorta) and grand fir (Abies grandis) are similar to terpene cyclases from angiosperms and fungi in respect of mass K for GPP and PI but differ in having an alkaline pH optimum are activated by K+,Rb' Cs' or NH,' but not Li' or Na' and for their M2+ cofactor require Mn2+ or Fe2' whilst Mg2' is not effective.Furthermore they are not protected against histidine-directed reagents by the substrate-M2+ complex. (-)-piperitone (-)-cis-piperitone oxide h h 57 piperitenone (+)-piperitone A 60 (-)-menthol (+)-cis-isopulegone 58 (+)-pulegone 59 (-)-rnenthone Ho*Q -O?2 (-)-trans-ca rveol (-)-cawone Scheme 16 Dewick The biosynthesis of CsCzs terpenoid compounds Four monoterpene cyclases were separated from the xylem of lodgepole pine and each of these transformed GPP into at least some of the major products of the oleoresin namely a-pinene 66 P-pinene 65 3-carene 52 sabinene 64 and P-phellandrene 51 in varying proportions.lo6 Like enzymes from peppermint and sage these monoterpene cyclases were inactivated by thiol-directed reagents but unlike the angiosperm enzymes they were not protected against inactiva- tion by co-incubation with the substrate and metal ion co- factor. Thus the gymnosperm enzymes appear to have at least some catalytically important thiol residues at a non-substrate-protectable region. Similarly the behaviour of gymnosperm and angiosperm enzymes to arginine-directed reagents was different. Conifers appear to have catalytically important arginine residues located at or near the active site and are protected against inactivation by pretreatment with the sub- strate and metal cofactor whilst angiosperms have some catalytically active arginines located at a non-substrate-protectable site.Like angiosperm enzymes the lodgepole pine monoterpene cyclases are able to catalyse both the isomeriza- tion of GPP to LPP and the subsequent cyclization. The products formed were predominantly enantiomerically pure and derived from (3S)-LPP but two of the enzymes yielded both ( -)-and (+)-a-pinene the latter which must result via the intermediacy of (3R)-LPP. The last step in the formation of a-pinene is loss of a proton from the pinyl cation 68 and what was originally C-4 of GPP [see Scheme 17 for (+)-a-pinene; consider a mirror image scheme for ( -)+pinenel.The stereochemistry of this proton GPP -Om HR 7 -Om (3R)-linalyl cation a-terpinyl cation 71 4/4-4 bornyl cation 68 pinyl cation 69 (+)-a-pinene isocamphyl cation (+)-p-pinene 70 (+)-carnphene Scheme 17 elimination has been studied using three monoterpene cyclases from sage (Salvia ~ficinalis).'~~ Cyclases I and I11 which both produce (+)-a-pinene 69 were both shown to retain label from (4S)-[4-2H,]GPP and lose the deuterium label from (4R)-[4- 2Hl]GPP (>94% loss; a small retention is due to a primary kinetic isotope effect). Cyclase I1 which produced ( -)-a-pinene 66 retained label from (4R)-[4-2Hl]GPP but a higher isotope effect showed in the loss of only 78% label from (4S)-[4-2Hl]GPP.This means that in all cases the proton lost was the axial hydrogen (see 71) which would represent maxi- mum overlap of the axial sp3 orbital with the emptyp orbital of the cationic centre. Since the diphosphate counterion is prob- ably located on the face opposite the gem-dimethyl bridge (Scheme 17) this anion may act as the base removing the proton. Samples of (+)-camphene 70 produced by cyclase I but not ( -)-camphene from cyclase 11 were shown to suffer loss of deuterium label by exchange with the medium. Label at C-10 of GPP was also vulnerable to exchange. Since there was no exchange in the case of (+)-a-pinene exchange must occur after the branch-point pinyl cation.There had also been earlier reports of the exchange of hydrogen at C-2 of GPP based on applications of natural abundance deuterium NMR and the importance of this has been investigated."* Studies with two of the monoterpene cyclases from sage using [2-2H 2-13C]- labelled GPP either alone or in admixture with unlabelled GPP confirmed the complete retention of deuterium and ruled out any transfer between GPP molecules. (+)-a-Pinene 69 ( -)-a-pinene 66 (+)-camphene 70 ( -)-camphene terpino- lene 67 and 1,8-cineole 54 [I &cineole cyclase coeluted with ( -)-a-pinene cyclase] figured amongst the products ana-lysed for deuterium exchange. In all cases GC-MS analysis indicated more than 98% deuterium retention. A presumptive active site peptide from a sage monoterpene cyclase has been identified.The monoterpene cyclase con- tained both (+)-a-pinene cyclase and (+)-bornyl diphosphate synthase since they are difficult to separate and purify in sufficient amounts for detailed study. The protein was sub- jected to irreversible binding with a labelled inhibitor the GPP analogue 44 and then cleaved with CNBr. This gave a covalently modified peptide mixture from which an abundant labelled 5 KDa peptide was isolated and sequenced. This was highly homologous through 22 residues with a segment of (4S)-( -)-limonene synthase from spearmint the only mono- terpene cyclase for which a complete amino acid sequence was known. A cytochrome P-450-dependent hydroxylating enzyme has been found in a microsomal preparation from the oil glands of hyssop (Hyssopus oficinalis) that converts ( -)-p-pinene 65 into the allylic alcohol (+)-trans-pinocarveol 72 (Scheme 18).'1° This product is then presumably the substrate for 65 (-)-p-pinene 72 (+)-trans-pinocarveol 73 (-)-pinocamphone + (OH 8 -6 66 (-)-a-pinene 75 (-)-rnyrtenol 74 (-)-isopinocarnphone Scheme 18 oxidation to the ketone then reduction of the double bond to give ( -)-pinocamphone 73 and ( -)-isopinocamphone 74 the major components of hyssop oil.The same preparation cata- lysed the hydroxylation of ( -)-a-pinene 66 to ( -)-myrtenol 120 Natural Product Reports 75 but at a slower rate. The major monoterpenoid of sage (Salvia oficinalis) leaves is (+)-camphor 76 but cell cultures accumulate only traces due to its very rapid catabolism in the cultures.Catabolism proceeds through 6-exo-hydroxycamphor 77 and 6-oxocamphor 78 (Scheme 19). An NADPH-0 depen-dent camphor hydroxylase has been demonstrated in a micro- soma1 preparation of sage cells and this has typical character- istics of a P-450 system.' '' The protein also cross-reacted with polyclonal antibodies raised against the P-450-dependent enzymes camphor-5-exo-hydroxylase from Pseudomonas putida and limonene-6S-hydroxylase from spearmint (Mentha spicata) suggesting it shares common properties with bacterial and other plant monoterpene hydroxylases. 76 (+)-camphor 77 6-exehydroxycamphor catabolism -78 6-oxocamphor Scheme 19 The irregular monoterpene chrysanthemic acid is found in esterified form in the pyrethrins from Chrysanthemum cinerariaefolium though details of its biosynthesis have been poorly defined.A US patent'12 now describes isolation of an enzyme chrysanthemyl diphosphate synthase which catalyses the conversion of DMAPP into (+)-trans-chrysanthemyl diphosphate 79 (Scheme 20) the gene sequence encoding for the enzyme and expression of this gene in yeast or E. coli. Transformed yeast cells carrying the gene are used to produce (+)-trans-chrysanthemyl alcohol 80 which may be oxidized chemically and converted into pyrethroids. / t -80 79 chrysanthemyl diphosphate Scheme 20 Iridoids are derived from geraniol33 via early hydroxylation of the %methyl (Scheme 21). A specific P-450-dependent hydroxylase has been demonstrated in catmint (Nepeta racemosa) and shown to transform both geraniol and nerol into their 8-hydroxy derivatives.' l3 Further a soluble enzyme from the leaves would oxidise 8-hydroxygeraniol 81 at both the alcohol functions to produce the dialdehyde 82.'14 8-Hydroxynerol was similarly oxidised whilst geraniol and nerol which were actually rather better substrates were oxi- dised to the corresponding monoaldehydes.The enzyme required NADP' as cofactor with NAD' being ineffective and was a dimer. A similar enzyme activity from Rauwolfia Dewick The biosynthesis of CsCzs terpenoid compounds 33 geraniol 81 8-h yd roxyge ranio I 83 iridodial 1 GoH-$yoH OGlc OH 86 deoxyloganic acid 85 deoxyloganic acid aglucone -HO 87 deoxyloganin 88 loganin Scheme 21 serpentina had also been shown to catalyse multiple oxidations but was a monomer.(Note that an alternative numbering system for geraniol is employed in these papers.) Feeding experiments with a range of 2H-labelled potential precursors of schismoside (=8-epi-lamalbid) 90 in shoots of Schismocarpus matudai have demonstrated good incorpora- tions of deoxyloganin 87 loganin 88 and 6P-hydroxyloganin 89 thus suggesting a sequence in which hydroxylations of loganin proceed in the order 6 7 and then 8 (Scheme 22)."' In loganin -HO -HO HO-89 6P-hydroxyloganin 90 schismoside Scheme 22 the case of the Forsythia iridoids forsythide 93 and 11-methylforsythide 94 modifications to the oxidation level appear to be effected on the acid rather than the ester.Thus feeding experiments in I;. viridissima and I;. europaea showed good incorporations of deoxyloganic acid 86 into adoxo- sidic acid 91 forsythide 93 and ll-methylforsythide 94 and adoxosidic acid was also a good precursor of the latter two 11 C02H C02H HO 86 deoxyloganic acid 91 adoxosidic acid 92 93 forsythide 94 11-methylforsythide H HO HO OGlc OGlc 95 capensioside 96 thunaloside 97 98 99 stilbericoside Scheme 23 geraniol -@o -c$ -GoH -HOGOH ' OH ' OH ' OGlc OGlc 100 8-epi-iridodial 101 8-epi-iridotrial 102 8-epi-deoxyloganic acid 103 mussaenosidic acid HO HO HO OGlc OGlc 108 catalpol 107 aucubin 106 bartsioside/6-deoxyaucubin 105 geniposidic acid 104 deoxygeniposidic acid I 112 antirrhinoside 111 1O-deoxycatalpol/5-deoxyantirrhinoside 110 linaridell O-deoxyaucubin 109 6,l O-dideoxyaucubin Scheme 24 metabolites.' l6 This supports a sequence involving successive oxidations at position 10 of deoxyloganic acid with methyla- tion at the C-11 carboxy group as the last step (Scheme 23).The observed loss of some of the label from C-10 of adoxosidic acid may be the result of reversible oxidation-reduction between alcohol and aldehyde 92. In Thunbergia data adoxo-sidic acid 91 is decarboxylated to capensioside 95 which serves 113 harpagide 114 tomentoside 129 secologanoside R = H 130 secoxyloganin R = Me as the precursor of stilbericoside 99 (Scheme 23).Il7 Since deoxyloganic acid 86 was also efficiently incorporated into thunaloside 96 in these experiments it is suggested that 7-hydroxylation of capensioside is likely to be the next step in the pathway and that the aldehyde 97 and the alkene 98 may then be involved as potential intermediates.A pathway via these two intermediates would be consistent with the obser- OGlc vation that label from H-6 H-7 and H-8 from deoxyloganic 4.. acid is retained during stilbericoside formation. 127 lilacoside R = CH~CH~ A further range of decarboxylated iridoid structures arise via 8-epi-iridodial 100 8-epi-iridotrial 101 and 8-epi-deoxyloganic 128 fliederoside R = CH2CH2aOH acid 102 (Scheme 24). The fonnation of the 8-epi-series neces- sitates generation of the different stereochemistry during the cyclization of the dialdehyde 82 (Scheme 21).Feeding exper- iments with deuterium-labelled substrates have been used to demonstrate the incorporation of 8-epi-iridodia1 8-epi- 8-epi-iridotrial was also well incorporated but this is likely to iridotrial and 8-epi-deoxyloganic acid into aucubin 107 and be hydrolysed before incorporation and may not be a direct harpagide 113 in Scrophuluria umbrosa. Is The glucoside of precursor since in the loganin series oxidation of the aldehyde 122 Natural Product Reports 85 deoxyloganic acid aglucone -84 iridotrial -83 iridodial J -HO--HO-kko OGlc OGlc 86 deoxyloganic acid 11 5 7-epi-loganic acid 116 7-epkloganin 1 J C02Me Me02C C02Me Xo OGlc 121 excelsioside 1 H02C C02Me Glc02C C02Me HO*oH o*oH-0$Ao -xo-xo / / H OGlc OGlc OGlc OGlc OGlc 124 loganic acid 117 7-ketologanic acid 118 7-ketologanin 119 11-methyloleoside 120 7-glucosyl-l l -methyloleoside J 1 HoTo7+o Hpo A OGlc O* OGlc OGlc OGlc 123 oleuropein 122 ligstroside 125 secologanic acid 126 8-epi-kingisidic acid Scheme 25 group precedes glucosylation.The later stages of the pathway to aucubin have also been defined by feeding experiments."' These registered excellent incorporations of deoxygeniposidic acid 104 geniposidic acid 105 and bartsioside (6-deoxyaucubin) 106 but not 6,lO-dideoxyaucubin 109. Dilution experiments also established the presence of geniposidic acid after feeding 8-epi-deoxyloganic acid. These experiments sup- port the pathway shown in Scheme 24 with decarboxylation occurring at the geniposidic acid stage and mussaenosidic acid 103 is postulated as an intermediate between 8-epi- deoxyloganic acid and deoxygeniposidic acid.In both Scutel- laria albida and Paulownia tomentosa aucubin is further transformed into the epoxide catalpol 108.'20 8-epi-Deoxyloganic acid 102 and bartsioside 106 were also shown to be excellent precursors and the role of aucubin was supported by its isolation from dilution experiments. 8-epi-Deoxyloganic acid was also incorporated into tomentoside 114 in P.tomen-tosa. However subtle variations to the catalpol pathway are observed in the biosynthesis of antirrhinoside 112 in Antirrhi- num majus (Scheme 24). Decarboxylation occurs at the deoxy- geniposidic acid 104 stage giving 6,lO-dideoxyaucubin 109 followed by 6-hydroxylation to linaride (1 0-deoxyaucubin) 110 epoxidation to 10-deoxycatalpol 111 and finally hydoxy- lation at the ring junction.'21' 122All intermediates from 8-epi- deoxyloganic acid onwards except for mussaenosidic acid 103 were shown to be well incorporated and the role of 103 was supported by dilution experiments.I2' The biosynthesis of a range of secoiridoids (oleosides) has been investigated in an extended series of feeding experiments using plants of the Olea~eae.'~~~'~~ Deoxyloganic acid 86 was incorporated into 11-methyl oleoside 119 ligstroside 122 and oleuropein 123 in Syringa josikaea and into 7-glucosyl 1 1-methyl oleoside 120 11-methyl oleoside ligstroside and excelsioside 121 in Fraxinus excelsior.123 7-epi-Loganic acid 115 also proved a good precursor of ligstroside and the subsequent involvement of 7-ketologanic acid 117 was pro- posed with the remaining compounds forming a logical sequence as shown in Scheme 25. 7-Ketologanic acid 117 was then proven to be well incorporated into excelsioside 121 in Fraxinus excelsior with significant incorporations into 11-methyl oleoside 119 and ligstroside 122.'24Iridodial83 was Dewick The biosynthesis of C5-CZ5terpenoid compounds incorporated into 7-ketologanin 118 whilst iridotrial 84 deoxyloganic acid aglucone 85 deoxyloganic acid 86 7-epi-loganic acid 115 and 7-ketologanin 118 were all incorporated into one or more oleosides predominantly excelsioside.From these results the precise sequence of the 7-oxidation and methylation steps could not be defined. The sequence postu- lated as in Scheme 25 was supported by further feeding experiments using Syringa josikaea and S. vulgaris tissues. It was noted that the sequence of steps between deoxyloganic acid and 7-ketologanin could vary with plant species and also season. In one series of experiments in Syringa vulgaris neither deoxyloganic acid 86 nor 7-ketologanic acid 117 were incor- porated into oleosides but instead were converted into 8-epi- kingisidic acid 126. At another time deoxyloganic acid proved a good precursor of the oleosides including oleuropein 123 lilacoside 127 fliederoside 128 and the dimethyl ester of oleoside.The oleosides are characterized by an 8,g-double bond and their origins appear to differ from those of the 'ordinary' secoiridoids such as secologanin by proceeding through the oxidised intermediates 7-ketologanic acid 117 or 7-ketologanin 118. In a series of feeding experiments the possible involve- ment of intermediates of the secologanin or 7-ketologanin type has been investigated. 25 Compounds such as secologanoside 129 and secoxyloganin 130 were not precursors of the Fraxinus and Syringa oleosides though secologanin particularly in Syringa josikaea was converted into 7-glucosyl 11-methyl oleoside 120. 11-Methyl oleoside 119 was by far the most efficient precursor of 7-glucosyl 1 1-methyl oleoside ligstroside 122 and oleuropein 123 and in combination with the earlier results it is concluded that 7-ketologanin 118 is likely to be the immediate precursor of 1 1 -methyl oleoside.This conversion is possibly a Baeyer-Villiger type mechanism (Scheme 26). Rupture of the peroxide bond followed by cleavage of the 7,8-bond and abstraction of H-9 would produce 1 1-methyl oleoside 119. Alternatively a similar reaction with abstraction of H-10 would produce secoxyloganin 128 and a Baeyer-Villiger oxidation with migration of the alkyl group would account for formation of 8-epi-kingiside 131. The origins of secologanic acid 125 have been investigated in leaves of Fontanesia fortunei and F. phillyreoides.126 This compound is 11 C02Me H I. o$-p;2 10 OGlc 118 7-ketologanin C02Me RO-0 9Ho*o -119 11-methyloleoside OGlc -130 secoxyloganin RO-04 I OGlc 131 8-epkkingiside Scheme 26 not derived from 7-epi-loganic acid 115 but instead from loganic acid 124(Scheme 25).Deoxyloganic acid 86 was also a good precursor. Furthermore deoxyloganic acid was shown to be hydroxylated at C-7 with retention of configuration and H-7 from loganic acid was retained during transformation to secologanic acid. This must therefore exclude involvement of 7-ketologanic acid. Larvae of the leaf beetles Phaedon amoraciae P. cochleariae Gastrophysa viridula and Plagiodera versicolora synthesize iridials chrysomelidial 133 and/or plagiodial 132 as defensive secretions. Feeding experiments have shown these arise from geraniol 33 via 8-hydroxygeraniol 81 and the dialdehyde 8-oxocitral82 with the early part of this pathway (Scheme 27) $OH -$: OH 33 geraniol 81 8-hydroxygeraniol 82 Phaedon sp/ Gastrwhysa \ Plagiodera versicolora / \ viridula Phaedon spp.D -. /-y LCHO 132 plagiodial 133 chrysomelidial Scheme 27 resembling the plant sequence to iridoids (Scheme 21).'27y Since the insects were able to transform 3-norgeraniol 134 and 8-hydroxy-3-norgeraniol 135 into norchrysomelidial 136 this substrate rather than the natural precursor was used as a 124 Natural Product Reports probe. It was established that in G. viridula [2H,]-8-hydroxy- 3-norgeraniol 134 was incorporated with no loss of label. On the other hand the Phaedon species transformed the substrate with loss of one H-4 deuterium suggesting that they first synthesize 1-norplagiodial 137 and that this is then isomerized to the more stable conjugated 1-norchrysomelidial (Scheme 28).In the case of Plagiodera versicolora the insect produced 3- D3C' 134 3-norgeraniol Gastrophysa viridula * :<:HO CD3 136 norchrysomelidial 135 D<o CHO Phaedon spp.* ' q Z H 0 I CD3 CD3 137 norplagiodial 136 norchrysomelidial 1 Plagiodera versicolora / CD3 1 38 139 Scheme 28 1 -norplagiodial and a further metabolite 2-norplagiolactone 138 each with four deuterium labels. Feeding experiments in Phaedon amoraciae with the chirally labelled [2H2]-3-norgeraniol 139 and its enantiomer established that the pro-S hydrogen was the one eliminated from position 4.Cyclization and proton loss are accommodated by the mechanism shown in Scheme 29 the process probably being initiated by Schiff base formation with the enzyme. Reviews describing the biosynthesis of thujane mono-terpene~,"~ genetic control of Mentha monoterpene biosyn- thesis,'30 the biosynthesis of rn~noterpenes'~'-'~~ and the role of geraniol 8-hydroxylase in iridoid bio~ynthesis'~~ have been published. CH-N-Enz H /-Hs+ \-Ha+ 1 \ -CHO isomerization CCHO CCHO Scheme 29 7 Sesquiterpenoids The simple acyclic sesquiterpene trans-p-farnesene 140 is elaborated from FPP by an enzyme purified from maritime pine (Pinus pina~ter).'~' The enzyme requires a divalent metal ion with Mg2' being preferred over Mn2'.The reaction presumably involves cation formation and proton loss (Scheme 30). Farnesoll41 is transformed into farnesall42 and FPP -140 trans-p-farnesene Scheme 30 3-hydroxy-2,3-dihydrofarnesal143 by protoplasts of the microalga Botryococcus braunii and both compounds were then efficiently incorporated into the triterpenoid hydro- carbons the botryoc~ccenes.'~~ Fatty acid esters of farnesol such as oleate palmitate palmitoleate and linolenate could be formed when fractions from homogenized cells were incubated with farnesol. 137Farnesol was incorporated into squalene and botryococcenes by cultures of the algae whereas the super- natant from cell homogenates converted FPP into squalene but not into the botryoc~ccenes.'~~ The pellet fraction was able to phosphorylate farnesol to give mono- and di-phosphate esters utilizing CTP as the phosphate donor.ATP was less effective than CTP and neither GTP nor UTP would partici- pate. It is suggested that farnesol can be phosphorylated by the cultures to give FPP which is then incorporated into squalene but the botryococcenes appear to be derived from farnesol rather than FPP. The farnesol-phosphorylating enzyme did not transform other isoprenoid alcohols such as geraniol and geranylgeraniol and furthermore would not phosphorylate farnesyl monophosphate. 139This may indicate the membranes are permeable to farnesol but not its monophosphate. OH 141 farnesol 147 142 farnesal Aristolochene synthase accomplishes the cyclization of FPP to the germacryl cation 144 and its subsequent rearrangement via the eudesmyl cation 145 to (+)-aristolochene 146 (Scheme 31).The gene coding for this enzyme has now been isolated from Penicillium roquefortii and expressed in E. coli as a fusion protein which has the appropriate sesquiterpene cyclase activity. The aristolochene synthase coding sequence has since been amplified and this has allowed overproduction of the protein to the level of about 40% of the bacterium's soluble pr~tein.'~' The 12-methylidene analogue 147 of FPP has proved to be an effective mechanism-based inhibitor of aristolochene synthase. 142 This substrate analogue may prob- ably be cyclized by the enzyme but it could then undergo Dewick The biosynthesis of CrCZ5 terpenoid compounds Fpp -(-p( -p...< + 144 germacryl cation I -H+ 146 (+)-aristolochene 145 eudesmyl cation germacrene A Scheme 31 Enzyme aristolochene synthase rearrangement or delocalization to place the positive charge in a region of the protein that does not normally encounter such charge perhaps resulting in alkylation of the protein.5-epi- Aristolochene 146a is produced in tobacco (Nicotiana taba- cum) and pepper (Capsicum annuum) as part of a sequence to sesquiterpene phytoalexins when the plant tissues are chal- lenged by fungi. Its formation presumably requires similar skeletal rearrangements as in the aristolochene pathway but a different conformation for the cyclizing FPP will be needed to attain the correct stereochemistry.In tobacco the 5-epi-aristolochene synthase enzyme appears to be encoded by a complex gene family. A full length cDNA sequence has been cloned into E. coli to allow production of the enzyme at a level of 5-8% of total soluble protein.'43 Enzymic reaction products from the recombinant protein were identical to those from the native enzyme. 5-epi-Aristolochene 146a is a good precursor of capsidiol 148 in arachidonic acid-treated green pepper seed- lings,'44 and a 3-hydroxylase enzyme catalysing the first step in this pathway (Scheme 32) has been i~olated.'~' This enzyme 147 5-epi-aristolochene i 148 capsidiol Scheme 32 was localized in the microsomes of elicitor-treated fruit and found to be a P-450-dependent system requiring 0 and NADPH cofactors.Alternative modifications to a eudesmyl cation can lead to vetispiradiene 149 and related compounds. Vetispiradiene synthase from Hyoscyamus muticus is encoded by a gene family of some 6-8 genes.'46 Genomic and cDNA clones have been isolated from cell cultures treated with a fungal elicitor preparation and bacterial expression of three of these showed they all coded for enzymes giving the same single reaction product. The deduced amino acid sequence was 77% identical to that of 5-epi-aristolochene synthase from tobacco though there were conspicuous differences suggesting several partial reactions were common but others were unique. Earlier studies have established that during the biosynthesis of pentalenene 150 the 9-pro4 hydrogen of FPP undergoes an intramolecular migration to the adjacent carbon probably by transfer to a basic group on the enzyme followed by its 125 f :B-Enz HgsCh-Enz L 150 pentalenene t I c HfJR H H 152 151 Scheme 33 Enzyme pentalenene synthase subsequent reincorporation becoming the 1 -pro-S hydrogen in pentalenene (Scheme 33).In more recent experiments the stereochemical features of the electrophilic allylic addition- elimination (S,,) reaction in which the C-4C-5 bond of pentalenene is formed have been investigated. 147 Incubation of (4R,8R)-[4,8-3H, 4,8-14C2]FPP with a pentalenene syn- thase preparation from Streptomyces UC53 19 gave pen-talenene retaining some 95% of the 3H label whilst only 53% of tritium was retained from the corresponding (4S,8S)- precursor.Thus the 8-pro-S hydrogen from FPP is lost. The implications of this are as follows. Cyclization of the cation 151 involves attack on the si face of the trans-double bond and the p-orbitals need to be aligned in a parallel orientation. If H, were then to be removed in the final deprotonation step this would require a 30" rotation of thep orbital in the pentalenyl cation 152 to get the necessary alignment for double bond formation. Since H, is actually lost the p orbital must therefore undergo a 90" rotation prior to deprotonation. It is hypothesized that a single base at the enzyme's active site could be responsible for deprotonation at C-9 reprotonation at C-10 and deprotonation at C-8 of the original FPP.Pentalenene synthase from Streptomyces has now been purified cloned and sequenced. 148 Expression of the gene in E. coli has allowed production of a recombinant protein with properties identical to those of the native enzyme and at levels accounting for more than 10% of the soluble protein. The recombinant protein has also been crystallized and studied by X-ray diffraction analysis. 149 The biosynthesis of trichothecene mycotoxins proceeds from FPP via the intermediate hydrocarbon trichodiene 154 and its origin through preliminary isomerization to nerolidyl diphosphate (NPP) 153 then cyclization and subsequent rearrangements has been well investigated (Scheme 34). The trichodiene synthase gene from Fusarium sporotrichioides has previously been cloned and expressed at low levels in E.coli and now amplification of the coding sequence has allowed the protein to be expressed at much higher levels accounting 126 Natural Product Reports I 153 (3R)-NPP I "d-r& 154 trichodiene Scheme 34 Enzyme trichodiene synthase for some 20-30% of the total soluble ~r0tein.l~' The sub- strate specificities of the recombinant and native enzymes have been compared and found to be identi~a1.l'~ Although NPP is known to be produced initially from FPP by allylic isomerization kinetic parameters show it to be a less attrac- tive substrate for the enzyme. This contrasts with mono-terpene cyclases where LPP is typically 2-7 times more effective than GPP as substrate.In the case of trichodiene synthase this may be the consequence of a slow conforma- tional change to the enzyme after it has bound NPP. Several FPP analogues were found to act as competitive inhibitors of trichodiene synthase with 1 0-fluoro-FPP 155 being the most 155 175 T-2 toxin effective. Reaction with thiol-directed reagents has suggested the presence of two cysteine residues at the active site of the F sporotrichioides enzyme. 152 The construction of mutants by site-directed mutagenesis has shown that C190A mutant (Cys replaced by Arg) retained partial activity whilst C 146A was essentially inactive. A hybrid enzyme constructed from amino acids 1-309 of F. sporotrichioides and amino acids 301-383 of F sambucinum had almost wild-type activity and allowed construction of a further series of mutants via the base-rich region 302-306.Three such mutants were over-expressed and purified. Two of these R304K and Y305T when incubated with FPP were found to accumulate both trichodiene and additional unidentified sesquiterpene hydrocarbons. During formation of the trichothecene skeleton trichodiene must be oxygenated at several positions and subjected to further cyclization. Isotrichodiol 161 is a proven intermediate and is suggested to be elaborated by the sequence shown in Scheme 36. Evidence supporting this comes from the obser- vation that the trichodiene epoxide 156 was partially trans- formed by cultures of Fusarium culmorum into the diepoxide 157 and the hydroxylated product 158 (Scheme 35).'53 The epoxidase activity appears to be an 0,-NADPH dependent P-450 system and is probably the same enzyme which intro- duces the epoxide function in the normal pathway.Since trichodiene is not transformed by a cell-free extract and the 2-hydroxy- 12,13-epoxy derivative (trichothecene numbering) 159 had previously been shown to be a trichothecene precur- sor the sequence is likely to be 159+160+161 which thus features two allylic oxidations (Scheme 36). For many years trichodiol 162 had been postulated as an intermediate in the A-h- 156 157 I 158 Scheme 35 biosynthesis of trichothecenes and more recently trichotriol 164 was added to the sequence. These compounds are now shown to be formed as artefacts from isotrichodiol 161 and isotrichotriol 163 respectively and 161 and 163 are proposed as true intermediates.’ 54 Both 9-epimeric forms of trichodiol can be isolated from Trichothecium roseum and of trichotriol from F.culmorum. Such epimers equilibrate on treatment with acid with cyclization then occurring if a 2-hydroxy group is present. All studies suggest trichodiol and trichotriol result from acid-catalysed isomerization of isotrichodiol and iso- trichotriol respectively via the allylic cation. Isotrichodiol was a much better precursor of the trichothecenes 3-acetyl- deoxynivalenol (3-AcDON) 172 and dihydroxycalonectrin (DHC) 170 in F. culmorum than trichotriol the latter prob- ably being incorporated after non-enzymic cyclization to trichodiene isotrichodermol 165.In competitive feeding experiments the incorporation of isotrichodiol was not influenced by simul- taneous administration of trichodiol and only marginally by trichotriol whereas isotrichotriol did repress incorporations. In later parts of the pathway DHC 170 and 15-deacetylDHC 171 were shown to be good precursors of 3-AcDON 172 indicating the last two steps are 15-deacetylation and then 8-oxidation.’53 Based on these and earlier studies the complete pathway is now believed to be as shown in Scheme 36. Pre-sambucoin 173 one of the products from acid-catalysed cyclization of isotrichodiol 161 was shown to be efficiently incorporated into sambucoin 174 a trichothecene-related metabolite in I;.culmorum (Scheme 37).lS4 Two or more genes which are involved in the biosynthetic pathway to trichothecenes in Fusarium sporotrichioides appear to be linked with the trichodiene synthase gene Tri.5 (=Tox~) in a gene cluster.’s5 One of these (Tri3) is assigned to a transacetylase which acetylates 15-deacetylCAL 167 to CAL 168 whilst Tri4 is suggested to encode a P-450 enzyme perhaps for the first oxygenation step in the pathway after trichodiene. 56 Transformants lacking a functional Tri4 gede were unable to synthesize trichothecenes but could convert isotrichotriol into T-2 toxin 175 suggesting all later enzymes were present. Tri6 from the gene cluster specifies a protein similar to Cys,His zinc finger proteins and disruption destroys the ability of Fusarium sporotrichioides to synthesize trichothecenes.lS7 However none of a variety of trichothecene precursors could be transformed into T-2 toxin and transcrip- tion of both Tri4 and Tri.5 was greatly reduced relative to the wild-type fungus.It is believed that Tri6 encodes a pro- tein involved in transcriptional regulation of trichothecene biosynthetic enzymes. 159 160 161 isotrichodiol 162 trichodiol 163 isotrichotrlol 164 trichotriol \ 168 CAL 167 15-deacetylCAL 166 isotnchodermin 165 isotrichodermol 1 ,OAc ___) q-HO HO HO \oic HO” OAc 169 7-hydroxyCAL 170 DHC 171 15-deacetylDHC 172 3-AcDON Scheme 36 Dewick The biosynthesis of CsC2 terpenoid compounds 161 isotrichodiol 173 pre-sambucoin I + 174 sambucoin Scheme 37 Feeding of the deuteriated mevalonates [4-2H2]- [5-2H2]- and [2-2H2]-MVA to callus cultures of Perilla frutescens var.crispa f. purpurea has exposed a number of hydride shifts in the biosynthesis of the constituent sesquiterpenes.' 58 The path- ways share a common FPP-derived bisabolyl cation 176 (Scheme 38).The labelling patterns in cuparene 179 demon-strate two concurrent pathways from 177 one involving a 1,4-hydride shift to 178 (compare trichodiene above) whilst the other features two 1,3-hydride shifts. Aromatic ring forma- tion then involves loss of one hydrogen from each of the methylenes in 178. For a-curcumene 180 a 1,2-hydride shift is demonstrated. P-Bisabolene 181 on paper should be formed merely by loss of a proton.Whilst this proton loss is con- firmed an unexpected change occurs in the cyclohexene ring implicating partial migration of the double bond from C-2/3 to C-3/4. As a consequence there is loss of a proton from C-4 and incorporation of a proton from the medium. Sesquiterpenes of the cadinane series arise by cyclization of NPP to a ten-membered ring followed by the formation of the decalin system with an accompanying 1,3-hydride shift (Scheme 39). A study of (+)-epicubenol 184 biosynthesis in a cell-free extract of Streptomyces sp. LL-B7 from [1-2H2]FPP confirmed the 1,3-hydride shift by the labelling of 184 at C-5 and C-1 1 [13-2H3]FPP produced [I 3-2H,]labelled epicube- nol. Incubation of [6-2H]FPP with the epicubenol synthase preparation was then used to confirm the predicted 1,2-hydride shift from C-10 to C-9 (Scheme 39).I6O Continuing these FPP -OQ A 176 bisabolyl cation FPP 153 (3R)-NPP 182 cis-germacryl cation )nJJ H20jyJ 1 -\ l5 "ri \ H 12.A./i\ 13 H 184 (+)-epicubenol 183 cadinyl cation 153 182 a 183 Scheme 39 Enzyme epicubenol synthase studies it was then established that the migrating hydride from C-1 of FPP was the 1-pro4 hydrogen the 1-pro-R hydrogen residing at C-5 in epicubenol.161 The intermediate role of NPP was explored by feeding both (3R)- and (3S)-( lZ)-[1-3H]NPP with only the former giving a labelled product. Both (3R)-(lZ)- [1-2H]NPP and (1 R)-[ 1 -2H]FPP gave essentially the same 2H H 177 / 178 179 cuparene 180 a-curcumene 181 P-bisabolene Scheme 38 128 Natural Product Reports distribution as analysed by GC-MS confirming the position of labelling as C-5.Thus initial isomerization of FPP to NPP must occur with suprafacial stereochemistry as seen with trichodiene synthase and also in the GPP to LPP isomeriza- tions for monoterpene cyclases. Attack of the ionized NPP by the C-1GC-11 double bond will be on the re face to generate the cis-germacryl cation 182 and the original 1-pro-S hydro- gen of FPP will be able to align with the vacant p orbital of the cationic centre. A second electrophilic cyclization gives the cadinyl cation 183 and finally capture of hydroxy group from water is in a syn sense with respect to the migrating hydride (Scheme 39).Enzyme preparations from upland cotton (Gossypium hirsutum) tissues infected with Xanthomonas campestris pv. malvacearum are able to convert labelled FPP into (+)-6-cadinene 185 (Scheme 40).'62 In the plant this material is then FPP A cadinyl cation 185 (+)-a-cadinene 186 lacinilene C A 187 2,7-dihydroxycadalene Scheme 40 incorporated into lacinilene C 186 its 7-methyl ether and 2,7-dihydroxycadalene 187 and may also be an early precursor of the cotton phytoalexins such as gossypol. A similar enzyme activity has been identified in extracts from G. barbadense infected with Verticillium dahliae. 163 By using [l-2H]-labelled FPP as substrate the formation of [5-,H]- and [l-2H]-labelled 6-cadinenes could be demonstrated thus supporting the 1,3-hydride shift.Challenging cell suspension cultures of G. arboreum with a preparation from I/. dahliae initiates pro- duction of gossypol and this is accompanied by increased mRNA levels. This has led to the isolation of two cDNA clones and the encoded proteins from these showed a signifi- cant degree of sequence identity with known plant terpene cycla~es.'~~ The resultant protein was expressed in E. coli and shown to cyclize FPP into (+)-6-cadinene. Again the con- version of this sesquiterpene into lacinilene C and 2,7-dihydroxycadalene by cotton plants was demonstrated. Under- standing of the biosynthesis of the binaphthyl bis-sesquiterpene gossypol 191 in Gossypium species is confused because of con- flicting experimental data.Thus one earlier study had shown that the folding of FPP generating the aromatic sesquiterpene hemigossypoll90 was in accord with the involvement of (Z,E)-FPP 189 (route A) whilst another had justified folding con- sistent with the use of (Z,Z)-FPP 188 (route B) (Scheme 41). In an effort to clarify this dichotomy gossypol biosynthesis in Thespesia populnea has been probed using a number of poten- tial precursors labelled with 3H and 14C.165 In this plant feedings with [2-I4C 5-3H2]MVA gave gossypol labelled in accord with route A and consistent with the 1,3-hydride migration typical of cadinane systems. The incorporations of [1-14C,l-3H2]geraniol [l-14C,l-3H2]farnesol and [2-14C,4-3H,]MVA also fitted route A but not route B and inferred that GPP is not isomerized to NPP nor is (Z,E)-FPP Dewick The biosynthesis of terpenoid compounds nerol ,B -\ CHO OH 188 (Z,Z)-FPP HO geraniol 190 hemigossypol 189 (Z,E)-FPF 'I Y HO OH HO A 191 gossypol Scheme 41 isomerized to (Z,Z)-FPP.However when [1-I4C,1-3H2]nerol was fed cyclization occurred according to route B. Accord-ingly this indicates that the normal route from GPP is via FPP (and then presumably NPP) to the cadinyl cation whereas if nerol is fed this is chain extended and can participate via route B. Gossypol is ultimately derived by oxidative coupling of two hemigossypol residues (or similar) but no details are yet available. Cultured cells of the liverwort Heteroscyphus planus accu- mulate a number of cadinane sesquiterpenes including 7-hydroxycalamenene 193 and 7-methoxy- 1,2-dihydrocadalene 194.The biosynthesis of these metabolites has been studied using a variety of ,H-and 13C-labelled MVAS.'