11 Biological Chemistry Biosynthesis By R. A. HILL Department of Chemistry University of Glasgow G12 800 1 Introduction The previous Report' on biosynthetic studies of secondary metabolites and related work covered the period 1986-88. This Report will cover the period 1989-91. The study of biosynthetic pathways continues to be a highly active area. Genetic approaches to biosynthetic work are still making a huge impact. Improvements in the instruments and techniques of NMR and mass spectrometry are allowing rapid advances in the sophistication of biosynthetic studies. Several reviews have appeared dealing with general aspects of biosynthetic tech- niques,2 mechanisms of enzymic glycosyl transfer,6 enzyme-catalysed allylic rearrangement^,^ and enzyme-catalysed carbon-carbon bond formation.' Comprehensive reviews of biosynthesis of the major groups of natural products appear regularly in Natural Product Reports.The papers presented at an international symposium on bioorganic processes have been published.' This report must be highly selective and aims to highlight some important advances in biosynthetic studies. 2 Fatty Acid and Polyketide Biosynthesis An excellent book covering all aspects of fatty acid and polyketide biosynthesis has been published." One of the most important advances in this area is the use of Electrospray Mass Spectrometry (ESMS) to determine the mass of proteins with high accuracy." This technique allows study of sequential steps of a biosynthetic pathway in which the intermediates remain bound to a protein.ESMS has been used to observe previously well-characterized enzyme bound intermediates12 and to T. J. Simpson Annu. Rep. Bog. Chem. Sect. E 1988 85 321. E. Leete Latinoamer. Quim. Supplement 1 1990 37. H. G. Floss and J. M. Beale Angew. Chem. Int. Ed. Engl. 1989 28 146. R. B. Herbert 'The Biosynthesis of Secondary Metabolites' 2nd Edition Chapman and Hall London 1989. ' J. A. Robinson Chem. SOC.Rev. 1988 17 383. M. L. Sinnott Chem. Rev. 1990 90 1171. 'J. M. Schwab and B. S. Henderson Chem. Rev. 1990 90 1203. R. Kluger Chem. Rev. 1990,90 1151. 'Molecular Mechanisms in Bioorganic Processes' ed. C. Bleasdale and B. T. Golding Royal Society of Chemistry Cambridge 1990. 10 D. O'Hagan 'The Polyketide Metabolites' Prentice-Hall London 1991.'' J. B. Fenn M. Mann C. K. Meng S. F. Wong and C. M. Whitehouse Science 1989 246 64. 12 R. T. Aplin J. E. Baldwin C. J. Schofield and S. G. Waley FEES Lett. 1990 277 212. 283 284 R. A. Hill monitor active site-directed inhibition of an enzyme.13 The acyl carrier protein (ACP) involved in fatty acid biosynthesis in Succhuropolysporu erythreu was used to establish that reactions could be monitored using the ESMS te~hnique’~ and a chemically prepared acyl derivative of the ACP was prepared and acylated intermediates produced by the action of fatty acid synthase were detected by ESMS.” This technique has great potential for the study of biosynthetic pathways. The degradation of linoleic acid (1) and linolenic acid in plants is an important factor that influences the flavour of fruit and vegetables.The action of lipoxygenases on linoleic acid (1) to give 9-hydroperoxyoctadeca-lO,l2-dienoic acid (2) has been well established. The hydroperoxydienoic acid (2)is transformed into colneleic acid (4) which itself can be degraded into short chain aldehydes. Studies16 using deuterium labelling have shown that the 8-(pro-R)-hydrogen of the hydroperoxydienoic acid (2) is lost in its transformation into colneleic acid (4). The divinyl ether oxygen of colneleic acid (4) was shown to be derived from oxygen not water.” These results are consistent with the intervention of an epoxy-carbonium ion intermediate (3). -COOH -0 13 P. Caffrey B. Green L. C. Packman B. J. Rawlings J.Staunton and P. F. Leadlay Eur. J. Biochem 1991 195 823. 14 A. M. Bridges P. F. Leadlay W. P. Revill and J. Staunton J. Chem Soc. Chem. Commun. 1991 776. l5 A. M. Bridges P. F. Leadlay W. P. Revill and J. Staunton J. Chem. Soc. Chem. Commun. 1991 778. 16 P. Fahlstadius and M. Hamberg J. Chem. Soc. Perkin 1 1990 2027. 17 L. Crombie D. 0.Morgan and E. H. Smith J. Chem. SOC.,Perkin 1 1991 567. Biological Chemistry Biosynthesis 285 Isotopic labelling studies" are consistent with the hypothesis that 13-hydroperoxy- linoleic acid (5) is converted into 13-hydroxy-12-oxooctadec-9-enoic acid (7) and 9-hydroxy- 12-oxooctadec-10-enoic acid (8) by nucleophilic attack by water on an allene epoxide intermediate (6) at carbons 13 and 9 respectively., HOO COOH J COOH OH J COOH 0 (7) OH COOH 13-Hydroperoxylinolenic acid (9) is converted in a similar manner to the corres- ponding a-and P-ketols and 12-oxophytodienoic acid (1 1).Isotopic labelling results are consistent with a route via antarafacial ring closure of a zwitterion (10) derived from an allene epoxide." 12-Oxophytodienoic acid (11) is further degraded to produce cyclopentanones such as jasmonic acid (12) that have plant growth regulatory properties. Many plants and insects are able to produce terminally unsaturated hydrocarbons from saturated fatty acids the elimination proceeds via an anti-elimination of the carboxyl group and the 3-(pro-S)-hydrogen of the precursor acid in the plant Carthamus tinctorius" and the insect Tribolium consusum.21 These results have been used to investigate the stereochemistry of the reduction of the enoyl system during fatty acid biosynthesis in the same organisms.Feeding experiments (Scheme 1) indicated that an anti-Re,Re addition of hydrogen occurs.22 This stereochemistry of addition is different from those observed in other organisms.23 8-Decanolide (13) and y-dodecanolide (14) are flavour components in fruit milk products and fermented foods. Their biosynthesis in plants is thought to be by 18 L. Crombie D. 0. Morgan and E. H. Smith J. Chem. SOC. Perkin 1 1991 577. 19 L. Crombie D. 0. Morgan and E. H. Smith J. Chem. SOC. Perkin 1 1991 581. 20 G. Gorgen and,W. Boland Eur. J. Biochem. 1989 185 237. 21 G.Gorgen C. Frossl and W. Boland Experientiu 1990 46 700. 22 C. Frossl and W. Boland J. Chem. SOC. Chem. Commun. 1991. 1731. S. A. Benner A. Glasfeld and J. A. Piccirilli Topics in Stereochem. 1989 19 193. 286 R. A. Hill OOH COOH 0- COOH Scheme 1 degradation of fatty acids. It was found that micro-organisms can be used to produce these flavour component^.^^ Thus Cludosporium suuueolens converted 13-hydroxy- octadeca-9,11 -dienoic acid (1 5) into 8-decanolide (13) and Yurrowiu lipolyticu con-verted 10-hydroxyoctadec-8-enoicacid (16) into y-dodecanolide ( 14). Labelling studiesz5 have shown that the addition of hydrogens to the diene system of 13- hydroxyoctadeca-9,ll-dienoicacid (15) in the biosynthesis of the 8-lactone (13) 24 R.Cardillo G. Fronza C. Fuganti P. Grassieli D. Pizzi G. Allegrone M. Barbeni and A. Pisciotta J. Org. Chem. 1991 56 5237. 