14 Biosynthesis By T. J. SIMPSON Department of Chemistry University of Edinburgh West Mains Road Edinburgh EH9 3JJ 1 Introduction This Report will discuss biosynthetic studies on secondary metabolites and related work. Despite the fact that this has been and continues to be an area of intense activity the last Report was in 1980.’ Thus this Report can only give a highly selective and inevitably personalized view of the main developments over the past five years. Highlights have included the continuing impact of n.m.r. methods in biosynthetic studies2p3 and in particular the use of isotope-induced shifts in 13C n.m.r. to monitor the biosynthetic origins of hydr~gen~?~ and so obtain and ~xygen~.~ information on the oxidation levels of biosynthetic intermediates; the study of the isolation and mechanism of terpenoid cyclases;’ the use of tissue cultures as sources of enzymes for alkaloid biosynthesis; and the application of genetic engineering techniques to biosynthetic studies.The appearance of several new book^^-^ on biosynthesis and secondary metabolism emphasizes the continuing importance of these areas. An undoubted highlight has been the appearance of the review journal Natural Product Reports to replace several of the established Specialist Periodical Reports titles and comprehensive coverage of the areas discussed here are to be found in the regular articles appearing therein. 2 Polyketide Biosynthesis P-Hydroxydecanoyl thioester dehydrase which is the pivotal enzyme in the biosyn- thesis of unsaturated fatty acids in anaerobic micro-organisms mediates the conver- sion of acyl-carrier-protein thioesters of (R)-3-hydroxydecanoic acid (l) (E )-dec-2-enoic acid (2) and (Z)-dec-3-enoic acid (3).The allylic rearrangement which interconverts (2) and (3) has been shown by 2H labelling and 2H n.m.r. methods to ’ J. R. Hanson Ann. Rep. Bog. Chem. Sect. B 1980 77 289. * T. J. Simpson in ‘Modern Methods of Plant Analysis’ ed. H. F. Linskens and J. F. Jackson Springer- Verlag Berlin 1985 p. 1. C. Abell in ‘Modern Methods of Plant Analysis’ ed. H. F. Linskens and J. F. Jackson Spnnger-Verlag Berlin 1985 p. 60. J. C. Vederas Can. J. Chem. 1982 60,1637. D. E. Cane Acc. Chem. Rex 1985 220. ‘R. B. Herbert ‘The Biosynthesis of Secondary Metabolites’ Chapman and Hall London 1981.’ P. Mannitto ‘Biosynthesis of Natural Products’ Ellis Horwood Chichester 1981. K. H. G. Torsell ‘Natural Products Chemistry’ Wiley Chichester 1983. E. Haslam ‘Metabolites and Metabolism -A Comment on Secondary Metabolism’ Clarendon Press Oxford 1985. 347 348 T. J. Simpson be an overall suprafacial process in which 4-( pro-4R)-hydrogen of (2) is removed and the enzyme-catalysed protonation occurs at the si face at C-2 of (2)." This finding is consistent with a 'one-base' mechanism in which an active site histidine acts as the base. 3-[2-13C]Decynoyl-NAC which is known to act as a reversible inhibitor of the enzyme has been synthesized and incubated with the enzyme." 13C{lH} N.m.r. analysis of the incubation mixture allows direct observation of the enzyme-inhibitor complex.Lipoxins A and B are formed from human leukocytes exposed to 15-hydroxyeicosatetraenoic acid.12 Their structures (4) and (5) have been assigned by syntheses based on biosynthetic speculation^'^ and evidence for their formation via the (5S),(6S) epoxide (6) has been ~btained.'