~~'~' The labelling patterns are consistent with a 1,3-hydride shift in the formation of the cadinyl cation then a 1,2-hydride shift prior to aromatization to calamenene 192 (Scheme 42). The hydrox- ylation of calamenene must occur without any NIH shift. The conversion of 7-hydroxycalamenene 193 into 7-methoxy- 1,2- dihydrocadalene 194 could occur by either of the two routes shown in Scheme 42 since the labelling patterns are consistent with either. The formation of (+)-epicubenol 184 and (+)-cubenene 195 in cell-free extracts from H.planus cultures has also been reported. 16' These experiments demonstrated the incorporation of (E,E)-[1-2H,]FPP but not (2,E)-[ 1 -2H,]FPP with both hydrogen labels retained though one was involved in the 1,3-hydride shift. Label from [6-,H2]FPP was incorpor- ated into C-9 providing evidence for the 1,2-hydride shift from C-10 (Scheme 42). Whether or not cubenene is a precursor of calamenene and other metabolites has not been established. Artemisinin (qinghaosu) 199 is a sesquiterpene derivative containing a peroxide linkage that has been shown to be crucial for the antimalarial activity displayed by this com- pound. Artemisinin is a modified cadinane derivative and both arteannuic acid ( =artemisinic acid) 196 and arteannuin B 197 are shown to be precursors.Labelled arteannuic acid was incorporated into arteannuin-B and artemisinin in both Artemisia annua plants and in a cell-free system from A. annua leaves.169 Use of oxidizing cofactors Fe2'-2-oxoglutarate or peroxidase-H,O enhanced incorporations in vitro. Artean- nuin B was converted into artemisinin using a crude extract from Artemisia annua leaves as well as in a semi-purified cell-free extract. 170 A possible mechanistic sequence has been proposed in which the last step is the reduction of the 14 cadinyl cation 192 calamenene 193 7-hydroxycalamenene 184 (+)-epicubenol 200 202 HOHO 0 qq 0 H02C 203 204 195 (+)-cubenene 194 (1S)-7-methoxy-1,2-dihydrocadaIene Scheme 42 and vellerol 212 when fed to freshly ground fruit bodies.'76 Velleral 211 is believed to arise from velutinal 209 by elimina- tion of a water equivalent (Scheme 45).The ring expansion process involved in the formation of velleral 211 was probed by reconstituting freeze-dried fruit bodies in 2H,0 but this did not lead to any deuterium labelling. This suggests the proton at C-3 required during the cyclopropane ring opening originates from velutinal and is not introduced from water. This is accommodated by a mechanism in which a C-4 proton migrates to C-3 as shown in Scheme 45. Involvement of the enol acetal 210 would also account for the formation of other identified metabolites such as piperdial and epi-piperdial 213. 11,13-double bond in artemisitene 198 (Scheme 43).In other studies 1 1,13-dihydroarteannuic acid 200 has been employed as a precursor of artemisinin. 171~172 Arteannuic acid and dihydroarteannuic acid were reported to be precursors of artemisinin in leaf homogenates of A. annua and arteannuic acid and epoxyarteannuic acid 201 the precursors of artean- nuin B.17*In another report 6-epi-deoxyarteannuin B 202 and 11,13-dihydro-6-epi-deoxyarteannuinB could be used as pre- cursors of artemisinin. 173 Oxygen was necessary for formation of artemisinin from the latter lactone by the leaf homogenates. The isolation of the cadinanolide 203 and the secocadinane 204 from A. annua may implicate these compounds as post-arteannuin B intermediates in the biosynthesis of artemisinin and a variant on the pathway shown in Scheme 43 has been put forward.174 The biosynthesis of guaianolide sesquiterpenes in hairy root cultures of blue-flowered lettuce (Lactuca JEoridana) has been investigated by feeding 3C-labelled acetate (single-and double-labelled) and MVA. 175 The labelling patterns in the carbon skeletons of 8-desoxylactucin 207 and 1 1,13- dihydrolactucin 8-0-acetate 208 were entirely consistent with the pathway shown in Scheme 44,and the germacranolides costunolide 205 and parthenolide 206 are suggested as intermediates. Fatty acid esters of the marasmane sesquiterpene velutinal 209 in the fruit bodies of the fungus Lactarius vellereus are converted into dialdehyde derivatives e.g. isovelleral 214 and the lactarane velleral 211 when the fungus is damaged.These dialdehydes act as a chemical defence system to protect against parasites but are then subsequently reduced to inactive alco- hols e.g. isovellerol 215 and vellerol 212. [12-2H3]Velutinal proved to be efficiently transformed into both isovellerol 215 130 Natural Product Reports cadinyl cation -0 196 arteannuic acid 197 arteannuin B (=artemisinic acid) i H02C 1 Hi. H+ H; 9 LO.. 0 c- 0 0 199 artemisinin 198 artemisitene Scheme 43 FPP cis-germacryl cation 205 costunolide I n W 0 0 206 parthenolide t 0 0 207 8-desoxylactucin 208 11,13-dihydrolactucin 8-Oacetate Scheme 44 Feeding experiments using [2-13C]acetate have been used to outline the biosynthetic origins of pinguisone 216 in cultures of the liverwort Aneura pinguis.177 The labelling pattern observed is consistent with the pathway outlined in Scheme 46. 13C-13C couplings for C-4/C-15 and C-8/C-12 indicated that two methyl migrations had occurred and the labellings also showed that bond cleavage of the main FPP chain was responsible for production of the indane system via a decalin skeleton. Hydride shifts as indicated will also be required in the proposed sequence. The drimane sesquiterpenoid cryptoporic acid I 218 from the fungus Ganoderma neojaponicum has a hydroxy group at C-3 and could in principle be derived by cyclization of epoxyFPP by a mechanism similar to that of steroid biosyn- thesis or alternatively may be formed by direct hydroxylation of cryptoporic acid H 217 (Scheme 47).The latter pathway is operative as demonstrated by the good incorporation of cryp- toporic acid H into 218.178Furthermore incorporation of 217 was inhibited when the P-450 inhibitor ancymidol was also employed. A group of so-called homoterpenes containing eleven carbons are in fact more correctly considered as degraded a: 2 3L -%H -&,-H~Q , *. 0 H 0’ HC H k/\‘O 15 11 7 216 pinguisone a = 13c Scheme 46 FPP 218 cryptoporic acid I 217 cryptoporic acid H Scheme 47 sesquiterpenes since they are formed by oxidative cleavage of four carbons. Thus 4,8-dimethylnona- 1,3,7-triene 224 is known to originate in a range of plants from nerolidol219. In a series of feeding experiments to explore the oxidative pro- cesses 2H-labelled precursors were fed to lima bean (Phaseolus lunatus) plantlets.179 Nerolidol 219 geranylacetone 223 the epoxide 220 the diol 222 and the trio1 221 were all converted into 224 indicative of two possible sequences as shown in Scheme 48. The chirality of the putative intermediates was established by feeding 1:l racemic mixtures where the enanti- omers had different labelling patterns and a high degree of enantioselectivity was observed. This sequence exhibits a for- mal analogy to the P-450 linked processes which are respon- sible for cleaving the side-chain of steroids e.g. cholesterol+ pregnenolone-,androstanes. The degree of enantioselectivity ti\ -a H\ I CHO . / CHO CHO CH20H HO H03 yb 209 velutinal H H+ 211 velleral 212 vellerol 1 210 \ CHO / CHO CHpOH H H 214 isovelleral 215 isovellerol 213 piperdiallepkpiperdial Scheme 45 Dewick The biosynthesis of CrCzs terpenoid compounds yC'"" \ / -FPP 21 9 (3S)-nerolidol 220 OH 222 221 223 geranylacetone x 224 225 Scheme 48 (96%) towards (38-nerolidol demonstrated by Phaseolus lunatus is not found in other plants and varies quite widely.180 Thus only a moderate degree of enantioselectivity (S:R= 66:34) was found in Gossypium herbaceum and Fragaria magna and other figures recorded were Spathiphyllum wallissi (94:6) Gerbera jamesonii (82 18) Lycopersicon esculentum (77:23) and Humulus lupulus (77:23) with 76:24 being observed for another cultivar of P.lunatus. The formation of these volatile homo terpenes is usually triggered by damage caused by insect infestation. It has also been shown that treatment of healthy undamaged plants of Lima bean with P-glucosidase leads to formation of volatiles predominantly 224 and the diterpene-derived 225 identical to those triggered by insect damage. 81 The irregular C,,-terpenoid dehydrogeosmin 226 the domi- nant component of the flower scent of the cactus Rebutia marsoneri is also a degraded sesquiterpene and its formation involves loss of the C side-chain from a eudesmane-type intermediate (Scheme 49).lS2 Farnesol has been shown to be incorporated into dehydrogeosmin and studies with a number of 2H-labelled farnesols provided evidence for the 1,2-hydride shift in Scheme 49.The plant growth regulator abscisic acid (ABA) 229 is produced from FPP by two main routes which involve either direct cyclization of the C precursor or alternatively initial formation of a C, carotenoid followed by oxidative metab- olism. The former direct pathway is utilized by several species of fungi whilst plants produce ABA by the indirect carotenoid route. Even in fungi routes vary and in Cercospora cruenta a pathway via y-ionylideneacetic acid 227 is favoured. Earlier feeding experiments with labelled 227 had demonstrated that it was converted into ABA via epimeric 4'-hydroxyacids 228 (Scheme SO) whilst two epimeric 3'-hydroxy acids 230 also formed appeared to fall outside the ABA pathway.In recent studies feeding 227 to C. cruenta actually gave four 3'-hydroxy I32 Natural Product Reports j OH cp'-226 dehydrogeosmin R= 227 y-ionylideneacetic acid 228 0 HO 229 ABA Scheme 50 acids the epimeric pair 230 and smaller amounts of the corresponding E analogues 231. Further feeding exper- iments with the 3'sepimer of 230 showed it to be metabolized by 1'-hydroxylation to 232 and then oxidation to the ketone 233 as well as partial isomerization to the 3's-epimer of 231. 232 233 In plants carotenoids with a suitable ring system are cleaved through the polyene chain to xanthoxin 234 which is then modified to ABA (Scheme 51). A cell-free system from Citrus sinensis has been shown to achieve ABA synthesis from P-carotene thus carrying out the necessary carotenoid ring modifications as well as the later step^."^ Indeed MVA was also utilized by the cell-free system giving ABA phaseic acid 236 and 1',4'-trans-abscisic acid diol 235.'85The diol 235 was the major product and was shown to be a metabolite of ABA rather than its precursor.Thus addition of unlabelled ABA as a cold-pool trap increased the incorporation of activity into ABA but reduced the levels in the diol and phaseic acid. The major metabolite of ABA in sunflower (Helianthus annuus) embryos was the 4'-O-glucoside of dihydrophaseic acid 237.lg6 Smaller amounts of phaseic acid 236 and dihydrophaseic acid were also formed. Phaseic acid is formed as a result of carotenoids 234 xanthoxin ABA-aldeh yde 1 235 1',4'-trans-ABA-diol 229 ABA I HO 237 dihydrophaseic acid 236 phaseic acid Scheme 51 hydroxylation of one of the gem-methyls followed by cycliza- tion.In tomato (Lycopersicon esculente) 1',4'-truns-abscisic acid diol 235 may also serve as a source of ABA.lg7 A number of reviews covering aspects of sesquiterpene biosynthesis have been published. These include discussions on the chemistry and genetics of trichothecene biosynthesis lS8 the biochemistry and regulation of trichothecene biosyn-thesis,189 macrocylic trichothecenes 190 the use of deuterium labelled precursors in the biosynthesis of sesquiterpenes in Perillu and Heteroscyphus 19' abscisic acid biosynthesis and metabolism 19*-93 and aromadendrene sesquiterpenoids.194 8 Diterpenoids The cembranoid diterpenes are the principal components of the gummy surface exudates of tobacco (Nicotiuna tubacum). Cell-free extracts from the trichomes of tobacco catalysed the conversion of geranylgeranyl diphosphate (GGPP) 22 into a-2,7,11-cembratrien-4-01 The 240 and its 4~-epimer.1g5 enzyme activity resembled other plant terpene cyclases in its solubility M,. pH optimum and requirement for Mg2+. That the products of cyclization were alcohols rather than the alkene cembrene 242 necessitates a revised pathway for the biosynthesis of the cembratriendiols e.g. 243 (Scheme 52) in which the initial cyclized cation 238 undergoes charge relocali- zation to 239 (probably via a 1,3-hydride shift) which is then quenched with water.Further hydroxylation gives compound 243. An earlier suggestion included cembrene 242 as an intermediate and derived this by ring opening of casbene 241. Casbene is formed from GGPP by the action of casbene synthase from the castor oil plant Ricinus communis. A near full-length casbene synthase cDNA clone has been isolated from elicited castor seedlings.19' The deduced amino acid sequence of the encoded protein had 42% identity with 5-epi- aristolochene synthase from tobacco and 3 1% identity with limonene synthase from spearmint indicating common fea- tures in the biosynthesis of mono- sesqui- and di-terpenes. The diterpenoid portion of the important anticancer agent Taxol (paclitaxel) 246 has its origins in GGPP and a soluble taxadiene synthase preparation from the stem bark and cam- bium of Pacific yew (Tuxus brevifoliu) has been is01ated.l~~ Activity levels in yew were low and could not be induced by stem wounding or elicitor treatment in marked contrast to the diterpene cyclase abietadiene synthase from lodgepole pine and grand fir (see below).The partially purified enzyme had similar properties to abietadiene synthase in respect of kinetic Dewick The biosynthesis of CsCzs terpenoid compounds 22 GGPP I ++ \ 7 238 241 casbene 11,3-shift 239 240 a-cembratrienol 242 cembrene 243 a-cembratriendiol Scheme 52 properties M, and a requirement for a divalent metal ion with Mg2+ being the best. It converted GGPP into a taxadiene but unexpectedly this was characterized as the 4(5),1 l(12)- diene 245 rather than the 4(20),11(12)-diene which had been predicted on the basis of many natural taxoids with double bonds in those position^.'^^ This structure has been confirmed by synthesis.lgg The formation of 245 is rationally formulated as shown in Scheme 53 via the bicyclic verticillene 244.Labelled taxadiene 245 was then shown to be a precursor of the complex taxoids by feeding experiments in Taxus brevifolia leaf discs where it was incorporated into several products including Taxol 246 and its 10,13-diol analogue 10-deacetylbaccatin 111. The common occurrence of taxoids with 4(20)-ene-5-oxy functionality and the lack of oxygenated derivatives with a 4(5)-double bond suggests hydroxylation at C-5 of taxadiene with allylic rearrangement may be the next step in the sequence.Then formation of the oxetane ring is formulated as via the epoxide with subsequent ring expansion (Scheme 54). A novel fungus Tuxomyces udreunae has been isolated from the inner bark of Tuxus brevifoliu and shown to synthesize Taxol and other taxanes de now in culture.200 Both 14C-acetate and I4C-phenylalanine were incorporated into Taxol. Unfortunately the yields of Taxol are very small and this does not yet provide a satisfactory system for Taxol produc- tion. Nevertheless the possibility that there has been genetic transfer between the tree and an associated fungus is most interesting. Cell-free extracts from trichome-bearing tissues of tobacco (Nicotiunu tubucum) convert GGPP into the bicyclic labdane diterpenoid cis-abienol 248 (Scheme 59.''' The enzyme activity was soluble was stimulated by Mg2+ and was un- affected by P-450 inhibitors.Similarly extracts from Nicotiunu w-w-m /L ' OW H+J I GGPP 244 verticillene GGPP I 1. H I 6 13 ?=$p 5 H 20 245 taxa-4(5) 1 1 (12)-diene taxenyl cation 1 1 OH 0 246 Taxol (paclitaxel) Scheme 53 Q5 20 245 taxadiene 1 Scheme 54 glutinosa converted GGPP into labdenediol 249 and sclareol 250,202exemplifying variations in the fate of the diphosphate 247 (Scheme 55). The two activities in N. glutinosa co-purified throughout the purification process. Cultured cells of the liverwort Heteroscyphus planus as well as producing sesquiterpenes of the cadalane type (see above) also synthesize a variety of diterpenes of the clerodane type.The biosynthesis of one of these heteroscyphic acid A 251 has been investigated by feeding 3C-labelled acetate and meval- onate prec~rsors.~~~~ 204 It was observed that there was prefer- ential labelling of the C units in the FPP-derived portion of the diterpene suggesting FPP from exogenous MVA was combining with endogenous IPP probably in the chloroplasts. In contrast the sesquiterpene C units were labelled equiva- lently. A similar non-equivalent labelling from MVA of the phytyl side-chain of chlorophyll a was noted though if glycine was used as precursor then equivalent labelling ensued.205 The labelling patterns in heteroscyphic acid A were consistent with Scheme 56 where folding of the GGPP precursor achieves a different stereochemistry to the labdanes in Scheme 55.The labelling confirmed the involvement of a 1,2-methyl migration as responsible for the introduction of the carboxy group C-20 134 Natural Product Reports I\ 247 248 cis-abienol I / OH 249 labdenediol 250 sclareol Scheme 55 GGPP t cis-clerodanes 10 251 heteroscyphic acid A Scheme 56 and also a 1,2-methyl migration of the 19-methyl from C-4 to C-5 in producing the trans-clerodane skeleton. The cis-clerodanes would be obtained by migration of the alternative C-4 methyl. The rice phytoalexins the oryzalexins are produced by a similar cyclization of GGPP as seen for the clerodanes above but proton loss generates copalyl diphosphate (PP) 252 (Scheme 57).Further cyclization initiated by loss of the diphosphate allows formation of (+)-sandaracopimaradiene (ent-isopimaradiene) 253 This is the likely substrate for oxgenation reactions beginning with the 3-hydroxy group. The conversion of the ent-isopimara-8( 14) 15-dien-3P-01 254 which is formed in UV-irradiated Oryza sativa leaves into ( -)-trachylobane 258 and (+)-sandaracopimaradiene 253 from copalyl PP reported some years ago in a cell-free extract have recently been resolved and partially purified. 207 (-)-Abietic acid 267 is a major component of the rosin GGPP 252 copalyl PP 8fl * +WH H H 253 (+)-sandaracopimaradiene 02,NADPH HO" HO" OH 254 ent-isopimaradienol 255 oryzalexin D I lozv NADPH HO" @y 256 oryzalexin E Scheme 57 oryzalexin D 255 and oryzalexin E 256 is catalysed by a microsomal fraction from the leaves.206 The conversion was dependent on 0 and NADPH and appears to be an inducible P-450 hydroxylase system.Diterpene cyclase activities in castor oil plant (Ricinus cornrnunis) are responsible for alterna- tive cyclizations of copalyl PP 252 (Scheme 58). The four activities generating ( -)-ent-kaurene 257 (+)-beyerene 259 -253 (+)-sandaracopimaradiene t I I 252 copalyl PP I 257 ent-kaurene 258 (-)-trachylobane 259 (+)-beyerene Scheme 58 Dewick The biosynthesis of CrC25 terpenoid compounds fraction of the oleoresin synthesized by grand fir (Abies grandis) lodgepole pine (Pinus contorta) and many other conifers as a defensive secretion against insect and pathogen attack.High levels of resin acid biosynthesis are induced in grand fir by stem wounding whereas lodgepole pine produces these materials constitutively. Abietic acid is derived from GGPP via ( -)-abieta-7(8) 13( 14)-diene 264 with subsequent oxidation of the C-18 methyl. Soluble extracts of lodgepole pine stem and mechanically wounded grand fir stem catalysed the Mg+-dependent cyclization of GGPP to ( -)-abietadiene 264 along with small amounts of pimaradiene 262 and its epimer ( -)-sandaracopimaradiene (isopimaradiene) 261 which are likely to be intermediates in the sequence (Scheme 59).,08 The product mixture from grand fir contained about GGPP 1 II 260 (+)-copalyl PP 262 pimaradiene 19 lBCH20H 264 (-)-abietadiene 265 abietadienol I C02H CHO 267 (-)-abietic acid 266 abietadienal Scheme 59 90% abietadiene whilst that from lodgepole pine contained more byproducts and yielded about 67% abietadiene.The grand fir enzyme was partially purified and had similar properties to other diterpene cyclases. In the metal ion require- ment Mg2+ was very much preferred to Mn2' which con- trasts to the monoterpene cyclases from these plants where Mn2' is more effective than Mg2+. There was no evidence for any separation of enzyme activities catalysing parts of the reaction sequence e.g. to (+)-copalyl PP 260 or pimaradiene 262 although these are logically implicated in the pathway (Scheme 59) the early parts of which mirror those of the oryzalexin pathway described above.Pimaradiene (or sandaracopimaradiene) undergoes a 1,2-methyl migration to generate the abietadiene skeleton via the abieta-8( 14)-enyl cation 263. Oxidation of abietadiene to abietic acid 267 is achieved by the successive action of three enzymes which have been demonstrated in cell-free extracts from both plants.209 The first two transformations abietadiene to abietadienol 265 and abietadienol to abietadienal 266 are catalysed by distinct P-450 systems requiring 0 and NADPH cofactors. Oxidation of the aldehyde 266 to the acid 267 is then accomplished by the action of an NAD+-dependent aldehyde dehydrogenase.The oleoresin secretion from grand fir consists of diterpene resin acids (rosin) and volatile monoterpenes (turpentine) which appear to act as a solvent for the diterpenes.210 This allows deposition of an antifungal rosin barrier to seal the injury. Monoterpene and diterpene cyclases the crucial enzymes required are coordinately induced in wounded stems reaching levels up to 100 times higher than in the uninjured stems. Interestingly the last enzyme in the abietic acid pathway the aldehyde dehydrogenase activity which is not inducible by wounding is constitutively expressed at a high level and therefore is not efficiently coupled to the earlier steps. Studies on the biosynthesis and metabolism of the gibberel- lins (GAS) always account for a considerable proportion of the terpenoid research literature.These diterpenoids with over 100 different structures now known play a significant role as plant growth hormones and have their origins in ent-kaurene 257. ent-Kaurene is produced from GGPP by the action of two enzymes firstly ent-kaurene synthetase A which cyclizes the substrate to copalyl PP 252 and then ent-kaurene synthetase B which accomplishes the further cyclization and subsequent rearrangement (Scheme 60). ent-Kaurene biosynthesis from GGPP has been observed using cell-free extracts from wheat (Triticum aestivum) seedlings pea (Pisum sativum) shoots and pumpkin (Cucurbita maxima) endosperm and in each case the activity was located in the plastids.211 A gene encoding for ent-kaurene synthetase A has been isolated from Arabidopsis thaliana and shown to express an active protein in E.coli.212 This gene would complement a dwarf gibberellin-responsive Arabidopsis mutant known to be blocked prior to ent-kaurene. Escherichia coli cells carrying both the ent-kaurene synthetase GGPP li 252 copalyl PP A gene and a gene from Erwinia urldovora which encoded GGPP synthase accumulated copalyl PP. The purification of ent-kaurene synthetase B from immature seeds of pumpkin has been reported.,' This activity was fully separated from ent- kaurene synthetase A and found to require a divalent metal ion such as Mg2+ Mn2' or Co2'. The hydroxylase enzyme catalysing the conversion of ent-kaurenoic acid 268 into ent-7- hydroxykaurenoic acid 269 in the fungus Gibberella fujikuroi has also been characterized and s~lubilized.~~~ This is a P-450- dependent enzyme requiring 0 and NADPH cofactors though FAD and monovalent metal ions stimulated the con- version.When ent-kaurene 257 and ent-kaurenoic acid 268 were fed to shoots of maize (Zea mays) GA,,-aldehyde 270 was elaborated but in addition several minor metabolites were detected.215 These have been identified as hydroxylated products 271 and 273 from ent-kaurene and 272 and 274 from ent-kaurenoic acid. The formation of 271 and 272 gives further examples of hydroxylation at an allylic position with re-arrangement of the double bond as seen with the biosynthesis of GA 302 from GA 300 (Scheme 61). The intensely sweet glucosides 276280 of the diterpenoid steviol 275 found in the plant Stevia rebaudiana are known to be formed from ent-kaurene 257.Three UDPGlc-dependent glucosyltransferase activities from the leaves have been frac- tionated and characterized.,16 One activity would accept steviol 275 and steviol monoside 276 as substrates but no other steviol glycosides. A second activity accepted only steviol whilst the third had rather broad substrate specificity accepting steviol steviol monoside steviol bioside 278 and stevioside 279. All the glucosyltransferases showed significant activity towards the flavonols quercetin and kaempferol. The conversion of stevioside 279 into rebaudioside A 280 by a partially purified enzyme from fresh leaves of S. vebaudiana has been reported and this enzyme will function when immobilized to DEAE-agarose gel.217 Scheme 61 gives the usual interrelationships of the most commonly encountered plant gibberellins as established from various experimental data.The stepwise conversion of GA,,- aldehyde 270 may proceed along the early 13-hydroxylation pathway via GA, 283 and GAa 286 or by the early non- hydroxylation pathway which involves GA, 282 and GA15 285. However many variations to this generalization are en- countered. In potato (Solanum tuberosum) shoots the early 13-hydroxylation pathway was confirmed by the conversion of labelled GA,,-aldehyde 270 and GA, 282 into GA, 283 GA 286 GA, 289 GA, 294 GA, 299 GA 295 and GA 290.,18 GA, 283 appears to be formed predominantly via GA, 282 rather than by GA,,-aldehyde 281 although the latter was produced by feeding GA,,-aldehyde 270.The 257 ent-kaurene 1 270 GA12-aldehyde 269 ent-7a-hydroxykaurenoic acid 268 ent-kaurenoic acid ent-kaurenal ent-kaurenol Scheme 60 Enzymes i ent-kaurene synthetase A; ii ent-kaurene synthetase B; iii ent-kaurene oxidase 136 Natural Product Reports 12 PH 270 GA12-aldehyde 281 GA53-aldehyde OH 282 GA12 283 GA53 I I 284 GA3 (open-lactone) 285 GA15 (open-lactone) 286 G& (open-lactone) 1 1 J 3-e-fl HOq HO HO C02H C02H C02H C02H C02H C02H CO2H 287 GA36 288 GA24 296 GAT 297 2,3-dehydro GAg 298 GA51 299 GAB 300 GAS 301 G& 302 GA3 Scheme 61 detection of endogenous GA, 298 suggested the non-conjugates.219A gene isolated from maize (Zeamays) encoding hydroxylation pathway was also active in this plant.In sor- a protein which appears to have significant sequence homology ghum (Sorghum bicolor) seedlings GA, 294 was metabolized to P-450enzymes is suggested to be involved in the early to GA 295 GA 290 and GA, 299 and possible glucosyl 13-hydroxylation step probably GA, 282+GA, 283.220 Dewick The biosynthesis of CrCZ5 terpenoid compounds 137 271 R=Me 273 R=Me 272 R=C02H 274 R=C02H C0,kl 275 steviol R1 = R2= H 276 s teviol monoside R1 = H; R2 = Glc 277 rubusoside R1 = R2 = Glc 2+1 278 steviolbioside Rl = H; R* = Glc-Glc 2+1 279 stevioside R1= Glc; R2 = Glc-Glc 2+1 280 rebaudioside A R1 = Glc; R2 = Glc-Glc I3+1 Glc Metabolism of GA, 294 to GA 295 has been demonstrated in both tall and dwarf forms of rice (Oryza sativa) and Arabi-dopsis thaliana though this was blocked in some mutants.221 Tall mutants also produced labelled GA29 299 from the feed- ings.Three 2-oxoglutarate-dependent dioxygenases have been detected in Cucurbita maxima separated and partially puri- fied.222 These were a 7-oxidase catalysing oxidation of the aldehyde group in GA,,-aldehyde 270 to give GA, 282 a 3P-hydroxylase converting GA15 285 into GA, 284 and a 20-oxidase which oxidized the 20-methyl in GA, to give GA15 285 and GA24 288. A crude enzyme preparation containing all three activities plus a 2P-hydroxylase was able to transform GAl,-aldehyde in the presence of "0 into ['802]GA4 292 and [180,]GA4 313 thus demonstrating that all oxygen atoms introduced after GA,,-aldehyde were derived from molecular oxygen.This 20-oxidase has been purified further and a partial amino acid sequence has been established.223 It was found to catalyse the transformation of GA, 282 into GA15 285 GA24 288 and also GA25 303 showing that it could oxidize C-20 sequentially to alcohol aldehyde and then acid (Scheme 62). GAS 283 was similarly oxidised to the alcohol GA 286 the aldehyde GA, 289 and then to the acid GA17 304 with small amounts of what was probably the C,,-gibberellin GA, 294. A 307 GA20-15-ene cDNA clone encoding the 20-oxidase was isolated and ex- pressed in E. The recombinant protein indeed catalysed the three step conversions of GA, to GA,, and GAS to GA17 but was also found to convert GA24 288 and GA, 289 into the C,,-gibberellins GA 293 and GA, 294 respectively.GA 295 was the product from incubation with the aldehyde GA23 305. A multifunctional gibberellin 20-oxidase has also been found in Arabidopsis thali~na.~~~ The gene was isolated by using the pumpkin cDNA as a probe and it was then expressed as a fusion protein in E. coli. The Arabidopsis protein had 55% amino acid identity with the pumpkin enzyme. This protein would convert GA, 283 into GA 286 with small amounts of GA19 289 and GA, into GA, 294 thus accounting for oxidation and loss of the C-20 methyl. In a parallel study three gibberellin 20-oxidase cDNA clones were isolated from Arabi-dopsis and expressed in E.coli as fusion proteins.226 Each of these oxidized GA, at C-20 giving predominantly GA via GA15 and GA24 with the acid GA25 303 being a minor prod- uct. GA, was similarly oxidized but less effectively than GA,,. It is inferred that the acids GA25 303 and GA17 304 are not intermediates in the formation of GA and GA,, but represent side-reactions (Scheme 62). A gibberellin 3P-hydroxylase from bean (Phaseolus vulgaris) converting GA, 294 into GA 295 and GAS 300 as well as the epoxidation of GAS to GA6 301 has been partially purified.,, A (2-20 hydroxylase converting GA, 282 into GA15 285 was also reported.*,' Variants on the pathways of Scheme 61 have been reported in several plants. In Brassica rapa GA 293 was converted into GA, 294 by a late 13-hydroxylation and GA 292 similarly into GA 295.229GA can apparently be produced by at least two metabolic pathways in this plant.The same conversions can occur in cultured cells of Raphanus sativus and a major pathway to GA is via GA and GA,.,,' Although GA, 294 may also act as a minor source of GA, the main metabolite of this gibberellin was found to be GA2,-15-ene 307. The 13- hydroxylation of GA giving GA has also been noted in several genotypes of Zea mays Oryza sativa and Arabidopsis th~liana.~~ ' Transformations in cell-free extracts of Cucurbita OH OH r HO& f\ C02H EO2H C02H C02H \ CO2H 'CC 282 GA12 -285 GA15 283 GA53 -286 G& ~ //304 -288 GA24 R=H -+ 289 GA19 R = OH ,\ 303 GA25 R = H GAIT R=OH 305 GA23 \ 306 GA20@ C02H \293 GAg R = H 295 GA1 294 GA20 R=OH Scheme 62 138 Natural Product Reports 282 GAl2 * 283 GA53 HO C02H C02H 288 GA24 * 287 GAS 310 12a-hydroxy-GA25 312 GA46 313 GA43 maxima which may be superimposed on those of Scheme 61 are outlined in Scheme 63.232,233Metabolism of GA, 282 in an endosperm system yielded GA13 309 GA, 313 and 12a-hydroxy-GA, 314 as major products with GA 292 GA, 291 GA, 311 and GA, 312 as minor products.Intermediates GA, 285 GA24 288 and GA, 303 accumulated at low protein concentrations. GA, 283 is formed from GA, by a micro- soma1 oxidase and converted predominantly into GA 295 GA23 305 GA2g 306 and GA 288. GA19 289 GA, 294 and GA, 315 are minor products. In further studies using an embryo preparation 12a-hydroxy-GA2 310 and 12a-hydroxy- GA, 308 were also detected in addition to those mentioned in the endosperm work.At low 2-oxoglutarate and ascorbate concentrations 3P-hydroxy products predominated whilst at high concentrations of cofactors 12a-hydroxy GAS were formed in increasing amounts. Only C19 GAS were 28-hydroxylated a characteristic of embryo systems. Glucosides and glucosyl esters are major products formed when gibberel- lins are fed to Dalbergia d~lichopetala.~~~ 294 and GAS GA, 300 give predominantly their glucosyl esters whilst GA 295 and GA 292 are converted into their 3-0-glucosides. Where modifications in the gibberellin portion occurred these were in accord with Scheme 61 and the product was sometimes bound as a glucoside.Dewick The biosynthesis of CrCzs terpenoid compounds 294 GA20 -292 GA4 \ 1 291 GAM 304 GA17 * 306 GAZE 290 GA8 311 GA39 I 314 12a-hydroxy-G& Scheme 63 The major gibberellin in Phaeosphaeria sp. L497 has been identified as GA 295 and it is formed in a pathway from GA,,-aldehyde 270 via GA 293 and GA 292 or via GA and GA, 294.,,*3 236 The pathway was confirmed by feeding exper- iments and is analogous to that seen in some plants (see above) but has not been noted previously in fungi. Retinoids possess a diterpenoid skeleton but are actually derived by oxidative cleavage of a C, carotenoid. Cleavage of P-carotene 316 may occur at the central double bond theoreti- cally giving two molecules of retinal 318 or by an excentric process liberating just one molecule of retinal.Retinal is then reduced to retinol (vitamin A,) 321 via an alcohol dehydro- genase activity an enzyme which is also required in the produc- tion of retinoic acid 319 from vitamin A via retinal (Scheme 64).Human alcohol dehydrogenase consists of a family of five related enzymes one of which denoted class IV is most active as retinol dehydrogenase. The gene for this enzyme has been cloned and sequenced and shown to be expressed principally in the human stomach but not in the liver.237 The mouse has a simpler alcohol dehydrogenase gene family than humans with only three types of enzyme though a'highly conserved class IV enzyme analogous to the human retinol dehydro- genase has been detected.,, Two retinol dehydrogenase activi- ties in microsomes of rat liver have been obtained by gene 317 9-cis-P-carotene 318 retinal 319 all-trans-retinoic acid C02H 320 9-cis-retinoic acid 321 retinol (vitamin A,) 322 11-cis-retinol \ 323 dehydroretinol (vitamin A2) 324 11&-retinal b0 Scheme 64 cloning and expression.2397 240 The proteins had amino acid sequences some 82% identical and both had higher activity with NADP' than NAD+.Both enzymes recognized retinol in its physiological form bound to cellular retinol-binding pro- tein. Enzyme activity was detected in liver brain kidney lung and testes though expression was in the liver. The biosynthesis of 1 1-cis-retinal 324 the chromophore of visual pigments requires isomerization of all-trans-retinol 321 to 1 1 -cis-retinol via a retinyl ester.The final step is catalysed in bovine retinal pigment epithelium by a membrane-associated 11-cis-retinol dehydrogenase which has been isolated and partially charac- teri~ed.,~~ This is a member of a family of short-chain alcohol dehydrogenases and is active with NAD+ but not NADP'. Dehydroretinol (vitamin A,) 323 constitutes some 2&25'%0 of the total retinoid content in human epidermis and its forma- tion directly from labelled retinol 321 has been demonstrated in cultures of human skin keratinocyte~.,~ No labelled retinal dehydroretinal retinoic acid or dehydroretinoic acid could be detected. An NAD' -dependent aldehyde dehydrogenase that cataly- ses oxidation of retinal to retinoic acid 319 has been purified from rat kidney.243 A cytosolic retinal oxidase activity from rabbit liver has been characterized as a metalloflavoenzyme containing two FADS eight iron and two molybdenum atoms.244 This enzyme required molecular oxygen for the conversion of retinal into retinoic acid but this oxygen was not incorporated into the product water supplying the new oxygen.A variety of biological effects appear to be mediated by retinoids and two families of receptors are recognized retinoic acid receptors (RARs) and retinoid X receptors (RXRs). 9-cis-Retinoic acid 320 is a specific ligand for RXR whereas both 9-cis- and all-trans-retinoic acid 319 activate the RAR family. The isomerization of all-trans-retinoic acid to 9-cis- retinoic acid and also 13-cis-retinoic acid has been observed in bovine liver membranes.245 The isomerization presumably occurs at the acid oxidation level since all-trans-retinol and all-trans-retinal were not converted into 9-cis-retinoids by the microsomes.The reaction is shown to be dependent on the presence of thiol groups in the protein and since isomerization was inhibited in the presence of a-tocopherol and ascorbic acid a free radical mechanism is ~urmized.~~~ An enzyme from rat kidney has been described which catalysed the oxidation of both 9-cis-retinal and all-trans-retinal into the corresponding acids suggesting that in this system isomerization occurs at the retinol oxidation level analogous to the 1 1-cis isomerization in the visual pigment pathway.247 Rat kidney enzyme also accepts both isomers of retinal as substrates with similar effi~iency.,~' Rapid isomerization of all-trans- and 9-cis-retinoic acids has been reported in rat serum.249 To add yet a further variant the conversion of 9-cis-'p-carotene 317 into both 9-cis-retinoic acid and all-trans-retinoic acid in human intestinal mucosa has been described.250 The addition of all-trans-p-carotene to the system increased the rate of all- trans-retinoic acid biosynthesis but did not affect the rate of 9-cis-retinoic acid formation indicating a direct pathway from 9-cis-p-carotene to 9-cis-retinoic acid.9-cis-j3-Carotene is not usually a constituent of fresh vegetables but processed vegeta- bles may contain up to 50% of the total j3-carotene as the 9-cis isomer.Recent reviews covering aspects of diterpenoid biosyn- thesis include discussions of taxol bio~ynthesis,~~ Taxol 1-253 production by fungi associated with Pacific yew,254 taxane diterpen~ids,~'~ biosynthesis and metabolism of gibberellins in higher plant^,^^^-^^^ and the genetics of gibberellin bio~ynthesis.~~'-~~~ 140 Natural Product Reports 1,!i-hydride shift, Me-C02Na --325 GFPP c- 327 astellatol 326 Scheme 65 9 Sesterterpenoids It is an unusual event to record biosynthetic studies on sesterterpenoids although many examples of this least common group of natural terpenoids are now known. The origins of the complex ring systems of astellatol327 in Aspergillus variecolor have been investigated by feeding ['3C2]acetate.260 The label- ling pattern supports a pathway via cyclization of geranyl- farnesyl diphosphate (GFPP) 325 and subsequent rearrange- ments but retaining all ten pairs of coupled carbons (Scheme 65).It is suggested that these include an early 1,5-hydride shift (as seen with other sesterterpenoids) then cyclization accompanying two 1,2-hydride shifts. Ring expansion of a cyclopropyl-containing cation 326 is suggested to account for the final ring structure. Note that the published scheme260 contains a number of errors and Scheme 65 is a corrected version. 10 References 1 P. M. Dewick Nut. Prod. Rep. 1995 12 507. 2 J. Gershenzon and R. Croteau in Lipid Metabolism in Plants ed.T. S. Moore CRC Boca Raton 1993 p. 339. 3 T. J. Bach Lipids 1995 30 191. 4 T. J. Bach in Plant Lipid Metabolism ed. J.-C. Kader and P. Mazliak Kluwer Dordrecht 1995 p. 321. 5 J. 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ISSN:0265-0568    