25 G. Fonza C. Fuganti P. Grassieli A. Mele G. Allegrone M. Barbeni and A. Pisciotta J. Chem. SOC. Perkin 1 1991 2977. Biological Chemistry Biosynthesis AoAo H 0 HO occurs on the same face of the molecule and that the addition of hydrogen in the y-lactone (14) shows the same steric course. The biosynthesis of lipoxins and related eicosanoids has been reviewed.26 The relationship between fatty acid and polyketide biosynthesis has been the subject of much research. The pathways appear to be very similar however during polyketide biosynthesis reduction of a carbonyl to give an alcohol can often occur with stereochemistry (S) opposite to that (R) thought to be general for fatty acid synthase enzymes.The stereochemistry of carbonyl reduction in fatty acid and polyketide biosynthesis in the same organism has been investigated using appropriate labelling studies. Oleic acid (17) is produced in the same organisms as cladosporin (18) from Cladosporium cladosporioide~~~ dehydrocurvularin (19) from Alternan'a cinerariae and antibiotic A26771 B (20) from Penicillium turbatum.** In each organism 26 K. C. Nicolaou J. Y. Ramphul N. A. Petasis and C. N. Serhon Angew. Chem. Int. Ed. Engl. 1991 30,1100. 21 B. J. Rawlings P. B. Reese S. E. Ramer and J. C. Vederas J. Am. Chem. SOC.,1989 111 3382. 28 K. Arai B. J. Rawlings Y. Yoshizawa and J. Vederas J. Am. Cbem. SOC,1989 111 3391.288 R. A. Hill the oleic acid biosynthesis involved placing acetate derived hydrogens in the pro-R position on the growing fatty acid chains whereas the corresponding polyketide leaves hydrogens in the pro4 position in the growing chain. Although only a small number of organisms have been studied it does appear that the stereochemistry of enoyl thiol ester reductase involved in fatty acid biosynthesis and polyketide forma- tion in fungi may be opposite. The structure and function of polyketide synthase complexes active in the produc- tion of antibiotics in Streptomyces species has been reviewed.29 The intermediates formed on the polyketide synthase from Aspergillus melleus have been studied with N-acetylcysteamine (NAC) thioesters.The intact incorporation of deuterium labelled NAC thioesters of crotonic 2,4-hexadienoic and 2,4,6-octatrienoic acids into aspyrone (21)30 support the hypothesis that aspyrone polyketide synthase resembles a fatty acid synthase in its mode of operation and that it follows a processive mode of operation. This hypothesis was further strengthened by labelling studies3* that showed that the NAC thioester of (R)-3-hydroxybutanoic acid is incorporated into aspyrone without loss of the hydrogen at C-3. The sequence of intermediates bound to the acyl carrier protein (ACP) of the aspyrone polyketide synthase is shown in Scheme 2. Dueterium labelled 3-hydroxydeca-4,6,8-trienoic acid (22) was incorporated intact into a~pyrone.~~ Studies using I7Olabelled acetate and I7O2 as precursors and 170 NMR spectroscopy to analyze the results have ACP.S 00 0 I 29 J. A. Robinson Phil. Trans. R. SOC.Land. B 1991 332 107. 30 J. Staunton and A. C. Sutkowski J. Chem. Soc, Chem. Commun. 1991 1110. 31 A. Jacobs J. Staunton and A. C. Sutkowski J. Chem. SOC.,Chem. Commun. 1991 1113. 32 J. Staunton and A. C. Sutkowski J. Chem. Soc. Chem. Commun. 1991 1108. 289 Biological Chemistry Biosynthesis .o -+.5? H HO Scheme 3 shown that aspyrone (21) asperlactone (24) and isoasperlactone (25) arise from a common diepoxide intermediate (23) (Scheme 3).33 The biosynthesis of pseudomonic acid A (26) was investigated using labelled acetate precursors and the results show that pseudomonic acid A (26) is formed from separate CI7and C9 moieties linked at the ester and that it is not formed by a Baeyer-Villiger type cleavage of a single long-chain ketone intermediate.The proposed involvement of 3-hydroxy-3-methylglutaricacid in the biosynthesis of pseudomonic acid A (26) was not verified.34135 OH The biosynthetic origins of the hydrogen atoms of the antibiotic nonactin (27) have been studied by following the incorporations of deuterium and I3C en-riched acetate propanoate and succinate using cultures of Streptomyces gri~eus.~~ 33 J. Staunton and A. C. Sutkowski J. Chem. SOC.,Chem. Commun. 1991 1106. 34 F. M. Martin and T. J. Simpson J. Chem. SOC.,Perkin 1 1989 207. 35 P. G. Mantle and K. M. Macgeorge J. Chem. SOC.,Perkin 1 1991 255.36 D. M. Ashworth C. A. Clark and J. A. Robinson J. Chem. SOC.,Perkin 1 1989 1461. 290 R. A. Hill Mechanisms are proposed to explain the formation of (+)-and (-)-nonactic acids from these precursors catalysed by a nonactin polyketide synthase multi-enzyme complex. The biosynthesis of this and other polyether ionophore antibiotics are covered in an excellent comprehensive review.37 Feeding studies with labelled precursors38 are consistent with solanapyrone A (29) from Alternaria solani being formed via an intramolecular Diels-Alder reaction of an intermediate such as (28). Further indirect evidence is provided by the isolation of solanapyrone D (30) from the same organism.39 Solanapyrone D (30) is apparently -OMe CHO H (29) I CHO H the result of an endo-Diels-Alder cycloaddition whereas solanapyrone A (29) is the result of an exo-addition.The previous proposal4' that betaenone B is biosynthe- sized via a Diels-Alder reaction is further strengthened by the isolation of probe- taenone 1 (32) from Phoma betae indicating a cyclization of an intermediate such as (31).41 Chirally labelled malonyl-CoA has been incorporated into 6-methylsalicylic acid (6-MSA)(34) using 6-MSA synthase from Penicillium patuZum?2 The results show that the hydrogen atoms removed from the two methylene groups at the 2- and 4-positions in the putative polyketide intermediate have opposite absolute stereochemistry. 37 J. A. Robinson Prog. Chem. Org. Nut. Prod. 1991 58 1. 38 H. Oikawa T.Yokota T. Abe A. Ichihara S. Sakamura Y. Yoshizawa and J. Vederas J. Chem. SOC. Chern. Commun. 1989 1282. 39 H. Oikawa T. Yokota A. Ichihara and S. Sakamura J. Chem. SOC., Chem. Commun. 1989 1284. 40 H. Oikawa A. Ichihara and S. Sakamura J. Chem. SOC.,Chem. Commun. 1988 600. 41 S. Miki Y. Sato H. Tabuchi H. Oikawa A. Ichihara and S. Sakamura J. Chern. Soc. Perkin I 1990 1228. 42 P. M. Jordan and J. B. Spencer J. Chem. SOC.,Chem. Commun. 1990 238. Biological Chemistry Biosynthesis 291 J FCOOH OH (34) An insight into the intriguing biosynthetic control of regiochemistry of intramolecular aldol reactions is provided with the molecular genetic study43 of the actinorhodin (35)gene cluster from Streptomyces coelicolor. Mutactin (36)is a shunt metabolite produced by a mutant strain.The results indicate that a polyketide aldolase specifies the correct cyclization of a complex oligoketide chain. 0 OH \ I I COOH OH (35) (36) D. H. Sherman M. J. Bibb T. J. Simpson D. Johnson F. Malpartida M. Fernandez-Moreno E. Martinez C. R. Hutchinson and D. Hopwood Tetrahedron 1991 47 6029. 