I1 Biosynthesis By R.A. HILL Department of Chemistry University of Glasgow Glasgow GI2 8QQ.UK I Introduction The previous Report' on the biosynthetic studies of secondary metabolites and related work covered the period 1989-91. This report covers the period 1992-93. The study of biosynthetic pathways continues to be a highly active area This report is a personal view of some of the highlights in the area of biosynthesis. The production of new microbial metabolites using unnatural precursors a technique known as precursor-directed biosynthesis,2 the use of plant cell culture^,^ and the biosynthesis of marine natural products4*' and isocyanides and cyanides6 have been reviewed. 2 Fatty Acid and Polyketide Biosynthesis Reviews have appeared on the biosynthesis of fatty acid and polyketide rnetabolite~~*~ and the angucucline antibiotics.' Deuterium labelling studies were used to show that theconversion ofpalmitic acid (1) into the sex pheromone (2) of the moth Mamestra brassicue occurs by a formal syn elimination of the 1 1-(pro-R)- and 1 Z(pro-R)-hydrogenatoms.lo This result indicates that the insect A'' desaturase is similar to the A9 desaturase found in mammals,plants and bacteria. Labelling studies have shown that pahitic acid (1) is converted into diabolic acid (3)by Butyrivibrio~brisolvenswithout the loss of label from either C-14 or C-16 ofpalrnitic acid (I)-" This rules out the possibility of the intermediacy of At'-or Al5-pa1mitic acids in the biosynthesis of diabolic acid (3). The degradation of the keto acid (4) to produce &decalone (5) in Sporubulumycesodorus involves inversion of the stereochemistry.This has been shown to be due to the intermediacy ofa ketone which is later reduced to give the opposite stereochemistry.' * Degradation of a-linolenic and ' R.A. Hill Annu. Rep. Prog. Chem. Sect. B,1991,&8 283. R. Thiericke and J. Rohr Nat. Prod. Rep. 1993 10 265. J-P. Kutriey Acc. Chem. Res. 1993 26 559. 'M.J. Garson Chem. Rev. 1993,93 1699. G. Cimino and G. Sodan Topics Current Chem. 1993,167 77. 'P.J. Scheuer Ace. Chem. Rex 1992 25,433. ' D. O'Hagan Nat. Prod. Rep. 1992 9,447. * D. O'Hagan Nat. Prod. Rep. 1993 10 593. J. Rohr and R. Thiericke Nat. Prod. Rep. 1992,9,103. lo W. Boland C. Frossl M. Schlottler,and M.Toth J-Chem. SOC. Chem. Commun. 1993 1155. It W. Fitz and D. Arigoni J. Chem. SOC. Chem. Commun. 1992 1533. *' W. Albrecht M.Schwarz 1. Heidlas and R. Tress] J. Org. Chem. 1992 57 1954. 31 I 312 R. A. Hill linoleic acids in green leaves produces a 'green odour'. This has been characterized as a mixture ofC aldehydes and alcohols. The biogeneration ofthe 'green odour' has been reviewed. Marine brown algae (Phaeophyceae) produce pheromones by metabolism of unsaturated icosanoic acids to give a series of C,,H, hydrocarbons. Deuterium labelling studies have been used to investigate their bio~ynthesis.'~ For example it has been shown that all-cis-icosapenta-5,8,11,14,17-eno~c acid (6) is metabolized to giffordene (7) by Gifordia mitchellae.The 1abeIling studies are consistent with a thermally-allowed antarafacial f l,7) hydrogen shift during the biosynthesis (Scheme 1 ). COOH OH Scheme 1 l3 A. Hatanaka Phyfochemistry 1993 34,1201. '* K. Stratrnann W. BoIand and D.G. Muller Termhedron 1993 49 3755. Biosynthesis 313 Studies with single and double-labelled 13C acetates have shown that thiarubine A (8) from Ambrosia artemisiifoh is formed from seven acetate units with the terminal acetate being ~1eaved.I~ All the carbons of oncorhyncolide (9) from the marine bacterial isolate MK157 have been shown to be derived from acetate." The methyl branches arise from C-2 of acetate. This type of branching has only been observed in a few biosyntbetic pathways. 4 H& s-s HOH2C ob 0 (10) 0 (9) Extensive labelling studies have shown that decarestricine €3 (10) from PenicilIiurn simplicissimum is a pentaketide.The epoxide oxygenis derived from molecular oxygen and the hydroxyl oxygen is derived from the medium. The biosynthetic relationships between the various members of the decarestricine family have been presented." There is more evidence for the processive mode of polyketide biosynthesis. The evidence has been extended to the polyether antibiotics with the incorporation of the N-acetylcystearnyl thioester of 5-oxo-2,4-dimethylhexanoic acid (I I) labelled with deuterium into rnonensin A (12) in Streptornyces cinnamonensis.18During this study it was found that the presence of 2,6-0-dimethyl-~-cyclodextrin and a P-oxidation l5 M.L. Gornez-Barrios,F.J. Parodi D. Vargas L. Quijano M. A. Hjortso,H. E. Flores and N. H. Fischer Phytochemistry 1992 31 2703. J. Needham R. J. Andersen and M.T. Kelly J. Chem. SOC.,Chem. Commun. 1992 1367. l7 M. Mayer and R. Thiericke J. Chem. SOC. Perkin Trans. I 1993,495. '' HAPatzelt and J. A. Robinson J. Chem. SOC.,Chem. Commun. 1993 1258. 3 14 R. A. Hill inhibitor during the fermentation was essential for successful incorporation. Further compelling evidence for the processive mode has also been provided with studies on methymycin (13)from Srreptomyces uenezueiue” and nargenicin (14) from Streptomy-ces aureus.20In the latter study the proposed (4+2) cycloaddition to form the octalin ring of nargenicin (14) is also supported.