DOI:10.1039/NP9971400111

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

Natura1 sesquiter penoids Braulio M. Fraga Instituto de Productos Naturales y Agrobiologia CSIC 38206-La Laguna Tenerfe Canary Islands Spain Covering 1995 Previous review 1996 13 307 1 Farnesane 2 Mono- and bi-cyclofarnesane 3 Trifarane 4 Bisabolane and majapolane 5 Sesquicamphane and sesquipinane 6 Trichothecane and laurane 7 Chamigrane thujopsane and omphalane 8 Carotane acorane cedrane duprezianane and anisatin group 9 Cadinane and indipane 10 Himachalane longipinane and longifolane 11 Caryophyllane modhephane isocomane silphinane and botrydiane 12 Humulane alliacane hirsutane precapnellane isocap- nellane lactarane isolactarane marasmane protoillu- dane illudane africanane astericane and tremulane 13 Germacrane 14 Elemane 15 Eudesmane lindelane valerane oppositane and iphio- nane 16 Vetisperane 17 Eremophilane bakkane and tridensane 18 Guaiane xanthalane pseudoguaiane and patchoulane 19 Aromadendrane bicyclogermacrane aristolane nar-dosinane and brasilane 20 Pinguisane and neopinguisane 21 Miscellaneous sesquiterpenoids 22 References 1 Farnesane The sesquiterpene ester (22 6E)-9-hydroxyfarnesyl oleate has been found in an extract of the aerial part of Urolepis hecatantha.Three farnesyl-homogentisic acid derivatives have been obtained from the seeds of Otoba parvifolia.’ Three new sesquiterpene hydroquinones rietone 1 8‘-acetoxy-rietone 2 and 8’-desoxyrietone 3 have been isolated from the South African soft coral Alcyonium Dictyodendrillins A B and C 46 are three new oxygenated sesquiterpenes which have been found in a Southern Australian marine sponge of the genus Di~tyodendrilla.~ The properties of a farnesol phos- phokinase obtained from a strain of Botryococcus braunii have been ~tudied.~ Several dimethylallyl diphosphate analogues having oxygen atoms in their alkyl chains were found to act as substrates in a reaction catalysed by pig liver farnesyl diphos- phate synthase.6 A novel inhibitor of ras farnesyl-protein transferase cylindrol A has been obtained from Cylindrocar- Abbreviations The following structural abbreviations are used in this article triflate in acetonitrile.” ipomeamarone12 and achieved.Boronia megastigma. l4 OR2 1 R’=OH; R2=H 2 R’ =OAC; R2=H 3 R‘=R2=H 4 5 OH 6 Syntheses of the phytoalexins (+)-( -)-(S)-my~porone’~ have been 2 Mono- and bi-cyclofarnesane The new sesquiterpene (E)-3-hydroxymegastigm-7-en-9-one7 and other compounds of this type have been obtained from Novel ionyl glycosides have been iso- lated from Alangium premnifolium 571 Maerua crassifolia,’ Urtica dioica” and Veratrum lobelianum2’ The occurrence composition and role as flavour precursors of the C ,-norisoprenoid glycosides in plant tissues have been reviewed.2 A new sesquiterpene-quinol riccardiphenol 8 has been isolated from a New Zealand collection of the liverwort Riccardia crassa.22 Another two compounds of this type cyclorenierin A 9 and cyclorenierin B 10 have been found in a sponge of the Haliclona genus collected in Vanuatu.23 The sesquiterpene apo-9‘-fucoxanthinone has been obtained from a cultured marine dinoflagellate of the genus Arnphidini~m.~ pon lucidum and its structure determined by X-ray analy~is.~ A mechanistically designed bis-cinchona alkaloid ligand serves Pistia stratiotes,” as a catalytic ligand in the OsO promoted enantioselec- tive dihydroxylation of the terminal isopropylidene group in farnesyl esters and other oligoprenyl derivatives.8 The bio- transformations of (f)-cis-nerolidol and nerylacetone using the plant pathogenic fungus Glomerella cingulata have been studied.A short synthesis of the hydrocarbon (32 6E)-a-farnesene has been described.” A biomimetic cyclization of farnesyl sulfone has been carried out using a preparation of mercury(I1) Fraga Nat ura1 sesquit erp en oids kuehneromycin B 28 have been isolated from fermentations of a Tasmanian Kuehneromyces specie^,^' whilst a novel anti- coccidial sesquiterpene fudecalone 29 has been obtained from a Penicillium Compound F1839-A 30 is one of the eight MeO 17 18 R=a-OMe 19 R=P-OMe Me0 OH h0rOOAcR 9 R=a-Me 11 R=H 10 R=P-Me 12 R=Ac 20 R=a-OMe 22 21 R=P-OMe 13 14 CHO 23 24 15 16 0 c,l"ppco2H The isolation has been reported25 of two ichthyotoxic sesqui- terpene glycerides tanyolide A 11and tanyolide B 12 from the nudibranch Sclerodoris tanya.Their structures were confirmed by synthesis. Five known brominated sesquiterpenes have been isolated from the red alga Laurencia karlae.26 Oceanapamine HO'.H02C 13 is a new antimicrobial sesquiterpene alkaloid which has C02Me been found in a Philippine sponge of the genus Oce~napia.~~ 25 26 Two novel cytotoxic sesquiterpenes arenaran A 14 and arenaran B 15 have been obtained from the sponge Dysidea arenaria which was collected in Thailand.28 The synthesis of (+)-(6R)-a-ionone has been achieved.29 A racemic synthesis of fulvanin 1 (sollasin A) has been de~cribed.~' The spirocyclic sesquiterpene ethers dactyloxene B and dactyloxene C have been synthesised in enantioselective 27 28 form.3' A short synthesis of (-)-chokol A has been reported.32 0 A rapid method has been developed for the simultaneous extraction and analysis of abscisic acid cytokinins and auxins.33 The isolation metabolism synthesis and biological activity of 3'-hydroxy-y-ionylideneaceticacid derivatives have been studied.Some of these compounds have been obtained from Cercospora cr~enta.~~'~' The synthesis biological activity and metabolism of several fluoroabscisic acid derivatives have also been de~cribed.~~.~~ Nine new phytotoxic sesquiterpenes named 3a-29 30 acet oxypol ygodial 16 3a-ace t ox ypol ygodial 1 2-dime thy1 acetall7,9-deoxymuzigadial 12a-acetal 18,9-deoxymuzigadial 12P-acetall9 3P-acetoxypolygodial 12a-acetal20,3P-acetoxy-novel sesquiterpenes obtained from the fungus Stachybotrys polygodial 12P-acetal 21 9-epideoxymuzigadial 22 3P-F-1839. These compounds have been shown to inhibit acetoxypolygodial 23 and muzigaal 24 have been obtained cholesterol e~terase.~~ from the leaves of Canella winter an^.^^ Limeolide 25 is a new Another fungus Antrodia cinnamomea contains the novel drimane sesquiterpene which has been isolated from Limeum sesquiterpene lactone antrocin 31.44 The structure of the The structure of haploporic acid A has been myoinositol monophosphatase inhibitor L-67 1,776 has been ptero~arpum.~~ determined as 26.This dimeric drimane sesquiterpene has been shown to be identical to that of the known ATCC 20928-B 32. The This compound has been found in two species of the Menno-found in the wood-rotting fungus Haploporus odor~s.~' biologically active sesquiterpenes kuehneromycin A 27 and niella genus.45 Nakijinol 33 is a new sesquiterpene with a 146 Natural Product Reports benzoxazole ring which has been obtained from an Okinawan sponge of the family Sp~ngiidae.~~ This same species contains two novel sesquiterpene quinones with a hydroxy amino acid residue.These compounds have been named nakijiquinone C 34 and nakijiquinone D 35 and exhibited inhibitory activity against c-erbB-2 kina~e.~~ Another two bioactive drimane derivatives wiedendiol A 36 and wiedendiol B 37 have been isolated from the marine sponge Xestospongia ~iedenmayeri.~' Another two sponges of the genus Hyrtios collected off the Hawaiian coast contain three novel sesquiterpenes which have been named 21-chloropuupehenol38 15-oxopuupehenol 39 and meloxinenone 40.49 The bioactive compound bis (su1fato)cyclosiphonodictyol A 41 is a new disulfated 31 f=N 0A 33 34 R=H 35 R=Me OH OH OH 38 39 40 41 sesquiterpene-hydroquinone which has been found in the sponge Siphonodictyon coralliphagum collected from the ocean in the Bahamas at a depth of 195 feet.50 The structure of 5'-acetylavarol has been confirmed by X-ray analy~is.~' The chemistry and sources of mono- and bi-cyclic sesquiter- penes isolated from the genus Ferula have been reviewed.52 Five sesquiterpene coumarins have been obtained from the roots of Ferula a~afoetida.~~ The structure of farnesiferol C which had also been isolated from this species has been confirmed by total synthesis.54 An efficient synthesis of wiedendiol A starting from com- mercial (+)-sclareolide has been described,55 whilst racemic Fraga Natural sesquiterpenoids albicanol has been used in the preparation of albicanal and drim-8(12)-ene.56 Several diterpenes have been utilised as starting materials in the syntheses of (+)-albicanol and (+)-bicyclofarnesol,57 drimadiene,58 polyg~dial~~~~~ and warburga- na1.60 The syntheses of natural ( -)-oblongolide6' and its enantiomer62 have been reported.Marine sesquiterpenes such as ( f)-isoa~anthodoral,~~ ( f)-arenarol,64 ( -)-ilimaq~inone,~~ ( f)-pallescensin A66 and ( f )+pi-pallescensin A67 have been synthesised. 3 Trifarane Trifarienols A-E (4246) are five trifarane sesquiterpenes which have been isolated from Cheilolejeunea trifaria. These compounds possess a novel skeleton named trifarane.It had 42 R1 = H; R2= P-OH H 47 43 R' = H; R2 = a-OH H 44 R' = Ac; R' = P-OH H 45 R1 = Ac; R2=a-OH H 46 R' = H; R2 = 0 48 49 50 been proposed that this originated from a pinguisane deriva- tive but now a more plausible biogenetic pathway via striatane-type sesquiterpenoids has been suggested.68 The synthesis of trifarienol A and trifarienol B have been carried 4 Bisabolane and majapolane Two new sesquiterpenes (+)-p-turmerone 47 and the bisab- olane endoperoxide 48 have been found in an extract of the aerial parts of Chrysoma pauclJEos~ulosa,~~ whilst the enol ether ligustilone 49 has been obtained from the roots of Ligusticum sinensis." A novel bisabolane glycoside has been identified as a constituent of the roots of Iostephane heterophylla a species used in Mexican traditional medicine.72 (-)-Curcuhydroquinone-1-monoacetate 50 is a novel bisabolane sesquiterpene which has been isolated from Barbadian Pseu-dopterogorgia arneri~ana.~~ The biotransformation of ( -)-a-bisabolol by the plant pathogenic fungus Glomerella cingulata has been Enantiocontrolled syntheses of (+)-j~vabione~~>~~ and (+)-epij~vabione~~ have been described.The termite trail attract- ants (a-a-bisabolene (2)-a-bisabolene and p-bisabolene have been prepared in racemic form.77 A synthesis of highly enantiomerically enriched (S)-( -)-bisabolane has been re-ported.78 The sesquiterpene 12-hydroxy-2-oxobisabol-3-ene has been ~ynthesised.~~ A total synthesis of the bisabolene sesquiterpenes (9-( +)-curcudiol (5')-(-)-curcupheno1 and (I?)-( -)-curcupheno1 have been described." Enantioselective total synthesis of the four stereoisomers of yingzhaosu C have been carried out." A concise synthesis of yingzhaosu C and epi-yingzhaosu C has also been developed.** A tetranorsesquiterpenoid which had been obtained from Heritiera ornithocephala has now been ~ynthesised.'~ A new chiral catalyst has been used in the enantioselective prep- aration of the chemotactic factor ~irenin.'~ The reaction of perezone and isoperezone with hydroxylamine has been described as a facile method for the introduction of an amino group into the quinone f~nctionality.'~ Seven new sesquiterpenes majapolene A 51 majapolene B 52 majapolone 53 majapol A 54 majapol B 55 majapol C 56 and majapol D 57 have been found in a Philippine collection 6 Trichothecane and laurane The first enantiocontrolled total synthesis of ( -)-debromofiliformin and ( -)-filiformin has been carried o~t.~~,~~ An asymmetric synthesis of (+)-laurene 61 has been described.95 7 Chamigrane thujopsane and omphalane A new chamigrane derivative 62 has been isolated from the digestive glands of the mollusc Aplysia dactylomela collected in Indian waters.96 The marine red alga Laurencia majuscula contains three brominated chamigrane sesquiterpenes which possess a novel pattern of antitumour activity.97 The novel sesquiterpenes mayurone epoxide 63 and thujopsenol epoxide How-,, .Ow-52 -, 51 53 54 62 63 64 cD--Br HO CI -0 Q HO OH 55 56 5PCOzMe 65 66 R=H 4 6-"' __ 67 R=hg 0 57 of the red alga Laurencia majuscula.All compounds except majapolene B 52 were isolated as inseparable diastereomeric mixtures.86 These compounds possess a cyclised bisabolane skeleton previously found in Laurencia obtusa (see Nut. Prod. Rep. 1985 2 149 structure 53) which we have now named majapolane. A racemic total synthesis of parvifolin and isoparvifolinone has been a~hieved.'~ 5 Sesquicamphane and sesquipinane The new sesquiterpenes cyclosantanal58 and epicyclosantanal have been obtained from Santalum album (sandalwood oil).'' ( -)-p-Santalene and (+)-epi-p-santalene have been prepared starting from (+)-nor~amphor.'~~~' A racemic total synthesis of the trinorsesquiterpene albene 59 has been devised." The structure of a sesquiterpene lactol 60 has been resolved by X-ray analysis.92 58 59 H 60 61 148 Natural Product Reports 68 R =Oleyl 69 70 0 woOH OH 71 72 64 have been found in an extract of Cupressus bakeri." A new sesquiterpene methyl omphalate 65 which possesses a rearranged chamigrane skeleton named omphalane has been obtained from the liverwort Omphalanthus filiformi~.~~ 8 Carotane acorane cedrane duprezianane and anisatin group The structures of fersorin and fersoridin have been determined as 66 and 67 respectively.These compounds have been obtained from the roots of a species of the Ferula genus."' A novel oleic acid ester of the carotane sesquiterpene, 14-hydroxy-CAF-603 68 has been isolated from cultures of the fungus Trichoderma virens.This compound possesses biological activity affecting the high conductance calcium- activated potassium channel. ''' A potentially useful precursor of carotane and tormensane derivatives 13-nor-2,6( 10)- carotadiene has been synthesised starting from ( f )-nerolidol.lo2 (+)-Acoren-'la-ol 69 is a new sesquiterpene which has been obtained from the liverwort Bazzania tri- dens.'03 The culture broth of the fungus Trichoderma koningii contains the novel sesquiterpene trichoacorenol 70.'04 The total synthesis of two sesquiterpenes with a duprezianane skeleton which had been isolated from Eremophila georgei has been achieved. '05 The biotransformation of (+)-cedrol and 8-epi-cedrol by the fungi Glomerella cingulata' O6 and Cephalosporium aphidi- cola,'O7 respectively have been studied.Cedrol has been shown to have PAF receptor binding antagonistic activity. lo* The molecular rearrangement of ( -)-a-cedrene into isomeric compounds with zizaene and patchoulane skeleta has been studied.'09 A new sesquiterpene lactone minwanensin 71 has been found in the pericarps of the fruit of Zllicium minwan- ense.' lo The structure of tashironin has been elucidated as 72. This presumed biosynthetic precursor of the anisatin-type of sesquiterpenes has been obtained from the wood of Zllicium tashiroi. '' 9 Cadinane and indipane Four new sesquiterpenes 7S76 have been obtained from the Taiwanese liverwort Reboulia hemisphaerica.' l2 Another four new cadinane derivatives ganomastenols A-D 77-80 have been isolated from the fungus Ganoderma mastoporurn.' l3 The essential oil of Solidago graminifolia contains the new sesqui- terpene 8,9-oxidocadalene 81.'l4 Inuloidin 82 is a novel plant growth inhibitory sesquiterpene which has been found in an extract of the dried flowers of Heterotheca inuloides,"5 The structure of macnabin has been determined as 83.This new secoamorphane triether has been isolated from the foliage of Cupressus macnabiana. 'l6 Another two novel cadinane ethers 84 and 85 have been found in Brazilian Vassoura oil which is prepared from the leaves of Baccharis dracunculifolia.' l7 10,12-Peroxycalamenene86 is a novel sesquiterpene with anti- malarial properties which has been obtained from the tubers of Cyperus rotundus a plant used in Thai popular medicine.' ' Shift reagent and 2D NOESY NMR methods have been applied to the structure elucidation of 4~,5a-dihydroxycubenol 87 which has been obtained from the tropical brown alga Dictyopteris delicatula. 'l9 Other new sesquiterpenes 88 and 89 have been found in the marine sponge Axinyssa aplysi-noides.120 (+)-&-Cadinene and another two novel cadinane sesquiterpenes 90 and 91 have been found in an extract of the cotyledons of cotton (Gossypium hirsutum) inoculated with Xanthomonas campestris. This system has been used to inves- tigate the biosynthesis of 6-cadinene from labelled farnesyl pyrophosphate. 121,122Another similar study using as elicitor Verticillium dahlia has been reported.123 The biosynthesis of (+)-cubenene (+)-epicubenol and (1S)-7-methoxy-l,2-dihydrocadalene by cell free extracts of cultured cells of the liverwort Heteroscyphus planus has been studied. 1243125 This species also contains the novel sesquiterpene (+)-5,8-dihydroxycalamenane 92.126 The sesquiterpene cadina- 4,10( 15)-dien-3-0ne 93 possesses chemosterilant activities against cattle tick Boophilus microplus and toxic action against adult Cylasformicarius a pest of sweet potato.'27 Enantioselective total syntheses of 7-desmethyl-2-methoxycalamenene' 28 and (+)-i~ozingiberene'~~ have been described. The phenolic derivative sesquichamaenol has been synthesised starting from ~alamenene.'~' A synthesis of the sesquiterpene lactone (+)-heptelidic acid has been reported.I3' An HPLC procedure for the rapid and simultaneous deter- mination in plant extracts of artimisinin and its biosynthetic precursors has been developed.' 32 The artemisinin content of tissue-cultured greenhouse-grown and field-grown plants of Artemisia annua have been e~aluated.'~~,'~~ A selection of this species with high yields of artemisin has also been carried out.35 The microbiological transformation of the antimalarial drug arteether by Cunninghamella elegans and Streptomyces lavendulae has been in~estigated.'~~ The 13C NMR assign-ments of a-and P-dihydroartemisinin have been carried The preparation of D-ring-contracted analogues of arti-misinin and their in vitro antimalarial activity have been Fraga Natural sesguiterpenoids R&: Hi R Hi A A 73 R=CHO 77 R=a-OH 74 R=CH20H 78 R=B-OH 75 R=CH~OAC 76 R=C02H OH i\ @io: H A 79 80 9 Ho*oH 0 A 81 82 83 84 85 86 ..q OH R ' H: A A 87 88 R=NC 90 89 R=NHCHO Hoqo #J OH i\ 91 92 93 94 95 96 97 R=Me 98 R= Pr investigated.138 Syntheses of 1l-a~aartemisinin'~~ and (+)-13- carbaartemisinin 140 have been reported.The structure of (+)-indipone has been determined as 94. This compound possesses a new carbon skeleton named indipane which probably derived from ( -)-epizonarene 95. Both substances together with the norsesquiterpenes 96-98 have been isolated from Cupressus bakeri.98 10 Himachalane longipinane and longifolane Sesquiterpenic hydrocarbons such as a-himachalane a-amorphane and germacrane A have been found in the larval 149 osmeterial secretion of Pachliopta aristolochiae.14' The struc- tures of two himachalene epoxides have been resolved by X-ray analysis. 142 Five new longipinane sesquiterpenes 99-103 have been obtained from Stevia errata.'^^ Another two com- pounds of this type 104 and 105 have been isolated from the -OR2 14'plished. 104 R =Ang 105 R=Tig roots of Stevia viscida. The molecular rearrangement in alkaline medium of a longipinane derivative rastevione mesylate has been studied.'45 A facile synthesis of (f)-isolongifolene and ( f)-isolongifolenedione has been accom- 11 Caryophyllane modhephane isocomane silphinane and botrydiane (+)-P-Caryophyllene has been identified in the essential oils of several liverworts by enantioselective gas chromatography.147 A new secocaryophyllene derivative 106 has been isolated from the leaves of Montanoa karwinskii. 14* The biotransformation of ( -)-caryophyllene by Chaetomium cochliodes and the antimite activities of their metabolites have been studied. 149 The rearrangement of (-)-P-caryophyllene with sulfuric acid in ether has been reinvestigated and fourteen hydrocarbons and four alcohols have been isolated and identified.15' New syntheses of (f)-isocaryophyllene ( f)-~aryophyllene,'~~ (f)-modhephene ( f)-epimodhephene and ( f)-isoco-mene152 have been described. 1 06 107 108 _-__-HO' OH 109 R = a-OMe H 111 112 110 R = p-OMe H 113 I50 Natural Product Reports A known silphinene derivative 107 has been shown to have antifeedant activity against Leptinotarsa decemlineata.This compound has now been found in Senecio palmensis.'53 Collybial 108 is a new antibiotic sesquiterpenoid which has been obtained from an American strain of Collybia con-fluens. 154 The three botrydiane sesquiterpenes 109-111 and the probotrydiane derivative 112 have been isolated from shake cultures of Botrytis ~inerea.'~~ This fungus also contains botrylactone,' 56 an antibiotic whose synthesis from D-glucose has permitted its structural revision to 113.'57 12 Humulane alliacane hirsutane precapnellane isocapnellane lactarane isolactarane marasmane protoilludane illudane africanane astericane and tremulane A stereoselective total synthesis of alliacol A alliacolide and 12-noralliacolide has been achieved.158 A short pathway for the synthesis of hirsutane derivatives has been developed. 159 Syntheses of the sesquiterpenes ( f)-ceratopicanol'" and (8R,1 1R)-( -)-precapnelladiene' " have been reported. Two novel sesquiterpenes 114 and 115 have been isolated from a species of the genus Buddleia. These compounds have a new skeleton which has been named isocapnellane. 162 A new lactarane sesquiterpene 116 has been obtained from the fungus Russula emetica. 163 The total synthesis of another compound of this type furanether B has been de~cribed.'~~ The antifeedant activity of several sesquiterpenes with lactarane marasmane and isolactarane skeleta against the storage pests Tribolium confusum Trogoderma granarium and Sitophilus granarius have been evaluated.These compounds or their chemical precursors had been isolated from species of Lactarius.'65 Two similar mushrooms of this genus have now been considered to be two different species Lactarius vellereus and Lactarius bertillonii considering their sesquiterpene content. 166 OH OH MeO" R 114 R=H 116 117 R=OH 115 R=OH 118 R=H OH OH H H 119 120 121 122 123 Tsugicolines A-D 117-120 are four protoilludane sesquiter- penes which have been found in still cultures of the fungus Laurilia ts~gicola''~ A new method of purification has been used in the isolation of the potent bracken carcinogen ptaqui- loside to prevent loss of activity.Its structure has now been confirmed by modern NMR spectroscopic techniques. 168 The aglycone of this glucoside ptaquilosin has also now been synthesised.169 Three new sesquiterpenes 121-123 have been isolated from Lippia integrifolia The compound 123 possesses a new skeleton while 121 and 122 have an africanane and an asteriscane framework re~pectively.'~' The total synthesis of (+)-dactyl01 has been a~hieved.'~' The synthesis of ( f)-8a-epi-tremulenolide has also been reported. '72 13 Germacrane The novel germacrane derivative 124 has been identified as a component of a strain of Streptomyces citreus. 173 The gerrna- crane alcohols 125 and 126 have been found in an extract of 124 125 R OAC 126 R = a-OH or p-OH 127 R= 2hosen the leaves of Juniperus comm~nis.'~~ Another compound of this type 127 has been obtained from the aerial parts of Salvia miltiorrhiza.75 The novel sesquiterpenoids (1 27,327-1 -acetoxygermacra-3,1O( 15)-diene and (1 S,32,5E)-l -acetoxy- germacra-3,5,10( 15)-triene have been isolated from the soft coral Nephthea chabroli. 176 The biotransformation of (+)-hedycariol and other germacrane sesquiterpenes by a suspen- sion of fresh chicory roots (Cichorium intybus) have been investigated.'77,178 A new route to the Mori's intermediate for the practical synthesis of (-)-periplanone B has been reported. 179 The new germacrane lactones which have been isolated from natural sources during 1995 are listed in Table 1.Structures 128-151 represent the new germacranolides whilst structures 152-161 have been assigned to the heliangolides and 162-166 to the melampolides. No lactones of the cis,cis-germacranolide type were isolated during 1995. There are several points to note in relation to these sesqui- terpene lactones. Known compounds of this type have been isolated from Carpesium glossophyllum 193 Centaurea aspera Centaurea malacitana '94 Centaurea napijolia,Ig5 Cnicus ben- edictus,'96 Heteranthemis viscedehirta 197Michelia nilagiri~a'~' and Neolitsea hiiranensis. 199 The sesquiterpene lactones pro- duced by hairy root cultures of Lactuca virosa transformed with Agrobacterium rhizogenes have been investigated.200 Melampolides have been used as chemotaxonomic markers in a study of Espeletia species.201 The bioactive sesquiterpene lactones parthenolide and costunolide have been extracted from the leaves of Magnolia grandflora by supercritical carbon dioxide and near-critical extraction with propane.202 The action of costunolide and dehydrocostunolide as inhibitors of the killing function of cytotoxic T-lymphocytes has been inve~tigated.~" The molecular structures and absolute configurations of the heliangolides tithonin204 and hirs~tolide~'~ have been con-firmed by X-ray analysis.The conformational analysis and the biological activity of leptocarpin and its acetate have been studied.206 The reaction of the molluscicide glaucolide B with bentonite has been in~estigated.~'~ A stereoselective and Fraga Natural sesquiterpenoids Table 1 Sources of germacrane lactones Source Ref.Germacranolides Allagopappus dichotomus Allagopappus viscosissim um Cladanthus arabicus 129 130-134 128 180 180 181 Helianthus sp. Mikania cordata 135 145-148 182 183 Mikania ypacarayensis Montanoa karwinskii 149 142-144 184 148 Neurolaena lobata 136-139 185 Perymenium berlandieri Tanacetum densum 140 141 150 186 187 Vicoa indica 151 188 Helianthus sp. Lychnophora rupestris Neurolaena lobata Heliangolides 152 153 154 182 189 185 Schkuhria pinnata Urolepis hecathanta 155159 160 161 190 1 Melampolides Enhydra fluctuans Mikania ypacarayensis Smallanthus sonchifolius 162 163 164 165 166 191 184 192 enantiospecific total synthesis of the cytotoxic furanohelian- golide eremantholide A starting from D-glucose has been achieved.208 14 Elemane The sesquiterpene 167 has been found in an extract of the leaves of Cryptomeria japoni~a.~'~ A plant growth regulator 8-desacylvernodanol 168 has been isolated from the fresh leaves of Vernonia auriculifera.210 The structure of zinnacitrin a new elemandiolide which has been obtained from the aerial parts of Zinnia citrea has been determined as 169.211The dilactones 170 and 171 have been found in the aerial parts of Mikania ypacarayensi~."~ The complete assignment of proton and carbon NMR spectra of elemol has been reported.212 The bioactive sesquiterpene lactones elema- 1,3,11 -trien-8,12-0lide and isoalloalantolactone have been isolated from the roots of Ratibida mexicana.213 The enantioselective total synthesis of p-elemene has been achieved.214 (+)-Limonene and (+)-2-carene have been used as starting material in the synthesis of (+)-a-elemene.215 The first asymmetric total synthesis of (+)-vernolepin has been described,216 whilst a (+)-vernolepin derivative has been synthesised starting from ~alonitenolide.~'~ 15 Eudesmane lindelane valerane oppositane and iphionane A new trinoreudesmane sesquiterpene 172 and a novel eudes- mane sesquiterpene 173 have been isolated from the liverworts Lophocolea bidentata2'8 and Plagiochasma rupestre,2'9 respect- ively.A norsesquiterpene norsesquibenihol 174 has been obtained from the essential oil of Chamaecyparis formo- Another six new norsesquiterpenes tehuetone 175 teutenone A 176 teuhetenone B 177 tephyllone 178 9-hydroxytephyllone 179 and 9-oxotephyllone 180 together with the sesquiterpene 7-epi-teucrenone 181 have been found in an ethanolic extract of the aerial parts of Teucrium hetero- phyllum,221 while another species of this genus Teucrium polium contained two novel eudesmane sesquiterpenes with the relative structures 182 and 183.222The new antibacterial sesquiterpene 184 has been isolated from Pluchea arg~ta.~~~ 151 128 129 R1 = Ang; R2 = (C0)Pr' 130 R1 = R2 = Mebu 131 R' = Sen; R2 = H 134 135 136 neurolenin E R1 = Val'; R2 = OH 137 neurolenin F R' = 5-OH-Tig; R* = H 132 R' = Sen; R2 = (C0)Pr' 133 R' = Tig; R2 = (C0)Pr' OR2 138 R=H 140 R' = H; R2 = OH 142 karwinsinolide A R' = Ang; R2 = Ac 145 139 R=Ac 141 R' =OH; R2= H 143 karwinsinolide 6 R' = H; R2 = Ang 144 karwinsinolide C R' = Ang; R2 = H Me0JpL0 Wo;::?in.0 HO (k 0 0 OH' 0 OAc 0 ' OH 0 146 147 R=H 149 150 151 vicolide E 148 R=OH to 'OH 0 ?Ac I' Roq+oH ofl Hoa ebu \ -'-0 \ \ OH \ 0 OH 0 OAc 0 0 0 152 153 154 lobatin C 155 R = Vali 0 156 R = 2-OH-Vali 158 R = 157 R=Ac 159 R='$Q C02Me AcO-a AcO-Q RO 0 0 0 160 161 164 R=H 166 162 R= JfL 165 R=Ac 163 R= The exudates of Hypochoeris radicata leaves stressed with gui~nensi.?~~ The sesquiterpene 192 has been isolated from copper@) chloride afforded the novel eudesmane derivative the oil of Baccharis dracunculifolia.'l7 Nine novel eudesmane 185.224 sesquiterpenes 19S201 have been identified as components of The acids 186 and 187 and the ester 188 have been identified the leaves of Cryptomeria jap~nica.~'~ A new sesquiterpene as components of Artemisia phae~lepis,~~' Laggera ptero- glucoside boarioside 202 has been found in the aerial parts of donta226and Tanacetum pr~eteritum,~~~ respectively. The root Maytenus b~aria.*~' Two brominated sesquiterpenes 203 and of Rhaponticum unlJorum228 contains the new eudesmane 204 have been found in the tropical green seaweed Neomeris derivative 189 whilst emmotin Z 190 and its methyl ether ann~lata.~~~ A new sesquiterpene alcohol 205 with antibiotic 191 have been found in the trunkwood of Poraqueiba properties has been obtained from an Antarctic sponge.232 152 Natural Product Reports Table 2 Sources of eudesmanolides Source Eudesmanolides Ref.256 OH Artemisia arborescens 210 257 HO Melanoselinum decipiens 211,212 167 168 258 Pluchea dioscoridis 213 227 Tanacetum praeteritum 214-216 Ho\ The total syntheses of ( f)-cua~htemone~~' and ( f)-0 eudesma-5,7( 1 l)-dien-8-one have been The eudes- :o 0 manolide artemisin has been used as starting material in the OEpang 0 preparation of 9-oxy-functionalised natural e~desmanes.~~' 170 Syntheses of a-di~tyopterol~~' have been and plat~phyllide~~~ 169 achieved. Ultrasounds have been used to enhance the rate of the reductive cleavage of sesquiterpene y-enonela~tones.~~~ Other eudesmane sesquiterpenes which have been synthesised o& C02Me during the coverage of this review were ( f)-6-eudesmen-4a-o1 ( f)-vetiselinene2& and ( f)-selina-3,l l-dien-9-01.~~~ 0 Many dihydroagafuran sesquiterpenes have been isolated 0 171 from members of the Celastraceae family Celastrus angula- t~s,~~~ Celastrus gemm~tus,~~' Celustrus flagellari~,~~~ Celas-trus hin~bii,~~~ Euonymus SP.,~~' Hippocruteu excels^,^^' Maytenus boari~~~~ The and Tripterigium ~iljordii.~'~*~~~ total synthesis of ( f)-euonyminol the sesquiterpene part of cathedulin K-19 has been achieved.255 New eudesmanolides have been obtained from different species (Table 2) and their structures shown to be 210-216.Known lactones of this type have been isolated from Achillea The 172 173 174 pratensi~.~~~ microbial transformations of 6a-and 6P-eudesmanolides by cultures of the fungus Rhizopus nigri- cans have been examined.260 A first total synthesis of (&)-diplophyllin has been devised261 and a highly stereoselective synthesis of ivangulin has been achieved.262 An approach to the skeleton of eriolanin has been devised.263 An unexpected 175 176 177 selective formation of a &lactone in the ozonolysis of a santonin derivative has been reported.264 Five novel dimeric sesquiterpenes shizukaols E-I 217-221 have been isolated from the roots of Chloranthus japonic~s.~~~ Another two lindelane sesquiterpenes chloranthalactone F and chloranthalactone A photodimer have been shown to have the same structure 222 which implied the revision of the structures previously assigned to these compounds.266 A new 178 R=H2 181 182 R = a-OH lactone with a valerane skeleton 223 has been obtained from 179 R = P-OH H 183 R=P-OH 180 R=O Senecio dens~jlorus.~~~ A catalytic asymmetric preparation of an intermediate in the OH synthesis of ( -)-oppositane and ( -)-prepinnarterpene has been devised.268 The synthesis of racemic cis-dracunculifoliol and trans-dracunculifoliol has been reported.269 The unnatural enantiomers of the oppositane sesquiterpenes of marine OAc origin ( -)-axamide-4 and ( -)-axisonitrile-4 have been syn- 184 185 the~ised.~~' The iphionane sesquiterpene lucinone 224 and the secoeudesmane glutinone 225 have been isolated from Jasonia glutinos~.~~' We propose changing the name glutinone to glunone to avoid confusion with the very well known triterpene glutinone.Three novel 10-desmethyl- 1 -methyl eudesmanes 206208 have been isolated from the unripe fruits of Ocotea corymbo~a.~~~ Other compounds of this type have been found as components 16 Vetisperane of Artemisia hedini.234 A short synthesis of the vetisperane sesquiterpenes ( f)-The structure of epoxyoplodiol 209 has been resolved by hinesol and ( f)-agarospirol via retro-benzylic acid rearrange- single crystal X-ray diffraction analysis.235 The major sesqui- ment has been a~hieved.~~~.~~~ terpene components of the oil of a Texas race of Gossypium hirsutum have been identified as a-selinene and P-~elinene.~~~ 17 Eremophilane bakkane and tridensane The known eudesmane sesquiterpene kogol and the aro-The an timicroalgal sesquiterpene bipolal has been obtained madendrane one globulol have been identified as components from a fungus of the genus Bipolari~.~~~ The structure of this of the essential oil of Murraya k~enigii.~~~ The assignment of compound is identical to that of the compound named KM-01 the 'H and I3C NMR spectra of a-eudesmol P-eudesmol and which had been isolated from the fungus Drechslera avenue y-eudesmol have been reported.These known compounds (see Nut. Prod. Rep. 1996,13,318 structure 267). Eremophila- have been isolated from Nerocallitropsis puncheri.2'2 9,11(13)-dien- 12-01 226 and another seven eremophilane Fraga Nutural sesquiterpenoids 153 0 0aH OH 186 187 1 88 189 190 R=H 191 R=Me R HO%OAC 192 193 1 94 195 R=OH 197 R=H 196 R=OAc 198 R=OH OAc I GlcO m ,OH %OH 199 R=OH %OH HOih/-GH 201 202 203 R' = Br; R2 = H 205 200 R=OAc 204 R' = H; R2= Br OH 206 207 208 209 OH '0 OH HOq+ 0 210 jordanolide 211 A3(4) 21 3 21 4 21 5 212 A4(I5) OH OH OH 0 -OH o*OH 0 0 0 0 21 6 21 7 218 R=H 220 221 219 R=OH 0 0 0 0 OH 222 223 224 225 I54 Natural Product Reports derivatives have been found in the essential oil of Aquilaria agallocha (agar~ood).~~~A revised structure 227 has been assigned to a benzofuran sesquiterpene which had been iso-lated from Ligularia ~agittu.~~~The roots of another species of HOJp+o 0m0 this genus Ligularia przewalskii contain eight novel eremophi-lane derivatives 228-235,277 whilst other compounds of this type 236-239 and 240 have been obtained from Gynoxys 241 R’ = H; R2 = a-H 242 R1 = OMe; R2 = a-H OAng 246 243 R’ = OMe; R2 = a-OMe 244 R1 = OTig; R2 = a-OH 245 R1 = OTig; R2 = P-OH 226 227 228 HO& HOmo 247 R’ = a-OH; R2 = a-H 248 R’ = P-OH; R2 = a-H 250 R= JyL 249 R’ = R2 = (3-OH 0 251 R=Epang C02H OAng 229 R= H 231 232 230 R=OH H(;:p.w H C O 2 M e HO 252 R’ = a-OMe; R2 = a-H 253 R’ = 0-OMe; R2 = P-H 0 254 C02Me C02Me 0 233 234 (11R) 235 (11S) AcO 255 256 R = a-OAc 258 257 R=P-OAc Sen0 236 R1 = R2 = Ang 240 Q@ &+ 237 R1 = R2 = Sen OAC 238 R1 =Ang; R2=Sen 239 R1 = Sen; R2 = Ang ‘-$ AcO‘ HO H acostae and Gynoxys buxifolia respectively.278The rhizomes from species of the genus Petasitis are a good source of 259 260 261 eremophilane sesquiterpenes.Thus petasine and other sesqui-terpenes have been obtained from Petasitis hybrid~s~~~,~~’ and fourteen eremophilenolides 241-254 have been isolated from Petasitis japonicus.281,282The first total synthesis of (+)-petasine and (+)-isopetasine has been reported.283 The new furanoeremophilane alloeophyllin 255 and the novel secoeremophilenolides 256-257 have been found in Senecio and Senecio macedonic~s,~~~ alloeophyll~s~~~ respectively. 262 263 R=H The effects of rishitin on potato tonoplast vesicle and 264 R=OMe vacuole proton transport have been evaluated.286Potato tuber tissues when infected by fungus generated hydrogen peroxide which elicited the accumulation of the phytoalexins rishitin and phyt~berin.~~’.~~~ Total syntheses of the bakkane lactones ( -)-homogynolide ~289 and 4-epi-bakkenolide A290have been carried out.The synthesis and the absolute configuration of the sesquiterpene C02Me aldehyde tridensenal 258 have been reported.291 This 265 compound had been isolated from the Taiwanese liverwort Bazzania tridens. ger~ldii,’~~ respectively. Another two compounds of this type, 261 and 262 have been found in an extract of the soft coral 18 Guaiane xanthane pseudoguaiane and patchoulane Nephthea chabroli collected at the Indian coast. 176 Karaferin The new guaiane sesquiterpenes 259 and 260 have been 263 and karaferinin 264 are two sesquiterpene esters which isolated from Liguluriu nel~mbifolia~~~ The exudates and Eriostemon Jitz-have been obtained from Ferula karakalen~is.~~~ Frugu Nutural sesquiterpenoids 155 Table 3 Sources of guaianolides ~~ Source Guaianolides Ref.Achillea nobilis 272 297 Artemisia glabella 270 298 Artemisia JiIatovii 276 299 Centaurea conifera 273 300 Cladanthus arabicus 266 181 Crepis pyrenaica 274 275 30 1 Curcuma aeruginosa 277 288 302 Hymenoxys richardsonii 286 303 hula thapsoides 279-285 304 Lychnophora rupestris 271 189 Saussurea lappa 267 268 305 Stevia gilliesii 269 306 of Hypochoeris radicata leaves stressed with copper(I1) chloride afforded the novel guaiane derivative 265.224The assignment of the 'H and 13C NMR spectra of guaiol and bulnesol have been reported.212 An enantioselective total syn- thesis of the marine sesquiterpene (+)-clavularin A has been achieved.295 The guaiane sesquiterpene alismol has been synthesised in racemic form.296 The sources of the new guaianolides that have been isolated during the period of coverage of this review are listed in Table 3 and the novel guaian-6a,l2-olides i.e.266-276 are listed in Table 4. The other new guaianolides are represented by structures 277-286. The structures of arglabin307 and handelin308 have been confirmed by X-ray analysis. These lactones have been obtained from Artemisia glabella and Handelia trichophylla respectively. Known guaianolides have been isolated from Achillea nobili~,~'~ lo Garberia heter~phylla,~ Hymenoxys rus-b~i,~'' Laserpitium lati- Lactuca saligna Lactuca vi~osa,~'~ foli~m~'~ The assignment of the and Zaluzania gra~ana.~'~ NMR spectra and the absolute stereochemistry of pyre-throidinin and parishin A have been reported.These known lactones have now been isolated from Tanacetum den~um.~'~ Table 4 Novel guaian-6a 12-olides Name Structure Position of double bond(s) Cladantholide B 266 2-3 10-14 Kauniolide derivative 267 34 1614 11-13 Kauniolide derivative 268 34 10-14 11-13 Ligustrin derivative Arglabin derivative Ereman t hin derivative 269 270 271 34 10-14 11-13 34 11-13 4-15 9-10 Anolide 272 10-14 11-13 Chlorohyssopifolin derivative Isolippidiol derivative Isolippidiol derivative Artefin 273 274 275 276 10-14 11-13 10-14 11-13 1614 11-13 0 0 277 zedoalactone A 278 zedoalactone B RQ 0 0 279 R=P-OH 282 280 R=cx-OH 281 R=cx-OAC HO' 0 0 R* 283 284 R' = OH; R2 = P-Me H 285 R' = H; R2 = CH2 0 286 R = Gk(3',6-A~) Biosynthetic studies of lactucin derivatives in hairy root cul- tures of Lactucafloridana have been carried A review on the sesquiterpenes and sesquiterpene lactones isolated from 14 6 3Q15 13 0 Substituents and configurations Ref.lP-OH 4P-OH 181 14-OXO 306 I5-OXO 306 8P-OR' 307 1P-OH 10a-OH 298 14-OAc 1lP 189 ~cz-OH, 4P-OH 297 3P-OH 4a-OH 8P-OR2 15-C1 300 ~P-OGLC, 8P-OH 4a 30 1 ~P-OGLC, 8P-OH 4a lla 301 1P 1 OP-EpOxy 3a-OH 4P-OH 299 156 Natural Product Reports the genus Tanacetum (Compositae) has appeared.317 Recent in vivo antitumour studies with parthenin and eupatoriopicrin and the antiulcer activity of dehydroleucodin have been reported.' The aerial parts of Xanthium strumarium contain the new xanthanolide 15-chloro-2-epi-xanthanol 287.319A total syn- thesis of ( -)-vulgarolide has been devised.320 This compound had been isolated from Tanacetum vulgare and erroneously named by us ( -)-vularolide (Nat. Prod. Rep. 1990 7 534). The new pseudoguaianolides 288-290 and 291-295 have been found in extracts of Hymenoxys subintegra303 and Arnica angu~tifolia,~~' respectively. Another two lactones of this type gaigranin 296 and gaigrandin 297,have been isolated from Gaillardia grandijl~ra.~~~ The crystal polymorphism of the lactones helenanin and erigerolide has been studied by X-ray analysis.These two known pseudoguaianolides have been Two new rearranged patchoulane sesquiterpenes 298 and 299,have been obtained from the roots of Valeriana fa~riei.~*~ A total synthesis of racemic patchouli alcohol has been devised.330 The root bark of Michelia champaca contains michampanolide 300 a sesquiterpene lactone with a new carbon skeleton which can be derived together with the guaianolide magnograndiolide from the germacranolide parthen~lide.~~~ 19 Aromadendrane bicyclogermacrane aristolane and brasilane The structure occurrence biosynthesis biological activity synthesis and chemistry of the aromadendrane sesquiterpenes have been reviewed.332 A novel aromadendrane sesquiterpene (+)-13-hydroxyspathulenol 301 has been isolated from Eri-respectively.The structures of an inuchinenolide C deriva-tive325 and isopa~litin~~~ have been determined by X-ray analysis. The optimisation of the retro-aldol conversion of tenulin to isotenulin has been An enantioselective synthesis of neoambrosin parthenin and dihydroisoparthenin has been reported.328 -0 HO-0 287 288 R=H 289 R=Ac "'0% 0 OMe 290 OH -0 0 ostemon brucei while other known aromadendrane derivatives isolated from Helenium species323 and from hula a~pera,~~~ have been identified as components of species of the genera Drummondita and Phebali~m.~~~ Another two compounds of this type 302 and 303,have been obtained from the soft coral Sinularia maxima.334 The structures of plagichiline L and plagiochiline M have been determined as 304and 305,respect-ively.These two secoaromadendrane derivatives have been found in an extract of the liverwort Heteroscyphus planus.'26 Aromadendrane secoaromadendrane and maaliane sesquiter- penes have been identified as components of the essential oil of another liverwort Plagiochila o~alifolia.~~~ The structure of plagiochiline A 306 has been confirmed by X-ray analysis and its conversion into hot-tasting compounds by human saliva has been investigated.336 The leaves of Eucalyptus globulus contain a new isopentyl phloroglucinol-sesquiterpene with antibacterial activity against cariogenic bacteria which has been named eucalyptone 307.337 A total synthesis of (+)-taylorione has been achieved.This product is the enantiomer of the natural compound which had been obtained from Mylia ta~lorii.~~~ A racemic HO4 isH 301 302 303 ACO AcO ?O 0 oa \ \ 304 R=H 306 305 R=Me CHO Lo 0 OHC :$ H 295 296 R= H 297 R=Ang CHO 307 308 'b. I H pay 298 R=H 300 &OH OH 299 R = AC 309 310 Fraga Natural sesquiterpenoids preparation of taylorione has also been reported.339 The biotransformation of (+)-aromadendrane and ( -)-alloaromadendrane by the plant pathogenic fungus Glomerella cingulata has been in~estigated.~" Aromadendrane sesquiter- penes are characteristic metabolites of the Mediterranean brown algae Taonia lacheana and Taonia atomaria f.ciliata. The chemical composition of Taonia atomaria from the Canary Islands is completely different suggesting the possi- bility that a botanical mis-identification has occurred with this last species. 341 Isolepidozene a diastereomer of bicyclogermacrane has been identified in several species of liver~orts.~'~ A revision of the structure of the sesquiterpene aldehyde 308 has been reported. This aristolane derivative has been isolated from the roots of Plectranthus hereroensis and Aristolochia debili~.~"~ A new nardosinane sesquiterpene 309 has been obtained from the gorgonian Phyllogorgia dilatat~.~" The absolute configuration of the brasilane sesquiterpene conocephalenol 310 has been determined by total synthesis.345 20 Pinguisane and neopinguisane The biosynthesis of pinguisone in an axenic culture of the liverwort Aneura pinguis has been A total synthesis of P-pinguisene has been reported.347 A novel sesquiterpene alcohol 311 with a new rearranged pinguisane skeleton that has been named neopinguisane has been obtained from the liverwort Dicranolejeunea yo~hinagana.~~~ // I 31 1 31 2 31 3 CHO / I 'OH 314 R=a-Me 316 315 R=fi-Me q0 OH 0 317 31a 21 Miscellaneous sesquiterpenoids Junicedranol 312 is a new sesquiterpene with a novel carbon skeleton which has been isolated from Juniperus oxy~edrus.~'~ The novel sesquiterpene cyclomyltaylan-5a-o1 313 has been obtained from the liverwort Reboulia hemisphaerica.I l2 A study of the production bioactivity and biogenesis of the sesquiterpenes epiraikovenal 314 secoepiraikovenal 315 raikovenal 316 and its putative biogenetic precursor preraiko- venal 317 in different strains of the marine ciliated protist Euplotes raikovi has been carried Pathylacton A 318 and parathylone are two new sesquiterpenes which have been found in an extract of Paralemnalia thyrsoides."' 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ISSN:0265-0568    