292 R. A. Hill The stereochemistry of hydrogen loss on formation of the vinyl group of ravidi- mycin (37) from Streptomyces ruvidus has been investigated by incorporation experi- ments using chirally labelled [2-2H,]propionate.44 The results indicate that the pro-S hydrogen of propionic acid is retained in the biosynthesis of the vinyl group of ravidimycin (37). OH OMe Sugar 0 (37) Anthraquinones such as austrocorticin (38) isolated from a toadstool of the Dermocybe genus have been shown to be derived from a propionate starter whereas norastrocorticin (39) and other anthracene metabolites from D.sanguine^^^ are derived from an acetate starter unit. MeOM (38) R=Me (39) R=H The biosynthesis of citreamicin (40) from Micromonosporu citreu has been studied using labelled precurs0rs.4~ A complex rearrangement of a polyketide intermediate is postulated and the '80-incorporation studies indicate some surprising results. The urdamycins are an interesting group of antibiotics from Streptomyces frudiae. They have been shown to be derived from a single decaketide chain.48 A series of non-enzymatic processes has been shown to occur in the elaboration of the urdamycin nucleus.Non-enzymatic condensation of urdamycin A (41) with 4-hydroxy- phenylpyruvic acid leads to urdamycin C (43).49 Urdamycin C (43) undergoes a novel non-enzymatic ring contraction to produce urdamycin H (44).50Urdamycin 44 R. F. Keyes and D. G. I. Kingston J. Org. Chem. 1989 54 6127. 45 M. Gill and A. GimCnez J. Chem. SOC.,Perkin 1 1990 1159. 46 M. Gill and A. GimCnez J. Chem. SOC.,Perkin 1 1990 2585. 47 G. T. Carter D. B. Borders J. J. Goodman J. Ascroft M. Greenstein W. M. Maiese and C. J. Pearce J. Chem. Soc. Perkin 1 1991 2215. 48 J. Rohr J. M. Beale and H. G. Floss J. Antibiot. 1989 42 1151. 49 J. Rohr J. Chem. SOC.,Chem. Commun. 1990 113. 50 J. Rohr Angew.Chem. Int. Ed. 1990 29 1051. Biological Chemistry Biosynthesis (41) X=H (42) X=SMe (43) RO (45) R = Sugar residues E (42) is formed by a non-enzymatic Michael-type addition (followed by oxidation) to urdamycin A (41).51Involvement of non-enzymatic processes may be considered for other biosynthetic schemes especially if there is a wide range of metabolic products. The antibiotic pradimicin A (45) from Actinomadura hibisca has been shown to be biosynthetically derived from 12 acetate units. D-Alanine is efficiently incorpor-ated into the side chain.52 3 Terpenoids Severalcomprehensive reviews on the biosynthesis of terpenoids have been published including the lower terpen~ids,~~-~~ sesquiterpene lac tone^,^^ mon~terpenoids,~~~~~ se~quiterpenes,~~ gibberellins,6' diterpene phytoalexins,6' marine natural products,62 triterpenoids and carotenoids,@ and biosynthetic methods.65 51 J.Rohr J. Chem. SOC.,Chem. Commun. 1989 492. 52 M. Kakushima Y. Sawada M. Nishio T. Tsuno and T. Oki J. Org. Chern 1989 54 2536. 53 M. H. Beale Nut. Prod. Rep. 1990 7 25. " M. H. Beale Nu?. Prod. Rep. 1990 7 387. 5s M. H. Beale Nut. Prod. Rep. 1991 8 441. 56 P. A. Vatakencherry and K. N. hshpakumari J. Sci. Ind. Res. 1989 48 31. 57 J. Gershenzon and R. Croteau Rec. Ado. Phytochem. 1990 24 99. 58 N. H. Fischer Rec. Adu. Phytochem. 1990 24 161. 59 D. E. Cane Chem. Rev. 1990 90,1089. 60 B. 0. Phinney and C. R. Spray Rec. Adv. Phytochem. 1990 24 203. 61 C. A. West A. F.Lois K. A. Wickham and Y.-Y. Ren Rec. Adv. Phytochem. 1990 24 219. 62 M. J. Garson Nut. Prod. Rep. 1989 6 143. 63 W. D. Nes Rec. Adu. Phytochem. 1990 24 283. 64 D. M. Harrison Nut. Prod. Rep. 1990 7 459. 65 J. Gershenzon D. McCaskill J. Rajaonarivony C. Mihaliak F. Karp and R. Croteau Rec. Adu. Phytochem. 1991 25 347. 294 R. A. Hill Advances have been made into the enzymology and genetics of the early stages of terpenoid biosynthesis. Studies on mevalonate-5-phosphate decarboxyla~e~~~~~ have indicated that the enzyme contains sulfydryl groups that are involved in substrate binding. The gene encoding isopentenyl diphosphate isomerase has been isolated.68 An investigation of this enzyme from Saccharomyces cerevisiae using site-directed mutagenesis has shown that the active-site residue Cyst-139 is involved in the catalytic action (Scheme 4).69 I-I PPO&-PPO& Scheme 4 Analysis of kinetic isotope effects using geranyl diphosphate (46) demonstrated that a-pinene (47) and P-pinene (48) are formed by the same enzyme in sage.70 Extensive studies on the biosynthesis of fenchol (49)71 and sabinene hydrate (50)72 have been reported.The Cll-homoterpene (51) and its C16-analogue (52) are found as the major constituents of the volatile oils of many plants. It has been shown that they are derived biosynthetically by the cleavage of a C4-unit from C15 and C20 precursors re~pectively.~~’~~ The recent work on the enzyme trichodiene synthase from Trichotheciurn roseurn has been ~urnmarized.~’ This enzyme converts farnesyl diphosphate into trichodiene 66 M.Alvear A. M. Jabalquinto and E. Cardemil Biochem. Biophys. Acta 1989,994 7. 67 A. M. Jabalquinto and E. Cardemil Biochem. Biophys. Acta 1989 996 257. M. S. Anderson M. Muehlbacher I. P. Street J. Proffitt and C. D. Poulter J. Biol. Chem. 1989 264 19169. 69 I. P. Street H. R.Coffman and C. D. Poulter Tetrahedron 1991 47 5919. 70 K. C. Wagschul T. T. Savage and R. Croteau Tetrahedron 1991,47 5919. 71 R. Croteau J. H. Miyazaki and C. J. Wheeler Arch. Biochem. Biophys. 1989 269 507. 72 T. W. Hallahan and R. Croteau Arch. Biochem. Biophys. 1989 269 313. ’3 W. Boland and A. Gabler Helv. Chem. Acta 1989 72 247. 74 A. Gabler W. Boland U. Preiss and H. Simon Helv.Chim. Acta 1991 74 1773. 7s D. E. Cane Pure Appf. Chem. 1989 61 493. Biological Chemistry Biosynthesis 295 (53) the isolation and sequence of the gene coding for this enzyme has been reported.76 Trichodiene (53) is the precursor of the trichothecene family of mycotoxins. The biosynthesis of 3-acetyldeoxynivalenol (54)from Fusarium cul-morurn has been reviewed.77 Post-trichodiene intermediates in the biosynthesis of 3-acetyldeoxynivalenol (54) from Fusarium culmorum have been isolated. Isotrichodiol (55)78 and 12,13-epoxy-9-trichothecen-2-o1(56) were incorporated into 3-acetyldeoxynivalenol(54)whereas 9-trichothecene-12,13-diol(57),although it was produced from trichodiene (53) by cultures of Fusarium culmorurn treated with the inhibitor ancymidol was not incorporated into the trich~thecenes.~~ (53) (54) Aristolochene synthase which has been isolated from cell-free extracts of Aspergil-lus terreus" and Penicillium roquefortii,'l catalyses the conversion of farnesyl diphos- phate into aristolochene (58).The mechanism of this cyclization has been studied using deuterium labelled precursors.82 The mechanism of this and other enzyme- catalysed allylic addition-elimination reactions in terpenoid biosynthesis has been reviewed.83 76 T. M. Hohn and P. D. Beremand Gene 1989 79 131. 77 L. 0.Zamir Tetrahedron 1989 45 2277. 78 A. R. Hesketh L. Gledhill D. C. Marsh B. W. Bycroft P. M. Dewick and J. Gilbert J. Chem. SOC. Chem. Commun. 1990 1184. 79 L. 0. Zamir K. A. Devor N. Morin and F.Sauriol J. Chem. SOC.,Chem. Commun. 1991 1033. D. E. Cane P. C. Prabhakaran E. J. Salaski P. H. M. Harrison H. Noguchi and B. J. Rawlings J. Am. Chern. SOC.,1989 111 8914. 