(13) (14) Incorporation of labelled acetate propionate butyrate and glucose into elaiophylin (15) from a Streptomyes species has established the biosynthesis of this rnacrolide. A detailed analysis of the fermentation time course of Streptouerticiliium baldacii subsp. netropse that produces the desertomycin family of macrolide antibiotics has provided details of the relationships between the various members of the family.*l Extensive labelling studies have established the polyketide origin of the [7.7]paracyclophanes ll-mga OH 0 OH 0 \\ sugar4 I 1 1 1 (15) such as nostocyclophane D (16)from the cyanobacteria Nostoc linckiu and Cylindros-permurnlicheniforrne.2 The experiments indicate that these [7.7]paracyclophanes arise by dimerization of nonaketide intermediates.The fungus Artropsis ~runcataproduces a range of interesting metabolites. Incorporation studies on one of these metabolites arthropsadiol A (17) have indicated that one of the terminal methyl groups (C-12) arises from the methyl group of rnethi~nine.’~ Methylation of a hexaketide intermedi- ate is proposed to explain the results. Labelled acetate and methionine were used to establish that spiciferone A (18) and spiciferin (19) from Cochliobolus spicifer are derived from a hexaketide with two C units.24 They probably both arise by oxidative cleavage of a bicyclic system (Scheme 2). l9 D. E. Cane R. H. Lambalot P.C. Prabhakaran and W. R. Ott 6. Am. Chem. Soc. 1993,115 522. D.E. Cane W. Tan and W. R. Ott J. Am.Chem. Soc. 1993,115 527. M. Mayer and R. Thiericke J. Cfiem. Sac. Perkin Trans. I 1993 2525. 22 S.C. Bobzin and R.E. Moore Tetrahedron,1993 49 7615. 23 W.A. Ayer and P. A. Craw Can.1.Chem. 1992,70 1348. 24 H.Nakajima R. Matsumoto Y. Kimura and T. Hamasaki J. Chem.Soc. Chern. Cornmun. 1992,1654; H. Nakajirna H. Fujirnoto R. Matsumoto and T. Hamasaki J. Org. Chem. 1993,58,4525. Biosynthesis 315 Scheme 2 Similar studies were used to establish the biosynthetic origin of zaragozic acid A (20) a squalene synthase inhibitor from an unidentified fungus (MF5443)." The remaining carbons arise from benzoic acid and succinic acid. Chirally labelied malonyl CoA has been incorporated into orsellinic acid using orsellinic acid synthase from Penicilliurn cyclopium.26 The results are similar to those obtained earlier for 6-methylsalicyclic acid.' The hydrogen atoms removed from the two methylene groups at the 2-and 4-positions of the putative polyketide intermediate have opposite stereochemistry. Doubly labelled acetate was used to show that melkin and 2,4-dihydroxyacetophenone are biosynthesized in the ant Rhytidoponera chalybaea in the same way as in fungi.27 Detaiied labelling studies using I3C 'H and "0 labelled acetates and "0,gas on LL-D253or (211 a metaboliteof Phoma pigmentiuora have shown that the metabolite is derived from two preformed polyketide chains.28 The label observed in the hydroxy- 25 K.M. Bryne B H. Arison M. Nallin-Ornstead and L. Kaplan J. Org. Chem. 1993 58 1019.26 J. B. Spencer and P.M. Jordan J Chem. Sac. Chem. Commun. 1992 645. '' C.-M.Sun and R. F. Toia J. Nut. Prod. 1993,56 953. 28 I. M. Chandler C. R. McIntyre and T.J. Sirnpson 1.Chem. SOC., Perkin Trans. 1 1992,2285. 316 R. A. Hill methyl side chain is randomized. This is explained by the involvement of a spirocyclopropyl intermediate. OH OH 0 OH HOPMeOmo* (21) (22) [1,2-' 3C,]Acetate feeding results have defined the chain folding of torosachrysone (22) from Dermatocybe rnushro~rns.~~ Incorporation experiments with simple and advanced precursors labeiled with I3C,'H and have shown that tajixanthone (23) is derived through cleavage of an octaketide-derived anthraquinone with the introduction of two dimethylallyl moieties. 30 Purpactin A (24) from Penicilliurn purpuragenurn has also been shown to be derived by oxidative cleavage of an octaketide-derived anthraquin~ne.~' Xanthoquinodin A (25) from Humicola sp.FO-88 has been shown to be produced from two octaketide chains one having been oxidatively ~leaved.~ A similar biosynthesis has been shown for Cercospora beticola toxin (CBT) (26).33 HO 29 M. Gill A. Gimenez and R. Watling 1.Nat. Prod. 1992,55 372. 30 S.A. Ahrned E. Bardshin C. R.McIntyre and T. J. Simpson Aust. J. Chem. 1992 45 249. 31 H. Nishida H. Tomoda S. Okuda and S. Omura 1. Org. Chem. 1993,57,1271. 32 H. Tabata H. Tomoda K. Matsuzaki and S. Ornura J. Am. Chem. Soc. 1993,115 8558. 33 A. Amone G. Nasini L. Merlini E. Ragg and G. Assante J. Chem. Soc. Perkin Trans.I 1993 145. Biosy nthesis 317 A useful folding code using E/Z terminology has been devised for the comparison of multicyclic polyketides and this has been used to compare their biosynthetic origins.34 The genetic understanding of polyketide synthesis continues to improve. This is aptly demonstrated by a study in which genes from the polyketide synthase (PKS)from Streptornyces roseufuluus,which produces both frenolicin B (27) and nanaomycin A (28) were expressed in Streptomyces colelicolor A3 with components of the actinor- hodin (29) gene cluster. The primary products of the recombinant strain are the new metabolites RM18 (30) RM18b (31) and the anthraquinone (32).35Analysis of the results confirms earlier conclusions regarding the protein determinants of chain length keto reduction and cyclization specificities.The biosynthetic model in which keto reduction occurs only after the complete poiyketide chain has been synthesized is supported. && \ 0 CO2H \ 0 CO2H (27) (28) Theangucyclin group ofantibiotics continues to provide interesting results. Blocked mutants of Streptomycesfiadias have produced five new products. Their relevance to postulated biosynthetic pathways is disc~ssed.