DOI:10.1039/NP9971400145

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

Fatty acids fatty acid analogues and their derivatives Marcel S. F. Lie Ken Jie,* Mohammed Khysar Pasha and M. S. K. Syed-Rahmatullah Department oj Chemistry The University of Hong Kong Pokfulam Road Hong Kong Covering 1988 to 1995 Previous review 1989 6 231 1 Periodicals books and reviews 2 Natural compounds occurrence and structure 2.1 Unbranched fatty acids 2.2 Branched-chain fatty acids 2.3 Cyclic fatty acids 2.4 Oxygenated fatty acids 3 Synthetic fatty acids 3.1 Synthesis of methyl-branched fatty acids 3.2 Synthesis of olefinic and acetylenic fatty acids 3.3 Synthesis of hydroxy c18 fatty acids 3.4 Synthesis of oxa 0x0 and epoxy fatty acids 3.5 Synthesis of unusual fatty acids 3.5.1 Carbocyclic and @heterocyclic fatty acids 3.5.2 Furan fatty acids 3.5.3 Nitrogen-containing fatty acid derivatives 3.5.4 Fatty acids containing sulfur selenium or tellurium 3.6 Halogenated fatty acids 3.7 Isotope-labelled fatty acids 3.8 Hydrogenation 4 Autoxidation peroxidation and reactions by enzymes 5 Physical properties 5.1 Thin layer and column chromatography 5.2 High performance liquid chromatography 5.3 Supercritical fluid chromatography and extraction 5.4 Gas chromatography 5.5 Infrared spectroscopy 5.6 Nuclear magnetic resonance spectroscopy 5.7 Mass spectrometry 6 Biosynthesis and biotechnology 7 References 1 Periodicals books and reviews Two new journals on fats and oils have appeared in circu- lation.Handbooks published include the second edition of The Lipid H~ndbook,~ prostaglandins and a lipid gl~ssary,~ related lipids,5 phospholipids,6 glycolipids phosphoglycolipids and sulfoglycolipids,7 and lipid bilayers and membranes.8 A new edition of the classic Fatty Acid and Lipid Chemistry by F. D. Gunstone has been published.' Books on different aspects of fats and oils include vegetable oils and oil crops,lo,ll marine lipids,I2 human milk,13 fatty acids in ind~stry,'~ micro-bial lipids ' carbohydrate polyesters as fat substitutes,16 bio- logically active ether lipid~,'~ waxes'* and insect lipids." A large number of books have appeared on the analysis method- ology and separation techniques of fatty acids the analysis of fats and ~ils,~O-~' chromato-supercritical fluid e~traction,~~ graphic technique^,^^ -38 mass spectrometry of lipids,39 and mass spectra of prostaglandins and le~kotrienes.~' Books on the technological aspects of fats and oils include technology of fats and -43 bleaching,# developments in oils and fats,45 technical applications of fats,46 the crystallisation and poly- morphism of fats,47 flavo~r~~,~~ and peroxidation of lipids,50p53 and detergents and s~rfactants.~~~~ on plant lipid Books metaboli~rn~~ fatty acid oxidation,62 and bi~chemistry,~~-~' lipid soluble anti-~xidants,~~ lipid and biopolymer mono-layers,64 biology of plant lipids,65 chemical synthesis of ether lipids66 and on applied lipid research67 have also been pub- lished.In the area of biotechnology the following books are available biotechnology of plant fats,68~69 single cell and bios~rfactants~~ and lipa~es.~~ Reviews have appeared that deal with the following topics lipids of Candida albic~ns,~~ lichens,76 skin,77 bry~phytes,~~ testis,78 yolk,79 ether lipids in biomembranes,80 y-linolenic acid,81,82 eicosanoid chemistry,83 fluorinated fatty acids,84 very-long-chain fatty acid~,~~,~~ lipid composition of adipose tissue,87 hydroperoxidation of unsaturated fatty esters,88 hy- drogenation of vegetable oil^,^'^^^ fats and oils as renewable raw material for the chemical industry," trans-fatty acids in n~trition,'~ lipids of ar~haebacteria,'~ and natural anti- oxidants from palm.94 Reviews on synthetic fatty acids and their derivatives in- clude the synthesis of hydroxy fatty acid~,'~,~~ cyclic fatty acids,97 Cl-Clg alkyl ole ate^,^' rare and unusual fatty acids,99 and biologically active ether lipids.loo The following reviews on some physical aspects separation and identification of fatty acids have been published crystal structures of fats and fatty acids,"' vibrational spectroscopic aspects of polymorphism and phase transition of fats and fatty acids,lo2 thin layer chromatography-flame ionisation detection of lipids,103 separ- ation of fatty acids by HPLC,'04,'05 HPLC of membrane lipids glycosphingolipids and phospholipids,106 separation of triacylglycerols by HPLC '07-'09 HPLC of chiral lipid molecules,' lo supercritical fluid chromatography,' '17112 gas chromatography of fatty acids,' l3 gas chromatographic analy- sis of plasma lipids,' l4 gas chromatographic separation of triacylglycerols,' development in the gas chromatographic retention index scheme,' l6 chromatography-mass spectrom-etry of lipids,'17-"9 high resolution I3C NMR12' and Fourier transform infrared spectroscopic studies of lipids.12' Reviews on the biochemical aspects of lipid molecules in- clude prostaglandins 122 thromboxanes,'22 leukotrienes 122-'26 hep~xilins'~~ and related arachidonic acid metabolites hydrox- yeicosatetraenoic acids (HETEs),'~~ fatty acid allene lipid oxidation in fish autoxidation of methyl lino- lenate,I3' P-oxidation of fatty oxidation of poly- unsaturated fatty acids by cytochrome P450,'35,'36 metabolism of fatty acids by platelet^,'^^ and function of skin lipids,'38 regulation of fatty acid bio~ynthesis,'~~ bilayer mem-branes '40,1 ' biotransformation '42 lipoxygenase pathway of plants 143 plant lip~xygenase,'~~ hydroperoxide isomerases 145 biosynthesis of fatty acid~,'~~,'~~ genetics of plant lipids148 and essential fatty acids in growth and development.149 2 Natural compounds occurrence and structure 2.1 Unbranched fatty acids The presence of 12:3(32,62,92)found in the female gametes of the alga Anulipus japonicus is a possible precursor of algal sex attractant^.'^' The pulp lipid extract of jujube (Zizyphus jujuba) fruit contains high levels of 14:1(52) 16:1(52) and 18:1(52) but none of these fatty acids are found in the seed 1i~ids.I~' A novel C, dienoic acid 18:2(92,15Z) was ident- ified in the pulp part of the mango (Mangifera indica) The same 18:2(52,92)fatty acid was also found distributed in the sn-1 and sn-2positions of the phosphatidylethanolamines isolated from the sponge Agkistrodon halys blomhofii.'53 The seed oils of Trichosanthes bracteata and T. nervifolia (Fam. Lie Ken Jie et al. Fatty acids fatty acid analogues and their derivatives Cucurbitaceae) contain punicic acid 18:3(92,11E,132) as the major fatty acid component. lS4 The seed oils of some Anemone species contain up to 20% of y-linolenic acid 18:3(62,92,122).'55 Stearidonic acid 18:4(62,92,122,152) and y-linolenic acid were found in the seed oil of Primula sp.156 and in the lipid extract of cornon borage 1ea~es.l~~ The structure of stearidonic acid in the seed oil of Ribes nigrum has been confirmed.58 A novel conjugated tetraene fatty acid 18:4(82,102,122,14E) occurs as one of the major components in Ixora chinensis seed A new pentaenoic fatty acid 18:5(32,62,92,122,152) was isolated from the lipids of cultivated microalga Gymnodinium kowa- levskii. 160 Two varieties of taramira (Eruca sativa) seeds were found to contain high levels (42%) of erucic acid 22 1(132).161 Several iso-polyunsaturated fatty acids were isolated from the sponge Plakortis halichondroides including two novel long- chain fatty acids viz. 12-methyl-18:1(52) and 20:2(6E,14E).'62 Moss species of the families Brachytheciaceae and Hypnaceae contain high levels of arachidonic acid 20:4(52,82,112 142) and eicosapentaenoic 20:5(52,82,112,142,172) (EPA) acid.163 The fatty acids isolated from the gametospores of the mosses Pogonatum urnigerum and Fissidens adianthoides con-sist of over 90% of 20:3(112,142,172).'64 Two unusual fatty acids 20:3(52,112,142) (4.9%) and 20:4(52,112,142,172) (10.5%) have been identified in Biota orientalis seed The occurrence of 18:2(62,11Z) and a novel 20:2(62,112) (9"/0) fatty acid in the phospholipid fraction of the marine sponge Euryspongia rosea suggests the presence of an active A6desatu-rase.Other fatty acids isolated from E. rosea include 20 1 (1 22) (1%) and 24:1(172) (4.5%).'66 The fatty acid profiles of extracts from seven Black Sea algae showed characteristic high levels of 16:O in the families Rhodophyceae Phaeophyceae and Chlorophyceae.Members of the Rhodophyceae family (viz. Corallina granifera and Phyllophora nervosa) possessed large amounts of 205 (30.3%) and 20:4 (4.3%) respectively while members of the Chlorophyceae family displayed only trace amounts of 20:4 205 and 22:6.'67 Two new polyunsaturated fatty acids were identified in marine algae 20:5(52,82 lOE 12E 142) from the coralline red alga Bossiella orbigniana'68 and 22:7(42,72,9E 1 lE 132,162,192) from Anadyomene stel- lata.'69 The major components of the fatty acids from the obligate plant pathogen Erysiphe graminis are 22 l(2E) and 24:1(2E)."' A purification method has been described for the isolation of 20:5 and 22:6 by low temperature urea crystallisation of the sodium salt from ethan01.l~~ Corey et al.have developed a convenient method for the recovery of 20:5(52,82,112 142,172) from cod liver oil via iodolactoni~ation.'~~ This method has also been applied to the isolation' of pinolenic acid 18:3(52,92,122)'73 and arachidonic acid.174 Two reviews have appeared on the occurrence of very long chain fatty acids (VLCFA) in plants and animal^.^^,^^ Several novel VLCFAs are reported originating from marine sponges 19:1(62) and 25:1(172),'75 23:1(162),176 26:1(182),'77 26:3 (5Z,9Z,19E),'78 28:1(52) and 27-methyl-28:2(5Z,92),179 and 30 1(232) 30:2(92,232) and 30:3(52,92,252);'80 from marine coelenterates 24:5(62,92,122,152,182) and 24:6(62,92,122 152,l 82,212);18' and a new cytotoxic fatty acid from the stem of Desmos cochinchinensis 24 1(72).18' The blubber of the ringed seals (Phoca hispida sp.) contains significant levels of polyunsaturated VLCFAs (c23-c28).83 The lipid extract from the horseshoe crabs (Tachypleus gigas and Carcinoscorpius rotundicauda) consists of an unusually high level of 26:O (>60Y0).'~~ A new family of very long chain a,o-dicarboxylic rhombifolia (Fam. Santalaceae)."' A series of new C23 acety- lenic fatty acids namely 23:4(4A,62,8E 194 18-bromo-23:5(4A,6A,8E 17E 19E) 16-hydroxy-23:5(4A,6A,8E, 174 194 and 18-bromo-16-hydroxy-23:5(4A,6A,8E,l7E,l9E) have been reported. 192 2.2 Branched-chain fatty acids Fatty acids with chain-lengths ranging between C, and C, are reported for five Great Barrier Reef sponges.One sponge (Pseudaxinyssa sp.) contains a relatively large amount of an isoprenoid acid (4,8,12-trimethyltridecanoic acid) and 18:2(52,92).'93*'94 7-Methyl-16:1(82) was identified in the marine sponge Desmapsama anchorata. Other methyl-branched fatty acids found in the phospholipid fraction of this sponge are 14-methyl-16:1(82) and 3-methyl-17:0.'95 Two isoprenoid fatty acids 4,8,12-trimethyl-l3:0 and 5,9,13-trimethyl- 14:0 have been isolated from the Senegalese and New Caledonian sponges Cinachyrella sp.19' The Senegalese gorgonian Leptogorgia piccola contains three novel branched fatty acids viz. 10-methyl-16 1(62) 7,9-dimethyl-16:1(62) 10-methyl-1 7:2(62,92) and a range of other known methyl branched fatty acids.197 Two very-long-chain methyl branched fatty acids 25-methyl-27:2(5,9) and 26-methyl-27:2(5,9) have been found in the Caribbean sponge (Ectyoplasia ferox) in addition to the following methyl-branched fatty acids 10,13-dimethyl- 14:0 9-methyl-16:1(112) and 15-methyl-16:1(1 12).198 23-Methyl- 25:2(52,92) was isolated from the sponge Cribrochalina vas- culum.199 Another new very-long-chain branched fatty esters viz. ethyl 23-methyl-250 was obtained from the stem bark of Cedrus deodara.200 Several new phytol-derived methyl- branched fatty acids 2,2,6,10,14-pentamethy1-15:0, 2,3,7,11 15-pentamethyl-16:O and 7,8-dimethyl-16 1(72) have been identified in the oil of redfish (Sebastes sp.) and in salmon (Oncorhynchus nerka).Also found in these fish oils including menhaden oil were the following monomethyl-branched fatty acids 11-methyl-14:0 11-and 13-methyl-16:0.201 Two rare unsaturated fatty acids 16-methyl-l9:1(9E) and 20:2( 102,142) were identified in the culture filtrates of the fungi Sporothrix JEocculosa and S. rugulosa.202 The structure of a cytotoxic constituent 19-methyl-20:2(5Z,92) of Turbinaria ornata was confirmed by synthesis.203 A stereoselective route to 18-methyl-20:2(52,92) has been described.204 The lipid of the bacterium Corynebacterium sepedonicum contains 12-methyl- 14:l(42) (6%) which provides a useful marker in distinguish- ing C. sepedonicum from other microorganism^.^^^ Several C23 C25 C27 and C29 methyl-branched aldehydes and acids have been reported from an alkadiene-producing strain of the green alga Botryococcus braunii.206 Lipid isolated from the ther- mophilic anaerobic eubacterium Thermotoga maritima con-tains 15,16-dimethyltriacontanedioicacid (diabolic acid) and a novel 15,16-dimethy1-30-glyceryloxytriacontanoic A methyl branched hydroxy C, fatty ester (methyl 3-methyl-8- hydroxy-4-decenoate) was isolated from Aspergillus niger and confirmed by synthesis.208 The lipid extract of the green alga Caulerpa racemosa yielded 3,7,11 -trimethyl- 10-0x0- 12:0.209 2.3 Cyclic fatty acids 9,lO-Methylenehexadecanoic acid and 1 1,12-methylene- octadecanoic acid were identified as the major non-polar fatty acids in the Gram-negative bacterium Pseudomonas cepacia.210 The relative configuration of a cyclopropanoid eicosanoid 1 was established by biomimetic synthesis from arachidonic acid acids (c2+30) Three acetylenic fatty acids were identified in the lipid fraction of the extract from the liverwort genus Riccia viz.18:2(6A,9Z) 18:4(6A,9Z 122,152) and 18:3(6A,92 122); A =acetylene.lg6 Acetylenic fatty acids were also found in mosses which include 18:4(6A,92,122,15Z) 20:3 (8A,112,142) and 20:4(5A,82,112 142).'s7-190 Nine acety- lenic fatty acids have been identified in the seed oil of Jodina 164 Natural Product Reports 18'from a eubacterium is reported. with an acetone extract of the georgonian Plexaura homoma- lla.21' A cyclopropanoid hydroxy-eicosanoid lactone 2 has been identified from the temperate red alga Constantinea An unusual cyclopropane-containing acetylenic acid was isolated from a deep-water New Caledonian sponge Cladocroce incurvata.213 A C18 cytotoxic azacyclopropane carboxylic acid ester 3 was isolated from the marine sponge Dysidea fragilis and the absolute configuration was confirmed principle isolated from the fungus Filoboletus was identified as OH OH n 8,13-dihydroxy-18:2(92,1 The extracts of needles of HO 1 2 O e < CCO-NH O H c 6 7 by circular dichroic spectral analysis of the corresponding 2-N-p-bromobenzamide of the a-amino ester formed by hydro- genation of compound 3.214A novel cyclopentenoid fatty acids 13-cyclopen-2-enyltridec-4-enoicacid was identified in the seed oil of Hydnocarpus anthelmintica as determined by mass spectrometric analysis.21 Dihydrohydnocarpic 4 and dihydrochaulmoogric 5 acid and two rare monounsaturated fatty acids [16 l(52) and 18 1(52)] were isolated from red algal species of the family Solieriaceae.2'6 Five cyclopentanoid fatty acids of the jasmonic acid type two of which contain an (5')-isoleucine moiety (e.g.compound 6) have been identified in the culture filtrate of the fungus Gibberella f~jikuroi.~~~ The sponge Fascaplysinopsis reticulate from Fiji contains a cyclohexenyl methyl branched polyunsaturated diacid 7 as identified by analysis of spectral data.218 2.4 Oxygenated fatty acids Fruit bodies of the edible mushroom Cantharellus tubaeformis produce 10-hydroxy-10 l(8E) acid in response to 13-Hydroxy-14:3(2E74E,8E) was identified in the culture fil- trate of Valsa ambiens and the monoglyceride of this fatty acid was shown to inhibit the growth of lettuce roots and the hypocotyls.220 A major component of cucumber (Cucumis sativus) cutin was confirmed as 8,16-dihydroxy-1 6:0.221 (2R)- Hydroxy-16:0 and 2-0x0-16:0 were isolated for the first time from marine algae Ulva pertusa and Porphyra sp.These gymnosperms (Pinus sylvestris and Juniperus communis) con-tain 9,16- and 10,16-dihydroxy-16:0 acids. 9,10-Epoxy-18- hydroxy-l8:O was isolated in significant amount from the leaves of Quercus petraea while 7-and 8-hydroxy-a,o-hexadecanedioic acids were found in the leaves of another angiosperm Corylus avellana.228 High levels of o-hydroxy 18 1 and 18:2 acids were found in the stigma of the tobacco plant (Nicotiana SP.).~~~ Two new oxygenated acetylenic acids 10- hydroxy- and 10-0x0-1 8:2(6A78E) were identified in the extract of the New Zealand thalloid liverwort (Monuclea f~rsteri).~~' An antifungal trihydroxy fatty acid 9,12,13-trihydroxy-18:l(lOE) was isolated from the tubers of taro (Colocasia antiquorum) which was inoculated with black rot fungus (Ceratocy~tisJimbriata).~~~ Three new and bioactive dihydroxy fatty acids (12R 13R)-dihydroxy-20:4(52,82, lOE 143 (12R 13R)-dihydroxy-20:5(52,82,10E,142,172) and (lOR,llR)-dihydroxy- 18:3(62,8E 123 were identified from the extract of the temperate red marine alga Farlowia mollis.These oxygen- ated fatty acids were found to modulate superoxide anion generation and inhibited the biosynthesis of lipoxygenase products and the functioning of the dog kidney Na'/K' ATPase.232,233Six novel branched a-hydroxy fatty acids viz. 2-hydroxy- 17-methyl- 18:0 2-hydroxy-2 1 -methyl-22:0 2- hydroxy-22-methyl-23:0 2-hydroxy-22-rnethyl-24:O and 2-hydroxy-24-methyl-25:O acids were identified in the sponge Smenospongia aurea. 234 The occurrence of a leukotriene and hepoxilin B from a plant (red marine alga Murrayella periclados) has been re- ported.235 The calcareous red algae Lithothamnion corallioides and L. calcareum contain 1 3-hydroxy-20:4(52,8Z7 1 12 143 13-hydroxy-20:5(52,82,112,142,172)and 8-hydroxy-13- oxo-20:4(52,9E 1 1E,1419.~~~ Three new hydroxy fatty acids were isolated from marine alga Gracilariopsis lemaneiformis viz.1O-hydroxy- 18:4(62,8E 122,152) 12-hydroxy-l8:3(52, 8E 1OE) and 12,13-dihydroxy-18-oxo-20:5(52,82,10E,142 1 6E).237 Hydroxy fatty acids isolated from barnacles Balanus balanoides include 10,11,12-trihydroxy-20:3(5,8,14),10,11,12-trihydroxy-20:4(5,8,14,17) 13,14,15-trihydroxy-20:4(5,8,11,17) and 12,13,14-trihydroxy-22:5(477710716,19).238 Four hydroxy isoprenoid eicosanoids cubensic acid 8 malaysic acid 9 ber-teric acid 10 and cameronic acid 11 have been identified as metabolites of the fungus Xylaria.239,240 (1 OR)-Hydroxy- 22:5(72,11E,132,162,19z> (8R)-hydroxy-20:5(52,9E, 1 12 142,172) and (8R)-hydroxy-20:4(52,9E, 1 12,142) were isolated in the free acid and ethyl ester form from the black coral (Leiopathes sp.) of Saint Paul Island in the South Indian Ocean.241 An inhibitor of 5-lipoxygenase and Na'/K' ATPase was identified as 11 -hydroxy- 16-oxo-20:5(5Z,82,12E 14E 17E) in the lipid extract of the marine alga Ptilota Jilicina (Fam.Ceramiale~).~~~ A physiologically potent eicosanoid (1 29- hydroxy-20:5(52,82,10E 142,172) has been isolated from the red marine alga Murrayella periclados which acts as an inhibi- tor of platelet aggregation and a mediator of inflammation.243 The acidophilic green alga Dunaliella acidophila produced two unusual hydroxy fatty acids (12R)-hydroxy-l8:3(92,13E, 152) and (9a-hydroxy- 18:3( 1OE,122,1523 when grown in a culture acids were shown to be biosynthetically derived from pal- medium of pH 0.2-2.5.244 The lipid extract of the leaves of mitic acid.222 Four novel homologous hydroxy fatty acids Glechoma hederacea contains a mixture of (9S)-hydroxy-were found in the fungus Pleurotus sp.viz. 7-hydroxy-18:2(IOE,1227 9-0~0-18:2( 10E,122) (9S)-hydroxy-l8:3( 10E 8,14-dimethyl-16 1(92) 7-hydroxy-8,16-dimethyl-18 The cutin of Limo-1(92) 7- 122,152) and 9-0~0-18:3(10~,122,152).~~~ hydroxy-8,18-dimethy1-20 16:0, l(92) and 7-hydro~y-8~2O-dimethyl-nia acidissima leaf contains 9,16- and 1O716-dihydroxy- 22 1(92).2239-Hydroxy-l8:2(10E,122),an aromatase inhibi- tor was isolated from the root of Urtica dioica.224 An efficient countercurrent distribution method for the large-scale isolation of methyl dimorphecolate methyl (+)-(9S)-hydroxy- 18:2( 1OE,12E) from Dimorphotheca phvialis seed oil has been developed.225 Two novel (9R)-hydroxy fatty acids viz.(9R)-hydroxy-18:3( lOE 122,152) and (9R)-hydroxy- 18:2( 10E 123 were obtained from the nitrogen fixing cyano- bacterium Ananbaena 30s-aquae J 30s-aquae. 226 An antiviral 10,20-dihydroxy-20:0 and 7-hydroxyhexadecane- 1,16-dioic A number of a-hydroxy very-long-chain fatty acids (VL-CFAs) viz. 2-hydroxy 20:O to 25:0 were found incorporated in the phosphatidyl fraction isolated from the Caribbean sponges Verungula gigantea and Aplisina archeri. Other non-oxygenated VLCFAs [29:2(5,9) 30:3(5,9,23) and 32 11 and an isoprenoid fatty acid (3,7,11,15-tetramethyl-16:0)were also identified in the extract of the sponge Aplisina ar~heri.~,~ The Lie Ken Jie et al.Fatty acids fatty acid analogues and their derivatives OH OH OH OH C02H 8 OH OH OH OH C02H 9 10 OH OH OH OH C02H 11 13 marine sponge Amphimedon compressa from the Puerto Rican coast furnished two a-hydroxy fatty acids 2-hydroxy 22:O and 2-hydroxy 23:0 and several VLCFAs [29:3(5,9,23) 30:3(5,9,23)].248 The cornified layer of human skin contains high levels of o-hydroxylated VLCFA such as o-hydroxy- 30:0.249 Several a-methoxy fatty acids 2-methoxy- 16 1(5Z) 2-methoxy- 16 l(62) and 2-methoxy-26:2(52,92) have been identified in the phospholipids of the Caribbean sponges Tropsentia roquensis Mycale laxissima and Spheciospongia cus-pidfera. 250,251 Two novel keto fatty acids 7-0x0- 18 1 (1 1Z) from Gardenia lucida seed and 9-0x0- 18 1(1 12) from Lagerstroemia speciosa seed oil have been isolated.253 Cibaric acid 14,18-dihydroxy-18:3(92,132,15E) was identified in damaged fruit bodies of the edible mushroom Cantharellus ~ibarius.,~~ Three unusual divinyl ethers (12-14) and a hydroxy fatty acid (13S)-hydroxy-l8:4(62,92,11E,152),were obtained from the brown alga Laminaria sp.255 Two enone ethers 11-methoxy-9-0x0- 19 1 (1OE) and 10-methoxy-8-0x0- 18 1(9E) were isolated from the root bark of Hibiscus rosa-sinensis.256 By using a bioassay-directed fractionation procedure 6-acetoxy-18:2(92,122) was identified as a pollen germination and growth inhibitor of Spatoglossum paclficum. 257 Peracetate methyl esters have also been found in the red alga Gracilariop-sis lemaneformis and were identified as (lOS)-acetoxy-18:4(62,8E 122,15Z) 12-acetoxy-l2:3(52,8E 1OE) and (12R 13S)-diacetoxy-l8-0~0-20:5(5Z,8Z, IOE,142 16E).258 Racemic cis-9,10-epoxy-18:0 was found in the lipid extract of human leukocytes when incubated with linoleic acid.259 Vernolic acid cis-12,13-epoxy-l8 1(9Z) is a major fatty acid component in the seed oil of Centratherum ritchiei260 and Vernonia galamen- The seed oils of the sea buckthorn and Hippophae rhamnoides contain high levels of 15,16-epoxy- 18:2(92,12Z) and 9,lO; 12,13-diepoxy- 1 8:0.262,263 A medium chain branched epoxy fatty acid 10,l l-epoxy-3,7,1 l-trimethyl-l2:2(2E,6E) was identified in the extract of the whole plant Cyperus iria (Fam.Cyperaceae). This epoxy fatty acid serves as a juvenile hormone for the grasshopper Melanoplus ~anguinipes.~~~ Minor epoxy fatty acids in Lesquerella fendleri seed oil have been identified as 15,16-epoxy-18:2(92,122) 9,lO-epoxy-18:1(122) and 9,10-epo~y-18:0.~~~ Ishii et al. have identified 17 new furan fatty acids among 30 furanoid fatty acids with chain lengths varying from C, to C, isolated from the hepatopancreas of the crayfish Procambarus clarkii. These unusual fatty acids account for 28% of the total sterol esters. Only small amounts of furan fatty acids were found in the triacylglycerol (0.5%) and phospholipid (0.7%) fractions. The most abundant furan fatty acid component was identified as 12,15-epoxy-l3,14-dimethyleicosa-12,14-dienoic acid 15 (1 1.9%).266 Furan fatty acids have also been found to Me Me C02H 15 occur in a wide range of plants in the fungus Agaricus bisporus and in yeast.267 Furan fatty acid containing a pentyl side chain was isolated from the alga Isochrysis sp.while a furan isomer with a propyl side chain was obtained from Phaeodac-tylum tricornutum. 268 A furan fatty acid 10,13-epoxy- 1 1 -methyloctadeca- 10 12-dienoic acid has also been detected in the intestinal bacteria of Six furan fatty acids have been identified (1621) in human and bovine blood plasma where these molecules are bound to choline and ethanolamine phospholipids. In the neutral lipid fraction of blood plasma the furan fatty acids occur only as cholesterol ester^.*^'.^^' Several new tetrahydrofuran fatty acids (22) have been found in human plasma mainly as the free acids but also in small amounts in the glyceride and phospholipid fractions.272 Introduction of methyl groups into the 3- and 4-position of the ring of 2,5-disubstituted furan fatty acids was achieved by incubation of the sodium salt of 2,5-disubstituted furan fatty acids with bovine liver homogenate in the presence of (9-adenosylmethionine and ~ysteine.,’~ Investigations of the biosynthesis of the furan fatty acids have ruled out linoleic acid as a possible biogenetic precursor of the furan fatty acids in vivo 166 Natural Product Reports in the rat.This study revealed that furan fatty acids were not produced de novo in the animal but appeared to have been accumulated in the tissues from nutrients.274 A number of a,o-dicarboxylic acids up to C, carbon atoms have been identified from the seed oil of Equisetum (horse- Bongkrekic acid 23 was identified as the toxic anti- biotic produced by Pseudomonas cocovenenans which was found in infected A novel antifungal triacetyl- enic hydroxydioxolone fatty acid 24 was isolated from the fermentation broth of Microbispora sp.277 3 Synthetic fatty acids 3.1 Synthesis of methyl-branched fatty acids Free radical addition reactions involving oleic acid to yield branched fatty acids have been reviewed.278 Sixteen branched fatty acids are produced by different synthetic methods includ- ing alkylation and hydrolysis of oxazolines to obtain 2-alkyl fatty acids desulfurisation of alkyl substituted thiophenecar- boxylic acids for 4- and 6-alkyl fatty acids and Kolbe reaction of dioic acids to give alkyl branches at different positions of the chain.279 7-Methyl-16 1(6E) a component of the marine sponge Arnphimedeon complanata was synthesised starting from methyl vinyl ketone by reaction with nonylmagnesium bromide.The resulting alcohol was subjected to Claisen orthoester rearrangement and then chain extended.280 The racemic mixture of tuberculostearic acid 10-methyl-1 8:0 was obtained by reaction of octylmagnesium bromide with 10-oxoundecanoic acid followed by elimination of the resulting hydroxy function,281 or by coupling of 1 -iodo-2-methyldecane with 1-hydroxyoct-7-yne to give a racemic mixture of 10-methyloctadec-7-yn- 1-01 which was hydrogenated and oxidised to the 10-methyl-1 8:O acid.282 The stereospecific syn- theses of 24:1(52) and ethyl 6-methyl-24:1(4E) have been accomplished to test their potential role in the biosynthesis of mycolic acids.Only 24:1(52) was found to act as a mycolic acid precursor.283 Racemic 2,4-dimethyl-22:0 a major acyl component of 2,3-di-O-acyl-a,a-trehalose glycolipid antigens isolated from Mycobacteriurn tuberculosis has been synthe- ~ised.~'~ A stereoselective synthesis of two marine fatty acids viz. 19-methy1-20:2(52,92) and 18-methyl-20:2(52,92) has been developed. The synthetic strategy was based on C-C bond formation via (2)-selective Wittig ~lefination.~'~ 3.2 Synthesis of olefinic and acetylenic fatty acids The synthesis of long-chain (3E)-alkenoic acids by the Kno- evenagel condensation of aliphatic aldehydes with malonic acid has been described.286 Methyl ester of 18:2(9E 1 1E) was prepared from methyl 9,12-dihydroxy-l8 1( 1OE) ester with chlorotrimethylsilane and sodium iodide in a~etonitrile.~~' Linolenic acid and the all 2-isomers of 20:5(5,8,11,14,17) 22:6(4,7,10,13 16,19)288,289 and 22:6(4,7,10,13,16,19)290were synthesised via the Wittig olefination reaction.The synthesis of 1 1-substituted linoleic acids (25-27) starting from a C acid has been reported.291 The synthesis of 5-and 6-methylidene arachidonic acid analogues (28,29) has been described by reaction of a lithio dienyne intermediate with an allylic chlo- ride synthon.2'2 Two analogues of arachidonic acid namely 20:4(52,82,1 lZ,13E) and 20:5(82,82,112,13E,15E),have been synthesised to study their reaction with porcine 12-1ipoxygena~e.~~~ The synthesis of 20:5(52,8Z 1 12,142,172) was performed by a sequence of selective reduction and Wittig reactions of synthons which afforded the (2)-skipped olefinic The syntheses of geometric isomers of C, poly-unsaturated fatty acids viz.20:5(52,82,1 lE 142,173 20:5(52,82,11 E,142,17E) 20:5(52,82,112,142,17E) and 22:6(42,72,102,132,162,19E) were also achieved by chain extension and similar olefination rea~tions.~~~,~~~ A general scheme for the preparation of non-conjugated unsaturated C02H 25 C02H 0 26 C02H OH 27 28 29 very long chain fatty acid methyl esters (viz.27:1 28:1 32:l and 32:4) with between one and four double bonds using the conjunctive reagent tosylmethylisocyanide has been devel- Syntheses of very long chain fatty acid methyl esters involving the treatment of o-iodo esters with the complexes formed from reactions of alkylcopper(1) and Grignard reagents furnished very long chain methyl alkanoates all-(2) alkenoates methylene-interrupted alkadienoates and alka-trien~ates.,~~ The synthesis of 26:2(5E,9E) was accomplished via copper- and palladium-mediated reactions using 1 -acetoxy- 4-chlorobut-2-ene as a building 24:1(15A) and 28:1(19A) were prepared by coupling of the lithium salt of dec-1-yne with the corresponding o-brom~acid.~~' An efficient synthesis route to methylene skipped diynes and triynes has been reported which employs the coupling of prop-2-ynylic halides or tosylates and 1-alkynes or a-acetylenic esters.301 A conjugated polyunsaturated fatty acid 18:3(5Z,7A,9A) was produced by coupling 1-decyne to a C enyne ester in the presence of Pd(PPh,),Cl, CuI and Et,NH in benzene.302 Long chain conjugated diacetylenic fatty acids and alcohols have been prepared from butadiyne.,' 3.3 Synthesis of hydroxy CI8 fatty acids This review does not cover the synthesis of prostaglandins thromboxanes and leukotrienes as these classes of compounds have been periodically reviewed.5~837122-127 Hydroxylation of oleic acid 18:1(92) in the allylic positions with selenium dioxide in tevt-butylhydroperoxide has been rep~rted.~~~.~~' 9,18- and 10,18-Dihydroxy- 18:O were prepared by condensa- tion of two apdifunctional synthons derived from suitable a,o-alkanedi~ls.~~~ The synthesis of 7,10-dihydroxy-l8 1(8E) was achieved by Wittig olefination reaction of appropriate synth~ns.~~~ (IOR)-Hydroxy-l8:1(8E) acid was prepared by a titanium(Ir1) mediated regiospecific reduction of a vinyl epoxy intermediate 30 which was obtained by Wittig coupling of 2,3-epoxyundecanal with ethyl 7-bromoheptan0ate.~~~ The key step in the enantioselective synthesis of ( -)-(8R)-hydroxy-18:2(5,6) the main antifungal constituent of Sapium japoni- cum involved the reduction of a conjugated acetylenic keto intermediate 31 with (S)-Alpine-b~rane.~'~ A similar approach was adopted for the synthesis of (8R)-hydroxy-18:2(92,122) and (8R)-hydroxy-l8 1(9z) which were found to induce pre- mature sexual sporulation in Aspergillus nid~lans.~~~ The syn- thesis of (10R)-hydroxy-l8:2(8E 122) (a fungitoxic compound found in the timothy plant Phleum pratense) was achieved by using D-xylose as the chiral source.311 Nicolaou et al.have reported a short stereocontrolled synthesis of methyl (1 2s)- hydroxy- 17:4(52,8E 10E) based on a Pdo-TlOH catalysed coupling reaction of a vinyl iodide and a vinylborane inter- mediate.312 A new synthetic route to optically active (10R)- hydroxy-l8:2(8E,122) has been de~cribed.~' Methyl Lie Ken Jie et al. Fatty acids fatty acid analogues and their derivatives 167 33 34 35 OH C02H 36 R = (CH&Me CH=CHCH2Me a-dimorphecolate (9S)-hydroxy- 18:2( lOE 12Z) was obtained via the bis(acetonitri1e)palladium dichloride catalysed re-arrangement reaction of an allylic acetate intermediate 1 1 -acetoxy- 18:2(9E 122) where the rearrangement occurred at the E-double bond.314 An enantiospecific synthesis of dimor- phecolic acid was reported by Sato's group via a vinyl hydroxy compound 32 as the key intermediate.315 The four stereo- isomers of methyl dimorphecolate were also synthesised by direct palladium catalysed coupling of the two corresponding olefinic parts of the conjugated diene In the synthesis of coriolic acid (13%-hydroxy-18:2(92,1 lE) the key intermediate (S)-oct-l-yn-3-01 was obtained by the asymmetric reduction of oct-l-yn-3-one with (S)-P-3-pinanyl-9-borabicyclo[3.3. llnonane.(3-Oct-1-yn-3-01 was treated with tributyltin hydride to give the (9-tributylstannyl-( lE)-octen-3-01 which was then coupled with (lOZ)-iododecenoic acid to furnish coriolic acid in 75% yield.317 Coriolic acid and its (1 3S)-N-tosylamino analogue were synthesised from D-mannitol as the chiral source.318 The optically active lactol 33 which was available in a two-step reaction from the hemiester obtained by an enantioselective enzymatic (pig liver esterase) hydrolysis of the exo-adduct of furan and dimethyl maleate was used as a key intermediate in the synthesis of coriolic acid.319 A new Horner-Wadsworth- Emmons reagent 34 has been developed which transformed carbonyl compounds into penta-2,4-dienals.This reagent was used in the synthesis of coriolic An efficient asymmet- ric synthetic route was developed to prepare y-hydroxy a$-unsaturated ketone or aldehyde intermediates for use in the synthesis of coriolic acid.32' A novel chemo-enzymatic enantio- specific synthesis of (9-coriolic acid mediated via immobilised alcohol dehydrogenase of baker's yeast has been described.322 15,16-Didehydrocoriolic acid [13-hydroxy-l8:3(92,1 lE 15Z)] was stereoselectively synthesised starting from pent-2-en-4- yn-1-01.~~~ In the synthesis of (6R)-hydroxyoctadeca-7,9-diynoic acid a HMG-CoA reductase inhibitor Sharpless epoxidation and acetylenic coupling were the key reaction steps of the pro- ced~re.~~~ Rao et al.reported the synthesis of ll-hydroxy- (1 2S 13S)-epoxy- 18:2(92,15Z) acid a constituent of rice plant infected with rice blast disease. The key intermediate was an epoxy benzoate 35 which was obtained by enantioselective (Sharpless) epoxidation of allylic prop-2-ynyl alcohol inter- mediates. Subsequent chain extension followed by partial hy- drogenation gave the required Another trihydroxy unsaturated fatty acid 36 from rice plants suffering from the rice blast disease has been synthesised from dimethyl (+)-tartrate as the starting chiral source.326,327 Four stereosiomers of 9,10,13-trihydroxy-l8 l(11E) were derived from methyl 9,l O-epoxy- 12-0ctadecenoate which was obtained by partial epoxidation of methyl linoleate. These trihydroxy C, fatty acids are potential anti-rice blast fungal substances.328 Other potential anti-rice blast fungal substances viz.(9S 12S 13%- and (9S 12R 1 5s)-trihydroxy- 18:2( 1 OE,152) acids have been synthesised from carbohydrate s~bstrates,~~~,~~~ (9R, 12S,13R)-and (9S,12S,13R)-trihydroxy-18:1(10E) acids were synthesised starting from tri-O-acetyl-~-glucal.~~~ (1 1 S,12S,13s)-Trihydroxy-l8:2(92,15Z) and (1 1R)-hydroxy-(1 2S 13S)-epoxy- 18:2(92,15Z) were obtained from (L)-(+)-and (D)-(-)-tartaric acid respectively,332 while (1 1s)- hydroxy-( 12S 13S)-epoxy-l8:2(92,15Z) was synthesised from manno nose.^^^ 3.4 Synthesis of oxa 0x0 and epoxy fatty acids The synthesis of ethyl 5-oxa-3,8,12-trimethyltridec-2-enoate isomers (insect juvenile hormone mimics) is reported using citronellol (3,7-dimethyloct-6-en-l-o1)as the starting Colneleic acid 37 was synthesised starting from 3-non~nal.~~~ The stereospecific removal of the pro-R hydro-gen at C-8 of (98-hydroperoxyoctadecadienoic acid in the biosynthesis of colneleic acid has been 9,12-Dioxostearic acid was obtained by treatment of ricinoleic acid 12-hydroxy-18 l(9Z) or iso-ricinoleic acid 9-hydroxy- 18:1(122) in the presence of catalytic amount of Hg(1r) acetate with Jones reagent.337 Oxidation of methyl dimorphecolate (+)-(9S)-hydroxy-l8:2( lOE 12E) using Oppenauer conditions furnished the corresponding %ox0 derivative.338 Ostopanic acid 7,12-dioxo-l8:2(8E IOE) was synthesised by a route based on the palladium catalysed isomerisation of an oxoacetylene intermediate obtained from pent-4-ynal as the starting material,339 or by using a P-chloro vinyl ketone as a key intermediate obtained from diethyl ~imelate.