81 T. M. Hohn and R. D. Plattner Arch. Biochem. Biophys. 1989 272 137. 82 D. E. Cane P. C. Prabhakaran J. S. Oliver and D. B. McIlwaine J. Am. Chem. Soc. 1990 112 3209. D. E. Cane C. Abell P. H. M. Harrison B. R.Hubbard C. T. Kane R. Lattman J. S. Oliver and S. W. Weiner Phil. Trans. R SOC.Lond. 1991 332 123. 296 R. A. Hill The biosynthesis of abscisic acid (59) in higher plants has been re~iewed.'~ Abscisic acid (59) originates by breakdown of carotenoid precursors in higher plants but has been shown to be formed directly from farnesyl diphosphate in several species of fungi.Further evidepce that abscisic acid (59) is produced from carotenoids in plants has been provided by the use of 1802 incubationss5986and 2H20studies." The biosynthesis of the clerodane skeleton in Tinospora cordifoh has been investigated using labelled mevalonic acid precursors." The results indicate that two 1,2-hydrogen shifts and two 1,2-methyl shifts occur during the formation of the ring system. The order of introduction of the hydroxyl groups in aphidicolin (60) using cultures of Phoma betae has been shown to be C-18 C-17 and finally C-3.89 Austin (61)90 and terretonin (62)9' are formed by a mixed terpenoid-polyketide pathway. Incorporation studies using labelled acetates methionine and 33- dimethylorsellinate (63) have shown that the biosynthetic pathways involve C- alkylation of 3,5-dimethylorsellinate (63) with farnesyl diphosphate an insight into the mechanisms of the biosynthetic steps that follow is provided with an analysis of the labelling patterns of these complex metabolites.Views on the evolution of terpenes to sterols have been presented.92 The biosyn- thesis of squalene has been reviewed.93 The enzyme that catalyses the cyclization of oxidosqualene to lanosterol has been purified from yeast and the enzymatic 84 R. A. Creelman Physiol. Pluntarium 1989 75 131. 85 J. A. D. Zeevaart T. G. Heath and D. A. Gage Plant Physiol. 1989,91 1594. 86 D. A. Gage F. Fong and J. A. D. Zeevart Plunf Physiol. 1989 89 1039. 87 R. W. Willows and B. V. Milborrow Phytochemistry 1989 28 2641.88 A. Akhila K. Rani and R. G. Thakur Phytochemistry 1991 30 2573. 89 H. Oikawa A. Ichihara and S. Sakamura Agric. Biol. Chem. 1989 53 299. 90 S. A. Ahmed F. E. Scott D. J. Stenzel T. J. Simpson R. N. Moore L. A. Trimble K. Arai and J. C. Vederas 1. Chem. SOC.,Perkin 1 1989 807. 91 R. McIntyre F. E. Scott T. J. Simpson L. A. Trimble and J. C. Vederas Tetrahedron 1989 45 2307. 92 G. Ourisson Pure Appl. Chem. 1989 61 345. 93 M. Y. Julia Chem. SOC.Rev. 1991 20 129. Biological Chemistry Biosynthesis properties de~cribed.9~ The cyclase of the protozoon Tetruhyrnenu pyriformis which normally converts squalene into the pentacyclic tetrahymanol (64) cyclized 2,3- dihydrosqualene into euph-7-ene (65) with an unexpected tetracyclic skeleton’ and backbone rearrangement.” Incorporation studies with [1 2l3C2]acetate have shown that the 3a-hydroxylanostane ganoderic acid S (66) is produced by the fungus Gunoderrna Zucidurn from (3s) -squalene 2,3-e~oxide.~~ 94 T.Hoshino H. J. Williams Y. Chung and A. I. Scott Tetrahedron 1991 47 5925. 95 I. Abe and M. Rohmer J. Chem. SOC. Chem. Commun. 1991 902. % M. Hirotani I. Asaka and T. Furuya J. Chem. SOC.,Perkin 1 1990 2751. 298 R. A. Hill An extensive study of the biosynthesis of cholesterol and lanosterol in isolated dog hepatocytes using 13C- and *H-labelled acetates and ethanol and exacting NMR studies have verified the earlier findings on cholesterol bio~ynthesis?~ Similar studies by the same group have elucidated the biosynthetic pathways to cycloartenol (67)98 and isofucosterol (68)99using tissue cultures of Physallis peruuiana.A number of papers have appeared concerning the details of the biosynthesis of the side-chains of sterols such as dinosterol (69) from Cryptothecodinium cohnii peridinosterol from Peridinium foliaceum gorgosterol from Cassiopea xamachana,'OO 24-propy-lidenecholesterol from Crysoderma mucosa,'o' cyclopropane-containing sterols of marine originlo2 and 24-ethylster0ls.'~~~'~~ The work on 24-ethylsterols contradicted earlier work on poriferasterol (70) but the earlier work was shown to be in error due to incorrect assignments of the I3C NMR spectra these errors have subsequently been corrected.'05 Studies on the biosynthesis of cardenolides in Asclepias curassavica did not support the hypothesis that the butenolide ring is formed by the condensation I 97 S.Seo H. Saito A. Uomori Y. Yoshimura K. Tonda Y. Nishibe M. Hirata Y. Takeuchi K. Takeda H. Noguchi Y. Ebizuka U. Sankawa and H. Seto J. Chem. SOC.,Perkin 1 1991 2065. 98 S. Seo A. Uomori Y. Yoshimura K. Takeda H. Seto Y. Ebizuka H. Noguchi and U. Sankawa J. Chem. SOC.,Perkin 1 1989 261. 99 S. Seo A. Uomori Y. Yoshimura H. Seto Y. Ebizuka H. Noguchi U. Sankawa and K. Takeda J. Chem. SOC.,Perkin 1 1990 105. 100 J.-L. Giner and C. Djerassi J. Org. Chem. 1991 56 2357. 101 J.-L. Giner and C. Djerassi J. Am. Chem. SOC.,1991 113 1386. lo* C. Djerassi and G. A. Doss in 'Studies in Natural Product Chemistry' ed.Atta-ur-Rahman Elsevier Amsterdam 1991 vol. 9 15. 103 I. Horibe H. Nakai T. Sato S. Seo K. Takeda and S. Takatsuto J. Chem. SOC.,Perkin 1 1989 1957. 104 S. Seo A. Uomori Y. Yoshimura K. Takeda H. Seo Y. Ebizuka H. Noguchi and U. Sankawa J. Chem. SOC.,Perkin 1 1989 1969. 105 D. Colombo F. Ronchetti G. Russo and L. Toma J. Chem. SOC.,Perkin 1 1991 962. Biological Chemistry Biosynthesis of a pregnane derivative with one molecule of acetate.'06 A study using l80-and 2H-labelled acetate and lSO2 has determined the mechanism of the side-chain cleavage of pregnenolone (7 1) (Scheme 5).'07 Labelled glucose has been incorporated i fi+ CH,COOH Scheme 5 into the side chain of bacteriohopanetetrol (72) and the results show that a ribose derivative is a precursor."* Labelled mevalonic acid was used to show that the aromatic ring D of nic-1 (73) from Nicandra physaloides is formed by ring D expansion of a steroid precursor with oxidative inclusion of the 18-methyl group'" and that it is formed from 24(28)-methylenecholesterol,details of the pathway have been elucidated."' / 106 H.W. Groeneveld A. Binnekamp and D. Seykens Phytochemistry 1991 30,2577. 107 S. L. Miller J. N. Wright D. Corina and M.Akhtar J. Chem. SOC.,Chem. Commun. 1991 157 548 and 792 (note that there are two corrigenda to this paper). 108 M. Rohmer B. Sutter and H. Sahm J. Chem. SOC.,Chem. Comrnun. 1989 1471. 109 H. K. Gill R. W. Smith and D. A. Whiting J. Chem. SOC.,Perkin 1 1990 2989. 1LO W.Andrews-Smith H. K. Gill R. W. Smith and D. A. Whiting J. Chem. Soc. Perkin 1 1991 291. 300 R. A. Hill 4 Shikimate and Related Metabolites An extensive review covering the molecular biology and biochemistry of the shiki- mate pathway has been published."' Regular comprehensive reviews have appeared covering all aspects of shikimate metabolite^."