~' The origins of the oxygen atoms in aquamycin (33) from Streptomyces fiadi~e~~ and antibiotic PD 116198 (34)38 from 34 J. Rohr J. Urg. Chem. 1992 57 5217. '' R. McDaniel S.Ebert-Khosta D. A. Hopwood and C. Khosla J. Am. Chem. SOC. 1993 115 11671. 36 J. Rohr M. Schonewolf,G. Udvarnoki K.Eckardt,G.Schumann C. Wagner J. M. Beale and S. D.Sorey J. Org. Chem. 1993 58 2541. 37 G. Udvarnoki T. Henkel R. Machinek and J. Rohr J. Org. Chem. 1992 57 1274. 38 S.J. Gould and X.-C. Cheng Tetrahedron 1993 48,11 135. 318 R. A. Hill Streptornyces phaeochrogenes WP 3588 have been studied (Scheme 3). Most of the angucyclins appear to be derived from a decaketide intermediate folding into an angular conformation; however labelling studies on antibiotic PD 115198 (34) are consistent with rearrangement of an initially-formed linear tetracyclic intermediate. Feeding experiments using Streptomyces murayamensis that produces dehyd-roabelomycin (35) and tetrangulol (36) indicate that deoxygenation occurs at a prearomatic ~tage.~ *OH HO HO (34) Scheme 3 Early intermediates on the biosynthetic pathway of tetracenomycin C from Streptomyces gluucescens have been identified in blocked mutants4' Labelling studies on esperamicin A from Actinornadura uerrucososporuhave indicated that the enediyne core (37)is derived from an octaketide intermediate with loss of the terminal carboxyl group.4l RO OH OR OH 0 R 0 (35) R= OH (36) R= H (37) 3 Terpenoids Reviews have appeared on HMG CoA reductase inhibitors:' the folding of ~qualene,~~sterol metabolism in the biosynthesis of marine sterol~,4~,~~ and 39 S.J. Gould X.-C. Cheng. and K.A. Halley J. Am. CAem. SOC. 1992,114,10066. 40 B.Shen H. Nakayarna and C. R. Hutchinson J. Nat. Prod. 1993 56 1288. 41 K.S. Lam J. A. Veitch J.Golik B. Krishnan S. E. KIohr K. J. Volk S. Forenza and T.W. Doyle J. Am. Chem. SOL 1993,115 12340. 42 A. Endo and K. Hasurni NQC.Prod. Rep. 1993,10 541. '' R. Bohlmann Angew. Chern. Int. Ed. Engl. 1992,31 582. 44 N. Ikekawa M. Morisaki and Y. Fujimoto Ace. Chem. Res. 1993,26 139. 4s B.J. Baker and R. G. Russel Topics Current Chem. 1993 147 1. 46 J.-L. Giner Chem. Rev. 1993,93,1735. 3iosy nthesis 319 the bacterioh~panoids.~’*~~ A new pathway for the biosynthesis of isopentenyl diphosphate in bacteria has been identified.49 Geranyl diphosphate synthase has been used to study the stereochemistry of the formation of geranyl diphosphate from dimethylallyl diphosphate and isopentenyl diph~sphate.~’ Detailed Iabelling studies have been conducted to investigate the biosynthesis of limonene (38) from geranyl dipho~phate.~ Deuterium labelled geranyl diphosphate has been used in biosynthetic studies of monoterpenes from Saluia of~cinalis.~~ These results cast doubt on the use of natural abundance deuterium NMR for biosynthetic studies.A OGlc Deoxyloganic acid (39)has been efficiently converted into oleoside methyl ester (40) using Fraxinus excelsior and Syringa j~sikaea.’~ Analysis of the results of feeding deuteriated rnevalonates to Perilla frutescens callus revealed that the biosynthesis of a-curcumene (41) involves a 1,2-hydride shift and the biosynthesis of cuparene (42) involves a 1,3-hydride shift.54 Deuteriated farnesols were incorporated into dehyd- rogeosmin (43) using flower heads of the cactus Rebutia m~rsoneri.~~ The results are consistent with dehydrogeosmin (43) being formed by loss of the three-carbon side chain of a eudesmane intermediate.Q$Q=Q+-(41) +% (42) Competitive feeding experiments indicated that isotrichodiol(44) and isotrichotriol ” G.Ourison and M. Rahmer Acc. Chem. Res. 1992,25 403. 48 M. Rohrner Pure Appl. Chem. 1993,65 1293. M. Rohrner M. Knani P. Simonin B. Sutter and H. Sahm Biochem. J. 1993 295 517. T. Endo and T. Suga Phytochemistry 1992,31 1565. H.-3. Pyun,R.M. Coates K. C. Wagschat P.McGeady and R. B. Croteau J. Org. Chem. 1993,58,3998. 52 D. Arigoni D. E. Cane J. H.Shim R. Croteau and K. Wagschal Phytochernistry 1993,32 623. ’’ S. Damtoft H. Franzyk and S. R. Jensen Phytochemistry 1993 34 1291.54 K. NabeIa K. Kawakita Y. Yada and H. Okuyama Biosci. Biotech. Biochem. 1992 57 792. 5s Z. Feng U.Huber,and W. Boland Hell. Chim. Acta 1993 76 2547. 320 R. A. Hill (45) rather than trichodiol(46) and trichotriol(47) are intermediates in trichothecene bio~ynthesis.~~ Labelled velutinal(48) is incorporated into the Iactaranes vellerol(49) and velleral (50) by bruised fruiting bodies of the mushroom Lactmius elle ere us.^^ [14C]Arteannuic acid (51) has been incorporated into both arteannuin €3 (52) and artemisinin (qinghaosu) (53) using Artemisia annua,suggesting that arteannuic acid (51)might be a common intermediate in the biosynthesis of (52) and (53).58 (44) R = H (46) R = H (45) R=OH (47) R=OH (49) R=CHzOH (50) R=CHO 0 0 (53) (52) The biosynthesis of ether lipids such as di-0-phytanylglycerol(54)has been studied in Ar~haebacteria.