~~' Selective epoxidation of the terminal double bond of 0-3fatty acids was achieved through the action of N-bromosuccinimide in aque- ous glyme followed by treatment with potassium hydroxide in methanol.341 Reaction of methyl santalbate 18:2(9A 1 lE) with mercury(r1) sulfate afforded methyl 9( lO)-oxo-l8 1(1la which upon treatment with rn-chloroperoxybenzoic acid fur- nished a mixture of methyl trans- l l 12-epoxy-9-oxo- l 8:O and methyl lO-oxo-18:1(1 The synthesis of methyl cis-9,1 O-epoxy- 1 3-oxo- 18:1(11E) and trans-9,1 O-epoxy- 13-0~0- 18:1(11E) was based on the stereoselective oxidation of linoleic acid by soybean lipoxygenase to produce the corresponding 13-hydroperoxide.The latter compound was reduced with sodium borohydride followed by oxidation esterification and epoxidation to yield the required cis- and trans-epoxides in an overall yield of 14 and 3% respectively.343 A concise enantioselective synthesis of 12(S) 13(S)-epoxy- 1 1 (S)-hydroxy- 18:2(92,152) was achieved by using Sharpless kinetic resol- ution of ally1 prop-2-ynyl alcohols.344 168 Natural Product Reports 3.5 Synthesis of unusual fatty acids 3.5.1 Carbocyclic and O-heterocyclic fatty acids A mild cyclopropanation procedure has been developed which utilises diazomethane and palladium(I1) acetate as the catalyst in the transformation of unsaturated fatty esters to the requi- site cyclopropane fatty ester derivatives.345 Concomitant ultra- sonic irradiation during the Simmons-Smith cyclopropanation reaction of unsaturated hydroxy 0x0 and epoxy fatty esters gave the corresponding cyclopropane fatty ester derivatives in good yields (57-75%).346 A novel tricyclo derivative 38 was also obtained from a furan fatty ester (methyl 9,12- epoxyoctadeca-9,11 -dienoate) by this procedure.346 Reaction of unsaturated fatty esters (methyl oleate linoleate ricin- oleate) with dichlorocarbene generated from aqueous sodium hydroxide in chloroform in the presence of triethylbenzylam- monium chloride gives the corresponding gem-dichloro- cyclopropane derivatives.347 The synthesis of a novel cyclo- propyl analogue of arachidonic acid 39 via a conver-gent synthesis that employed methyl (lR,2S)-2-formyl-cyclopropanecarboxylate in conjunction with the ylide from (3Z,6Z)-pentadeca-3,6-dienyl(triphenyl)phosphonium iodide has been reported.348 Cyclopropene fatty acids have been synthesised from acety- lenic intermediates using ethyl diazoacetate as the cyclopro- penating agent,349,350 or by alkylation of the corresponding 1-lithiocyclopropenes.35’Another new approach to cyclopro- pene fatty acids has been developed for the synthesis of methyl sterculate 40 and methyl 2-hydroxy-sterculate which involved the 1,2-deiodination of 1,2-diiodocyclopropanes with butyl- lithium at low temperat~re.~’~ The synthesis of deuteriated cyclopropene fatty esters structurally related to palmitic and myristic acids has been reported.353 A number of 1,2-disubstituted cyclopentane fatty esters viz.methyl 9-(2-butylcyclopentyl)non-8-enoate,methyl 7-(2- hexylcyclopentyl)hept-6-enoate and methyl 5-(2-octyl-41 S COH O 2 H 42 cyclopentyl)pent-4-enoate were synthesised by the Michael addition of alkylmagnesium bromide to an unsaturated cyclic ester.354 Methyl 9-(2-butylcyclopentyl)nonanoateand 10-(2- propylcyclopenty1)decanoate were also obtained by Wittig reaction between 2-alkylcyclopentanones and the correspond- ing o-carbomethoxyalkyltriphenylphosphoniumbromides.355 The total synthesis of chromomoric acids e.g. 41 was achieved via coupling of enone intermediates and retro-Diels-Alder rea~tions.~~~~~’’ The racemic mixture of a cyclopentenone fatty acid 42 produced by duckweed which inhibits algae growth was synthesised by Michael addition of nitromethane to 2-methylcyclopent-2-enone followed by carbonyl protection and the Nef reaction to give an aldehyde which was further elaborated by Wittig reaction and reduction.The cyclopenten- one double bond was subsequently introduced by selena-ti~n.~” Isomeric cyclopinolenic acids 43 and 44 (components of tall oil) were synthesised by means of an intramolecular Diels-Alder reaction involving 18:3(52 lOE 1219.~~~ 12-Oxophytodienoic acid 45 was prepared starting from cyclopent-3-ene-l,2-diaceticacid.360 A versatile route to 1,2-disubstituted aromatic arachidonic acid analogue 46 involved the stepwise cross-coupling of alkynes to 1,2-dibr~mobenzene.~~’ Rigidified arachidonic acid analogues 47 and 48 were synthesised by application of the Julia’s olefination proced~re.~~~,~~~ 0 <? I> Me(CH2)5-C-CH2 -CH2 -C-(CH2)7C02Me 49 R1/R2 50 R1 = Me(CH2)s; R2 = (CH&C02Me RVR~ 51 R’ = Me(CH2)S; R2 = (CH2)7C02Me Me(CH2)&&CH2)&OOMe 52 Me Me Me C02H ?H c5H11m 43 44 OH 54 0 k C45 2 H 46 Me(CH2)7H(CH2)7COOMe e(C H2)6 (C H2)7COOM e CN*0 0QO 0 55 56 Treatment of keto fatty esters with trimethylsulfonium methylsulfate gave the corresponding 2,2’-epoxy derivatives 47 (49) via a two-phase methylene transfer reaction.364 Reaction Lie Ken Jie et al.Fatty acids fatty acid analogues and their derivatives of compound 49 with boron trifluoride-methanol complex furnishes a mixture of 2,5-tetrahydropyranyl derivatives (50) and when treated with boron trifluoride-diethyl ether com- pound 49 gives compound 51.36s A 2,5-disubstituted c18 tetrahydrofuran fatty ester 52 was obtained from methyl ricinoleate by addition of bromine to the isomerised substrate followed by hydrogenation over palladium on The total synthesis of a cholesterol biosynthesis inhibitor 53 was accomplished where the key step involved a highly diastere- oselective aldol condensation of a chiral crotonate imide to introduce the stereogenic centres at the ring carbons.367 Three approaches have been reported for the total synthesis of pseudomonic acid C 54.368 Free radical and Lewis acid induced reactions involving the double bonds in unsaturated fatty acids have been conducted by Metzger et al.which has resulted in the production of a large number of functionalised cyclic and branched fatty acid derivatives (e.g. 55 56).369p3g2 3.5.2 Furan fatty acids 2,5-Furandicarbaldehyde was used as the starting material in the preparation of 2,5-disubstituted furan fatty acids 57 via / C02H 58 59 the Wittig olefination reaction.383 A novel method for the introduction of a methyl group into the furan ring of a 2,5-disubstituted C, furan fatty acid via a malonic acid function has been de~eloped.'~~ The 3- and 4-positions of the furan ring of a 2,5-disubstituted furan fatty ester were meth- ylated by reaction with dimethyl acetylenedicarboxylate to give the bicyclo Diels-Alder adduct of the substrate.Retro-Diels- Alder reaction of the partially hydrogenated bicyclo intermedi- ate gave a furan fatty acid derivative containing carbomethoxy groups at the 3- and 4-positions of the furan ring. Reduction of the ring ester functions yielded the required 3,4-dimethyl substituted furan fatty ester.38s The synthesis of furan fatty esters containing a phenyl substituent at the 3- or 4-position of the furan ring was also reported which involved methyl 9,10-epoxy-12-0xostearateas a key The mono- di- and tri-acylglycerols of a c,8furan fatty acid were prepared by chemical and enzymatic means.387 Furanoacety- lene phytoalexins (wyerone 58 and dihydrowyerone 59) were prepared in multigram quantities from f~rfural.~'~ Incubation of a 2,Sdisubstituted furan fatty acid (viz.10,13- epoxyheptadeca- 10,12-dienoic acid) with bovine liver homoge- nate at pH 8.65 in the presence of (9-adenosylmethionine and cysteine showed the formation of 3,4-dimethyl-substituted furan derivatives in addition to other metabolic The oxidation of furan fatty acids by soybean lipoxygenase-1 required the presence of linoleic acid as a lipoxygenase sub- strate to produce dioxoenoic acid.390 The effects of soybean lipoxygenase-1 on phosphatidylcholines containing furan fatty acids have been studied. The results suggest that furan fatty acids play a strategic role in the antioxidative processes in plant cells.391 The biosynthesis of furan fatty acids in Saccharum sp.was found to be Ca2' ion dependent.392 170 Natural Product Reports 3.5.3 Nitrogen-containing fatty acid derivatives c 8 unsaturated fatty esters containing L-amino acid residues (glycine alanine valine leucine and phenylalanine) were derived from methyl 12-amino- 18:l(9Z) and their properties compared to the corresponding methyl N-stearoylamino acids.393 Reaction of nitrene generated in situ by lead tetra- acetate oxidation of 3-amino-2-methyl-4-oxoquinazoline with olefinic and hydroxy-olefinic fatty esters furnished the corresponding aziridine derivatives.394 Reaction of 1-mesyloxynonane with methyl 8-aminooctanoate gives methyl 9-azastearate. Treatment of methyl 9,lO-azidohydroxystearate and methyl 10-azido- 12-hydroxystearate with triphenylphos- phine furnished the corresponding aziridine 60 and azetidine M~(CH~),G(CHZ)~CO~M~ 63 65 Ri = (CH2)7Me; R2 = (CH2)7C02Me HN-N-~2 66 R' = (CH2)12; R2 = H C6H5 or MeC6H4 HNqo 0 68 61 derivatives re~pectively.~~' Vicinal nitroso-oxo fatty deriva- tives were obtained by reaction of azirine fatty esters with m-chloroperoxybenzoic Isomers of methyl 1-pyrroline fatty esters 62 and 63 were derived from methyl ricinoleate and iso-ricinoleate respe~tively.~~~,~~~ The physical properties of a large number of N-substituted pyrrolinium and pyrrolidine derivatives obtained from methyl 1-pyrroline fatty ester were ~tudied."~,~'' The synthesis of a trisubstituted c,8 pyrrole fatty ester 64 containing a methyl group at the 3-position of the ring was rep~rted.~" Similar long-chain pyrrole fatty esters have been obtained by the reaction of lipoxygenase with methyl 9,10-epoxy-13-oxo-18:1(11E)and 12,13-epoxy-9- 0x0-18:1(1OE) in the presence of butylamine and ly~ine.~'* Reaction of methyl oleate with ethyoxycarbonylfonnonitrile oxide or nitrile oxide gave positional isomers of isoxazoline derivatives 65.403,404 Reaction of methyl 4-0x0-18 l(2E) with hydrazines provided the corresponding pyrazoline derivatives 66 which exhibits antifungal and antibacterial a~tivities.~'~ An efficient method has been developed for the preparation of pyrazole fatty ester derivatives 67 by the reaction of 10,12-dioxostearate with hydrazine and substituted hydra-zines in water under ultrasonic irradiati~n.~'~ Reaction of epoxy fatty esters with glycine in DMF in the presence of AlCl yields 2-morpholinone derivatives 68 in quantitative yields.407 3.5.4 Fatty acids containing sulfur selenium or tellurium Ten methyl dithiastearate isomers e.g.69 containing 04 methylene groups between the two sulfur atoms were synthe- sised using a one-pot synthesis approach by coupling of 71 72 0 s'i 0 C02H 73 bromoalkanes and o-bromoalkanoic acids to alkanedithi-01s.~'~ Raman studies of positional isomers of thialaurates and thiastearate have been cond~cted.~'~ Various biochemical reactions involving thia fatty acids have been carried out which include the sulfoxidation of 9-thiastearate and aromatic thia fatty acids with baker's yeast (Saccharomyces ~erevisiae),~" 414 metabolism of 3-thia fatty acids in rat liver and other and inhibition of protozoan cyclo- propane fatty acid synthetase by phosphatidylethanolamines containing thia fatty acid moieties 70.423,424Reaction of 2-alkylthiophene with malonate esters and ceric ammonium sulfate gave the thienylmalonic acid esters which were converted to the thiophene fatty acid derivatives.425 Treat- ment of 4-0x0-18 1 (2E) with 3-mercaptopropan-l,2-diol (1-thioglycerol) in boron trifluoridediethyl ether afforded a mixture of C, oxathiolanes 71 and thioethers 72.426A rapid and high-yielding two-step synthesis of fatty thioacids has been devised which involves the reaction of an acid chloride with thioacetic acid followed by deacetylation with propylamine or b~tylamine.~~' Thioether isomers 73 of long chain fatty esters are prepared from methyl 9,12-dioxo-18:1( 1OE) with mercap- toacetic and mercaptopropionic Fatty esters contain- ing a thiosulfinate group 74 were obtained in high yield when a mixture of the sulfoxides of 2-thia fatty esters (75 and 76) was chromatographed on a silica gel column.429 Bis-oxathiolane sulfides of fatty esters 77 were prepared by the reaction of dioxo fatty ester with P-mercaptoethanol in the presence of boron trifluoride-diethyl ether in acetic Reaction of epoxy fatty esters of methyl undec-10-enoate with p-fluorophenyl isothiocyanate furnished a mixture of 1,3-oxazolidine-2-thione 78 and 1,3-0xathiolane 79 derivative^.^^' Spiro[oxathiolane-2,2'-dihydrotetrazoles]80 were obtained by ? ? Me( CH2)9 -S-S-(CH2)&02Me Me(CH&X02Me 74 75 n s'b Me(CH2,,-(CH2)7C02Me S MeO2C(CH2)4SC02Me ? P 76 77 S N-R 78 R = H or pfluorophenyl 79 R = pfluorophenyl N=N /v -C02Me 80 R = phenyl or fluorophenyl 81 82 83 n = 0,5or a; m = a,7 or 10 to the corresponding hexahydro-3-alkyl-6-thioxo-1,2,4,5-tetrazine-3-alkanoate esters 83 with thiocarb~hydrazide.~~~,~~~ A number of epithio C,* fatty esters (84-88) were prepared by reaction of the corresponding epoxy fatty esters with dimethylthi~formamide.~~~ Reaction of methyl 9,l O-epoxy- 12- oxostearate with dimethylthioformamide in trifluoroacetic acid gave a mixture of epithio oxostearate furan and thiophene 89 fatty ester derivatives.437 The synthesis of positional isomers of methyl selenalaurate 90 and methyl telluralaurate 91,438 unsaturated and hydroxy alkylseleno fatty acids 92,4392,5-disubstituted selenophene 93 and tellurophene 94 fatty esters have been rep~rted.~',~~~ Selenium dioxide oxidation of long chain semicarbazones affords 4,5-disubstituted 1,2,3-selenadiazoles 95.442 Reaction of methyl ricinoleate and iso-ricinoleate with phenylselenenyl chloride gave rise to phenylseleno-substituted tetrahydrofuranyl fatty esters 96 and 9,12-epoxy-13-phenylselenenyloctadecanoate 97 respecti~ely.~~ 3.6 Halogenated fatty acids A large number of brominated polyunsaturated fatty acids have been isolated from the marine sponge Xestospongia sp.which include 20-bromo-20:4(5E,l lE,15E,19E) 14,14-dibromo- 14:3(4,6,1 3),"4 18-bromo-l8:4(7A,9Z,15A,l7E) 18-bromo-l8:5(5A,7A,9E 1~4,17E),~~ bromo-l6:4(5A,7A,9E 199 18-bromo-l8:4(5A,7A,9E 17E) 18-brorno-l8:5(5A,7A 9E 15A 17E) 18-bromo-l8:5(5A,7A,9E 15E 17A) 18-bromo- 18:5(5A,7E,13E,15A,17E), 18-bromo-18:6(5A,7A,9Z,13E,15A, reaction of epoxy fatty esters with dihydrotetra~olethiones.~~~17E),446 6-brom0-28:3(5E,92,242),~~ and 18-bromo- 18:4 Reaction of 10-hydroxyundecanoate with phosphorus oxy- (5474 15A 1713.~~' Several 6-bromo-5,9-dienoic acids [such chloride followed by treatment with aminoethanol and ethyl- as 6-bromo-28:2(5E,9Z) 6-bromo-23-methyl-24:2(5E,9Z)] enediamine gave rise to methyl lo-(1,3,2-oxazaphospholidin-2-were found in the extract of the Caribbean sponge Agelas 0ne)undecanoate 81 and methyl lo-( 1,3,2-diazaphospholidin-sp.,449 Petrosia SP.~~'and Amphimedon terpenen~is.~~'~~~~ 2-0ne)undecanoate 82.433Keto fatty esters were transformed Seven antifungal brominated C,8 acetylenic acids including Lie Ken Jie et al.Fatty acids fatty acid analogues and their derivatives 171 S I\ Me(CH2)7CH-CH(CH2)7C02Me 84 S /\ Me(CH2)4CH-CHCH2C H=CH(CH2)7C02Me 85 R /s\ Me(CH& CCH2 CH- CH(CH2)7C02Me 87 Me(CH2)5CCH2C HzCH(CH~)~CO~M~ 88 C02Me 89 Me(CH&,-Se-(CH&CO2Me Me(CH&,-Te-(CH2),C02Me 90 m+n=9 91 m+n=9 R' seLC02Me 92 R = Me Et,Pr Ph Me(CH2),,jjseL(CH,),C02Me Me(CH2)mATeL(CH2)nC0,M8 93 m+n=&12 94 m+n=7-12 99 JePh synthesised using silver fluoride diethylaminosulfur trifluoride and dichlorofluoromethane as fluorination agents respect- i~ely.~'~ The following o-fluorine labelled fatty acids have been synthesised using K18F in the presence of Kryptofix 2.2.2 [1 7-18F]-3-methyl- 17:O and [17-18F]-3,3-dimethyl- 1 7:0,460 [1 6-18F]-3-methyl- 16:0,461 several [o-18F]-3,5-dimethyl alka- noic acid hom~logues,~~~ and [2-I8F]-fatty acids from the corresponding 2-bromo fatty 3.7 Isotope-labelled fatty acids The synthesis of trideuteriated methyl branched fatty acids was achieved by successive reduction of a carboxylic ester group of 2-alkyl-w-hydroxy carboxylic acid intermediates with lithium aluminium de~teride.~~ Deuterium-labelled pristanic (2,6,10,14-tetramethylpentadecanoic) and phytanic (3,7,11,15- tetramethylhexadecanoic) acids have been synthesised.The key reaction a Favorski rearrangement generated the methyl branch while incorporating the deuterium labels.465 Labelled [16,16 16-2H3]- 16 1( 11A) and [15,15,16,16,1 6-2H5]- 16:2( 1 12,132) acids were synthesised where the deuterium atoms were introduced by reaction of iodoalkynes with (CD,),CuLi and alkylation of a terminal diyne with CH3CD21 respectively.466 Multigram quantities of methyl [12,13-2H2]linoleate were obtained by deuteriation of methyl 18:2(92,12A) (isolated from Crepis alpina) in the presence of Lindlar catalyst. The total synthesis of the other labelled geometric isomers of methyl linoleate involving labelled inter- mediates via the Wittig olefination reaction was also rep~rted.~~~,~~~ The syntheses of chiral isotopic labelled lino- leic acid isomers viz.98 and 99 have been described. The -C02H > 98 -C02H 2 18-bromo-l8:4(5A,7A,15A,172) were isolated from a Japanese marine sponge Petrosia sp.453 An Indonesian sponge Oceanapia sp. contained two new brominated acetylenic acids [14,16-dibrorno-16:5(5A,7E,9E,l3E,152) and 6,14,18-tribromo- 16:5(52,7E,9E 13E 1 52).454 A new long-chain fatty acid 6-bromo-26:2(5E,9Z) was identified in the phospholipid fraction of a Hymeniacidonidae marine sponge. Incorporation experiments with radiolabelled precursors revealed that bromi- nation occurred during the final step in the biosynthesis process.455 A new threo-18-fluoro-9,1O-dihydroxystearicacid was identified in the seed oil of Dichapetalum toxicarium.456 The chlorination of linoleic acid resulted in a mixture of tetrachlorostearic acid trichlorohydroxystearic acid and dichlorohydrins of linoleic Camps et al.have reported a high yielding Wittig condensation of trifluoromethyl ketones with non-stabilised phosphorus ylides in the preparation of trifluoromethyl alkenols. Bromination of the latter followed by chain extension resulted in trifluoromethyl substituted branched-fatty Several monofluorinated fatty acids viz. 2- 3- and 4-fluoropalmitic acid and 5-fluoro-16 l(42)were A 100 key intermediate (15')-[l-2H]oct-2-yn-1-01 (96% ee) was obtained by reduction of [1 -2H]oct-2-ynal with fermenting bakers' yeast.Subsequent chain extension and semi-hydrogenated furnished compound 98. For the synthesis of 99 the starting block was (85')-8-hydro~y-[8-~H]octanoic acid.469 [14,14-2H2]Linolenic acid was synthesised to study the bio- synthesis of 12-oxophyto- 10,15-dienoic acid 45 in plants.470 The synthesis of [14,14,15,15,17,1 8-2H6]linoleic acid by successive Wittig reactions starting from [2,2,3,3,5,6-2H6]heXanal has been described.471 Using similar strategies Rakoff has synthesised a number of polyunsaturated fatty acids which include [15,15,16,16-2H4]-18:3(6,9,12) [12,13, 15,16-2H,]-18:4(6,9,12,15),472 [3,3,4,4,8,8,9,9-2H,]-20:3(1 1 14,17),473 [8,8,9,9-2H4]-20:3( 11,14,17) and [8,9-2H2]-20:4 (8,11,14,1 7),474 and [8,8,9,9-2H4]-20:4(5 11,14,1 7).475 Luthria and Sprecher have used Grignard coupling reactions to obtain [19,19,20,20-2H4]-20:4(5,8,11,14) and [19,19,20,20-2H4]-20:3 (8,l 1,14).476 Partial deuteriation of methyl linoleate in the presence of Wilkinson's catalyst tris(tripheny1phosphine) chlororhodium complex furnished equimolar mixture of methyl [9,10-2H2]-18:1(122) and [12,13-2H2]-18 1(9Z) which were separated by silver resin HPLC chr~matography.~~~ 172 Natural Product Reports Wilkinson's catalyst has also been successfully used in the deuteriation of unsaturated trigly~erides.,~~ Methyl [15,16- 2H,]-18:3(92,122 152) was obtained by Wittig olefination involving methyl 12-0x0- 12 l(9Z) and (32)-[3,4-2H2]-hexenyltriphenylphosphonium bromide.The latter compound was prepared from hex-3-ynol by using Lindlar's catalyst and deuterium gas to introduce the deuterium labels. A similar synthesis route has been adopted for the preparation of methyl [6,6,7,7-2H4]-1 8:3(92,122,15Z) by coupling 3,6-nonadienyl- triphenylphosphonium iodide with methyl [6,6,7,7-,H4]-9- ox~nonanoate.~'~ The all-trans parinaric acid [18:4(9,11,13,15)] with all vinyl positions deuteriated has been prepared by Wittig coupling of a diene phosphorane with an a,P-unsaturated aldehyde-ester. The deuterium labels were introduced via lithium aluminium deuteride reduction of a substituted propynoic ester intermedi- ate.480 Sequential Wittig coupling reactions were also used to prepare C, deuteriated unsaturated fatty acids and leuko- trienes including methyl [19,l9,20,20,20-2H5]-20:4(5Z 82,112 142),481 [19,l9,20,20,20-2H5]-LTA4,481 [20,20,20-2H3]-20:4(52,82,1 1Z,142),482 [I 1,12,14,15-2H4]-LTA~83 and [11,12,14,15-2H4]-LTA4 LTC, LTD and LTE,.484 A number of fatty acids containing "C- 13C- and ''C-labels have been synthesised for medical and biochemical studies.[l-llC]Palmitic acid and 3-methyl-[l-' 'Clheptadecanoic acid were produced by carbonation of the Grignard intermediate with '1C02.485 For the preparation of [l-'3C]linoleic acid the lithium enolate of methyl linoleate was reacted with diethyl dibromomalonate to form methyl a-bromolinoleate which was nitrilated with Na13CN. The resulting cyanoester was further elaborated to give [1-'3C]linoleic Esterification of [l-'3C]oleic acid and [4-'3C]cholesterol was successfully accomplished using oleic anhydride in the presence of dimeth- ylaminopyridine (DMAP) in chloroform or directly using oleic acid and cholesterol in the presence of dicyclohexylcarbodiim- ide (DCC) and DMAP in ~hloroform.~~~,~~~ 3-Methyl-15-phenyl[ l-'4C]pentadecanoic acid was obtained by a one-carbon extension with Na14CN from the methanesulfonate A ester of 14-methyl-1-phenyl-tetrade~anol.~~~ similar chain extension procedure has been applied to 1,16-diiodohexadecane to yield 17-iodo[ 1 -14C]heptadecanoic acid.490 The synthesis of [l-14C]arachidonic acid was achieved by carbonation of the corresponding Grignard intermediate with 14C0,.491 The syntheses of (92,12E)- and (9E,12Z)- [l-'4C]linoleic acid and (52,82,1 1Z,14E)-[l-'4C]arachi- donic (92,122,15E)-and (9E 122,152)-[ 1 -14C] octadecatrienoic acid have also been described.493 3.8 Hydrogenation The use of polymer-supported palladium (Pd-styrene) and nickel catalysts for a continuous hydrogenation system for soybean oil has been studied.494 Decalin in the presence of 10% palladium on carbon was shown to be an efficient hydrogen- transfer agent in the hydrogenation of unsaturated fatty esters where no external source of hydrogen was Aque-ous sodium formate in the presence of palladium was also found to be an efficient hydrogen donor in the hydrogenation of soybean Hydrogenation of soybean and rape oil on palladium on alumina gave partially hydrogenated fat with low trans-i~omers.~~"~~~ Low pressure and low temperature hydrogenation of castor oil over nickel catalyst yielded a saturated waxy product rich in hydroxystearic acid.4y9 Hydrogenation of cottonseed oil in the presence of chromium modified nickel on silica suppressed the formation of stearate completely.500 The rate of hydrogenation of soybean oil in the presence of nickel catalyst increased by five times when the process was conducted under concomitant ultrasonication (20 kHz 375 W cm -2).501 Electrocatalytic hydrogenation of soybean oil was accomplished without exter- nal supply of hydrogen by using active Raney nickel powder as a cathode surface in the electrochemical reduction of water molecules.The hydrogenated oil has a high stearic acid content but consists of very low levels of trans-is~mers.~'~ A homogeneous chromium catalyst from Cr(CO) showed low hydrogenation activities but produced very low (<1%) levels of trans-is~mers.~~~ Hydrogenation of jojoba wax and castor oil was achieved using an intercalated ruthenium complex R~C~,(H),(ASP~,),.~~~ Oleic acid was reduced by hydrogena- tion over ruthenium-tin-alumina to give a mixture of (Ha- octadec-9-en- l -01.505s06 Regioselective hydrogenation of nona-3,8-dienoic acid to non-8-enoic acid was accomplished by adding water to RhCl[P(p-tolyl),] catalyst in benzene while in the absence of water the hydrogenation furnished non-3-enoic acid Partial reduction of unsaturated fatty esters was achieved by the diimide generated from hydroxylamine-ethyl acetate under nitrogen.508 4 Autoxidation peroxidation and reactions by enzymes A number of analytical methods have been developed or modified to assess the degree of autoxidation which include GC analyses of short-chain aldehydes and their 2,4,6-trichlorophenylhydrazone derivative^,^'^-^' ' ethane and pen- tane in free oxygen radical-induced lipid per~xidation,~~ 4-hydro~yalkenals;~'~ TLCAensitometric analysis of alde-hyde~,~~~ colorimetric method of the 2,4-dinitrophenyl hydra- zones derived from triphenylphosphine reduced peroxide^,^' and thiobarbituric acid assays.51G520 The red pigment formed in the thiobarbituric acid method was shown to be due to aldehyde species other than mal~naldehyde.~~~ The structures of two of the red pigments formed during autoxidation of methyl arachidonate were identified as 3-(5-hydroxy-3-oxo-2-pentylcyclopentyl)prop-2-enal and methyl 4-[2-(2-formylviny1)-3-h ydrox y-5-0xocyc1open tyl] bu tanoate.522 Methods to determine the amount of hydroperoxides in fats and oils by HPLC,523,524 spectrophotometric triiodide assay,525 electron spin resonance spectroscopy.526 A micro- computer based system for data acquisition has been devised for oxygen uptake with a Warburg respirometer for lipid oxidation experiments.527 A sensitive method through the application of potentiometry to conventional peroxide value tests for the micro-determination of lipid hydroperoxide has been developed.528 A sensitive method to detect linoleic acid hydroperoxide involves its conversion with 2-methyl-6-phenyl- 3,7-dihydro-imidazo-[ 1,2-a]-pyrazin-3-one to a chemilumines- cent produ~t.~~~,~~' Treatment of methyl linoleate hydroperox- ides with 1-aminopentane in the presence of haem formed fluorescent derivatives which were readily detected and quantified.531 Autoxidation of methyl linoleate was readily initiated by the ozonide of all~lbenzene.~~~ The major volatile components generated from the oxidation of arachidonic and eicosapentae- noic acids by gill 12-lipoxygenase are mainly the c8 and C9 unsaturated alcohol and aldehydes.533 The mechanisms of free radical oxidation of unsaturated lipids have been reviewed534 Peroxidation of polyunsaturated fatty acids was directly initi- ated with a cobalt porphyrin complex.535 Homolytic and heterolytic scissions of hydroperoxidised unsaturated fatty acids with iron(II1) complexes gave rise to keto-unsaturated Photooxidation of unsaturated fatty acids by oxygen proceeded efficiently in the presence of a riboflavin derivative (riboflavin-2',3',4',5'-tetraacetate) via a singlet oxy- gen mechanism.537 Based on '80-labelling experiments the generation of a-hydroxy aldehydic compounds in the course of lipid peroxidation of linoleic acid has been e~plained.~~~.~~~ The perhydroxyl radical (HOO-) generated chemically (from KO,) or enzymatically (xanthine oxidase) is a good initiator of fatty acid peroxidation in linoleic acid in ethanol-water dispersions where the superoxide serves only as the source of HOO- radicals.540 The synthesis of E,E-diene hydroperoxides via iodine-mediated photoisomerisation of protected hydroper- oxides (as the 2-methoxypropyl peroxyketal) is reported.541 An enzymatically derived intermediate y-peroxy-a,P-unsaturated Lie Ken Jie et al.Fatty acids fatty acid analogues and their derivatives 173 aldehyde 100 was used in the synthesis of (13S)-hydroperoxy- 9(Z) 1 1 (E)-octadecadienoic Studies on the autoxid- ation of sea urchin lipids and meadowfoam oils (both contain- ing unsaturated fatty acids with the double bond at the 5-position) showed that such oils were more stable to autoxid- ation than the oils containing normal monoenoic and dienoic The autoxidation of polyunsaturated triacylglycerols fatty acids with allene oxide ~ynthase.’~~ The enzymic con- version of 13-hydroperoxylinoleic acid into 13-hydroxy- 12- oxo-l8:1(9Z) and 9-hydroxy-12-oxo-18:l(lOE)was shown to proceed via an allene epoxide inte~mediate.’~’ Crombie et al.have demonstrated by isotopic labelling experiments that the divinyl 9-ether oxygen of colneleic acid 37 originated from oxygen and not water from a study of (99-has been intensively studied by Frankel and co-~orkers.’~~~~~~ hydroperoxylinoleic acid with potato tuber enzyme.571 Incu- The effects of tocopherols in the spontaneous oxidation of methyl linoleate were examined. Result showed the inhibition of trans,trans-hydroperoxidesduring the induction period but when present in high concentration a-tocopherol acted as a pr~oxidant.~~’ 9-Hydroperoxy-y-linolenic acid is selectively obtained by treatment of y-linolenic acid with soybean lipoxy- gena~e.~~~ Lipid peroxyl radicals resulting from peroxidation of polyunsaturated fatty acids by soybean lipoxygenase were directly detected by rapid-mixing continuous-flow electron paramagnetic resonance (EPR) The photo- lysis of (13S)-hydroperoxy-l8:2(92,11E) gave a complex mixture of oxygenated fatty acid derivative^.^^' Fatty acid hydroperoxide isomerase in Saprolegniu parasitica isomerised (13S)-hydroperoxy-l8:2(92,1lE) to an a,p-epoxy deriva-tive (1 lR,12R)-epoxy-( 13S)-hydroxy-18 1(92)? Incubation of [14-2H2]linolenic acid with a flax enzyme preparation showed the formation of 12-oxophyto-10,15-dienoicacid 45 via an allene epoxide intem~ediate.”~ Incubation of (13S)-hydroperoxy-18:3(92,1 lE,15Z) with corn (Zea muys) hydroperoxide dehydrase gave an unstable allene oxide inter- mediate which was further converted to 12-0x0-13-hydroxy- 18:2(92,15Z) and 12-oxophyto- 10,15-dienoic 12-Oxophyto- 10,15-dienoic acid from (1 3S)-hydroperoxy- 18:3 was found to consist of a mixture of enantiomers where bation of a C s disubstituted furan fatty acid with lipoxidase in the presence of SH-containing compounds (cysteine or glutath- ione) furnished an unstable unsaturated dioxo compound viz.10,13-dioxo-l8:1 (1 1 Incubation of a number of pos- itional isomers of methyl thiastearates with yeast Saccharo- myces cerevisiae has been conducted.Methyl 9-thiastearate gave the sulfoxide derivative while methyl 6- 7- 12- and 13-thiastearate yielded the corresponding thia~leates.~~~ 5 Physical properties 5.1 Thin layer and column chromatography The facile flash chromatographic separation of 1 -mono- 1,3- di-and tri-oleoylglycerols has been dernon~trated.’~~ An improved method for the separation of lipid esters by argen- tation thin layer chromatography (TLC) or by circular reversed-phase TLC has been developed for ethyl and 2-chloroethyl esters of unsaturated fatty acids.57’ A method for the impregnation of silver nitrate on silica TLC plates is re~ommended.’~~ Unsaturated diacylglycerols were success-fully separated by argentation TLC and by reversed-phase TLC in the presence of silver Mixtures of lipids and phospholipids were separated by centrifugally accelerated TLC the major enantiomer has the (9S,13S)-~onfiguration.~~~~~’~ on a preparative scale on a Chromatotron (model 7924T A mechanism has been proposed for the cleavage of the hydroperoxide 0-0 bond of (13S)-hydroperoxy- 18:3 (92,ll E,152) by soybean peroxygenase which results in the formation of (13s)-hydroxy-l8:3 and an epoxy isomer 15,16- cis-epoxy-l3S-hydroxy-18:2(92,1 lE).’56 A fatty acid hydro- peroxide lyase from germinating soybean seedlings is reported to cleave 13-hydroperoxides of linoleic and linolenic acids to form hexanal and cis-hex-3-ena1 respectively.’’7 The effects of polyunsaturated free fatty acids and esterified linoleoyl deriva- tives on the oxygen consumption and C aldehyde formation with soybean seed homogenates have been st~died.~” 9- and 13-Hydroperoxides and the corresponding ketodienes were produced from linoleic acid with oxygen in the presence of lipoxygenase isozymes isolated from kidney bean and pea a-Linolenic acid was converted to (9s)- 16- dihydroperoxy- l8:3(92,122,15Z) by soybean lipoxygenase whereas (9S)-16-dihydroxy-l8:3(92,12E,15Z)was obtained when incubated with potato tuber lipoxygenase.’60 Potato tuber lipoxygenase caused double hydroperoxidation of a-linolenic acid to yield 9,16-dihydroperoxy-l8:3(10E 122,14E) which was subsequently converted to the corre-sponding dihydroxy- 18:3 13-Hydroperoxylinoleate 10-oxodec-8-enoic acid and oct- 1-en-3-01 are the metabolic products of linoleic acid formed by a mycelial-pellet hom- ogenate of the fungus Pleurotus pulmonarius.’69 Eight stereo- isomeric 9,10,13- and 9,12,13-trihydroxyoctadecenoicacids arising from the peroxidation of linoleic acid by soybean lipoxygenase have been identified.563 A kinetic study of the conversion of (13S)-hydroproxy-l8:2(92,1lE) to the cor-responding hydroxy fatty acid by potassium hydroxide has been conducted.564 Mono and polyunsaturated fatty acids are efficiently epoxidised by soybean peroxygenase in the presence of alkylhydroperoxides as co-s~bstrates.~~’ Soybean epoxide hydrolase efficiently catalyses the hydration of the two positional isomers of linoleic acid monoepoxides into their corresponding vi~-diols.~~~ Two reviews on the biosynthesis and conversions of fatty acid allene oxides are a~ailable.~~~~~~~ Allene oxides and epoxy alcohols were obtained from hydroperoxy polyunsaturated I74 Natural Product Reports Harrison Research Palo Alto CA).579 The separation of ethyl esters of unsaturated fatty acids from cereal and fish oil by centrifugal partition chromatography has been reported.580 Prepacked silica Sep-Pak columns were ideal for the separation of cholesteryl esters from triglycerides but were not efficient for the separation of phospholipid^.^^' High performance TLC (HPTLC) of mono- di- and tri-glycerides in cosmetics phar- maceuticals and food products offered an efficient and quick method for the quantitative assessment of such materials.’s2 Nanogram quantification of nonpolar lipid classes was achieved by HPTLC.58’,’s4 TLC coupled with flame ionisation detection (Iatroscan TLC-FID) permits the accurate quanti- tation of triglycerides free fatty acids phosphatidyleth-anolamine and phosphatidylcholine in fish lipids.’85 The same method was applied to the quantitation of 1,2-diacylglycerol in rat heart586,587 and the quantitation of phospholipids in animal 5.2 High performance liquid chromatography A practical guide on high performance liquid chromatography (HPLC)Sg9 and a review on the separation and detection of triglycerides by HPLCS9’ have been published.Quantitative and qualitative normal phase (silica) HPLC analyses were performed on animal fats and seed oils with separation of triacylglycerols according to their degree of ~nsaturation~~’~~~~ and configuration of the double The amounts of mono- di- and tri-acylglycerols in edible fats were also deter- mined by this technique.594 Normal phase HPLC with a fluorometric post-column detection system has been developed for the analysis of hydroperoxides of triglycerides and choles- terol esters.595 Under reversed-phase conditions (using Spherisorb ODs-2) mixtures of mono- di- tri-acylglycerols and free fatty acids were readily ~eparated.~~~.~’~ Similar analyses (on C ,-bonded silica columns) were conducted on the triacylglycerols of the alga Chlorella ke~sleri,~~ cereal lipids,599 bovine and human milk,600- 602 and fish oils.603,604 The retention behaviour of saturated triacylglycerols has been studied with results indicating that triacylglycerols interact with the stationary phase (C,,-bonded silica) by having one two or three acyl chains penetrating into the bonded layer.60s Application of HPLC in triacylglycerol analysis allowed the detection of castor oil in adulterated vegetable oil by monitor- ing the presence of triri~inolein.