^-^^^ Studies on the enzymes in chorismate metabolism and the enterobactin biosynthetic pathway have been detailed."' Dehydroquinate synthase catalyses the conversion of 3-deoxy-~-arabino-heptulosonate-7-phosphate (DAHP) (74) into 3-dehydroquinate (75). The enzyme has been isolated and purified to homogeneity from Streptomyces coelicolor and has been found to be extremely thermostable."6 The enzyme has also been purified from Escherichia coli and has been shown to be a monomeric metalloenzyme containing one tightly bound Co2+."' The metal-free apoenzyme is not catalytically active activity is fully restored with Co2+ and to about half this level with Zn2+.However in vivo the enzyme is believed to contain Zn2+ rather than Co2+ the latter being introduced during the purification procedure. Dehydroquinate synthase also binds one molecule of NAD+. The overall enzyme mechanism has been well studied and the findings reviewed."* _. HxoH 0-OH I OH IOH (74) (75) Shikimate-3-phosphate (76) is converted to 5-enolpyruvylshikimate 3-phosphate (EPSP) (78) by EPSP synthase. Further evidence for the existence of the tetrahedral intermediate (77) has been provided."' Synthetic analogues of this intermediate have been shown to be potent inhibitors of the enzyme from Petunia hybrida.12' An extensive review of EPSP synthase has been pubiished.121 EPSP (78) is converted into chorismate (79) by chorismate synthase.This enzyme has been isolated and purified from a cell culture of Corydalis sempervirens.'22 Debate over the mechanism of action of this enzyme continues but an anti-1,4-elimination seems to be fa~0ured.l~~ Chorismate (79) is converted into isochorismate (80) by isochorismate synthase. This enzyme has been purified and ~haracterized.'~~ The hydroxyl group 111 R. Bentley Crit. Rev. Biochem. Mol. Biol. 1990 25 307. P. M. Dewick Nut. Prod. Rep. 1989 6 263. 113 P. M. Dewick Nut. Prod.Rep. 1990 7 165. 114 P. M. Dewick Nut. Prod. Rep. 1991 8 149. C. T. Walsh J. Liu F. Rusnak and M. Sakaitani Chem. Rev. 1990 90 1105. 116 P. J. White J. Young I. S. Hunter H. G. Nimmo and J. R. Coggins Biochem J. 1990 265 735. 117 S. L. Bender S. Mehdi and J. R.Knowles Biochemistry 1989 28 7555. 118 J. R. Knowles Aldrich Actu 1989 22 59. P. N. Barlow R. J. Aplleyard B. J. 0.Wilson and J. N. S. Evans Biochemistry 1989 28 7555. 120 D.G.Alberg and P. A. Bartlett J. Am. Chem. Soc. 1989 111 2337. 121 K. S. Anderson and K. A. Johnson Chem. Rev. 1990 90 1131. 12* A. Schaller V.Windhofer and N. Amrhein Arch. Biochem. Biophys. 1990 282 437. 123 S. Balasubramanian C. Abell and J. R. Coggins J. Am Chem Soc. 1990 112 8581. 124 J. Liu N. Quin G. A.Berchtold and C. T. Walsh Biochemistry 1990 29 1417. Biological Chemistry Biosynthesis COOH COOH @o" OH I OH OH (76) (77) I COOH COOH OH OH (79) (78) \ COOH of isochorismate is labelled by H2180 indicating that a 1,5-addition/elimination sequence occurs.125 Phenylpropanoid metabolism and molecular biology associated with the biosyn- thesis of flavonoids lignans and coumarins have been reviewed.'26 Simple coumarins from higher plants are normally derived by cyclization of a cinnamic acid precursor however 4-hydroxy-5-methylcoumarin(8 1) found as its glucoside in Gerbia jarnesonii has been shown to be of polyketide rigi in.'^' The incorporation of phenylpropanoids into cell walls including lignins has been reviewed,'** lignan biosynthesis has been covered in recent and three reviews on lignin biosynthesis have a~peared.'~'-'~~ Natural lignans are normally enantiomerically pure and arise from a stereocon- trolled coupling process.Evidence for the conversion of coniferyl alcohol (81) into 125 S. J. Could and R. L. Eisenberg Tetrahedron 1991 47 5979. 126 K. Hahlbrock and D. Scheel Annu. Rev. Plant Physiol. Plant Mol. .BioL 1989 40,347. 12' T. Inoue T. Toyonaga S. Nagumo and M. Nagai Phytochemistry 1989 28 2329. 128 E. Yamamoto G. H. Bokelmam and N. G. Lewis ACS Symp. Ser. Plant Cell Wall Polymers Biogenesis and Biodegradation 1989 399 68. 129 P. M. Dewick in 'Studies in Natural Product Chemistry' ed. Atta-ur-Rahman 1989 5 459 Elsevier Amsterdam. 130 D.C.Ayres and J. D. hike 'Lignans-Chemical Biological and Clinical Properties 1990 Cambridge University Press Cambridge. 131 N. G. Lewis and E. Yamamoto Annu. Rev. Plant Physiol. Plant Mol. Biol. 1990 41 455. 132 T.Higuchi Wood Sci. TechnoL 1990 24 23. 133 C. Haertig D. Meyer and K. Fischer 2. Chem. 1990 30,233. 302 R. A. Hill CHO 6 'OMe pinoresinol (82) in a direct stereochemically controlled coupling by a crude enzyme preparation from Forsythia intermedia has been pr0~ided.I~~ The stereospecific coupling of coniferyl alcohol (81) to produce secoisolariciresinol (83) was also demonstrated using a cell-free extract of Forsythia intermedi~.'~' The biosynthesis of flavonoids has been reviewed'36 and the biosynthesis of proanthocyanidins has been covered in a recent b00k.l~~ Chalcomoracin (84) and H Meovg HO \ H HO 1 OH OH Y (83) (84) other chalcone dimers e.g.the kuwanones have been isolated from various species of mulberry (Moms spp.). Evidence is presented that they arise by an enzymic Diels-Alder coupling of chalcone precursor^.'^^ The conversion of flavanones into isoflavanones has been well studied. It is catalysed by the enzyme isoflavone synthase which is a cytochrome P-450-dependent enzyme requiring NADPH and O2.139 Details of the mechanism of the conversion of the flavanone (85) into the 2-hydroxyisoflavanone (86) have been pre~ented.'~' The cyclization of isoprenyl side- chains in flavonoids and isoflavonoids is catalysed by cyclase enzymes. The stereochemical aspects of the cyclization of rotenoic acid (87) to deguelin (88) in Tephrosia vogellii have been in~estigated.'~' 134 T.Umezawa L. B. Davin E. Yamamoto D. G. I. Kingston and N. G. Lewis J. Chem. Soc. Chem. Commun. 1990 1405. 13' T. Umezawa L. B. Davin and N. G. Lewis Biochem. Biophys. Res. Commun. 1990 171 1008. 136 G. Hrazdina and R. A. Jensen UCLA Symp. Molecular Cellular Biol. New Series 1990 133 27. 137 'Chemistry and Significance of Condensed Tannins' ed R. W. Hemmingway and J. J. Karchesy 1989 Plenum Press New York. 138 Y. Hano T. Nomura and S. Ueda J. Chem. SOC.,Chem. Commun. 1990 610. 139 T. Hakamatsuka H. Noguchi Y. Ebizuka and U. Sankawa Chem. Pharm. Bull. 1990 38 1942. 140 T. Hakamatsuka M. F. Hashim Y. Ebizuka and U.Sankawa Tetrahedron 1991 47 5969. 141 P. Bhandari N. van Bruggen L. Crombie and D. A. Whiting J. Chem. Soc. Chem. Commun. 1989,982. Biological Chemistry Biosynthesis __* 0 (85) OMe (87) The origin of the skeleton of mangostin (89) from Garcinia mangostana has been shown to be benzoate and ma10nate.l~~ Cinnamic acid is readily incorporated into mangostin (89) but it undergoes side-chain degradation prior to incorporation. The biosynthesis of caldariellaquinone (90) from Sulfolobus acidocaldarius has been in~estigated.'