~ Other interesting papers discuss the possible routes for formation of the rethrolone segment of the pyrethroid in~ecticides;'~ and jasmonic acid (8) has been shown to be formed from linolenic acid via 12-oxo-phytodienoic acid (7) in tissue sections of several higher plant species.16 (CHACOzHro2H Me G M e / OH (6) 0 (7) (8) n n = 7; 9,14-dehydro = 1 Incorporation studies" with [1-13C]propionate and 13C-labelled acetates indicate that the antibiotic furanomycin (lo) a metabolite of Streptomyces threomyceticus is derived from the triketide precursor (9).Colletodiol (14) a macrocyclic dilactone isolated from Colletotrichium capsici and a Cytospora sp. has been shown by a LO J. M. Schwab and J. B. Klassen J. Am. Chem. SOC.,1984 106 7217. J. M. Schwab W. Li C.-K. Ho C. A. Townsend and G. M. Salituro J. Am. Ckem. SOC.,1984,106,7293. 12 C. N. Gerham M. Hamberg and B. Sarnuelsson Biochem. Biophys. Res. Commun. 1984 118 943. l3 J. Adams B. J. Fitzsimmons Y. Girard Y. Leblanc J. F. Evans and J. Rokach J. Am. Chem. SOC. 1985 107 464. 14 B. J. Fitzsimmons and J. Rokach Tetrahedron Lett. 1985 26 3939. 15 L. Crombie and S. J. Holloway J. Chem. SOC.,Perkin Trans.I 1985 1393. 16 B. A. Vick and D. C. Zirnmerman Plant Physiol 1984 75 458. 17 R. J. Parry and H. P. Buu J. Am. Chem. SOC.,1983 105 7446. Biosy n thesis 349 O'H (14) combination of 13C 'H and l80 labelling studies18 to be formed via the macrocyclic triene (13) which is itself formed via the triketide and tetraketide precursors (11) and (12). Oxidative ring-cleavage of preformed aromatic intermediates results in formation of many unusual structures. 13C-Labelling methods and studies with advanced intermediates have demonstrated the formation of astepyrone (1 5) in Aspergillus terreus" and of the mycotoxin botryodiplodin (18) in Penicillium roquefortii,20 via M&o -R&02H M$Me __* HO OH Ho 0 (16) R = OH ' (18) (15) (17) R = H orsellinic acid (16).The formation of patulin (19) via ring-cleavage of 6-methyl- salicylic acid (17) has been intensively studied for many years. Cell-free preparations of a patulin-minus mutant of Penicillium urticae convert isopatulin (20) into both (E)-and (2)-ascladiol (21). However only the (E)-isomer is further converted into patulin.21 '80-Labelling studies have shown that only the carbonyl oxygen is derived from acetate the others are introduced by oxidative processes.22 (17) -8' go / d H#' / -/ HOCH2 CH20H HO 0 0 OH 18 T. J. Simpson and G. I. Stevenson J. Chern. SOC.,Chern Cornmun. 1985 1822. 19 K. Arai T. Yoshimura Y. Hatani and Y. Yamamoto Chern. Pharrn. Bull. 1983 31 925. 20 F. Renauld S. Moreau and A.Lablanche-Combier Tetrahedron 1984 40 1823. 21 J. Sekiguchi T. Shimamoto Y. Yamada and G. M. Gaucher AppL Environ. Microbiol. 1983,45 1939. 22 H. Tijima H. Noguchi Y. Ebizuka U. Sankawa and H. Seto Chern. Pharm. Bull. 1983 31 362. 350 T. J. Simpson Comparison of the incorporation of 2H from [ 1-13C,2H3]- and [l-13C,2Hl]acetate into 6-methylsalicylic acid by both ’H n.m.r. and 2H-isotope-induced shifts in 13C n.m.r. show that there is no observable isotope-eff ect and so the necessary deproton- ations required for aromatization of the intermediates from cyclization of the enzyme-bound polyketide precursor must be enzyme-mediated and presumably stereo~pecific.~~ Full details of 14C and 13C-labelling studies on aspyrone (24) and asperlactone (25) have appea~ed.’~ 180-Labelling studies have shown surprisingly that no acetate- derived oxygen is retained in these metabolites.Alternative cyclizations of the epoxy-carboxylic acid (23) itself derived from the trienone (22) were proposed to HoY+Me 0 account for the co-occurrence of these compounds in Aspergillus rnelleu~.