~~~ Adulteration of olive oil by as low as 1% of linoleic-rich vegetable oil level was determined by HPLC.6"7 An evaporative light scattering detector was used for the analysis of wheat flour lipids and triglycerides of avocado oil.h08.60y The phenacyl esters of 22 saturated monounsaturated and polyunsaturated fatty acids (6:O-22:6) were analysed by reversed-phase HPLC.Several critical pairs of these esters were successfully resolved. The results were comparable to those obtained by GC analysis.61" HPLC offered a specific method for the analysis of the phenacyl esters of very-long- chain saturated fatty acids (18:O-28:O) in tall oil rosin.611 Enrichment of cyclic fatty acid monomers in oils and very- long-chain fatty acids with more than 23 carbon atoms by reversed-phase HPLC for subsequent GC-MS analysis has been described."' 2-l4 The determination of a-tocopherol free cholesterol cholesterol esters and triacylglycerols in human plasma by reversed-phase HPLC is rep~rted."~ HPLC analyses have been conducted on the following fatty acid derivatives amides6I6 and hydroxy derivatives of arachi- donic acid,"17 and dimers formed in thermally oxidised or used fats and To separate unsaturated triacylglycerols according to the degree of unsaturation argentation chroma- tography has been adapted to HPLC.Pre-HPLC separation was achieved when the sample was chromatographed on silver nitrate impregnated silica,62' or with commercially available solid-phase extraction (SPE) tubes packed with cation-exchange material.62' Special silver nitrate impregnated stationary phases (including bonded phenylsulfonic moieties in the silver ion form silver-ion saturated XNlOlO resin) have been developed and successfully used for the separation of unsaturated triacylglycerols and fatty esters.624p63s Sixteen cyclic fatty acids formed from linolenic acid in heated linseed oil were fractionated by argentation HPLC and their structure identified by GC-MS."' Resolution of racemic 2-hydroxy C,-C fatty acids as their 3,5-dinitrophenylurethane and methyl ester derivatives was achieved by HPLC using a fused silica capillary column containing a chiral phase viz.N-(S)-2-(4-chlorophenyl)isovaleroyl-u-phenylglycineionically bonded to silica gel.h37Separation of the sn-1 from ,vn-3 monoacylglyc- erol on a chiral column (Sumichiral OA-4100 from Sumitomo Chemical Co.. Osaka Japan) for the stereospecific analysis of sn-triacylglycerols was reported.638 Monoacylglycerol enanti- omers as their di-3,5-dinitrophenylurethane derivatives were separated on N-(R)-1-(a-naphthy1)ethylaniinocarbonyl-(a-valine bonded to silica gel stationary phase.639 Diacylglycerols have been separated as their (S)-(+)-or (R)-( -)-(1-naphthy1)ethyl urethane derivatives by HPLC on a silica gel (Hypersil) column.640 Enantiomers of I-alkyl-2-acyl-rac-glycerol 1-alkyl-3-acyl-rac-glycer01,~~' alkyl glycerol ethers,63' 1,3-glyceryl diethers"' and epoxy fatty esters644 were also successfully separated by HPLC on chiral stationary phases.5.3 Supercritical fluid chromatography and extraction Two reviews are available on supercritical fluid chromatog- raphy and extracti~n."~~'~~~ The effect of carbon dioxide (CO') flow rate on the separation of triacylglycerols by capillary supercritical fluid chromatography has been Enrichment of polyunsaturated fatty acids (20:5 and 20:6) from fish oil was achieved by extraction with supercritical CO2.64X 652 Isolation of fungal lipid by supercritical fluid tographic resolution of mixtures of homologous fatty acids (1 2:0-20:0).6s6 Capillary supercritical fluid Chromatography was applied to the separation of a-keto unsaturated methyl fatty esters,658 triacylglycerol components in butterfat fish and vegetable triacylglycerols from butterfat coupled to a flame ionisation detector or a double focusing mass spectrometer,660 and mono- and di-acylgly~erols.~~~ Sep-aration of methyl esters of 18:0 18:l and 18:2 was achieved with CO containing 4% of ethyl acetate as an entrainer on a silica gel-supported silver nitrate column or on silver nitrate doped silica Separation of fatty acid methyl esters according to the degree of unsaturation using a permanganate- treated anion exchanger packed capillary supercritical fluid column was reported.664 Unsaturated triacylglycerols were separated and quantified by supercritical-fluid argentation chromatography using packed microcolumn and a miniatur- ised evaporative light-scattering dete~tor.~~'.~~~ The quantita- tion of cholesterol in milk fat by supercritical fluid chromatog- raphy was found to be a more accurate than by gas liquid chr~matography.~~~ 5.4 Gas chromatography A practical guide on gas chromatography of lipid^,^' and a review on GC analysis of plasma lipids have been published.668 A computer-aided approach to the GC analysis of the ozonolysis products of non-conjugated positional isomers of 18:2 has been reported.669 An accurate and reproducible method has been developed for calculating the equivalent chain length (ECL) values of saturated and unsaturated fatty acids for identification purpose^."^ Christie has reappraised the equivalent chain length (ECL) values of series of positional isomeric methyl octadecadienoates on different modern stationary phases (Carbowax 20M Silar 5CP CP-Si1 84 and Phenyl-methyl silicone).671 In the analysis of fatty acids con- taining a double bond at either the A4 or A5 position of the alkyl chain GC-MS analysis of the iodolactone derivatives permitted the identification of such positional isomers.672 The structural isomers of saturated C, cyclic fatty acid methyl esters from heated vegetable oils were identified by using high resolution capillary GC columns followed by mass spectro- metric analysis.67' Improved GC high performance gel per- meation and TLC-FID methods have been reported for the determination of monomer dimer and trimer fractions from heated vegetable Wijesundera et al.have resolved the artifacts which were observed during GC analysis of distilled methyl and ethyl esters of fish oil as due to the presence of 20:5(5E,8Z. 1 1Z,142,179 20:5(52,8E 112,14Z 172) and 20:5 (5Z,8Z71 1Z,142,17E).67s The structure of 18:5(32,62,92 122,15z) isolated from cultured unicellular alga Prorocentrurn mininiurn was confirmed by GC analysis of the partially hydrazine reduced substrate.676 A short review on the preparation of fatty acid methyl esters for GC analysis of lipids in biological materials has been presented.677 Two new methods for the preparation of methyl esters from vegetable oils have been reported.Tetramethylgua- nidine and trimethylsulfonium hydroxide in methanol are efficient reagents for this p~rpose.~~'.~~" A rapid transesterifi- cation method using tetramethylammonium hydroxide was described for identification of fatty acids in nut oils as their methyl esters.680 Methylation of conjugated C I dienoic acids was best performed using sodium methoxide in methanol or tetramethylguanidine in methanol for quantitation purpose.681 Free fatty acids reacted with trimethyl-(a,a,a-trifluoro-rneta-to1yl)amnionium hydroxide in hexane to form the correspond- ing quaternary ammonium salts which were converted on-column to the methyl esters for GC analysis,6x' extraction using CO and other gases has been ~tudied.~~'?~'~ The fatty acid composition of triacylglycerols was accurately The retention behaviour of free fatty acids has been deter- determined by conversion of the triacylglycerols to the corre- mined by supercritical fluid chromatography on an inert sponding pyrrolidide derivatives6*' A GC method has been packed column of ODs-silica gel using supercritical CO as the developed for the determination of the absolute configuration mobile phase."' Water saturated CO improved the chroma- of the two chiral carbon atoms of epoxy fatty acids.The Lie Ken Jio et al. Futty ucids futty acid analogues and their derivutives method involved the conversion of the saturated epoxy ester into a pair of regioisomeric allylic alcohols by consecutive treatments with selenophenoxide anion and hydrogen perox- ide oxidative ozonolysis on the ( -)-menthoxycarbonyl derivatives of the allylic alcohols and steric analysis of the resulting two 2-hydroxy acids as the ( -)-menthoxycarbonyl ester derivatives.684 A standardised method for the GC deter-mination of mono- and di-acylglycerols in fats and oils is available.685 5.5 Infrared spectroscopy Fourier transform infrared spectroscopy (FTIR) was used in the quantitative measurement of isolated non-conjugated trans fatty acid contents in fats and Using the peak area with a baseline drawn between 998 and 944 cm -' of the FTIR spectrum the amount of trans fatty acids was found to be within 4% of the value determined by capillary gas liquid chromatographic analysis.691 A non-invasive short-wavelength near-infrared (700-1 100 nm) spectroscopic method to estimate the crude lipid content in the muscle of rainbow trout has been described.The measurements were performed in the diffuse reflectance mode by conveying the light to the intact whole fish over an optical fiber bundle.692 Dimer and polymer triacylglycerols and the acid value of used frying fats and oils were determined by near-infrared reflectance spectroscopy (NIRS) in the transflection mode.693 An improved Raman spectroscopic procedure for the determination of the total unsaturation in oils and margarines using Fourier transform Raman spectroscopy has been reported.694 5.6 Nuclear magnetic resonance spectroscopy The use of a new lanthanide shift reagent viz.tris(tetraphe-nylimidodiphosphinato) praseodymium [Pr(tpip),] permitted the complete resolution of the 'H NMR signals of lauric When arachidonic acid [20:4(52,82,112,142)] was analysed by this method the position and configuration of the olefinic bonds and the preferred conformation of the carbon skeleton were determined.695 The 'H spin-lattice relaxation time (Tl) measurements and one-dimensional difference nuclear Overhauser enhancement (NOE) studies of the struc- tures of a-and P-cyclodextrin complexes of linoleic and arachidonic acids showed the carboxy arms of both acids to be present within the cyclodextrin cavity while the double bonds were partly exposed or buried.696 Unsaturated fatty acids isolated from the heart tissue were quantified from the inten- sities of the signals arising from the shifts of the methylene protons adjacent to the olefinic bond.697 Proton magnetic resonace imaging of lipids in pecan (Carya illinoensis) embryo provided a useful technique to study the lipid distribution in oil seeds.698 The melt crystallisation of trilaurin and trimyristin was also investigated by magnetic resonance imaging which showed that crystallisation in the emulsified state required an extended period of time.699 The analysis of the conformation of the glycerol structure of glycerides by 'H NMR spectros- copy allowed the fractional populations of the conformers of each glyceride to be st~died.~" Extensive I3C NMR studies have been carried out on the positional isomers of methyl esters of thialauric and thiastearic acids,70' azide-containing fatty derivative^,^'^ allenic fatty sulfinyl and sulfonyl derivatives of methyl la~rate,~'~ genated soybean The effects of bis-homoallylic and -homoallylic hydroxy substitution on the olefinic ' reso-nance shifts in fatty acid methyl esters were investigated719 and the shifts of the olefinic carbon atoms of several such substi- tuted fatty esters were unequivocally confirmed by Lie Ken Jie and Cheng.720,72' High resolution 13C NMR permitted the distribution of fatty acids in the sn-1 and sn-2 position in plant glycoglycerol lipids to be accurately determined.722 This tech- nique also allowed the amounts of mono- di- and tri-acylglycerols in glycerol ester mixtures and seed oils to be measured.723,724 The quantitative triacylglycerol analysis of whole vegetable seeds by 'H and I3C magic angle sample spinning NMR spectroscopy has been reported.725 13CNMR analyses have been carried out on fish oils with emphasis on the spectral assignment of signals for the 0-3 group of distribution of 20:5 and 22:6 acids between the a-and P-glycerol and free fatty acids released after fish thermal processing.733 The I3C NMR spectra of 28 branched- chain acids wool wax acids and isostearic acid have been recorded and assigned.734 Lie Ken Jie and Lam have conducted an intensive study of a large number of synthetic symmetrical and asymmetrical tria- cylgly~erols,~~~ triacylglycerols containing monoacetylenic and ethylenic groups,736 739 and fatty acids and triacylglycerols containing a diacetylenic sy~tern.~~~,~~' The carbon shift sig- nals of unsaturated carbons in triacylglycerols were evaluated as rational functions.742 The 13C NMR spectra of some synthetic glycerol esters have been recorded.743 Detection of specific carbon nuclei in 2H enriched long chain fatty acids by 13C NMR spectroscopy was employed in the study of fatty acid metabolism in rat liver.744 Analysis of monoacylglycerols as the isopropylidene derivatives by I3C NMR spectroscopy allowed 1-and 3-acyl-sn-glycerols and 2-acyl-sn-glycerols to be identified.This method is of use for determining the regio- chemistry of monoglycerides which are produced by enzy- matic hydrolysis of triacylgly~erols.~~~ Quantitative analysis of mixtures of acylglycerols and the enantiomeric purity of such compounds were determined by using Eu(hfbc),-d shift reagent.746 5.7 Mass spectrometry A one-step conversion of branched fatty acids to the 4,4- dimethyloxazoline derivatives has been developed.747 These derivatives were useful for locating the position of methyl branches in the alkyl chain of fatty acids in the uropygial secretion of ~uc~s,~~~,~~~ double bond sites in fatty acids from palm kernel double bonds in polyunsaturated C, fatty aCidS,751-754 conjugated diene and triene systems in fatty hydroxy functions in hydroxylated fatty cyclopropene ring in malvalic and sterculic a~id,~~~,~~~ and elucidating the structure of cyclopentenyl fatty acids in seed oil of Flacourtiaceae sp.759 The position of double bonds in unsaturated fatty acids were also determined by mass spectro- metric analyses of the dimethyl sulfide ad duct^.^^'-^^^ M ass spectral analyses of the picolinyl esters of positional isomers of dimethylene interrupted diacetylenic fatty conjugated diacetylenic fatty deuteriated acetylenic and olefinic fatty acids have been condu~ted.~~~,~~~ The fatty acid compo- sitions of Biota orientalis and borage oils were determined by mass spectrometric analysis of the picolinyl esters of the fatty The positions of the double bonds in polyunsatu- tellurium dichloride derivatives of methyl tell~ralaurate,~~~ a~ids.~~~,~~~ long chain 0-3 polyene nitriles amides and acetates rated fatty acids (20:5) from eel lipids were determined by of fatty and polyol esters and ethers.708 Many seed oils and fat products have been studied by I3CNMR spectros- copy.709,710 Th is technique allowed the detection of cis-vaccenic acid [18:1(1 la]in palm 18:3(5E,92,122) and other A5 unsaturated acids in meadowfoam (Limnanthes alba) and pine seed y-linolenic acid in evening primrose borage blackcurrant in the seed oil of ja~anicus,~' a-linolenic acid in heat abused epoxy fatty acids in Vernonia galamensis seed and trans-fatty acid isomers in hydro- GC-MS of the methylthiolated derivatives of the monoenoic acids obtained by partial reduction with hydra~ine.~~~ The position of the cyclopropane ring in long chain fatty acids was identified by mass spectral analysis of the 2-substituted ben- zoxazole derivative^.^^' Piperidyl and morpholinyl esters are suitable derivatives for determining the structure of un-saturated and methyl-branched fatty acids.These derivatives gave stable ion fragments during mass spectral analysis which allowed the position of the methyl branch and double bonds to 176 Natural Product Reports be Mass spectral analysis of triazolopyridine derivatives of branched and unsaturated fatty acids has been Location of the position of epoxy rings in epoxi- dised linoleic arachidonic and docosahexaenoic acids by mass spectrometry was achieved by analysis of the corresponding 3-pyridinylmethyl esters.Characteristic cleavage occurred at the C-C bond at the epoxy ring.773 Allylic nitrite and nitro derivatives of methyl linoleate and methyl linolenate by nega- tive chemical ionisation mass spectrometry were used to iden- tify the products of autoxidation of polyunsaturated fatty acids initiated by nitrogen Hydroxy and hydroper- oxy fatty acids are converted to the 3-methyl-7-methoxy-1,4-benzoxazin-2-0ne derivatives which are characterised by liquid chromatography-mass spectrometry.775 The use of tandem mass spectrometry for determining structure of biologically interesting molecules including fatty acids and complex lipids has been reviewed by The application of GC-tandem mass spectrometry for the analysis of very low quantities of mycobacterial lipids has been accom- pli~hed.~~~ Selective ion monitoring (SIM) mass spectrometry has been applied to the peak assignments of the triacylglycerols in vegetable oils by selecting certain characteristic ions with the same retention time on the SIM profile.778 Electrospray and tandem mass spectrometric analyses were carried out on mixtures of mono- di- and tri-acylgly~erols.~~~ Triacylglycer-01s were also investigated by atmospheric pressure chemical ionisation (APCI) mass spectrometry where minimal frag- mentation occurred resulting primarily in diacylglycerol [M -RCOO]' and [M + 11' protonated molecular species to be formed.780 Application of negative ion chemical ionisation tandem mass spectrometry to the analysis of low erucic acid rapeseed oil (Brassical campestris) provided information on the fatty acid composition in positions sn-2 and sn-1/3 of the tria~ylglycerol.~~ ' Triacylglycerols of human milk were ana- lysed by ammonia negative ion tandem mass spectrometry which showed the most abundant triacylglycerol as sn-18 1-16:O-18 1 ( Molecular mass determination of methyl esters of mycolic acids using thermospray mass spectrometry has been accomplished.783 6 Biosynthesis and biotechnology A study of the biosynthesis of triacylglycerols containing very long chain monounsaturated fatty acids in seeds of Lunaria unnua shows that 22:l and 24:l are found exclusively in the sn-l,3-p0sitions.~*~ A6-Desaturation of [''C]linoleoyl or [14C]oleoyl-CoA which leads to the formation of y-linolenic acid has been studied in vitro with microsomal fractions from developing seeds of borage (Boragu uficinulis L.).785 The incubation of deuterium labelled oleic acid with a culture of the protozoan Tetrahymena yielded the corresponding deu- teriated 18:2(62,92) which was useful for 2H NMR studies of membranes and lipid metabolism.786 In the study of the biosynthesis of 15,16-dimethyltriacontanedioicacid (diabolic acid) from [16-*H3] and [14-*H,]palmitic acids no loss of deuterium occurred during formation of diabolic acid from these labelled fatty acids in Butyrivibrio Jibrisolvens.This result ruled out claims of the involvement of A14 or A'' unsaturated fatty acids as intermediate^.^^^ ~~~k~68,69,73 and review articles on lipases describing the general aspects and optimisation of enantioselective lipases in organic solvents,788 developments in biotechnology of fats and from the 2- or 4-methyl branched isomers during lip~lysis.~~~ Hydrolysis of the single-cell oil from Mortierella sp.with lipase from Candida cylindracea allowed arachidonic acid to be enriched from a 25% to a 50% level in the tria~ylglycerol.~~~ Candida cylindracea lipase also showed resistance to acyl groups composed of 22:6 in triacylglycerol molecules. 798,799 The A5unsaturated fatty acid 20:4(52,112,142,172) in Biota orientalis seed oil was strongly discriminated against when the mixture of free fatty acids of the seed oil was reacted with butanol in hexane in the presence of Candida cyclindracea lipase.800 Candida antartica lipase catalyses the reaction between diesters and amino alcohols to give products contain- ing a peptide linkage with a terminal ester and a hydroxy function.These compounds were subsequently cyclised in the presence of the lipase to yield macrocycles.801 0-Acylation of L-homoserine or L-serine with free fatty acids was achieved with lipases from Candida cylindracea and Rhizopus delemar. The emulsifying activity of 0-oleoyl-L-homoserine was shown to be higher than those of conventional surfactants (e.g. casein or sodium oleate).802 Methyl esters of fatty acids containing an N-heterocyclic (1 -pyrroline or pyrrolidine) system were readily hydrolysed in the presence of lipases from Candida cylindracea or Rhizomucor miehei under ultrasonic irradiati~n.~'~ A study of the lipolysis of evening primrose and borage oil showed that the rate of lipolysis of triacylglycerol species containing two or three molecules of y-linolenic acid was significantly slower than those containing only one molecule of y-linolenic acid.804 In the hydrolysis of triacylglycerols containing palmitoyl linoleoyl or linolenoyl groups pancreatic lipase showed pref- erential hydrolysis (1.62.4 times) of monohydroperoxide of linoleoyl and linolenoyl acyl groups in the 1,3-position of the triacylglycerol over non-hydroperoxidised acyl Rabbit gastric lipase has been shown to be sn-3 specific in the hydrolysis of chiral triacylglycerols while lipase from the fungus Pseudomonas aeruginosa is sn-1 specific.806 The commercially available immobilised fungal lipase from Rhizomucor miehei on macroporous support (Lipozyme@) showed high enzyme activities for the esterification of satu- rated unsaturated straight-chain or branched-chain fatty acids with a wide variety of alcohols.807 This immobilised enzyme was used in the biorefining of high acid rice bran oil by converting the major portion of the free fatty acid to neutral glycerides when glycerol was added.808 Lipozyme discriminates 22:6 (DHA) from other unsaturated fatty acids originating from fish oils making DHA a poor substrate either as a free fatty acid in lipase-catalysed esterification with methanol or in the hydrolysis of the methyl ester of DHA to the free fatty acid.809 Preparation of triacylglycerols enriched with 205 (EPA) and DHA by interesterification of cod liver oil and free fatty acid or ester concentrates via the lipase from Mucor miehei (Lipozyme) has been described.810 Lipozyme was used in the preparation of N-lauryloleoylamide which showed the enzyme's versatility to produce an amide bond.81 Fatty hydroxamic acids a new class of amphiphilic molecules were prepared by reacting hydroxylamine with fatty acids or their methyl esters using the lipase of Mucor miehei.*12 The immobilised lipases from Mucor miehei on Duolite ES 562 resin (Rohm and Haas Philadelphia PA) hydrolysed triacyl- glycerols of 12:0 14:0 16:O and 18:l more rapidly than those containing short chain fatty acids (4:0 6:0 8:O and 10:O).813 In the esterification of polyunsaturated fatty acids with propanol other lipids,789379o purification and structure of lipa~es,~~' lipase ~electivity~'~ and their applications in lipid biotransfor- mation have been published.793 A lipase from Candida rugosa has been immobilised on polyethylene glycol to yield a modi- fied lipase which is stable in water and favours reaction with oleic versus stearic Candida rugosa lipase immobilised by absorption on swollen Sephadex LH-20 hydrolyses olive oil efficiently in is~octane.~~~ The lipases of the fungi Candida rugosa and Aspergillus niger discriminate the 3-methyl branched 2-mercaptoethanol S-esters of methyloctanoic acid the lipase from Rhizomucor miehei exhibited preference for 205 (EPA) over 20:6 (DHA) fatty acids.814 Enzymatic trans- esterification of palm olein with nonspecific and 1,3-specific lipases has been studied where the effects of transesterification and hydrolysis of palm olein by the various lipases resulted in changes in the overall degree of saturation of the triacyl- glycerol components.815 Interesterification of polyunsaturated seed oils (peanut sunflower safflower soybean and linseed) with methyl oleate in hexane in the presence of Mucor miehei lipase furnished high oleic acid oils.816 Incorporation of 20:5 and 22:6 fatty acids into trilinolein was achieved by Lie Ken Jie et al.Fatty acids fatty acid analogues and their derivatives 177 interesterification of the ethyl esters of 20:5 and 22:6 with trilinolein and vegetable oils with Mucor miehei and Candida antarctica lipase~.*~~-~*~ Transesterification and alcoholysis reactions catalysed by Mucor miehei and Humicola lanuginosa lipases are 173-specific when the reactions are carried out in diethyl ether but not in hexane.821 A polyester of 10-hydroxydecanoic acid (a 52mer M,=9346 and M,= 12 065 polydispersity of 1.29) was prepared by incubation of the monomer in hexane with lipase from Candida cylindracea.822 The lipase isolated from Rhizopus delemar discriminates petroselinic acid 18 l(62) and y-linolenic acid against other positional Lesquerolic acid 14-hydroxy-20 1(1 1Z), and auricolic acid 14-hydroxy-20:2( 1 lZ,172) in the seed oil of Lesquerella fendleri were selectively released during the also used for the isolation of 22:6 from tuna fish Lipases in Carica papaya latex causes the specific hydrolysis of the sn-3 acyl group in triacyIglycerols.861 In esterihcation reactions carried out in hexane lipase from oilseed rape (Brassica napus) discriminates against polyunsaturated fatty acids containing a (42)- or (62)-double bond.862 Lipase from Vernonia galamen- sis seed showed a high degree of selectivity for trivernolin (triacylglycerol containing unsaturated epoxy fatty acids vernolic Lipase isolated from germinating rape seeds (Brassica napus) was used in enriching y-linolenic acid from evening primrose fatty acids by esterificati~n.~~~~“~ However the same lipase was unable to hydrolyse wax esters contained in jojoba oil and orange roughy (Hoplostethus atlanticus) hydrolysis of the oil catalysed by Rhizopus arrhizus lipa~e.*~~ The lipase of the fungus Geotrichum candidunz is noted for its strong preference for fatty acids that contain a (92)-double bond.825-828 It also discriminates strongly against fatty acids with more than 18 carbon atoms such as 22:l and 22:6 fatty acids during esterifi~ation.*~~-*~~ The y-linolenic acid present in borage oil free fatty acids was concentrated (to 95%) during the esterification reaction cata- lysed by the lipase of Geotrichuni candidum.833 Other lipases (Candida cylindracea and Lipozyme) were not as discriminat- ing as that from G.c~ndidum.~~~’~~ Lipase from Pseudomonas JEuorescens bound to a cyanuric chloride derivative of mono- methoxypolyethylene glycol becomes soluble in various hydro- phobic media (benzene toluene and chlorinated solvents) and exhibits high enzymic activities in these organic solvent^."^ A large-scale production of immobilised lipase of Pseudoinonas JEuorescens biotype I on macroporous weak-anion exchange resin has been reported.This lipase was used to liberate 20:5 and 22:6 fatty acids from sardine The commercial lipase of Pseudomonas cepacia (Amano) has been purified characterised and immobilised on Sepharose which remains stable in organic solvents and in supercritical carbon dioxide.839 Resolution of (R)-and (9-hydroxyalkanoic esters was achieved by selective hydrolysis of the racemic mixture of the acetoxy esters by the lipase of Pseudomonas sp.840 Ester synthesis with a crude lipase preparation from Pseudomonas fragi in hexane favoured primary over secondary alcohols while tertiary alcohols were not e~terified.’~’ Lipases from Pseudomonas sp.were found to be most effective in hydrolysing tallow and palm oil to give 700/0 (mass) of the monoacyIgIyceroI.s42 Solvent-free enzymatic glycerolysis was carried out on cod liver oil and oils from blubber of harp seal and minke whale with lipase AK from Pseudomonas sp. to give 42-53% of monoacylglycerols.843 A new method for the enzy- matic synthesis of monoacylglycerols by lipase-catalysed (from Pseudomonas cepaciu) transesterification reactions between fatty acid vinyl esters and glycerol or 1,2-0-isopropylidene-rac-glycerol with or without organic solvent has been devel- Enzymatic syntheses of specific mono- di-and tri-acylglycerols with various lipases have also been described.8458’3 Estolides were successfully formed from 14-hydroxy-20 I( 1 1Z) (lesquerolic acid) and oleic acid when catalysed by Pseudornonas sp.lipase. The product consisted of a mixture of monoestolide with two lesquerolic acyl groups and die~tolide.,’~ An extracellular lipase isolated from the fungus Pythium uftimum strain no. 144 showed preference for triacylglycerols containing fatty acids having the (2)-olefinic un~aturation.~’~ Lipase G from Penicillium sp. catalyses the esterification of 18:l(9Z) with glycerol to yield predominantly monooleoylglyc- er01.*’~3*~~ Lipases isolated from Staphylococcus aureus showed a high specificity for the acyl group in the sn-1 position (S-configuration) which was hydrolysed by a factor of ten faster than that in the sn-3 position of the triacylglycerol.”58 Lipase of Chromobacterium viscosurn discriminates fatty acids containing a A5 double bond in the alkyl chain thus allowing such positional isomeric fatty acids to be concentrated from meadowfoam (Lirnnanthes alba) seed This lipase was Two lipoxygenase isoenzymes (CL-I and CL-2) were puri- fied from chickpea (Pedroxillano sp.).The CL-2 isoenzyme produced only hydroperoxides from linoleic acid while CL- 1 simultaneously formed hydroperoxides and carbonyl com-pounds and exhibited high cooxidative activity with carotene and retinol substrates.867 An arachidonic acid 15-lipoxygenase enzyme in the gill tissues of teleost fishes has been identified.The enzyme is active toward polyunsaturated fatty acids present in the tissue producing hydroxylated metabolites from C,, C, and CZ2 fatty acids at carbons 13 15 and 17 respectively.868 The mycelia of the fungus Mortierella alpina produce 20:5(52,82,1 lZ,142,172) (13.5% of the total fatty acid content) when grown at low temperature (6-1 6 0C).869,870 Addition of oils which contain a-linolenic acid to the growth medium of Mortierella alpina enhanced the production of 20:5.’” On cultivation with sesame seed oil the fungus pro- duced dihomo-y-linolenic acid and 20:3( 82,112,142) in sig- nificant quantity (23% of fatty acid content).872 Linoleic acid was converted into y-linolenic acid by the fungus Mortierella ramanniana var.unguli~pora.~~~ Mortierella alpina IS-4 a mutant defective in 12-desaturase produced fatty acids of the 0-9 family i.e. 18:2(62,92) 20:2(82.112) and 20:3 (52,8Z,1 lZ).874 This mutant produced high levels of odd chain polyunsaturated fatty acids 19:4(52,8Z 1 1Z,142)”’ and converted a-linolenic acid to 20:5(52,82, 1 12,142,172) which accounted for ca. 20% of the total mycelial fatty The effects of aging Mortierella mycelium on the production of 20:4(52,8Z,112,142) and 20:5 have been The fungus Saprolegnia sp. and Thraustochytrium aureum produced 20:5 and 22:6 fatty acids respectively.8783879 Two microalgae Monodus subterraneus and Prophyridium cruentum have been found to produce 20:5 in about 20% and 44% of the total fatty acids respectively.880388’ Bacteria iso- lated from mackerel intestine were found to produce high levels of 20:5.’** ( 13S)-Hydroperoxy-9(2) 1 1 (a-octadecadienoic acid was obtained when linoleic acid was incubated with soybean li- poxygenase (LOX 1) in a biphasic octane-water ~ystem.~*”~*~ Linoleic acid (1 3S)-hydroperoxide was converted via an allene epoxy intermediates to (1 3R)-hydroxy- 12-0~0-(92)- octadecenoic acid and 9-hydroxy- 12-0xo-( 109-octadecenoic acid by the isomerase enzyme isolated from flax seeds.886 Incubation of linoleic acid with an enzyme isolated from the red alga Lithothamnion corallioides yielded (1 1 S)-hydroxy- 18:2(92,122) and 9-hydroxy-l8:2(10E 122) 13-hydroxy-18:2(92,11E) and 1l-ket0-18:2(92,122).~~~ Enzymatic oxi-dation of y-linolenic acid in a preparation of the red alga Lithothamnion coraflioides gave rise to conjugated tetraene fatty acids and bis-allylic hydroxy acids as the main prod- ucts.*** A hydration enzyme isolated from Flavobacterium sp.DS5 transformed oleic and linoleic acid to their corresponding 10-hydroxy and 10-keto fatty a~ids.~~~~*~” Oleic acid was also biotransformed in the presence of Staphylococcus sp. to 10-ketostearic acid8” and to 10-hydroxystearic acid with rest- ing cells of Nocardia chlester~licuni.~~~ A novel fatty acid 7,10-dihydroxy-18 1(8E) was obtained from oleic acid when incubated with bacterial strain PR3 (isolated from a water ~ample)’~~.’~~ Bioconversion of oleic acid with Bacillus 178 Natural Product Reports pumilus gave a mixture of 15- 16-and 17-hydroxy-9-octadecenoic acids.89s The isomerase enzyme from the fungus Saprolegnia parasitica converted (1 3S)-hydroperoxy-(9Z 11Z)octadecadienoic acid to (1 lR,12R)-epoxy-( 13s)-hy- droxy-(9Z)-octadecenoic acid and (9S 10R)-epoxy-( 1359- hydroxy-( 11E)-octadecenoic acid.(99-Hydroperoxy-( 1OE 12Z)-octadecadienoic acid was biotransformed to (lOR 1 1R)-epoxy-(9S)-hydroxy-( 12Z)-octadecenoic acid and (12R 13s)- epoxy-(9S)-hydroxy-( 10E)-octadecenoic acid by the same enzyme.896 A 12-lipoxygenase was isolated from the temperate red alga Gracilariopsis lemaneformis which produced 12-hydroxy-20:4 from 20:5.897 8,15-Dihydroxy-20:4 was formed by the sequential action of the 15- and 12-lipoxygenases when arachidonic acid was incubated with tissue homogenates from fish (rainbow trout and marine rockfish) gills.898 12- Hydroxystearic acid was converted to 5-hexyl-tetrahydro-furan-2-acetic acid (43%) 4-hydroxydecanoic acid (1 8%) and y-decanolactone (1 5%) by Bacillus lent~s.~~~ Two oxygenases (a cytochrome P-450 and a peroxygenase) associated with microsomes prepared from germinating seeds of Euphorhia lagascae were found to convert linoleic acid into cis-12,13-epoxy-9(Z)-octadecenoicacid (vernolic acid).”’ The epoxy derivatives of 20:5 formed by incubation of the substrate with cytochrome P-450 epoxygenase were characterised by mass spectral analysis of the diol product^.^'^ A single cyto- chrome P-450 isoenzyme from Vicia sativa microsomes oxi- dised the omega carbon of 7- 8- 9- and 10-dodecenoic acids to give corresponding hydroxy C, analogues and also epoxidised 1 1-dodecenoic acid.902 9(Z)- and 9(E)-Dodecenoic acids were epoxidised by a microsome preparation from Jerusalem arti- choke tuber to form cis-and trans-epoxydodecanoic acids respectively.The absolute configuration of the epoxy products was determined.903 Bioreduction of keto fatty acids or esters with bakers’ yeast (Saccharomyces cerevisiae) gave the corresponding hydroxy fatty acids or esters of high enantiomeric purity in good chemi- cal yields.904 The presence of sulfur atoms at the A6 and A13 positions of the alkyl chain of stearic acid did not prevent the desaturation of the chain at the C-9 position of the dithia analogue when incubated in yeast (Saccharomyces cere-visiae).”” Enzymatic hydrolysis of synthetic isomers of methyl 5- 9- and 12-thiastearate with Candida cylindracea or porcine pancreatic lipase gives high yields of the corresponding thia fatty acids.Hydrolysis was very slow in the case of methyl 3- and 4-thiastearates (1 5-25% yield) while methyl 2-thiastearate was not hydr~lysed.~’~ Reaction of 2-mercaptoethanol with ethyl alkanoates in the presence of porcine pancreatic lipase yielded exclusively the O-acyl ester while other lipases (from Candida cylindracea and Pseudomonas sp.) were found to be less effe~tive.~’~ Wax esters were prepared via a solvent-free esterification process involving fatty acids with stearoyl alcohol in the presence of Lipozyme (immobilised lipase of Mucor miehei).”’ Two immobilised lipases from Candida antartica (SP382) and C.cylindracea were shown to catalyse the interes- terification of acetylated glucose fatty acid esters by reaction of glucose pentaacetate and vegetable Methods for pre- paring non-polar glyceryl ether lipids highly enriched with 20:5 or 22:6 by immobilised lipases have been described.”’ The authors wish to thank Ms Kalluri Prabhavathi Mr M. 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Lie Ken Jie et a]. Fatty acids fatty acid analogues and their derivatives