~~~~' The results indicate that there is a cornrnun pathway for the biosynthesis of these lipids in both methanogenic and halophilic bacteria and that di-0-phytanylglycerol (54) is formed in two distinct steps from glycerol and geranyl- geranyl diphosphate in Methanobacterium thermoautotrophicum.Feeding experiments with [l,2-13C,facetate and [2-' 'C,'H,facetate in Amsonia ellipticahave provided details of the biosynthesis of the side chains of campesterol(55) and dihydrobrassicasterol (56)?' The results are consistent with the biosynthesis 56 A. R Hesketh B. W.Bycroft P. M. Dewick and J. Gilbert Phytochemistry 1993,32 105.57 T.Hansson Z.Pang and 0.Sterner Acfa Chem. Scad. 1993,47,403. 58 R.S.Sangwan K. Aganval R. Luthra R. S. Thakur and N. Singh-Sangwan Phytochernistry 1993,34 1301. 59 D.Zhang and C. D. Ponlter J. Org. Chem. 1993,58 3919. 60 D.Zhang and C. D. Poulter J. Am. Chem. Soc. 1993,115 1270. 61 S.Seo A. Uomori Y. Yoshimura K. Takeda H.Noguchi Y. Ebizuka U. Sankawa and H. Seto,3. Cfiem. Suc. Perkin Trans. I 1992 569. Biosynthesis 321 involving regiofacial specific reduction of a C-24 double bond which is formed by double bond isomerization of a A24‘28’-sterol (Scheme 4). Detailed studies on the biosynthesis of estrogens by human microsomal placental arornatase have shown that / \ Scheme 4 the biosynthesis proceeds through several different pathway^.^',^^ Incorporation of 13C-labelled acetate and formate into citreohybridone 3 (57) by a hybrid strain derived from Penicilliurn citreo-uiride indicated that citreohybridone B (57) is formed by a meroterpenoid pathway similar to that for terret~nin.~~ 4 Shikimate and Related Metabolites Reviews have appeared on the biosynthesis of shikirnate metabolite^,^^,^^ phenyl-62 E.Caspi H.R.W.Dharmaratne E. Roitman and C.Shackleton J. Chem. Soc. Perkin Truns. I 1993,1191. 63 H. R. W. Dharrnaratne,J. L. Kitgore E. Roitman C. Shackleton and E. Caspi,J.Chem.Soc. Perkin Trans. 1 1993 1529. 64 S. Kosernura H. Miyata K. Matsunaga and S. Yamamura Tetrahedron Lett. 1992 33 3882. 65 P.Dewick Nat. Prod. Rep. 1992 9 153. 322 R. A. Hill propanoids and isoflavonoids in alfaifa,67 isoflavone and pterocarpan phytoaIexins,68 and phenylpr~panoids.~~ The enzymes of the shikimate pathway have continued to cume under extensive scrutiny and have been re~iewed.~' The dehydration of 3-dehydroquinate (58) to 3-dehydroshikimate (59) is catalysed by 3-dehydroquinase.There are two distinct classes of 3-dehydroquinase which are quite different in physical and biochemical propertie~.~' The active site Iysine that is known to form a Schiffs base with the ketone carbonyl of 3-dehydroquinate (58) is found in Type I enzymes but not in Type I1 enzymes. The properties of isochorismate hydroxymutase which catalyses the interconversion of chorismic acid (60) and isochorisrnic acid (61) have been described.'2,73 OH (59) C02H C02H t [l-3C]PhenyIaIanine was incorporated into the phenylpropanoid (62) without 66 P.Dewick Not. Prod. Rep. 1993 10 233. 67 R. A. Dixon A. D. Choudhary K. Dalkin R. Edwards T. Fahrendorf G. Gowri M.J. Harrison C.J. Lamb G. L. Loake C. A. Maxwell J. Orr and N. L. Paiva Recent Adu. Phyrochern. 1992,26,81. 68 W. Barz and R. Welle Recent Adv. Phytochem. 1992,26 139. 69 C.J. Douglas M. Ellard K. D. Hauffe E. Molitor M. Moniz de Sa S. Reinold R. Subramaniam and F. Williams Recent Adv. Phytochem. 1992 26 63. 70 B.K. Singh D. L. Siehl and J. A. Connelly Oxford Suro. Plant Mol. Cell Bid. 1991 7 i43. 71 C. Kleanthous R. Deka K. Davis S. M. Kelty A. Cooper S.E. Harding N. C. Price A. R. Hawkins and J.R. Coggins Biochem. J. 1992 232 687.72 P.M.M.Schaaf,L. E. Heide E. W. Leistner Y. Tani M. Karas and R. Deutzmann J. Not. Prod. 1993,56 1294. 73 P. M. M. Schaaf L. E. Heide E. W. Leistner Y. Tani and M. M.El-Olemy J. Nut. Prod. 1993,56 1304. Biosynthesis 323 rearrangement of the side chain.74 Labelled l-j4-methoxyphenyl)prop-I-ene (63) was incorporated into epoxypseudoisoeugenol 2-methylbutyrate (64)in tissue cultures of Pimpinella anisum;the labelling of the metabolite indicated that an NIH shift of the side chain had occurred during the introduction of the aromatic hydroxyl group.75 A similar migration is probably involved in the biosynthesis of the tyrosine kinsse inhibitor erbstatin (65) from a Streptomyces species as iabeiled shikimic acid phenylalanine and tyrosine are incorporated efficiently into erbstatin (65).'5 Biosyn-thetic studies on axenornycin D (66) show that the aromatic moiety is derived from a metabolite of the shikimate pathway and that the biosynthesis is different to that of the menaq~inones.~~ I OMe (43) OH I I The role of salicyclic acid as a pIant hormone has been reviewed.78 The biosynthetic pathway ofsaIicycIic acid from cinnamic acid is nut clear at present.Levels ofsalicyck acid in plants appear to be controlled by conjugation with Sporopellenin from Curcurbita maxima is a biopolymer that is resistant to chemical physical and biological degradation. Although its structure is not known [U-'4C]-phenylalanine has been shown to be efficiently incorporated into sporopollenin.80-82 Labelling studies using [W-'3C]glucose [3-I3Cfphenylalanine and [1,2-'3C J-phenylacetic acid (67)into thiotropocin (68)from Pseudomonas CB-104 are consistent with a shikimate origin involving symmetrical phenylpyruvate and phenylacetate 74 K.H. Horz and J. Reichling Phytochemistry 1993 33 349. 