~~ Both the methyl and sulfur of the methylthio group are derived from methionine but the experiments clearly show that the methylthio group was not incorporated intact. Both D-and L-tyrosine have been incorporated into caldariel- laquine (90) indicating that the cells can readily convert D-tyrosine into L-tyrosine; labelling results have shown that the C-3 pro3 hydrogen of tyrosine is retained in the biosynthesis.14 The origin of the isocyanide carbons of xanthocillin X monomethyl ether (91) from Dichotomomyces cejpii remain obscure.Recent studies have eliminated many obvious candidate^.'^^ (90) (91) 142 G. L. Bennett H.-H. Lee and N. P. Das J. Chem. SOC.,Perkin 1 1990 2671. 143 D. Zhou and R. H. White J. Chem. SOC.,Perkin 1 1990 2346. 144 D. Zhou and R. H. White J. Chem. SOC.,Perkin 1 1991 1335. 145 K. M. Cable R. B. Herbert A. R. Knaggs and J. Mann J. Chem. SOC.,Perkin 1 1991 595. 304 R. A. Hill 5 Alkaloids and other Amino-acid-derived Metabolites The range of investigations into alkaloid biosynthesis is increasing.Some excellent reviews have appeaared on alkaloid biosynthe~is,'~~~'~~ pyrrolizidine alkaloid biosyn- thesis,'48 indole and bisindole biosynthesis in plant cell cultures,'49 alkaloid biosyn- thesis in plant cell cult~tres,'~~ and antibiotic bio~ynthesis.'~' The substrate tolerance and mechanism of action of diamine oxidases from pea seedlings have been Ornithine has been symmetrically incorporated into hyoscyamine (92)in root cultures of Hyoscyarnus ~lbus'~~ in contrast to the well established unsymmetrical incorporation of ornithine into hyoscyamine (92) in Datura species. No evidence for a pathway involving 6-methylornithine could be found in Hyoscyarnus ~lbus.'~~ Further evidence for the symmetrical incorporation of ornithine into nicotine (93)in Duboisia leichhardtii has been pr~duced.'~~ The recent work on these alkaloids has been reviewed.'" Trachelanthamidine (94)rather than isoretronecanol (95)is incorporated into echinatine (96)in Cynoglossum o_tfi~inaZe'~* and emiline (97)in Erniliaflarnrne~.'~~ The involvement of the immonium ion (98)in the biosynthesis of several pyrrolizidine alkaloids has been established.16' The necic acid portion of dicrotaline (99)has been shown to be derived from isoleucine and not from acetate mevalonate or 3-hydro~y-3-methylglutarate.'~~ 146 R.B. Herbert Nut. Prod. Rep. 1990 7 105. 147 R. B. Herbert Nut. Prod. Rep. 1991 8 185. 148 D. J. Robins Chem.SOC.Rev. 1989 18 375. 149 J. P. Kutney Nu?. Prod. Rep. 1990 7 85. M. H. Zenk Rec. Adv. Phytochem. 1989 23 429. 151 H. G. Floss and J. M. Beale Angew. Chem. Znt. Edn. EngZ. 1989 28 146. 152 J. E. Craig R. B. Herbert and M. M. Kgaphola Tetrahedron Lett. 1990 31 6907. 153 A. M.Equi A. M. Brown A. Cooper S. K. Ner A. B. Watson and D. J. Robins Tetrahedron 1990 47 507. 154 Hashimoto Y. Yamada and E. Leete J. Am. Chem. Soc. 1989 111 1141. 155 T. Hashimoto Y. Yukume and Y. Yamada PZunru 1989 178 123 and 131. 156 E. Leete T. Endo and Y. Yamada Phytochernistry 1990 29 1847. 157 E. Leete PZuntu Med. 1990 56 339. 158 E. K. Hunec and D. J. Robins J. Chem. Soc. Perkin 1 1989 1437. 159 H. A. Kelly E. K. Hunec M. Rodgers and D.J. Robins J. Chem. Res. (S) 1989 358. 160 A. A. Denholm H. A. Kelly and D. J. Robins J. Chem. SOC., Perkin 1 1991 2003. 161 A. A. Denholm and D. J. Robins J. Chem. Soc. Chem. Commun. 1991 19. Biological Chemistry Biosynthesis OXNH2 Me N Anosmine (100) from Dendrobiurn parishii has been shown to be derived from two lysine units one via cadaverine and the other presumably via pipecolic acid.16* Labelling studies have shown that acetate and methionine are incorporated into harzianopyridone (101) from Trichoderma harzianurn however the biosynthetic origin of the remaining C3N unit remains A similar study on spiro- staphylotrichin A (102) from Staphylotrichum cocosporurn demonstrated that acetate and methionine are incorporated as shown and the remaining C3N unit is derived from aspartic Methionine A CH,-COOH Me0 A H (1011 A OMe %@\ (102) The previous reports that both D-and L-phenylalanine are incorporated into cytocholasine D (103) have been investigated.It now appears that D-phenylalanine is incorporated via phenylpyruvic acid and ~-phenylalanine.'~' The enzymes 162 T. Hemscheidt and I. D. Spencer J. Chem Soc. Chem. Commun. 1991,494. 163 J. M. Dickinson J. R. Hanson P. B. Hitchcock and N. Claydon J. Chem. Soc. Perkin 1 1989 1885. 162 P. Sandmeier and Ch. Tamm Helv. Chim Actq 1989 72 774. A. Hadeuer P. Roth and Ch. Tamm 2. Naturforsch. Teil C 1989 44 19. 306 R A. Hill Me0 ..MH Me0 II "PkJMe 0 catalysing the oxidative coupling of reticuline to salutaridine (104) and berbamunine have been characterized and shown to be cytochrome P-450 linked NADPH and O2 dependent microsomal bound enzymes.'66 The incorporation of C-2 of tryp- tophan into the imino carbon of spirobrassinin (105)from Brussicu carnpestris implies a molecular rearrangement (Scheme 6).'67 H 'N H H H Scheme 6 (105) Labelled acetate glycine and 1802 have been incorporated into manumycin (106) from Streptornyces p~rvulus.'~~ The C7N unit is not formed from an aromatic precursor and may be formed from dihydroxyacetone and succinate or closely 0 0 NH 166 M.H. Zenk R. Gerardy and R. Stadler J. Chem. SOC. Chem. Commun. 1989 1725. 167 K. Monde and M. Takasugi J. Chem. SOC.,Chem. Commun.1991 1582. 168 R. Thiericke A. Zeeck J. A. Robinson J. M. Beale and H. G. Floss J. Chem. SOC.,Chem Commun. 1989 402. Biological Chemistry Biosynthesis related precursors. An insight into the biosynthesis of manumycin (106) is provided by studies with unnatural precursor^.'^^ Deuterium labelling studies have shown that the proton at C-5a of roquefortine (107) from Penicillium roqueforti is derived from the 2-hydrogen of tryptophan"' contrary to an earlier rep01t.l~~ This observation implies that no 2-substituted indoles can be intermediates in the biosynthesis. Incorporation studies have shown that cyclo-(L-phenylalanyl-L-seryl) (108) is an efficient precursor of gliotoxin (109) from Gliocladium uirens and hyalodendrin (1 10) from a Hyalodendron species.'72 The cyclic dipeptide (108) is not cleaved prior to incorporation and is incorporated into epidithiodioxopiperazines with opposite sulfur bridge stereochemistry.Obaflorin (1 11) from Pseudomonasfluorescens is an interesting p-lactone antibio- tic. Earlier work has shown that p-aminophenylalanine (1 12) is incorporated into carbons 3 to 10 of obaflorin (lll).173 Carbons 1 and 2 were shown to be derived from glyoxylate (113) itself derived from glycine once the conditions for incorpor- ation were determined.'74 The labelling experiments showed interesting labelling of both carbons 1 and 2 of obaflorin (111) when [2-13C]glycine was fed this is a result of glycine metabolism producing glyoxylate (1 13) labelled at both carbons. Unlike obaflorin (1 11) in which the nitro group is derived by oxidation of an amino group 169 R.Thiericke H.-J. Langer and A. Zeeck J. Chem. SOL,Perkin I 1989 851. 170 B. Bhat D. M. Harrison and H. M. Lamont J. Chem. SOC.,Chem. Commun. 1990 1518. 171 C. R. Pulham P. T. Brain A. J. Davies D. W. H. Rankin and H. E. Robertson 1.Chem. SOC.,Chem. Commun. 