ISSN:0265-0568    

DOI:10.1039/NP9971400163

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

Diterpenoid and steroidal alkaloids Atta-ur-Rahman and M. Iqbal Choudhary H. E. J. Research Institute of Chemistry (International Centre for Chemical Sciences) University of Karachi Karachi- 752 70 Pakistan Covering Mid-1994 to the beginning of 1996 Previous review 1995 12 361 I 1.1 1.2 1.2.1 1.2.2 1.2.3. 1.2.4 1.2.5 1.2.6 1.2.7 1.2.8 1.2.9 1.2.10 1.2.11 1.2.12 1.2.13 1.2.14 1.2.15 1.3 1.4 1.4.1 1.5 1.5.1 1.6 2 2.1 2.2 2.2.1 2.2.2 2.2.3 2.2.4 2.2.5 2.2.6 2.2.7 2.2.8 2.2.9 2.2.10 2.2.11 2.2.12 2.2.13 2.2.14 2.2.15 2.2.16 2.3 2.4 2.4.I 2.4.2 Diterpenoid alkaloids Introduction Phytochemical studies Alkaloids of Aconitum carmichaeli Debx.Alkaloids of Aconitum hemslevanum Pritz var. peng-shinese W. J. Zhang Alkaloids of Aconitum nagarum var. heterotrichum Fletcher et Lauener Alkaloids of Aconitum vilmorinianum Kom. Alkaloids of aphids Brachycaudus aconiti Mordv. and B. napelli Schrk. Alkaloids of Consolida ambigua L. P. W. Ball and V. H. Heywood Alkaloid of Delphinium albzjlorum DC. Alkaloids of Delphinium andersonii Gray Alkaloids of Delphinium cardiopetalum DC. and Delphinium cossonianum Batt. Alkaloids of Delphinium elatum L. Alkaloids of Delphinium munzianum P. H. Davis & Kit Tan Alkaloids of Delphinium nuttallianum Pritz. Alkaloids of Delphinium peregrinum var. elongutum Alkaloids of Delphinium potaninii W. T. Wang Alkaloids of Spiraea japonica var.incisa Yu Spectral studies Other studies N-Oxides of some norditerpenoid alkaloids Synthetic studies Synthesis of the A/E/F tricyclic section of methyl- 1ycaconi tine Pharmacology Steroidal a1 kaloids Introduction Phytochemical studies Alkaloids of Astragalus polycanthus Royle Alkaloids of Cephalodiscus gilchristi Alkaloids of Fritillaria maximowiczii Freyn. Alkaloids of Fritillaria siechuanica S. C. Che Alkaloids of Fritillaria taipaiensis L. Alkaloids of Fritillaria yuminensis L. Alkaloids of Holarrhena pubescens Buch. Ham (H. untidvsentrica) Alkaloids of Lycopersicon pimpinellifolium (Jusl.) Mill. Alkaloids of Ritterella tokioka Alkaloids of Sarcococca vagans A1 k alo id of Solan um abut ilo ides Alkaloids of Solanum robustum Wendl Alkaloid of Solanum triste Jacq.Alkaloids of Veratrum oblongum Loes. f. Alkaloids of Veratrum viride Ait. Alkaloids of Vitex canescens Kurz Spectral studies Other studies a-Tomatine contents of fruits of Lycopersicon esculen- tum Production of steroidal alkaloids by hairy roots of Solanum aviculare and effect of gibberellic acid 2.4.3 Oxygen transfer and culture characteristics of self-immobilised Solanum aviculare aggregates 2.5 Synthetic studies 2.5.1 Synthetic studies towards batrachotoxin 2.6 Pharmacology 3 References 1 Diterpenoid alkaloids 1.1 Introduction This review covers the literature published or abstracted from the middle of 1994 to the beginning of 1996 on diterpenoid (C,,) and norditerpenoid (C19) alkaloids.Plants of genera Aconitum and Delphinium remained targets of vigorous phyto- chemical investigations and a large number of new C, and C, diterpenoid alkaloids were isolated from these plants. Feng- Peng Wang has reviewed the advances in the field of diterpe- noid alkaloids in China between 1986 and 1992. The review includes both isolation studies and synthetic investigations.’ An interesting article highlighting various classes of diterpe- noid alkaloids their botanical sources medicinal importance and toxicology has been published recently which contain 148 important references on this topic2 1.2 Phytochemical studies 1.2.1 Alkaloids of A conitum cavmichaeli Debx. Phytochemical investigations on the ethanolic extracts of the lateral roots of A.carmichaeli Debx. (Ranunculaceae) collected from Sichuan Province China have resulted in the isolation of a new norditerpenoid alkaloid aldohypaconitine 1 along with OH 1 r ‘0H Me0 1 aldohypaconitine seven known alkaloids hypaconitine mesaconitine aconitine neoline fuziline songorine and s~ngoramine.~ The ‘H NMR spectrum of alkaloid 1 did not show any signal for NCH or NCH,CH3 protons while the IR spectrum exhibited absorp- tion of a formyl group. Compound 1 did not react with Dragendorff s reagent indicating the amidic nature of the nitrogen. Structure 1 was confirmed by its synthesis from h ypaconi tine. 1.2.2 Alkaloids of Aconitum hemslevanum Pritz var. pengshinese W.J. Zhang The fresh roots of A. hemslevanum of Chinese origin have yielded a new C, alkaloid 1-epicrassicaudine 2 and six known norditerpenoid alkaloids indaconitine chasmaconitine ludaconitine chasmanine talatisamine and fre~hitine.~ The A tta-ur- Rahrnan and Choudhary Diterpenoid and steroidal alkaloids Me0 2 1-epicrassicaudine structures were elucidated on the basis of spectroscopic tech- niques (FD and EI MS ‘H NMR I3C NMR). Compound 2 was found to be the 1-epimer of crassicaudine by comparison of the ‘H NMR and 13C NMR data of crassicaudine. Treat- ment of 2 with 10% solution of NaOH in methanol afforded 14-0-debenzoyl-8-0-deacetyl crassicaudine. The change of configuration of the methoxy group at C-1 affects the 13C NMR chemical shift values of C-1 C-2 C-3 and C-5.4 1.2.3 Alkaloids of Aconitum nagarum var.heterotrichum Fletcher et Lauener Chinese workers have isolated two known diterpenoid alka- loids denudatine and songorine and three known norditerpe- noid alkaloids aconitine deoxyaconitine and yunaconitine from the roots of A. nagarum. Their structures were identified by spectral analysis and chemical method^.^ 1.2.4 Alkaloids of Aconitum vilmorinianum Kom. Continuing their phytochemical investigations on the roots of the Chinese plant A. vilmorinianum Ding et al. have isolated a novel diterpenoid alkaloid vilmoridine 3 along with the five 0 \. ‘H 3 vilmoridine known bases acoforine columbidine yunaconitine sachaconi- tine and 14-0-acetylsachaconitine.Compound 3 has a new car- bon skeleton with ring B cleaved between C-6 and C-7. Structure 3 was determined by spectroscopic studies (IR HREI MS ‘H NMR I3C NMR DEPT) and comparison with the known alkaloid vilmorrianone which has C-6 C-7 carbonyl functions.6 1.2.5 Alkaloids of aphids Brachycaudus aconiti Mordv. and B. napelli Schrk. A novel C,,-norditerpenoid alkaloid brachyaconitine 4 has been isolated from the pentane extract of aphids B. aconiti and B. napelli (Aphididae) feeding and living on Aconitum napellus “OH HO 5 432 1 OCCH2C H=CHCH2Me E Me0 4 brachyaconitine L. A number of known diterpenoid such as lipoaconitine 14-O-acetylneoline isotalatizidine virescenine senbusine A 12-epi-napelline 12-epi-dehydronapelline songorine and 14-0- acetylsenbusine have also been isolated from both aphids.Compound 4 has a rancid odour like the aphids. Structurally it possesses an unusual C-8 trans-hex-3-enoyl side chain which was determined by spectroscopic data (FAB MS COSY NOESY ROESY HETCOR COLOC DEPT) and by semi- synthesis from aconitine. Spectral data of previously known 8-0-acetyl- 1 Sa-hydroxyneoline and complete ’H NMR assign- ments for the known alkaloid 14-0-acetylneoline have also been reported. The origin of the alkaloid is still unclear i.e. whether the aphids take up brachyaconitine 4 from the plant or whether they produce it from aconitine taken up earlier by biotrans- formation. However the authors suspect it to be the result of biotransformation since it contains an unusual molecular structure not common in plants.The alkaloid patterns in B. aconiti and B. napelli were found to be very similar and both species of aphids contained aconitine as the major base.7 1.2.6 Alkaloids of Consolida ambigua L. P. W. Ball and V. H. Heywood (syn. Delphinium ajacis L.) Pelletier et al. continuing their work on diterpenoid alkaloid bearing plants have now isolated a new diterpenoid alkaloid 13-0-acetylvakhmatine 5 and a known alkaloid vakhmatine ACO HO.. 5 13-acetylvakhmatine from the ethanolic extracts of the seeds of C. ambigua (Ranun- culaceae) by using high performance centrifugal partition chromatography (HPCPC). The structure of the new alkaloid was established by spectroscopic studies (HREI MS IR COSY HETCOR DEPT SINEPT) and chemical correlation.Alkaline hydrolysis of 5 afforded vakhmatine identical with an authentic sample.8 1.2.7 Alkaloid of Delphinium albijforum DC. The first ever phytochemical investigation on aerial parts of D. albiflorum of Turkish origin has resulted in the isolation of a new alkaloid deacetylheterophylloidine 6 along with the 0 \?3 6 deacetylheterophylloidine known bases hetidine and lycoctonine. The structure of 6 was established on the basis of detailed spectroscopic data (HREI MS IR ‘H and 13CNMR) and chemical correlation by the alkaline hydrolysis of heterophylloidine.’ ‘H and I3C NMR 192 Natural Product Reports chemical shift assignments for various carbons and protons in hetidine have been revised on the basis of SINEPT and DEPT NM R experiments.1.2.8 Alkaloids of Delphinium andersonii Gray D. andersonii is a shrub grown in the 'Wild Cat Hills' (1000 m) of Utah USA. Cattle deaths due to grazing on this plant have been observed. Working on the aerial parts of the plant Pelletier et al. isolated a minor hetisine-type diterpenoid alkaloid andersobine 7 through droplet counter current chromatography (DCCC). The structure of 7 was determined p++o*c 7 andersobine by detailed NMR spectroscopic studies (HREI MS COSY HETCOR SINEPT NOESY etc.) while stereochemical assignments were made with the help of 1D NOE." 1.2.9 Alkaloids of Delphinium cardiopetalum DC. and Delphinium cossonianum Batt. A slightly functionalised hetisine-type diterpenoid alkaloid cossonidine 8 was isolated from D.cardiupetalum (a plant OH 8 cossonidine widely distributed in Spain and the south of France) and D.cossonianum (taxon endemic to Morocco). The structure was inferred from its 'H and 13C NMR spectra (ROESY COSY HMQC HMBC DEPT) and chemical derivatisation. 1.2.10 Alkaloids of Delphinium elatum L. D. elatum var. 'black night' is a rich source of norditerpenoid and diterpenoid alkaloids. Continuing their investigations on various parts of D. elatum Pelletier et al. have isolated two new lycoctonine-type norditerpenoid alkaloids blacknine 9 and blacknidine 10 along with six known alkaloids I4-deacetylnudicauline delectinine delelatine delpheline methyllycaconitine and ajaconine from the whole plants.The EtN 'c structures of the new bases were determined from spectro- scopic data (IR mass 'H NMR 13C NMR DEPT COSY HETCOR and selective INEPT).I2 1.2.11 Alkaloids of Delphinium munzianum P. H. Davis & Kit Tan Collaborative work of Spanish and Turkish scientists on the aerial parts of D. munzianum has resulted in the isolation of five new norditerpenoid alkaloids. The endemic perennial plant was collected from north-east Anatolia. These C, alkaloids include the new aconitine-type bases 14- 0-benzoylperegrine 11 14-0-acetylperegrine 12 munzinanone 13 munzianine 14 ' R1 I M eO 11 14-0-benzoylperegrine R' = a-H P-OAc; R2 = Me; R3 = Bz; R4 = H 12 14-0-acetylperegrine R1 = a-H P-OAc; R2 = Me; R3 = Ac; R4 = H 13 munzinanone R1 = 0;R2 = Me; R3 = R4 = H 14 munzianine R1 = a-H p-OH; R2 = R3 = R4 = H 15 10-hydroxyperegrine R1 = a-H P-OAc; R2 = Me; R3 = H; R4 = OH and 10-hydroxyperegrine 15 along with the known alkaloids peregrine and 14-0-methylperegrine.The structures of the new alkaloids were determined through extensive spectro- scopic studies (IR MS 'H NMR I3C NMR HMBC HMQC NOESY) chemical derivatisation and partial syn- thesis. The authors proposed that the norditerpenoid alkaloids isolated from D.munzianum may be biogenetically derived from munzianine 14. D. munzianum afforded a number of aconitine-type norditerpenoid alkaloids with an oxygen func- tion at C-68 but did not afford lycoctonine-type alkaloids as found in other Delphinium species.1.2.12 Alkaloids of Delphinium nuttallianum Pritz. In their phytochemical investigations on the diterpenoid alkaloids of D. nuttallianum (cow-poisoning plant of the interior rangelands of British Columbia Canada) Benn et al. have isolated seven new and seven known diterpenoid alka- loids as minor constituents. The new alkaloids include four norditerpenoid alkaloids karasamine-8-U-acetate 16 128-hydroxykarasamine 17 128-hydroxykarasamine-8-0-acetate 18 and 1-epi-128-hydroxykarasamine 19 and three diterpenoid alkaloids delnuttaline 20 delnuttidine 21 and delnuttine 22. The seven known bases were browniine browniine 14-O-acetate condelphine 14-dehydrobrowniine 14-dehydrodelcosine leroyine 14-0-acetate and hetisine 1 1-0-acetate. l4 The structure of a norditerpenoid alkaloid nuttallianine previously reported by the authors from the same plant was revised to 23.The revision relates to the stereochemistry of the C-6 hydroxy group. A consequence of this revision is that all the norditerpenoid alkaloids reported from D. nuttallianum by the authors having hydroxylation or methoxylation at C-6 must have the 6P-configuration for this substituent. The struc- tures of all these bases were elucidated through extensive spectroscopic techniques (HREI MS COSY COSY DQF-LR NOESY HMQC COLOC DEPT et~.).'~,'~ The structure of delnuttaline 20 [(68 98 138)-13-acetoxy-6,9- dihydrohetisan-2-oneI was also confirmed by single-crystal 9 blacknine 10 blacknidine X-ray diffraction studies. l6 A tta-ur- Rahman and Choudhary Diterpenoid and steroidal alkaloids 20 delnuttaline R1= OH; R2 = OAc 22 delnuttine 21 delnuttidine R' = H; R2 = OH 1.2.13 Alkaloids of Delphinium pevegrinum var.elongaturn De la Fuente and Ruiz-Mesia continuing their phytochemical studies on the epigeal parts of D. peregrinum of Moroccan origin have isolated three new aconitine-type norditerpenoid alkaloids dehydrobicoloridine 24 bicoloridine alcohol 25 and ,OAc 24 dehydrobicoloridine 25 bicoloridine alcohol re 26 peregridinine peregridinine 26 together with nine known alkaloids 14-0-acetylperegrine peregrine alcohol peregrine bicoloridine nudicaulidine dihydrogadesine hetisinone hetisine and atisinium ch10ride.l~ The structures of the new alkaloids were determined by extensive NMR spectroscopy (COSY I3CNMR DEPT HMQC) and partial synthesis.The struc- ture of dehydrobicoloridine 24 was confirmed by partial syn- thesis from bicoloridine. The NMR data of aconitine-type compound 26 lack an N-ethyl group. The methine carbon resonance at 6 168.5 (HMQC 6 7.20 s) was attributed to the existence of a C(19)=N- azomethine group in 26. The struc- ture of peregridinine 26 was further confirmed by synthesis from the known alkaloid peregrine. The known alkaloids 14- 0-acetylperegrine peregrine alco- hol peregrine bicoloridine nudicaulidine dihydrogadesine hetisinone hetisine and atisinium chloride were identified by comparison with authentic samples (TLC mp IR MS 'H and 13CNMR). The authors concluded that D.peregrinum produces aconitine-type norditerpenoid alkaloids with a P-oxygen function at C-6 together with lycoctonine-type alkaloids.1.2.14 Alkaloids of Delphinium potaninii W. T. Wang Continuing phytochemical investigations on the roots of D. potaninii used in Chinese traditional medicine for the treatment of rheumatism and neuralgia have resulted in the isolation of three new lycoctonine-type diterpenoid alkaloids potanine 27 potanidine A 28 and potanidine B 29.19Their structures were 27 potanine ,OMe C=O 0'"' 28 potanidine A R1= OMe; R2= OH; R3 = NHCOCH(Me)CH2COO(CH2)3Me 29 potanidineB R1 =OH; R2=OAc; R3 = -0 established on the basis of spectral data (EI and FD MS 'H and 13C NMR) and chemical methods. Compound 27 is the second reported lycoctonine-type C,,-diterpenoid alkaloid having a C-8 methoxy group.The known alkaloids methyllycactonine delsemines A and B delvaines A and B and takaosamine have also been isolated from this plant." 1.2.15 Alkaloids of Spiraea japonica var. incisa Yu Hao et al. continuing their work on the phytochemistry of the roots of 5'. japonica (Rosaceae) of Chinese origin have isolated three more new atisine-type diterpenoid alkaloids spiramines P 30 Q 31 and R 32 along with two known alkaloids spiramines A and B. The structures of these compounds were based on spectroscopic (EI MS 'H and 13CNMR NOESY) and chemical evidence. Mass spectrometry H NMR (NOESY) and 13CNMR of 30 (R)and 31 (S) indicated that they are a pair of epimers at C-16.2" 1.3 Spectral studies The structure and relative configuration of the atisine-type C, diterpenoid alkaloid delnuttaline 20 isolated from D.nuttalli-anum Pritz. was also determined by X-ray diffraction methods and found to be 6,9-dihydroxy-2-oxohetisan-13-yl acetate. The crystal structure analysis showed the presence of two 194 Natural Product Reports 30 spiramine P R1 = Me; R2 = OH 32 spiramine R 31 spiramine Q R1 = OH; R2 = Me independent molecules in the asymmetric unit. The structure is stabilised through a network of hydrogen bonds involving nitrogen and hydroxy groups with 0-0 and 0.-N separations in the range 2.688(7)-2.903(8) A.16 1.4 Other studies 1.4.1 N-Oxides of some norditerpenoid alkaloids Preparation and complete NMR assignments of eight new N-oxides of eight norditerpenoid alkaloids aconitine 33 ajacine 34 delphinine 35 delphisine 36 deltaline 37 heterati-sine 38 lappaconitine 39 and N-deacetyllappaconitine 40 have been reported.These N-oxides were prepared by carrying out the reactions at room temperature with meta-chloroperbenzoic acid (mCPBA) and fairly good yields were obtained.2' The 'H and I3C NMR chemical shifts of the N-oxides and their parent alkaloids were compared and remarkable differ- ences in their chemical shifts were observed due to the presence of the N-oxide group in these substances. All the carbons (i.e. C-17 C-19 and C-20) directly attached to the N-atom experi- enced downfield chemical shifts of ca. 12 to 20ppm. The structures of these N-oxides were determined on the basis of their spectroscopic data ('H I3C NMR DEPT COSY HETCOR SINEPT).21 1.5 Synthetic studies 1.5.1 Synthesis of the AIEIF tricyclic section of meth yll ycaconitine The major C, norditerpenoid alkaloid methyllycaconitine 41 has been isolated from many plants of the genera Delphinium and Aconitum.The compound has shown a variety of biologi- cal activities. It exhibits high toxicity to mammals acting on the neuromuscular junction inhibiting the neurotransmission pro- cess and inducing paralysis. It is regarded as the most potent non-protein antagonist of the nicotinic acetylcholine receptor. Methyllycaconitine 41 also exhibited powerful insecticidal activity and is an ideal candidate for structure-activity rela- tionship studies.However this requires larger quantities of the alkaloid which can be obtained only through synthesis. Baillie et al. ,22 in the first phase of their attempt to synthesise methyllycaconitine 41 synthesised a tricyclic amine 42 with Ring Ad 41 methyllycaconitine 42 five stereogenic centres representing the rings A E and F of the bNHAC alkaloid. Schemes 1 and 2 present the proposed retrosynthetic 33 aconitine N-oxide R1 = Et; R2 = OH 35 delphinine N-oxide R1 = Me; R2 = H 34 ajacine N-oxide analysis and the synthetic sequence adopted respectively. The retrosynthetic strategy (Scheme 1) involved an initial disconnection of the C-N bond in 43 to the cis-fused 6,5- bicarbocyclic alkamine 44in which the N and ethereal 0 were viewed as relating to the isoxazolidine 45.Compound 45 was viewed as the product of a 1,3-dipolar cycloaddition reaction from the nitrone 46 which can be obtained involving a Diels-Alder reaction.22 36 delphisine N-oxide 37 deltaline N-oxide A H I43 44 0- ONHR 38 heteratisine N-oxide 39 lappaconitine N-oxide R = Ac 40 N-deacetyllappaconitine N-oxide \ " 46 45 R=Me Scheme 1 Retrosynthesis of 43 A tta-ur- Rahman and Choudhary Diterpenoid and steroidal alkaloids 195 toxicologically assessed these chemical constituents through a mouse bioassay.23 These data define the potential mammalian toxicity of a wide range of norditerpenoid alkaloids. The study established that lycoctonine-type norditerpenoid alkaloids containing a tertiary nitrogen (N-ethyl bicyclo substituted tertiary alkaloid nitrogen atom) and/or a C-18 anthranilic acid ester substituent are generally toxic.Two other structural features i. e. functionality at the anthranilic acid amine T OH 77% &HO ,,/ 92% fR nitrogen and at C-14 also enhance the toxicity. For vi example MSAL-type alkaloids [N-(methylsuccinimido) . anthanoyllycoctonine norditerpenoid alkaloids] such as nudi- 0..- ,," /' cauline 14-deacetylnudicauline methyllycoconitine elatine /\ CH(OMe)2 CH(OMe)2 etc. were found to be highly toxic (LD, 2.7-10 mg g- I). A; R = C02Me (80%) C; R = H (83%) .G B; R = CHO (91%) D; R = Me (63%) vii J/ E; R = H (94%) G; R = H (80%) F; R=Me(92%) H; R = Me (82%) J I; R = H (47%) R I J; R = Me (67%) 42 Scheme 2 Synthesis of the tricyclic amine 42 Reagents and conditions i MeC(OEt), EtCO,H 142 "C 3.5 h; ii KOH MeOH; iii 4 equiv.CH,=C(Me)CHO H,O NaHCO, room temp. 24 h; iv HC(OMe), MeOH PTSA; v DIBAL toluene -80 "C; vi MeNHOH HCl or EtNHOH TFA Et,N benzene reflux 3.5 h; vii NiCl, 6H,O NaBH, MeOH; viii (a) 5 mol dm- HCl; (b) buffer pH 5.5; (c) NaCNBH,; ix NaH THF Me1 Scheme 2 presents the synthesis of the tricyclic amine 42 which contains the rings A E and F of methyllycaconitine 41 with five stereogenic centres. The synthesis was achieved in nine steps from the commercially available penta- 1,4-dien-3-01 with a 16% overall yield.22 1.6 Pharmacology Completing their phytochemical investigations on the diterpe- noid alkaloids of D.nuttallianum Benn et al. have isolated 45 diterpenoid alkaloids which include 36 aconitine or lycoctonine-type norditerpenoid and nine hetisinoid diterpe- noid alkaloids. The authors concluded that the predominant alkaloid methyllycaconitine (MLA) (12% of total bases) has the highest mammalian toxicity of all the alkaloids isolated from the plant; it may therefore account for the poisonous properties of the plant. MLA was also found to be a potent competitor of a-bungarotoxin (aBGT) binding to nicotinic acetylcholine receptors (nACHR) in the nanomolar range in insects. Toxicity studies on these alkaloids indicated that the tertiary N-atom and anthranilic acid substituent are two important structural features to impart toxicity.l4 Poisonous larkspurs (Delphinium) species are the major cause of cattle poisoning in the grazing lands of western United States. Phytochemical investigations on various types of larkspur have resulted in the isolation of a large number of norditerpenoid and diterpenoid alkaloids. Manners et al. have 196 Natural Product Reports Many of these alkaloids have been identified from larkspur species (D. brownii D. bicolor D. nuttallianum D. nudicaule D. andersonii D. glaucescens D. occidentale D. barbeyi) which are indigenous to the western United States and Canada and which are commonly associated with cattle poisoning.23 2 Steroidal alkaloids 2.1 Introduction This review covers the literature published or abstracted during the period mid-1994 to the beginning of 1996 on steroidal alkaloids.The plants of Solanum Veratrum Fritil- laria and family Buxaceae (Bums Sarcococca) continue to be rich sources of steroidal and triterpenoidal alkaloids. The majority of phytochemical investigations were conducted on Fritillaria and Veratrum species. Interestingly a large number of structurally complex and biologically active steroidal alkaloids have also been isolated from marine invertebrates (Ritterella tokioka and Cephalodiscus gilchristi). Vieira and de Carvaiho have published a comprehensive review covering the distribution of steroidal alkaloids in vari- ous species of Solanurn plants. The review includes several tables containing information on the distribution of different glycoalkaloids and non-glycoalkaloids in various plants.24 Gaffield and Keeler have published a comprehensive review on the structure-activity relations and implications of plant steroidal alkaloids as teratogens.The review contains many useful references on the 2.2 Phytochemical studies 2.2.1 Alkaloids of Astragalus polycanthus Royle The steroidal alkaloid zygadenine has been isolated for the first time as its 3-@P-~-glucoside 47 from A. polycanthus (Leguminoseae). The structure of the compound was deter- mined on the basis of spectral data (MS 'HNMR GASPE) and chemical transformations. The enzymatic hydrolysis of 47 with p-glucosidase yielded glucose identified by paper and thin-layer chromatography thereby supporting a P-linkage of the glucose moiety.26 2.2.2 Alkaloids of Cephalodiscus gilchristi Continuing their work on cytotoxic metabolites of the tiny tube-inhibiting marine worm Cephalodiscus gilchristi (Phylum /N !i r-MOH OH 47 zygadenine glucoside 27 'OH 26 L 27' 48 cephalostatin 12 R = H 49 cephalostatin 13 R = OH H OH 50 cephalostatin 14 R = H 51 cephalostatin 15 R = Me Hemichordata class Pterobranchia) which occurs in the Western Indian Ocean and was collected from off the coast of Southeast Africa Pettit et ul.have isolated four new cytotoxic disteroidal alkaloids cephalostatins 12 48 13 49,27 14 50 and 15 51.28Cephalostatin 12 48 is the first example of a symmetri- cal disteroidal alkaloid while cephalostatin 13 49 is the unusual C-1' hydroxylated product derived from it.The UV spectrum of cephalostatin 12 48 showed absorptions at 288 and 308 nm suggesting the presence of a pyrazine ring. The FAB MS indicated the formula C,,H,,N,O, (M' + 1) but the I3C NMR spectrum (APT) showed only 27 signals suggest- ing a twofold symmetrical axis. The I3C NMR spectrum of cephalostatin 13 49 showed one less methylene carbon and an additional methine signal at 6 74.4 indicating that cephalostatin 13 is a hydroxy derivative of cephalostatin 12 48. Cephalostat-ins 13 49 and 14 50 were found to contain an epoxide function- ality between C-14' and C-15' as evident from the chemical shift of the C-15' methine proton which resonated at 6 3.75 and 6 57.5. The structures of these complex metabolites were determined with the help of detailed spectroscopic experiments such as HRFAB MS COSY TOCSY HMQC HMBC NOESY ROESY etc.The cephalostatins significantly inhibited the growth of animal and human cancer cell line^.^'-,^ 2.2.3 Alkaloids of Fvitillavia rnaxirnawiczii Freyn. Qian and Nohara working on the phytochemistry of the bulbs of F. muximowiczii (Liliaceae) (Rinyon-Baimo) of Chinese origin isolated three new steroidal alkaloids 23-isokuroyurinidine 52 15,16-seco-22aH,25PH-solanida-5 14-dien-3P-01 0-p-D-glucopyransoyl-( 1-+4)-P-~-xylopyranoside 53 and hapepunine 3-O-~-cellobioside 54 along with a known jerveratrum-type alkaloid k~royurinidine.~' The structures were largely determined by exploiting spectroscopic techniques such as FAB MS COSY NOE 13CNMR etc.Cleaved sugar configurations were determined by GC analysis. Enzymatic hydrolyses were carried out to analyse sugar moieties. Unlike other Fritilluriu species E rnaxirnowiczii does not contain ceveratrum-type alkaloids. Pharmacological studies on the ethanolic extract of the plant have indicated that it can be used to treat coughs. Atta-ur-Rahrnan and Choudhary Diterpenoid and steroidal alkaloids OH 52 23-isokuroyurinidine c@=*- \ @! OH HO OH 53 15,16-seco-Z2aH 25PH-soIanida-5,14-dien-3P-ol OP-D-glUCOpyranOSyl-( 1 +4)-P-D-xylopyranoside OH OH 54 hapepunine 3-OP-cellobioside 197 2.2.4 Alkaloids of Fritillaria siechuanica S. C. Che Me A new steroidal alkaloid siechuantine 55 was isolated from the bulbs of Fritillaria siechuanica of Chinese origin.The structure H'bO H of the new compound was determined by spectroscopic technique^.