75 R. Martin and J. Reichling Phytochemistry 1992 31 51 1. '' Y. Tabata M. Imoto and K. Umezawa J. Antibiot. 1992 45 1382. " V. Firese A. Boos H.-J. Banch and E. Leistner Phytochemistry 1993,32 613. '' I. Raskin Annu. Rev. Plant Physiol. Plant Mol. Biol. 1992 43 439. 79 N. Yalpani N. E. Blake and M. Schultz Plant Physioi. 1992 100 11 14. 8o S. Gubatz and R. Wiermann Bot. Acta 1992 105,407. 81 S. Gubatz and R. Wiermann Z. Naturforsch. C Biosci. f993,48 10.S. Wilmesmeier S. Steuernagel and R. Wiermann 2.Nnturforsch. C Biosci. 1993 48,697. 324 R. A. Hill 5 intermediate^.^^ PhenyIacetic acid (57) presumably undergoes oxidative ring expan-sion followed by further oxidation and incorporation of sulfur. The biosynthesisof ansatrieneA (59)from Streptumyces cullinus has been extensively studied.868 The results indicate that the cyclohexanecarboxylic acid portion is derived from shikimate. Full details of the biosyntheticstudies on rotenone (70) and amorphigenin (71) by Arnorpha jkuticosa seedlings have been published.8s-g0 A mechanism involving a spirodienoneintermediate has been proposed fur the migration of the phenyl group in the biosynthesis of isoflavon~ids.~ 0 OMe 1 HoY 0 (49)(49) (70)(70)RR == HH (71)(71) R=OHR=OH The biosynthesis of the aromatic side chains of taxol (74) has been studied.92The results indicate that phenylalanine is incorporated via B-phenylalanine (72) and phenylisoserine (73).The benzoyI moieties are also derived from 8-phenylalanine(72) either directly or via phenylisoserine (73) (Scheme 5). 83 D.E. Cane 2. Wu and J. E. Van Epp J. Am. Chem. Soc. 1992 I14,8479. 84 K. A. Reynoids P. Wang K. M. Fox and H. G. Floss J. Antibiot. 1992 45,411. 85 K.A. Reynolds J. Nut. Prod. 1993 56 175. 86 K. A. Reynolds N. Seaton K.M. Fox K. Warner and P. Wang J. Nut. Prod. 1993,545 825. 87 3.S. Moore H. Cho R. Casati E. Kenedy K. A. Reynolds U. Mocek J. M. kale and H. G. Floss,J.Am. Chem. Soc. 1993 115 5254. 88 P.Bhandari L.Crombie P. Daniels 1. Holden N. Van BrUggEn and D.A. Whiting J. Chem.Soc. Perkin Trans. 1 1992 839. 89 P.Bbandari L. Crombie G.W. Kilbee S.J. Pegg G. Proudfoot J. Rossiter M. Sanders and D.A. Whiting 1.Chem. Soc. Perkin Trans. I 1992 851. 90 P.Bhandari L. Crombie M. F. Harper J. Rossiter M. Sanders and D. A. Whiting J. Chem. Soc. Perkin Trans. 1 1992 1685. 91 L. Crornbie and D. A. Whiting Tetrahedron Lett. 1992 33 3663. 91 P.E. Flemming U. Mocek and H. G. Floss J. Am. Chem. Soc. 1993,115 805. Biosynthesis 325 Scheme 5 5 Alkaloids and Other Amino-acid-derived Metabolites Reviews have appeared on the biosynthesis of plant alkaloids and nitrogenous micro bid metabolites. 3,94 Labelled serine is a better precursor than alanine for the carbons and nitrogen of the dehydroalanyl portion of valanimycin (75) from Streptomyces uiridifaciens.Iso-butylamine and isobutylhydroxylamine are incorporated into the remaining carbons and nitrogen the latter very efficiently (Scheme 6).95It is not known how the N-N bond is formed in valanimycin (75). Labelling studies have established that ornithine is the precursor of avicin (76)from Streptomyces auiceusg6and that domoic acid (77)from Nitzscia pungens is derived from a geranyl precursor and a metabolite of the citric acid pathway.97 The hydroxycyclopentenone portion of reductiomycin (78) from Srrepto-myces xanthochromogenus has been shown to be derived from 5-arninolevuIinic acid 93 R.B. Herbert Nar. Prod. Rep. 1992 9 507. 94 R.B.Herbert Nut. Prod. Rep. 1993 LO 575. 95 R.J. Parry Y.Li and F.-L. Lii J. Am.Chem. Soc. 1992 114 10062. 96 S.J. Godd and S. Ju J. Am.Chem. Soc. 1992 114 10 166. 97 D.J. DougIas U.P.Ramsey J. A. Walker and J. L.C. Wright J. Chem. Soc. Chem. Comun. 1992 714. 326 R. A. Hill whereas the right-hand portion is derived by ring cleavage of a symmetrical product of the shikimic acid pathway.98 ,OH Several groups have been involved in biosynthetic studies of the tropane alkaloids. [l-13C]Acetate has been efficiently incorporated into ttopane alkaloids using hairy root cultures of Hywcyamus alb~s.'~ Intermediate trapping and competitive feeding experiments have established that phenyllactic acid (79)is a precursor of the tropic acid moiety of hyoscyamine and scopolamine (hyoscine) (81) from Dams str~rnoniurn.'~~-~~~ Transformed root cultures of a Datum hybrid were used to demonstrate that the 5a and 7cr hydrogens of hyoscyamine (80) are retained in the formation of scopolamine (81).Io3 OH (79) Transformed root cultures of Nicotiuna rustica have been used to convert the unnatural precursor N-ethylputrescine (82) into (S)-N-ethylnornicotine (831 with an 98 H.Cho,b.M.Beale,C.Graff U.Mocek A. Nakagawa S.Urnura,and H. G.Floss,J. Am. Chem.Suc. 1993 115 12296. 99 M. Sauerwein K. Shimomura and M. Wink Phytochemistry 1993,32,905. loo M. Ansarin and J. G. Wooley Phytochemistry 1993 32 1183. lo' M,Ansarin and J.G. Wooley J. Not. Prod. 1993 56 1211. lo' Y.Kitamura S. Nishima H. Miura and T.Kinoshita Phytochemistry 1993,34 425. lo' A. B. Watson I. K.A. Freer D.J. Robins and N. J. Walton J. Nut. Prod. i993,56 1234. Biusynthesis 327 H2N H-NEtI -+ N (82) (83) efficiency similar to that of the natural system which uses N-rnethylputre~cine.'