1990 177. 172 M. I. Pita Boente G. W. Kirby G. L. Patrick and D. J. Robins J. Chem. SOC.,Perkin 1 1991 1283. R. B. Herbert and A. R. Knaggs Tetrahedron Lett. 1988 29 6353. 174 R. B. Herbert and A. R. Knaggs Tetrahedron Lett. 1990 31 7515. 308 R. A. Hill c1 cJg&l COOH OVO (113) the nitro group of dioxapyrrolomycin (114) is derived from direct biochemical nitration in Streptomycesfum~nus.'~~ The organism was grown on a medium contain-ing K'5N'803as the sole source of nitrogen the ratio of l80to 15Nin the dioxapyr-rolomycin (114) was found to be the same as in the nitrate precursor.The origin of the cyano group in kinamycin D (115) from Streptomyces muray-amaensis has been established as C-2 of acetate and that the cyanide carbon was originally part of the polyketide Details of the 6 steps in the biosynthesis of antibiotic LL-C10037a (1 17) from 3-hydroxyanthanilic acid (116) in a Streptomy-ces species have been e1~cidated.l~~ 0 QH Recent work on the mechanism of pyridoxal-5'-phosphate dependent decarboxy-lase and transaminase enzymes has been de~cribed.'~~ A collection of 25 papers concerned with the biosynthesis of branched chain amino acids has been pub-1i~hed.I~~ An insight into the biosynthesis of the coronamic acid (120) portion of coronatine from Pseudomonas species has been achieved.'*' Labelling studies have shown that isoleucine (1 18) is first converted into alloisoleucine (119) and that only one hydrogen is lost from the methyl group of alloisoleucine (119) when it is converted to the cyclopropyl bridge of coronamic acid (120).The hydrogen at C-3 of alloisoleucine (119) is retained during the biosynthesis and 6-hydroxyal-loisoleucine is not incorporated. These results imply that an oxidative cyclization process is probably taking place in the biosynthesis of coronamic acid (120). H2N9@tH ""\ HOOC -HOOC '-HOOC 175 G. T. Carter J. A. Nietsche J.J. Goodman M. J. Torrey T. S. Dunne M. M. Siegel and D. B. Borders J. Chem. Soc. Chem. Commun. 1989 1271. P. J. Seaton and S. J. Gould J. Antibiot. 1989 42 189. 177 S. J. Gould B. Shen and Y. G. Whittle J. Am. Chem. SOC,1989 111 7932. 178 D. Gani Phil. Trans. R. Soc. Lond. B 1991 332 131. 179 'Biosynthesis of branched chain amino acids' ed. Z. Barak D. M. Chapman and J. V. Schloss BCH New York 1990. R. J. Parry M.-T. Lin A. E. Walker and S. Mhaskar J. Am. Chem. Soc. 1991 113 1849. Biological Chemistry Biosynthesis Considerable progress continues to be made in the area of p-lactam antibiotics. Excellent reviews have appeared on the biosynthesis of penicillins and cephalo- sporins,181 mechanistic studies on isopenicillin N synthase (IPNS)18* and the molecular biology of the bio~ynthesis.'~~ The biosynthetic route to the penicillins and cephalosporins is summarized in Scheme 7.The first step in the biosynthesis involves the formation of the LLD-ACV tripeptide (121) by ACV synthase. This enzyme has been purified from Aspergillus nidulan~,'~~ Cephalosporium acremonium and Streptomyces ~lauuligerens.'~'~'~~ The nucleotide sequence of the gene in Penicil- lium chryosogenum which codes for ACV synthase has been determined.ls7 Labelling studies with [4-2H6,'802]valine have shown that both one and two valine oxygens are subject to intracellular exchange prior to formation of the ACV tripeptide (121).'88 Studies with analogues of ACV as substrates for IPNS have given more H I I Y H I COOH COOH -H2NY-q(NEA-s I H2NY-yp$ 0 0 I H H I I Scheme 7 181 J.E. Baldwin J. Heterocyclic Chem. 1990 27 71. 182 J. E. Baldwin and M. Bradley Chem. Rev. 1990,90 1079. 183 S. W. Queener Antimicrobial Agents Chemotherapy 1990 34 943. 184 J. van Liempt H. von Dohren and H. Kleinkauf J. Biol. Chem. 1989 264 3680. 185 J. E. Baldwin J. W. Bird R. A. Field N. M. O'Callaghan and C. J. Schofield J. Antibiot. 1990,43,1055. 186 J. Zhang and A. Demain Biochem Biophys. Res. Commun. 1990 169 1145. 187 D. J. Smith A. J. Earl and G. Turner EMBOJ. 1990 9 2743. 188 J. E. Baldwin R. M. Alington J. W. Bird and C. J. Schofield J. Chem. SOC.,Chem. Commun. 1989,1615. 310 R A. Hill evidence about the mechanism of a~tion.'*~-'~~ The IPNS enzyme catalyses two ring closures the P-lactam ring is formed first however synthetic (126) was not incorpor- ated into isopenicillin N (122) using enzyme extracts from Cephalosporium acremonium or Streptomyces ~Zauuligerus,'~~ similar results were obtained with syn- thetic (127) and IPNS from S.clauuligerus. These results imply that the intermediate is enzyme bound. Evidence to support an insertion-homolysis mechanism for the events leading to C-S bond formation has been pre~ented.'~~ Genes for various isoenzymes of IPNS have been expressed to a high level in soluble form in E. ~oli.'~' H COOH (126) R= L-a-aminoadipoyl (127) R= Ph CHZ-C-II The ligands coordinated to iron in IPNS have been Isopenicillin N epimerase which converts isopenicillin N (122) into penicillin N (123) has been isolated from Nocardia la~tamdurans.'~~ The epimerase gene from Streptomyces cZavuZigerus has been mapped and ~equenced.'~~ Penicillin N (123) is converted into the cephalosporins by DAOC/DAC synthase which catalyses the ring expansion to deacetoxycephalosporin C (DAOC) (124) and hydroxylation to deacetylcephalop- sporin C (DAC) (125).The stereochemistry of the ring expansion has been studied using enzymes from Cephalosporium acremonium.200 Both ring expansion and hydroxylation require a-ketoglutarate as cofactor. Labelling studies have shown that in each step the a-ketoglutarate is converted into succinate and C02 and that label from 1802 is incorporated efficiently into succinate.201 The incorporation of labelled oxygen into DAC (125) was only about 50% indicating that oxygen exchange is probably occurring at a putative iron-oxygen intermediate.When C-3 deuterated penicillin N (123) is incubated with DAOC/DAC synthase the shunt metabolite (128) is produced giving further insight into the ring expansion mechanism.202 189 J. E. Baldwin C. J. Schofield and B. D. Smith Tetrahedron 1990 46 3019. 190 J. E. Baldwin M. Bradley S. D. Abbott and R. M. Adlington J. Chem. SOC. Chem. Commun. 1990,1008. 191 J. E. Baldwin R. Adlington C. J. Schofield and H-H. Ting Tetrahedron 1990 46 6145. 192 J. E. Baldwin G. P. Lynch and C. J. Schofield J. Chem. SOC. Chem. Commun. 1991 736. 193 A. I. Scott R. Shankaranarayan and S.-K.Chung Heterocycles 1990 30,909. 194 J. E. Baldwin R. M. Adlington N. P. Crouch J. W. Keeping S. W. Leppard J. Pitlik C. J. Schofield W. J. Sobey and M. E. Wood J. Chem. SOC.,Chem. Commun. 1991,768. 195 J. E. Baldwin J. M. Blackburn J. D. Sutherland and M. C. Wright Tetrahedron 1991,47 5991. 196 L.-J. Ming L. Que Jr. A. Kriauciunas C. A. Frolik and V. J. Chen Znorg. Chem. 1990 29 1111. 197 V. J. Chen A. M. Orville M. R. Harpel C. A. Frolik K. K. Surerus E. Munck and J. D. Lipscomb J. Biol. Chem. 1989 264 21677. L. Lhiz P. Liras J. M. Castro and J. F. Martin J. Gen. Microbiol. 1990 136 663. 