^' 9 OQH H HO H 55 siechuantine 0 56 taipaienine 2.2.5 Alkaloids of Fritillaria taipaiensis L. Phytochemical investigation on the bulbs of Fritillaria taipai- ensis collected from the mountains of Liupanshan Ningxia province China has resulted in the isolation of a new 12-nor- 17a-homo-steroidal alkaloid taipaienine 56 along with five known alkaloids chuanbeinone imperialine verticinone per- missine and isoverticine. Their structures were elucidated by IR MS and various ID- and 2D-NMR spectral analyses and by comparison of the physical data with those of authentic samples. Taipaienine 56 has an unusual structure with a hydroxy substituent at C-25 of the (22R)-trans-quinolizidine moiety and a nitrogen lone pair with an a-orientation (trans-ring junction between rings E and F).32 2.2.6 Alkaloids of Fritillaria yuminensis L.Zhang et al. continuing their work on the bulbs of Fritillaria yuminensis have isolated six more alkaloids. The new alkaloids yubeinine (5a,14a-cevanine-3a-hydroxy-6-one) and yubeiside (5a,14a-cevanine-3-one-6~-O-~-glucoside) along with the known alkaloids imperialine delavinone tortifolisine and adenosine were identified by means of spectral analysis and chemical reactions.33 2.2.7 Alkaloids of Holarrhena pubescens Buch. Ham (H. antidysentrica) Chemical investigations of the bark of Holarrhena pubescens (Apocynaceae) have resulted in the isolation of two new steroidal alkaloids of the conanine series holamide 57 and pubescinine 58 and a known alkaloid norconessine.Their structures were established through spectroscopic studies (IR mass 'HNMR COSY 13CNMR DEPT HMQC). The structures of the new compounds were determined to be N-(N'-methylacetamido)-3-oxo-l,4,-conadiene for holamide 57 and 1 1 a-acetoxy-N-dernethyl-3-oxo-l,4,17(20)-conatriene for pubescinine 58. Both compounds showed hypotensive activity in rats at a dose of 3 mg kg- 198 Natural Product Reports 57 holamide 58 pubescinine 2.2.8 Alkaloids of Lycopersicon pimpinelliflium (Jusl.) Mill. Ripperger and Porzel have reported the isolation and structure elucidation of two new steroidal alkaloids pimpifolidine 59 and 22-isopimpifolidine 60 from the roots of wild tomato H 59 pimpifolidine fi 0 'OH Ho H 60 224sopimpifolidine Lycopersicon pimpinellifolium (Solanaceae) grown in the fields in Halle (Saale) Germany.Two known bases tomati- dine and soladulcidine have also been isolated from this plant. The structures of the new compounds were elucidated as 22,26-epimino-16P,23-epoxy-5a,22aH,25aH-cholestane-3P 23a-diol for 59 and 22,26-epimino- 16P,23-epoxy-5a,22pH 25aH-cholestane-3P,23a-diol for 60 with the help of detailed spectroscopic studies (COSY APT NOED etc.). The stereo- chemistry at various asymmetric centres and conformations of the rings were determined by NOE difference measurements and coupling constants between coupled protons.35 2.2.9 Alkaloids of Ritterella tokioka Fukuzaura et al.continuing their search for cytotoxic sub- stances from Japanese marine invertebrates have isolated thirteen new minor dimeric steroidal alkaloids ritterazines A-M (61-73) from the lipophilic extracts of the tunicate Ritterella tokiok~~~~~ by chromatography on octadecylsilane (ODS) and Sephadex LH-20. The animals were collected from off the coast of the Izu Peninsula. The dimeric steroidal alkaloids ritterezines exhibited remarkable cytotoxicity. They are closely related in their structures to cephalostatins which were isolated from an Indian Ocean hemichordate Cephaludiscus gilchristi. Occur-rence of these compounds in different phyla may indicate their microbial origin.The gross structures of these rather complex looking molecules were determined by interpretation of spec- troscopic data such as HRFAB MS COSY DQF-COSY i+ H HO HO 62 ritterezine B I..-HO 63 ritterezine C 64 ritterezine D R = H; 22s 65 ritterezine E R = Me; 22s .OH )'I H. I ' H (OF 1O' HO-HO-'F " 66 ritterezine F R1 = OH; R2 = R3 = H; 22s 67 ritterezineG R1 =OH; R2=H; AI4; 22s 68 ritterezineH R1,R2=0; R3= H; 22R 69 ritterezine I R1,R2= 0; R3 = OH; 22s HMQC HMBC NOESY ROESY DEPT etc. The relative stereochemistry of each steroid unit was deduced by 'H-'H coupling constants and NOESY data. 2.2.10 Alkaloids of Sarcococca vagans Three new steroidal alkaloids vaganins A-C (74-76) along with the three known bases have been isolated from Sarco-coccu vagans (Buxaceae) collected in Xishuangbanna China.Their structures were determined through chemical and spectroscopic techniq~es.~~ 2.2.1 1 Alkaloid of Solanum abutiloides Japanese investigators have recently isolated a 26-aminocholestanol derivative abutiloside A 77 from the roots of Solanum abutiloides (Solanaceae). The structure was assigned on the basis of NMR spectroscopic techniques. Compound 77 is regarded as a novel key biogenetic intermediate to spirosolane- and solanidane-types of alkaloid^.^' 2.2.12 Alkaloids of Solanum robustum Wendl. A new glycoalkaloid N-hydroxysolamargine 78 along with the known compounds solamargine solasonine and N-hydroxyrobustine have been isolated from the leaves of 70 ritterezine J R1 = R2 = OH; 22R 71 ritterezine K R1 = H; R2=OH; 22R 72 ritterezine L R1 = R2 = H; 22R 73 ritterezine M R1 = R2 = H; 225 S.robustum grown in a field in Halle (Saale) Germany. The structure of the new base 78 has been elucidated as (25R)-3p-{ 0-a-L-rhamnopyranosyl-(1 +=2)-O-[a-~-rhamnopyranosyl-6Ac 74 vaganineA 75 vaganineB R=H 76 vaganineC R=Ac Atta-ur-Rahman and Choudhary Diterpenoid and steroidal alkaloids . 'HO+O' OH 77 abutiloside A OH HO Ho 78 Khydroxysolamargine (1 +4)]-~-~-glucopyranosyloxy} Sol--22aN-spirosol-5-en-N-01. amargine solasonine and N-hydroxyrobustine were identified by LSI'MS (liquid secondary ion mass spectrum) and spectroscopic comparisons with literature values.The 13CNMR signals of the aglycone of 78 agreed with those of N-hydrorobustine indicating the aglycone to be N-hydroxysolasidine while the sugar part corresponded to the signals of ~olamargine.~' On the basis of literature precedents and their own experience the authors concluded that Solanum species often contain complex mixtures of steroidal alkaloid glycosides which pose separation problems. They therefore suggested that many of the older publications which used less sophisticated techniques are worth repeating. 2.2.13 Alkaloid of Sofanum triste Jacq. Reynolds et al. have isolated a new steroidal alkaloid (22R,25R)-3P-amino-5-spirosolene79 and its previously synthesised dihydroderivative (22R,25R)-3P-amino-5a-spiro-solane 80 from the methanolic extracts of the aerial parts of Solanurn triste collected in Trinidad.This was the first phyto- chemical investigation on this plant of restricted distribution. 79 (22R,25R)-3P-amino-5-spirosolene; A5z6 80 (22R,25R)-3P-amino-5a-spirosolane 81 oblonginine The structures were determined by use of the combination of 2D-NMR technique^.^' 2.2.14 Alkaloids of Veratrum oblongurn Loes. f. Veratrum oblongurn (Liliaceae) of Chinese origin is used to prepare the Chinese crude drug 'Li-ld. The first ever phyto- chemical investigation on the roots of this plant by Kadota et al. has resulted in the isolation of a new steroidal alkaloid oblonginine 81 and five known steroidal bases vanilloyl- zygadenine veratroylzygadenine angeloylzygadenine cevadine and shin~nomenine.~~ Compound 81 is the 22-epimer of veramiline and its structure was determined to be (22R,25S)- 22,26-epiminocholest-5-en-3P-olby 'H NMR (COSY) 13CNMR (DEPT HETCOR long-range HETCOR) and other spectroscopic techniques.Compound 81 was shown to be identical with a compound obtained by the degradation of ~olanidine.~~ 2.2.15 Alkaloids of Veratrum viride Ait. Phytochemical investigations on the ethanolic extracts of the roots and rhizomes of Veratrum viride have led to the isolation of two new steroidal alkaloids rubivirine 82 (12P-hydroxyrubijervine) and veramivirine 83 (I2P-hydroxyvermaline). The structures of these new compounds were determined mainly by spectroscopic studies (thermo- spray LC-MS 'H and 13CNMR COSY NOESY TOCSY HETCOR HMQC HMBC).44 2.2.16 Alkaloids of Vitex canescens Kurz Vitex canescens is a medium-sized tree scattered throughout the countryside of Thailand.Suksamrarn et al. have isolated a new ecdysteroid canescensterone 84 from the ethanolic extract of the dry bark of I? canescens collected from Pipoon district Thailand. The structure of compound 84 was found to be pinnatasterone 24-0-(pyrrole-2-carboxylate)as determined by spectroscopic experiments (EI MS IR D,O-exchanged 'H NMR) and chemical derivati~ation.~' 2.3 Spectral studies The known steroidal alkaloid imperialine 0-N-oxide 85 has been isolated from the bulbs of Fritilluria siechuanica S. C. Che 200 Natural Product Reports Me Plants grown in this valley are very bitter due to the high levels of tomatine content.The authors found that this does not adversely affect the natives of this valley among whom the bitter types are popular.47 HO 6 82 rubivirine U HO 83 veramivirine 3' 4' 04 canescensterone flH HO H 0 85 imperialine P-Koxide of Chinese origin. Its structure was identified on the basis of chemical and spectroscopic methods (FAB MS COSY DEPT COLOC NOED NOESY). The 'H and 13CNMR signals have been unambiguously assigned and the chemical shifts of a number of carbons were revised.46 2.4 Other studies 2.4.1 a-Tomatine contents of fruits of Lycopersicon esculentum Rick et al. discovered that a variant of Lycopersicon esculen- tum var.cerasiforme contains very high levels (500-5000 pg g -of dry weight) of the bioactive glycosteroidal alkaloid a-tomatine in its ripe tomatine is toxic to certain fungi vertebrate and anthropods and can serve as a source of steroids for synthesis of sterols. Gas chromatography was used for the quantitative analysis and identification of the alkaloid was confirmed by GC-MS technique. This high alkaloidal level at the ripe fruit stage is quite unusual in this species and is only confined to plants grown in a tiny enclave in the valley of Rio Mayo Department San Martin Peru. Generally toma- tine is present in very high concentrations in earlier stages of fruit development in L. esculentum the levels of which then decrease rapidly during the mid-period and finally diminish gradually to near zero at maturity.2.4.2 Production of steroidal alkaloids by hairy roots of Solanum aviculare and effect of gibberellic acid Solanum aviculare hairy root cultures established after trans- formation with Agrobacterium rhizogenes A4 were found to contain significantly higher levels of steroidal alkaloids. In shake flask culture the maximum alkaloid yield (measured as solasodine equivalent) was 32 mg g -dry weight and in a three litre air-driven bioreactor the yield was 29 mg g-'. This represents a five-fold increase as compared to the previously reported in vitro yields. The production of steroidal alkaloids in these in vitro cultures was found to be growth associated and limited only by oxygen ma~s-transfer.~~ 2.4.3 Oxygen transfer and culture characteristics of self-immobilised Solanum aviculare aggregates Doran et al.have recently measured oxygen transfer charac- teristics of self-immobilised S. aviculare cells using aggregates 3.0 to 12.5 mm in diameter. This work suggests that oxygen delivery is facilitated in living cell aggregates by mechanisms which depend on metabolic activity and which do not function in deactivated cells.49 2.5 Synthetic studies 2.5.1 Synthetic studies towards batrachotoxin Batrachotoxin 86 is a steroidal alkaloid isolated from frogs of the genus Phyllobates which is known to exhibit remarkable biological activity. Grinsteiner and Kishi contemplated the synthetic strategy of batrachotoxin mainly by using an intramolecular Diels-Alder process to construct the A-D rings of the molecule.They envisioned the coupling of a furan- annelated A-B ring with a suitable alkene-bearing portion. Thus the coupling of furan A with aldehyde B using anion chemistry yielded two possible diastereomers of furfuryl alcohol C. The unstable enal of compound C when allowed to stand in a dilute solution at room temperature underwent a high-yielding selective Diels-Alder reaction to afford com- pound D. Compound D has a highly functionalised steroidal framework. This route (Scheme 3) allows the rapid installation of substituents at some of the more inaccessible positions on this n~cleus.~' 2.6 Pharmacology Cephalostatins 12 48 and 13 49 isolated from the marine worm Cephalodiscus gilchristi were screened against a panel of 60 human cancer cell lines at the National Cancer Institute Bethesda USA.The compounds showed substantial growth inhibitory activity against many of the human tumour cell lines. Cephalostatins 12 48 and 13 49 yielded average GI, values of about 400 nM and > 1000 nM re~pectively.~~ Two other novel dimeric steroidal alkaloids cephalostatins 14 50 and 15 51 isolated from the same worm have also been found to be cytotoxic and antineoplastic. They both inhibited the growth of P388 lymphocytic leukaemia and a selection of other human cencer cell lines. The cytotoxic potencies of cephalo- statins 14 and 15 were in the range of 100 nM and 68 nM respectively.28 The dimeric steroidal alkaloids ritterezines A-M (61-73) isolated from the tunicate Ritterella tokioka have shown potent toxicity against P388 murine leukaemia cells.Ritterezines B 62 F 66 and G 67 which have 5/5 and 5/6 spiroketal rings were found to be the most toxic irrespective of the C-22 stereochem- istry or the presence of A14 olefin while oxidation of the OH-12 to a ketone (ritterezines H 68 and I 69 resulted in a considerable loss in cytotoxic activity. Ritterezines A 61 D 64 A tta-ur-Rahman and Choudhary Diterpenoid and steroidal alkaloids 201 HO. 86 batrachotoxin A B 1 i-iii H C 1 iv v D Scheme 3 Reagents and conditions i Bu”Li THF -78 ‘C followed by addition of B then HPLC separation; ii NaH RX THF; iii TBAF THF; iv MnO, CH,Cl,i v hexanes room temp.8 h and E 65 which have rearranged steroidal skeletons have activity similar to the 12-keto derivatives. In these compounds the stereochemistry at C-22 or methylation at C-24 did not affect the cytotoxic activity. Ritterezine J 70 in which C-26 is oxidised and accordingly possesses two 5/6 spiroketal groups is less active than ritterezine F 66. Since ritterezines J-M (7C73) have similar activity the hydroxy groups at C-7 and C-17 are apparently not important for cytotoxic activity. Further investigation of the structure-relationships of the ritterezines is in progress.38 Holamide 57 and pubescinine 58 two conanine-type ster- oidal alkaloids were isolated from Holarrhena pubescens of Pakistani origin through bioassay-directed isolation.These compounds showed hypotensive activity in rats at a dose of 3 mg kg -’. At this dose holamide 57 was slightly less active than pubescinine 58 causing reduction in blood pressure by 29.33 f3.81% and 34.98 f2.0% re~pectively.~~ Toxicity and mutagenic potential of a number of steroidal alkaloids were assessed in impregnated CD2 transgenic mice. These mice contain multiple copies of a hgtlO/acZ construct integrated into the genome of each cell. A predetermined estimated maximum tolerated dose of several steroidal alka- loids (Solanum glycoalkaloids from potato a-chaconine and a-solanine; aglycones solanidine and solasodine and a Veratrum alkaloid jervine) were given to these mice. Observa- tions were made of dams and foetuses for indications of toxicity and/or terata; some dam livers and foetuses were assayed for mutagenicity using the lacZ gene.Other dams were gavaged with a single dose of 75 mg all-trans-retinol kg -to serve as a reference teratogen. Surprisingly this dose of retinol 202 Natural Product Reports was not clearly teratogenic. The results of both positive and non-positive selection systems showed that the mutation fre- quencies in the livers of the dams dosed with a-chaconine a-solanine and solanidine were three to four times higher than normal in the livers of this transgenic mouse strain.51 Cyclopamine 87 a jervine-type alkaloid isolated from many species of Veratrum (e.g. skunk cabbage) is shown to be a 87 cyclopamine potent mammalian teratogen.Dunn et al.,working on embryo of Xenopus laevis as a model have now proved that cyclopamine directly effects the survival of cranial neural crest cells. However it was discovered that cyclopamine may not cause growth arrest. This study evaluates the embryological basis of teratogenesis of cyclopamine. 52 Gaffield and Keeler studied the teratogenic effects of solani- dine alkaloids in Syrian hamsters. They discovered that oral administration of a-solanine and a-chaconine and their agly- con solanidine induce craniofacial malformations (exen-cephaly encephalocele and anophthalmia). The teratogenicity of solanidine N-oxide a putative metabolite suggests that N-oxidation is not an effective mammation detoxification pathway.53 Gaffield and Keeler have also recently contributed another review on the struct ure-activity relationship of plant steroidal alkaloid teratogen~.~~ A new ecdysteroid canescensterone 84 isolated from Vitex canescens has exhibited high moulting hormone activity in the Musca domestica bioassay which might be due to the presence of the pyrrole-2-carboxylate moiety at the 24-po~ition.~~ 3 References 1 F.-P.Wang Youji Huaxue 1994 14 359. 2 R. Seeger Dtsch. Apoth. Ztg 1994 134 Jahrg. Nr. 29.21.7 2749. 3 X. K. Wang T. F. Zhao and S. Lai Chin. Chem. Lett. 1994 5 671. 4 F. P. Wang X. P. Dai J. Z. Wang and R. Zhang Chin. Chem. Lett. 1995 6 109. 5 D. Lisheng W. Feng and C. 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Benn Acta Crystullogr. Sect. C. 1993 49 1871. 17 G. de la Fuente and L. Ruiz-Mesia Phytochemistry 1995 39 1459. 18 H. Y. Pu and F. P. Wang Chin. Chem. Lett. 1994 5 939. 19 H. Y. Pu and F. P. Wang Acta Pharm. Sin. 1994 29 689. 20 X.-J. Hao X. Hong X.-S. Yang and B.-T. Zhao Phytochemistry 1995 38 545. 21 Y. Bai H. K. Desai and S. W. Pelletier J. Nat. Prod. 1995 58 929. 22 L. C. Baillie J. R. Bearder and D. A. Whiting J. Chem. SOC. Chem. Commun. 1994 2487. 23 G. D. Manners K. E. Panter and S. W. Pelletier J. Nar. Prod. 1995 58 863. 24 R. F. Vieira and L.D. A. Freire de Carvaiho Rev. Bras. Farm. 1993 74 97. 25 W. Gaffield and R. F. Keeler Plant-Assoc. Toxins Proc. Int. Symp. Poisonous Plants 4th 1994 333 CAB International Wallingford UK (Chem. Abstr. 1994 124 109 037). 26 R. K. Gupta J. Singh and D. D. Santani Indian J. Chem. Sect. B 1995 34B 1021. 27 G. R. Pettit Y. Ichihara J. Xu M. R. Boyd and M. D. Williams Biorg. Med. Chem. Lett. 1994 4 1507. 28 G. R. Pettit J. Xu Y. Ichihara M. D. Williams and M. R. Boyd Can. J. Chem. 1994 72 2260. 29 G. R. Pettit Y. Kamano M. Inou C. Dufresne M. R. Boyd C. L. Herald J. M. Schmidt and D. L. Doubek Can. J. Chem. 1986,67 1509. 30 Z.-Z. Qian and T. Nohara Phytochemistry 1995 40 979. 31 F. Wang R. Zhang J. Wang and X. Tang Nut. Prod. Res. Dev.1994 6 1. 32 R. Feng W. H. Lin and M. S. Cai Chin. Chem. Lett. 1994,5 383. 33 J.-X. Zhang A.-N. Lao and R.-S. Xu Zhiwu Xuebao 1993 35 963. 34 B. S. Siddiqui S. B. Usmani S. Begum S. Siddiqui K. Aftab and A. Gilani Heterocycles 1995 41 267 35 H. Ripperger and A. Porzel Phytochemistry 1994 35 813. 36 S. Fukuzawa S. Matsunaga and N. Fusetani J Org. Chem. 1994 59 6164. 37 S. Fukuzawa S. Matsunaga and N. Fusetani J. Org. Chem. 1995 60 608. 38 S. Fukuzawa S. Matsunaga and N. Fusetani Tetrahedron 1995 51 6707. 39 M. Qiu Z. Li D. Cao and R. Nie Zhiwu Xuebao 1993 35 885. 40 E. Ohmura T. Nakamura R. H. Tian S. Yahara H. Yoshimitsu and T. Nohara Tetrahedron Lett. 1995 36 8443. 41 H. Ripperger Phytochemistry 1995 39 1475. 42 A.Maxwell M. Seepersaud R. Pingal D. R. Mootoo and W. F. Reynolds J. Nat. Prod. 1995 58 625. 43 S. Kadota S. Z. Chen J. X. Li G.-J. Xu and T. Namba Phytochemistry 1995 38 777. 44 K. A. El Sayed J. D. McChesney A. F. Halim A. M. Zaghloul and M. Voehler Phytochemistry 1995 38 1547. 45 A. Suksamrarn C. Sommechai P. Charulpong and B. Chitkul Phytochemistry 1995 38 473. 46 F.-P. Wang R. Zhang X.-Y. Tang and J.-Z. Wang Youji Huaxue 1994 14 548. 47 C. M. Rick J. W. Uhlig and A. D. Jones Proc. Natl. Acad. Sci. USA 1994 91 12 877. 48 M. A. Subroto and P. M. Doran Plant Cell Tissue Organ Cult. 1994 38 93. 49 I. Ananta M. A. Subroto and P. M. Doran Biotech. Bioengin. 1995 47 541. 50 T. J. Grinsteiner and Y. Kishi Tetrahedron Lett. 1994 35 8333. 51 L. Crawford and B. Myher Food Chem. Toxicol. 1995 33 191. 52 M. K. Dunn M. Mercola and D. D. Moore Dev. Dyn. 1995,202 255. 53 W. Gaffield and R. F. Keeler Chem. Res. Toxicol. 1996 9 426. A tta-ur-Rahman and Choudhary Diterpenoid and steroidal alkaloids

ISSN:0265-0568    

DOI:10.1039/NP9971400191

出版商:RSC    

年代:1997

数据来源: RSC

摘要:

Hot off the press Robert A. Hill" and Andrew R. Pittb aDepartment of Chemistry Glasgow University Glasgow UK G12 8QQ. E-mail bobh@chem.gla. ac.uk bDepartment of Pure and Applied Chemistry Strathclyde University Thomas Graham Building 295 Cathedral Street Glasgow UK GI 1 XL. E-mail a.r.pitt@strath. ac. uk Reviewing the recent literature on natural products and bioorganic chemistry C. H. Heathcock tells the story of Some of his studies on the synthesis of Duphniphyllum alkaloids in an inaugural article (Proc. Natl. Acad. Sci. USA 1996 93 14323). The route based on a biomimetic strategy forming seven a-bonds and five rings in one reaction was improved by a serendipitous mis- labelling of a reagent to give an efficient route to the skeleton (Scheme 1) and some hints on a possible biosynthesis.(i) MeNH2 (ii) HOAc A OHC4 \ Scheme 1 Callipeltoside A 1 is a cytotoxic constituent of a marine sponge (Culipelta sp.) that contains a novel aminodeoxy sugar callipeltose and a dienyne cyclopropane moiety (L. Minale and co-workers J. Am. Chem. SOC.,1996 118 11 085). The marine hydroid Tridentuta marginata contains tridentatol A 2 f with a dithiocarbamate functionality which deters feeding by a predator (N. Lindquist et al. Tetrahedron Lett. 1996 37 9131). Dictyosphaerin 3 from a South Australian green algae Dictyosphaeria sericea has a novel bicylic skeleton presumably jyNfe HO 2 OH 3 derived from a fatty acid (R. J. Capon and co-workers J. Nat. Prod. 1996 59 1154).Lissoketal 4 is a new spiroketal from Lissoclinum voeltzkowi (C. Hopmann and D. J. Faulkner Tetrahedron Lett. 1997 38 169). Nudenoic acid 5 from the Taiwanese liverwort Mylia nuda has a new sesquiterpenoid skeleton that is postulated to be formed from a spirovetivane precursor (Chia-Li Wu and co-workers Tetrahedron Lett. 1996 37 9307). I 4 5 Rarisetenolide 6 is one of three sesquiterpenoids with a new skeleton isolated from Euplotes ,rariseta (F. Pietra and co-workers Helv. Chim. Acta 1996 79 2180). The new skeleton is related to that of the diterpenoid focardin from E. focardii [see 'Hot off the press' Nut. Prod. Rep. 1996 13(4) iii]. Hyalic acid 7from Hyalis argentea is presumably derived by a Baeyer-Villiger oxidation of a kaurane precursor (M.I. Ybarra et al. Phytochemistry 1997,44,479). A number of new Me0 I 1 6 7 ... Hill and Pitt Hot 08the press 111 variants on the rare ring-A 3,4-secocycloartanes that have cytotoxicity towards mammalian cancer cell lines have been isolated for the first time from two Gardenia species (G. L. Silva et al. Tetrahedron 1997 53 529); caronolide 8 is an example. A meroterpenoid setosusin 9 from the Ascomycete Corynascus setosus has been shown to have tremorogenic properties (M. Yamazaki and co-workers Chem. Pharm. Bull. 1996 44 1843). ==$f 0 8 9 The tetrahydrofuran functionality found in the acetogenins from the Annonaceae are postulated to be formed by epoxi- dation of a V-diene system followed by ring opening and reclosure.Muridienin 1 10 from Annona muricata contains the V-diene system which when treated with mCPBA and then with HClO produces the typical functionality of the acetogenins (Scheme 2) (B. Figadkre and co-workers 10 HO Scheme 2 Tetrahedron Lett. 1996 37 9301). This supports the postu- lated biogentic pathway. The pyrrolecarboxylic acids 11 and 12 makaluvic acids A and B respectively from the marine sponge Zyzzya fulginosus probably arise biogenetically from oxidative cleavage of a tyrosine-derived alkaloid (F. J. Schmitz and co-workers J. Nut. Prod. 1996 59 1104). The unusual alkaloid angustimaline 13 from Alstonia angustifolia is iv Natural Product Reports 11 R=H;R'=Me 12 R = Me; R' = H probably derived from a macroline-type precursor such as alstophylline 14 which also occurs in the plant (T.-S.Kam et al. Tetrahedron Lett. 1997 38 477). 0 13 Me 14 Evidence for the involvement of an intermolecular Diels- Alder reaction in the biogenesis of macropho& acid 15 by Macrophoma commelinae cultures has been presented (H Oikawa et a/. Chem. Commun. 1997 97). The authors propose that an intermediate such as 16 is involved (Scheme 3). The intermediate analogue 17 was found to inhibit the conver- sion however an anion-based condensation is not rigorously excluded. OMe 0 15 Scheme 3 /p Me02C& 17 Further evidence for the formation of a tertiary cyclopropyl carbinyl cationic intermediate in the conversion of presqualene diphosphate to squalene has been provided by M.B. Jarstfer et al. (J. Am. Chem. SOC.,1996 118 13 089). Using the partially reduced NADPH analogue of NADPH they were able to isolate the alcohol previously suggested as an inter- mediate by Rilling strongly supporting the intermediacy of the cation. R. E. Hill et al. have used extensive labelling studies to probe the biosynthesis of vitamin B 18 (pyridoxol) in Escherichia coli (J. Biol. Chem. 1996 271 30 426). They have shown conclusively that it is constructed from 1 -deoxy-D-xyulose and 4-hydroxy-~-threonine alone. Feeding experiments have demonstrated that C-3 of oospoglycol 19 a metabolite of from 4-hydroxy-L-threonine 18 "YOH Po OH 0 19 Gloephyllum abietinum is labelled from [3-'3C]serine or ['3C]formate (J.Sonnenbichler et al. Phytochernistry 1997,44 267). This implies that C-3 of oospoglycol 19 is derived from the tetrahydrofolate C pool. The remaining carbons are derived from acetate. The leaves and stems of the wood plant Aspalathus linearis have been shown to contain the enolic glucoside of phenylpyruvic acid 20 (C. Marais et al. J. Chem. SOC. Perkin Trans. I 1996 2915). This is the first isolation from woody plants of a derivative of phenylpyruvic acid which is a postulated intermediate in the biosynthesis of a-hydroxychalcones. OGlc PhAO*H 20 21 An answer to the debate on the protonation state of thiamine pyrophosphate 21 (TPP) during catalysis has been provided by the elegant work of D. Kern et al.(Science 1997 275 67). Measurement of thermodynamic and kinetic depro- tonation at C-2 of TPP using NMR hydrogen-deuterium exchange and the use of TPP analogues and site directed Hill and Pitt Hot ofl the press mutants for pyruvate decarboxylase and transketolase show that a glutamate interacts with the 1'-N activating the 4'-NH to act as a proton acceptor. This occurs at a rate faster than the overall rate of the reaction showing that the protein causes rapid deprotonation at C-2 to give a short lifetime carbanion rather than this being the resting state. Isotopic labelling studies have been used to determine the biosynthetic origin of all the structural features of sulfomycin (U-102408) 22 (G. D. Fate J. Am. Chem. SOC. 1996 118 11 363).OH HN '*OH 22 The determination of the X-ray crystal structure of Escherichia coli dihydrodipicolinate synthase (DHDPS) with substrate substrate analogue inhibitor and the feedback inhibitor lysine-bound and the identification of (48-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipocolinicacid 23 as the HO T H C OO -coo -23 only product of the transformation has allowed S. Blickling et al. to propose a simple mechanism using tyrosine-1 33 and lysine-161 of the enzyme (Biochemistry 1997 36 24). A general review of the work on the modular polyketide syn- thase 6-deoxyerythronolide B synthase has been published (R. Pieper et al. Chem. SOC. Rev. 1996,25 297). It covers the organisation function and the synthesis of 'unnatural' natural products.In the same issue W. Buckel and B. T. Golding have published a review of their studies on glutamate and 2-methyleneglutarate mutases as paradigms of vitamin B 12 dependent enzymes (Chem. SOC.Rev. 1996 25 329). This contains a discussion of possible mechanisms in the light of spectroscopic isotopic and modelling studies. A mild method for the conversion of hydroxy nitriles into the corresponding lactones using the microorganism Rhodococcus rhodochrous has been developed by S. K. Taylor and co-workers (J. Org. Chem. 1996 61 9084) (Scheme 4). 0. Rotthaus et al. have reported the extension of the use of 0 Scheme 4 Saccharomyces cereviciae in organic solvents for the reduction of keto esters (Tetrahedron 1997 53 935). They generally see the same high yields and ees as for aqueous conditions but some solvents cause an altered enantiospecificity.The strength of the n-facial hydrogen bond has been studied in a series of neat experiments by the Hunter group (Chem. Commun. 1996 253 1). Using chemical mutation removing a n-facial hydrogen bond from a zipper complex they estimate that it is worth -4.5 f0.5 kJ mol-'. As transient features are not apparent for the time-averaged structures that are obtained from X-ray crystallographic and NMR spectroscopic studies L. Young and C. B. Post have used the study of thermal fluctuations in a molecular dynamics study to attempt to probe these features (Biochemistry 1996 35 15 129). Fluctuations in the structure of NADH are affected by binding to the NADH binding site of lactate dehydrogenase compared to the free state giving rise to structures similar to the putative transition state.This agrees with the long accepted suggestion that enzymes can funnel conformations towards the transition state to reduce the entropic barrier. H. Wagner et al. have reported the synthesis of a series of oligo tetrahydrofuran peptides and their behaviour as mimics of ion pores in membranes (Angew. Chem. Int. Ed. Engl. 1996 35 2643). Structures of the type 24 when n=9 form a helical array that can span the membrane and conductance measurements confirm that this can act as an ion pore. hOBn n 24 vi Natural Product Reports

ISSN:0265-0568    

DOI:10.1039/NP997140iiib

出版商:RSC    

年代:1997

数据来源: RSC