~~ Detailed labelling experiments using benzoicacid benzaldehyde l-phenylpropane- 1,Zdione (84),and cathinone (85) to investigate the biosynthesis of the Ephedra alkaloids such as ephedrine (86) have been reported.'05 The results indicated that benzoic acid rather than benzaldehyde is involved in the biosynthetic pathway and that two carbons are added from pyruvate to produce l-phenylpropane-l,2-dione (84) and hence by transamination into cathinone (85) (Scheme 7). I Scheme 7 The stereochemistry of imidazoleglycerol phosphate dehydratase was studied using deuterium labelled precursors.lo' The study concluded that the reaction (Scheme 8) proceeded with inversion of configurationat C-3 of imidazole glycerol phosphate (87) and that the hydrogen added to C-2 comes from the medium. Scheme 8 Anatoxin a(s) (88) is a potent antichohesterase produced by Anabaena flosaquae. Feeding experiments using 'C-IabeHed precursors demonstrated that the carbon lo4 H. D. Boswell A. B. Watson N.J. Walton and D. J. Robins Phytochemistry 1993,34 153. '05 G.Grue-Ssrensen and I. D. Spenser J. Am. Chem. Soc. 1993 115 2052. lo' J. A. Moore A. R. Parker V. 1.Davisson and J. M. Schwab J. Am. Chem. Soc. 1993,115 3338 328 R. A. Hill atoms of the main skeleton are derived from arginine and the three methyl groups come from the C The labelled thiol(89) is incorporated into biotin (90)without loss of label; this finding gives sume insight into the introduction of sulfur into biotin.''' Full details of the biosynthetic studies on obafluorin (91) from Pseudomonas fluorexens have been published.'0g-' lo The labelling studies indicated that 4-aminophenylalanine 2,3-dihydroxybenzoic acid and giycine (which supplies carbons 1 and 2) are precursors... "K" +NH2 '",P/oMe+o -0' Labelling studies have shown that the quinolizidine (92) is an effective precursor of vertine (93) and lythrine (94) in Decodon uerticillaatus.' Labelled lysine was incorporated into ring A of these alkaloids and the results indicate that a symmetrical intermediate is involved.Full details of the biosynthetic studies on anosmine (95)from Dendrobium anosltturn have appeared. A detailed study on the biosynthesis of streptazolin (96)from a Streptomyces species has revealed that it has a polyketide origin with the carbonyl carbon being derived from the C pool.' The hydroxyl oxygen comes from molecular oxygen which suggests an epoxide intermediate. Feeding experiments using [1,2-'3C,]acetate to Lycopodiurn tristachyum indicated that the acetate derived C units of Jycopodine (97) are formed ID7 B. S. Moore 1. Uhtani C. 3.de Konig R. E. Moore and W. W. Carmichael Tetrahedron Lett. 1992.33 6595. *** A. Marguet F. Frappier G. Guillerm M. Azaulay D. Florentin and J.-C.Tabet,J. Am.Chern. SOC.,1993 115,2139. *09 R. B. Herbert and A. R. Knaggs J. Chem. Soc. Perkin Trans. I 1992 103. *lo R. 3. Herbert and A. R. Knaggs J. Chem. SOC.,Perkin Trans. I 1992 109. 'I1 S.H. Hedges R. B. Herbert,and P.C. Wormald J. Nar. Prod. 1993,56 1259. 'I2 T. Hemscheidt and 1.D. Spencer J. Nat. Prod. 1993 56 1281. M. Mayer and R. Thiericke J. Org. Chem. 1993 58 3486. Biosynthessis 329 though a symmetrical intermediate.' The phenazine antibiotic saphenamycine (98) from Streptomyces antibioticus has been shown to be derived from metabolites of the shikirnic pathway.'" 0 6Me (95) (93) R = aH (94) R= BH Oxazolomycin (99) has been shown to have a very interesting biosynthesis."6 Glycine is incorporated as the starter unit of two separate polyketide chains.Propionate is not incorporated; all the methy1 groups including the gsm-dirnethyl groups,are derived from methionine. The biosynthetic origin of the remaining carbons is still to be established. *Methionhe H3C-C02H 'I4 T. Hemscheidt and I.D. Spencer J. Am. Chem. Soc. 1993,115 3020. 'lS C. W.Van't Land U. Mocek and H. G. Floss J. Urg. Chem. 1993 58 6576. U. Grafe H. Kluge and R. Thiericke Leibigs Ann. Chem. 1992 429. 330 R. A. Hill Further details of the incorporation of glycerol into the rn-C,N unit of asukarnycin (100) from Streptomyces nodosus ssp. asukaensis have been published.' Biosynthetic studies on the aporphine and protoberberine alkaloids pruduced by cell cultures of Peumus boidus and Berberis stolorrijiera indicate dernethylation and remethylation occur readily via the C pooI.' l8 The biosynthesis of the cularine alkaloids in Corydalis claviculata and Sarcapnos crassifoh plants has been studied using 13C and I8O-labelled intermediates.l Precursor-directed biosynthesis of unnatural ergot alkaloids in Ciauiceps purpurea has been reported.Me I The biosynthetic pathways of a range of diketopiperazine alkaloids have been studied. N-Methylalbonoursin (101)from a Streptornyces species has been shown to be derived from leucine and phenylalanine."' Labelling studies using acetate and tyrosine have shown that all nine carbons of tyrosine are incorporated into sirodesmins A (1021 B (1031 and C (104)in Sirodemiurn diuersum.'22 The studies indicate that Claisen-type rearrangement of an 0-(3,3-dimethylallyl )tyrosine derivative is involved in the biosynthesis.