199 S. Kavacevic M. B. Tobin and J. P. Miller J. Bacten'ol. 1990 172 3952. 200 J. E. Baldwin R. M. Adlington N. P. Crouch N. J. Turner and C. J. Schofield J. Chem. SOC.,Chem.Commun. 1989 970. 201 J. E. Baldwin R. M. Adlington C. J. Schofield W. J. Sobey and M. E. Wood J. Chem SOC,Chem. Commun. 1989 1012. 202 J. E. Baldwin R. M. Adlington N. P. Crouch C. J. Schofield N. J. Turner and R. T. Aplin Tetrahedron 1991,47 9881. 19' Biological Chemistry Biosynthesis H RN Progress towards the understanding of the biosynthesis of clavulanic acid (132) from Streptomyces cluuuligerus continues to be made (Scheme 8). Proclavaminic acid (129) has been synthesized and its absolute stereochemistry has been deter- mined.203 The four electron oxidative cyclization of proclavaminic acid (129) to clavaminic acid (131) is carried out by an a-ketoglutarate dependent and iron containing oxygenase clavaminic acid synthase (CAS).Labelling experiments have shown that the oxygen of the hydroxyl group of proclavaminic acid (129) gives rise to the oxygen of the oxazolidine ring of clavaminic acid (131)204and that the ring closure goes with retention of configuration at C-4' of proclavaminic acid (129).205 Dihydroclavaminic acid (130) has been isolated as an intermediate in this cycliz- atioa206 Possible mechanisms for this oxidative cyclization have been dis-I COOH COOH (132) (131) Scheme 8 Labelling studies have shown that aristeromycin (133) from Streptomyces citricolor arises from a normal adenyl biosynthesis that the cyclopentane ring arises from glucose by formation of a bond between C-2 and C-ti209and that the amine (134) is an intermediate.210 Labelled forms of ATP and adenosine when administered to 203 K.H. Baggaley S. W. Elson N. H. Nicholson and J. T. Sime J. Chem. Soc. Perkin I 1990 1513 and 1520. 204 W. J. Krol A. Basak S. P. Salowe and C. A. Townsend J. Am. Chem. SOC.,1989 111 7625. 205 A. Basak S. P. Salowe and C. A. Townsend J. Am. Chem SOC.,1990 112 1654. 206 J. E. Baldwin R. M. Adlington J. S. Bryans A. 0.Bringhen J. B. Coates N. P. Crouch M. D. Lloyd C. J. Schofield S. W. Elson K. H. Baggaley R. Cassels and N. Nicholson Tetrahedron 1991,47,4089. 207 C. A. Townsend and A. Basak Tetrahedron 1991,47 2591. 208 S. P. Salowe E. N. Marsh and C. A. Townsend Biochemistry 1990 29 6499. 209 R. J. Parry V. Bornemann and R. Subramanian J. Am. Chem. Soc. 1989 111 5819. 210 R.J. Parry K. Haridas R. de Jong and C. R. Johnson J. Chem. SOC.,Chem. Commun. 1991 740. 312 R A. Hill cell-free extracts of Streptomyces griseofus produced sinefungin (135) with evidence that both precursors had been significantly degraded before incorporation.211 The ribose ring of adenosine was incorporated into sinefungin (135) intact without loss of label from C-5’. Experiments suggested that the C-C bond formation between arginine and ribose preceded attachment of the adenine ring. HOHzC rl A. HO OH HO OH HOOC NH2 6 Porphyrins A comprehensive review of biosynthetic experiments concerned with porphyrins chlorophylls and vitamin B12has appeared.212 Full details of the work that led to the discovery of the dipyrrolic cofactor essential for the action of hydroxymethyl- bilane synthase (HMBS) (or porphyrobilinogen deaminase) that converts por- phobilinogen into hydroxymethylbilane (HMB) have been p~blished.~~~,~’~ This work has also been re~iewed.~” Details of the mechanisms of HMBS and uropor- phyrinogen I11 synthase (or cosynthetase) that converts HMB into uro’gen I11 have been reviewed.216 Uro’gen I11 synthase mechanism molecular biology and bio- chemistry is covered in another review.217 Further evidence for the involvement of a spiro-intermediate between HMB and uro’gen I11 has been presented.218 The extensive investigation into the biosynthesis of vitamin B12has been covered in two The biosynthesis of vitamin B12from uro’gen I11 involves inter alia 8 C-methylation steps.Precorrins 1,2 and 3 have been identified as the first three intermediates. Recent work on the details of these steps has been pub- lished.221-223 Evidence that norcorrins are involved in the biosynthesis of cobyrinic 211 R. J. Parry and S. Ju Tetrahedron 1991 47 6069. 212 F. J. Leeper Nut. Prod. Rep. 1989 6 171. 213 A. D. Miller F. J. Leeper and A. R. Battersby J. Chem. SOC.,Perkin 1 1989 1943. 214 G. J. Hart A. D. Miller U. Beifuss F. J. Leeper and A. R. Battersby J. Chem. SOC.,Perkin 1,1990 1979. 215 P. R. Alefounder G. J. Hart A. D. Miller U. Beifuss C. Abell F. J. Leeper and A. R. Battersby Bioorganic Chem. 1989 17 121. 216 A. R. Battersby and F. J. Leeper Chem. Rev. 1990,90 1261. 217 N. Crocket P. R. Alefounder A. R. Battersby and C.Abell Tetrahedron 1991 47 6003. 218 M. A. Cassidy N. Crockett F. J. Leeper and A. R. Battersby .IChem. SOC.,Chem. Commun. 1991,384. 219 A. R. Battersby Pure AppL Chem. 1989 61 337. 220 A. I. Scott Pure Appl. Chem. 1989 61 501. 22 1 R. D. Brunt F. J. Leeper I. Grgurina and A. R. Battersby J. Chem. SOC.,Chem. Commun. 1989,428. 222 C. L. Gibson and A. R. Battersby J. Chem. SOC.,Chem. Commun. 1989 1223. 223 G. W. Weaver F. Blanche D. Thibaut L. Debussche F. J. Leeper and A. R. Battersby J. Chem. Soc. Chem. Commun. 1990 1125. Biological Chemistry Biosynthesis acid has not been A new intermediate has been isolated and named precorrin 6x.226 The site of reduction of precorrin 6x by NADPH has been determined.227 7 Miscellaneous Metabolites The mechanism of oxidative reactions catalysed by P-450and related iron-containing enzymes has been reviewed.228 A generally valuable and sensitive method for determining the configuration of chiral methyl groups has been developed.The method depends on the observation that the diastereotopic protons on C-7 of (136) have different NMR chemical shifts.229 (138) R= NH2 (139) R=OH (140) Incorporation studies have shown that label from 1802 does not appear in the oxirane oxygen of fosmycine (137) from Streptomyces fradi~e.~~' Both (2-aminoethy1)phosphonic acid (138)231 and (2-hydroxyethy1)phosphonicacid( 139)232 are incorporated into fosmycin (137). The methyl carbon is derived from the methyl of methionine (S)-(2-hydroxypropyl)phosphonic acid (140) was efficiently incor- porated into fosmycin (137).233 224 J.Kulka C. Nussbaumer and D. Arigoni J. Chem. SOC. Chem. Commun. 1990 1512. 225 I. Grgurina S. Handa G. Weaver P.A. Cole and A. R.Battersby J. Chem. SOC.,Chem. Commun. 1990 1514. 226 D. Thibaut L. Debussche and F. Blanche Proc. Natl. Acad. Sci. USA 1990 87 8795. 227 G. W. Weaver F. J. Leeper A. R. Battersby F. Blanche D. Thibaut and L. Debussche J. Chem. SOC. Chem. Commun. 1991 976. 228 M. Akhtar and J. N. Wright Chem Rev. 1990 90 1079. 229 F. A. L. Anet D. J. O'Leary J. M. Beale and H. G. Floss J. Am. Chem. SOC., 1989 111 8935. 230 F. Hammerschmidt G. Bovermann and K. Bayer Liebigs Ann. Chem. 1990 1055. 23 1 F. Hammerschmidt H. Kahlig and N. Muller J.Chem. SOC. Perkin 1 1991 365. 232 F. Hammerschmidt and H. Kahlig J. Org. Chem. 1991 56 2364. 233 F. Hammerschmidt J. Chem. Soc. Perkin 1 1991 1993.