[2,4,5,6,7-'H,]Tryptophan was incorporated into roquefurtine (105)by Penicilliurn roqueforti and into aszonalenin (106) by Aspergillus zonatus with retention in each case of all five deuterium atoms.123 The Sa-hydrogen of both metabolites is retained; this precludes the involvement of any 2-substituted indole intermediates in the biosynthesis. Incorporation studies using labelled acetate and rnevalonoIactones have established that the C-22 methyl groups become scrambled during the biosynthesis of fumitremorgin B (107) and verruculogen (108) by Penicilliurn uerrucutoswn.'24 It is proposed that the scrambling of the methyl groups occurs during H. Cho 1.Sattlet J. M. Beale A. Zeech and H.G. Floss J. Org. Chem. 1993 58 7925. ' B. Schneider and M. H. Zenk Phytochernisrry 1993 32 897. 119 M.J. Muelfer and M. H. Zenk Leibigs Ann. Chem. 1993 557. N. C. Perellino J. Malyszko M. Ballabio B. Gioia and A. Minghetti J. Nat. Prod. 1992 55 424. K. A. Gurney and P.G. Mantle J. Nut. Prod. 1993 56,1194. J. D. Bu'Lock and L. E. Clough Aust. J. Chem. 1992,45 39. 123 B. Bhat D.M. Harrison and H. M. Lamont Tetrahedron 1993 49 10663. "* R. Veggaar R. M. Horak and V. J. Maharaj J. Chem. SOC.,Chem. Commun. i993 274. Biosy nthesis 33 1 the formation of ring c. Evidence from feeding studies indicated that brevianamide A (109)irom Penicilliumbreoicompacfurnis probably nut formed via an indole derivative by a (4 + 2) cycloaddition.'2'~'z6 (102) n=2 (103) n=3 (104) n=4 (108) (1W The biosyntheses of the modified peptides thiostreptin from Streptornyces uzureus and Streptomyces luurentii,' nosiheptide from Streptornyces c~ctuosus and the destruxins from Metarhizurn anisopliae' 29 have been investigated.125 J. F. Sanz-Cervera T. Gliaka and R. M. Williams J. Am.Chem. Soc. 1993 115 347. 126 J. F. Sam-Cervera,T. Gliaka and R. M. Witliams Tetrahedron 1993,49 8471. 127 U. Mocek,Z. Zeng D. O'Hagan P. Zhou,L.-D.G. Fan J. M. Beale and H.G. Floss J. Am. Chem. Soc. 1993 115 7992. 128 U.Mocek,A. R. Knaggs R. Tsuchuja T. Nguyen J. M. Beale and H.G. Floss J. Am. Chem. SOC.,1993 115,7557. I29 A. Jegorov P. Sedmera and V. Matha Phytochemistry 1993 33 1403.332 R. A. Hill More results have been published in the area of B-lactam antibiotic biosynthesis. Full details of the studies using labelled valine in the formation of the ACV tripeptide by ACV synthase have been rep~rted.'~~-'~' The incorporation of '*O from both molecular oxygen and water into #l-Iactams using deacetoxy and deacetylcephalos- porin C synthase is interpreted as the first example of water exchange of intermediates in a non-haem iron a-ketoglutarate dependent di0xygena~e.I~~ Illuminating studies on the biosynthesis of clavulanic acid (10) in Streptornycas clavuligerus have been reported.'331135 The pathway has been elucidated and is shown in Scheme 9. The enzyme that catalyses the conversion of proclavaminic acid (I 11)into HO-..fJ H 0$YOk02H Scheme 9 130 J.E. Baldwin R.M. Adlington J.W. Bird R.A. Field N.M.O'Callaghan and C.J. Schofield Tetrahedron. 1992,48 1099. 131 J.E. Baldwin M. F.Byford R. A. Field C.-Y. Shiau W. J. Sobey,and C.J. Schofield Tetrahedron.,1993 49 322 1. 132 J. E. Baldwin R. M. Adlington N. P. Crouch I. A. C. Pereira,R. T. Aplin and C. Robinson J. Chem. Suc. Chem. Commun. 1993 i05. 133 B. P. Valentine C.R.BaiIey A. Doherty J. Moms S.W. Ebon K. H. Baggaky and N. H. Nicholson J. Chem. SOC.,Chem. Commun. 1993 1210. 134 S. W. Elson,K.H. Baggaley M. Fulston,N. H. NichoIson J. W. Tyler J. Edwards,H. Holms 1. Hamilton and D. MousdaIe J. Chem. Soc. Chern. Comun. 1993 121 t. 135 S. W. Elson K. H. Baggaley M. Davison M. Fulston N. H. Nicholson G.D.Risbridger and J. W. Tyler J. Chern. Soc. Chem. Commun. 1993 1212. Biosynthesis 333 clavaminic acid (112) has been well st~died.'~~-'~' Unnatural precursors have been used to probe the mechanism of this enzyme. 6 Porphyrins A book covering the biosynthesis of both linear and cyclic tetrapyrroles has been p~blished.'~' Phycobilins are open-chain tetrapyrroles that function as chromophores of light-harvesting chromoproteins in some photosynthetic organisms. The biosyn- thesis of these phycobilins has been re~iewed.'~' Genetic studies have had a great impact on studies of the biosynthesis of the tetrapyrr01es.l~~ Further evidence for the involvement of a spiro intermediate in the formation of uroporphyrinogen 111 from a linear hydroxyrnethylbilane has been presented.144 A synthetic spirolactam related to the proposed spiro intermediate was shown to be an inhibitor of cosynthetase (uroporphyrinogen I11 synthase). The studies of the biosynthesis of vitamin BI have been progressing and the pathway is now very well understood (Scheme The fate of the oxygen atoms in precorin-2 (112) when it is converted enzymatically into cobyrinic acid has been The results indicated that the carboxyl group of the C-2 acetategroup (*) in precorrin-2 (113) loses one labelled oxygen atom. This is consistent with the involvement of this carboxyl group in lactone formation. The structure of precorrin-3A (114) has been ~0nfirrned.l~~ Precorrin-3A (114) is converted into the hydroxy y-lactone precorrin-3B (3x) (115) by the enzyme encoded by gene CobG from Pseudornonas denitrificans.1487149 This enzyme contains iron and incorporates molecu-lar oxygen into precorrin-3B (115).150The ring contraction of precorrin-3B (1 15) is catalysed by the enzyme corresponding to gene CobJ. The product of this transform- ation was identified as precorrin-4 (116)' 51 Other workers identified factor IV the 35 J. E. Baldwin R. M. Adlington J. S. Bryans M. D. Lloyd T. J. Sewell,C. J. Schofield,K. H. Baggaley and R.Cassels J. Chem. Soc. Chem. Commun. 1992,877. 137 J. E. Baldwin K. D. Merritt C.J. Schofield S. W. Elson and K. H. Baggaley J. Chem. SOC. Chem. Commun. 1993 1301. 13' D. B. McElwaini and C. A. Townsend 1.Chem SOC.,Chem. Commun. 1993,1346. 139 D. Iwata-Reuyl A.Basak L. S. Silverman C.A. Engle,and C. A. Townsend,J. Nat. Prod. 1993,56,1373. 14* J.E. Baldwin V. Lee M. D. Lloyd,C. J. Schofield,S. W. Elson and K. H. BaggaIey,J.Chem. SOC.,Chem. Cornmun. 1993 1694. 14' 'Biosynthesis of Tetrapyrroies' ed. P.M. Jordan Elsevier Amsterdam 1991. S.I. Beak Chem. Rev. 1993,93,785. A.I. Scott Tetrahedron. 1992,48 2559. loo W.M. Stark C.J. Hawker G.J. Hart A. PhiIippides P. M. Petersen J. D. Lewis F. J. Leaper and A. R. Battersby .I.Chem. Soc. Perkin Trans. 1 1993,2875. 14' A.R. Battersby Acc. Chem. Res. 1993,26 15. 146 R. A. Vishwakarma S. Balachandran A. I. D. Alanine N. f.J. Stamford F. Kiuchi F. J. Leeper and A. R. Battersby J. Chem. SOC.,Perkin Trans. 1 1993,2893. 147 M. J. Warren C. A. Roessner S.-I. Ozaki N. J. Stolowich P.J.Santander and A. I. Scott Biochemistry 1992 31 603. A. I. Scott C. A. Roessner N. J. StoIowich J. B. Spencer C. Min and S.-I. Ozaki FE5S Lett. 1993,331 105. L.Debussche,D. Thibaut M. Danzer F. Debu D. Frkchet F. Herman F. Blanchqand M. VuiIhorgne,l. Chem. SOC.,Chem. Commun. 1993,1100. 150 J. B. Spencer N. J. Stolowich C.A. Roessner C. Min and A. 1.Scott J.Am. Chem. Soc. 1993,115,11610. A. I. Scott C. A. Roessner N. J. Stolowich J. B. Spencer C. Min and S.-I. Ozaki FEBS Lett. 1993,331 105. 14' 334 R. A. Hill IR' (115) R=H (114) R=CH3 Scheme 10 Biosynthesis 335 R' = CH,CH,CO,H; R2 = CH2C0,H Scheme 10 oxidation product of pre~orrin-4.'~~*'~~ The next step in the biosynthetic pathway is the addition of a methyl to C-11 of precorrin-4 (116) to produce precorrin-5 (117).'54 The acetyl group of precorrin-5 (117) is replaced by a methyl group to produce precorrindA (fix) (1 18).i55Precorrin-6A (6x)reducta~e'~~*'~~ (encoded by the gene 1S2 D.Thibaut L. Debussche D. Frechet F. Herman M. Vuilhorgne and F. Blanche J. Chem. SOC. Chem. Commun. 1993 513. 153 L. Debussche D. Thibaut M. Danzer F. Debu D. Frechet F. Herman and M. Vuilhorgne J. Chem.SOC. Chem. COmmUR. 1993 1100. 154 C. Min,B.P. Atshaves,C. A. Roessner N.J. StoIonwich J. B. Spencer,and A. I. Sc0tt.J. Am. Chem. Soc. 1993 115 10380. 155 F. Blanche M. Kodera M. Couder F.J. Leper D. Thibaut and A. R. Battersby J. Chem. Soc. Chem. Commun. 1992 i38. 156 F. Kiuchi D. Thibaut L. Debussche F.J. Leeper F. Blanche and A. R. Battersby J. Chem. Soc. Chem. Commun. 1992 306. 157 K. Ichinose M. Kodera F.J. Leeper and A. R. Battersby J. Chem. Soc. Chem. Commm. 1993 515. 336 R. A. Hill CO~K'~~) catalyses the reduction of precorrin-6A (118) to form precorrin-63 (6y) (119).lS9The enzyme that is encoded by CobLi6' catalyses the introduction of methyl groups at C-5 and C-15 of precorrin-6B and decarboxylation of the acetate side chain on C-12 to produce precorrin-8x (120).'61The transformation of precorrin-8x (120) into hydrogenobyrinic acid (121)is cataIysed by the enzyme encoded by CU~H'~~ and involves the migration of the methyl group at C-11to C-12 in a [1,5]-sigmatropic shift. The pathway to vitamin B, in anaerobic bacteria such as Salmonellatyphimuriurn is parallel but n0nidentica1.l~~ The methyl group of methionine has been shown to be the source of the methy1 groups at C-2 and C-7 of the porphyrin of cytochrome c3 in the anaerobic bacterium Desurovibrio vutgaris.164 7 Miscellaneous Metabolites Reviews on the biosynthesis of bacterial cell wall peptidogly~an'~' and the use of chiral methyl groups in biosynthetic studies'66 have been published.Further details of the biosynthesis of fosfumycin (122)from Streptomycesfiadiue have been p~bIished.'~~~~~~ The C-3 deoxygenation of pentopyranine C (123) from Streptornyces griseoch-romogenes has been studied. 69 Labelling studies using Streptomyces subrutilus which produces I-deoxynojirimycin (124) and I-deoxyrnannonojirimycin (125) have shown that the biosynthetic pathway involves the isomerism of glucose to fructose followed by oxidation of C-6 to an aldehyde transamination and cyclization.70 OH (124) R= aOH (125) R = f3OH F. Blanche D. Thibaut A. Famechon L. Debussche B. Cameron and J. Crouzet J.Bucteriol. 1992,174 1036. 159 F. Blanche F. Kuichi L. Debussche F.J. Leeper F. BIanche and A. R. Battersby J. Chem. SOC. Chem. Commun. 1992 139. F. Blanche A. Famechon D. Thibaut L. Debussche B. Cameron,and J. Crouzet,J. Bacreriot. 1992,174 1050. 16' D. Thibaut F. Kiuchi L.Debussche F. Blanche M. Kodera F. 3. Leeper and A. R. Battersby J. Chem. SOC.,Chem. Commun.,1992 982. 162 D. Thibaut M. Couder A. Famechon L. Debussche B. Cameron J. Crouzet and F. Bianche J. Bacteriol.1992 174 1050. 163 J. R. Roth J. G. Lawrence M. Rubenfield,S. Kieffer-Higgins and G.M. Church J.Bacreriol. 1993,175 3303. H. Akutsu J.4. Park and S. Sano J. Am. Chem. Soc. 1993 115 I2 185. 16' T.D.H. Bugg and C.T. Walsh Not. Prod. Rep. 1992 9 199. H. G. Floss and S. Lee Acc. Chem. Res. 1993,26 116. IC7 F. Hammerschmidt Leibigs Ann. Chem. 1992 553. ''* F. Hammerschmidt and H. Kahlig Leibigs Ann. Chem. 1992 1201. S.J. Gould and J. Guo J. Am. Chem. SOC. 1992 114 10 176. "* D. J. Hardick D. W. Hutchinson S. J. Trew and E. M.H. WelIington Tetrahedron 1992 48 6258.