*~ This provides a simple model for the more complex epoxide-mediated cyclizations proposed to occur in monensin and other polyether antibiotics. The formation of cryptosporiopsonol (26) uia dihydroisocoumarin intermediates in Periconia mucros- pinosu26has been confirmed. Incorporation of [2-13C,2H3]acetate into mellein (27) in Aspergillus melleus was studied by both 2H n.rn.r. and edited 13C n.m.r. using a pulse sequence which allows only isotopically shifted signals to be ob~erved.~’ The simultaneous incorporation of two deuterium atoms at C-4 provides compelling evidence along with similar observations for brefeldin A,28 rnon~cerin,~~ and c1 AMe hMe & OH OH 0 23 C.Abell and J. Staunton J. Chem. SOC.,Chem. Commun. 1984 1005. 24 R. G. Brereton M. J. Garson and J. Staunton J. Chem. SOC.,Perkin Trans. 1 1984 1027 and preceding papers. 25 S. A. Ahmed T. J. Simpson J. Staunton A. C. Sutkowski L. A. Trimble and J. C. Vederas J. Chem. SOC.,Chem. Commun. 1985 1685. 26 G. B. Henderson and R. A. Hill J. Chem. SOC.,Perkin Trans. 1 1982 3037. 27 C. Abell D. M. Doddrell M. J. Garson E. D. Laue and J. Staunton J. Chem. SOC.,Chem. Commun. 1983 694.28 C. R. Hutchinson Acc. Chem. Res. 1983 16 7. 29 F. E. Scott T. J. Simpson L. A. Trimble and J. C. Vederas J. Chem. SOC.,Chem. Commun. 1984 756. Biosynthesis 351 colletodiol," that reductive modification of polyketide precursors takes place con- comitant with and not after chain elongation. The tetronic acid multicolic acid (28) is derived by oxidative modification of 6-pentylresorcylic acid. 180-Labelling studies establish details of the mechanism of ring cleavage and lactone ring f~rmation.~' Similar 180-labelling studies established that all the oxygens in griseofulvin (29) are acetate-derived to discount the proposed intermediacy of hydroquinone intermediates in formation of the grisan ring system.31 Biosynthetic studies of the chromanone ~~-D253cu (31) showed that the carbons of the hydroxyethyl side-chain become equivalent at some point in the biosynthesis.The cyclopropane intermediate (30) was proposed to account for this.32 1 OH (31) 2H-and 180-Labelling studies show that biosynthesis of palitantin (32) in Penicil-Zium brefeldianum does not involve aromatic intermediate^.^^ The origins of fulvic acid and citromycetin have long been a subject of speculation. In a careful study in Penicilfiumfrequentans it has been shown that citromycetin is in fact an artefact and the true metabolite is polivione (33).34 "0-Labelling studies35 show that the oxygen at C-4 is derived from the atmosphere to provide the first definitive evidence that this group of compounds are formed via a ring-cleavage process from a heptaketide-derived intermediate related to fusarubin (35).H02C OH 0 HO" CHZOH HO ' 0 0 (32) (33) 30 J. S. E. Holker E. O'Brien R. N. Moore and J. C. Vederas J. Cfiern.SOC.,Cfiern. Cornmun. 1983 192. 31 M. P. Lane T. T. Nakashima and J. C. Vederas J. Am. Cfiern.SOC.,1982 104 913. 32 C. R. McIntyre T. J. Simpson L. A. Trimble and J. C. Vederas J. Chem. Soc. Cfiern.Cornmun. 1984 706. 33 A. K. Demetriadou E. D. Laue and J. Staunton J. Cfiern. Soc. Cfiern. Cornmun. 1985 1125. 34 A. K. Demetriadou. E. D. Laue F. J. Leeper and J. Staunton J. Cfiern.Soc. Cfiern.Comrnun. 1985,762. 352 T. J. Sirnpson 5-Deoxyfusarubin (34) is a yellow pigment which has been isolated from a mutant strain of Nectria haematococca which normally produces fusarubin.The strain used was a double crossover-producing mutant designated Ann A*58.yelJl derived from the Ann A"58 mutant which showed higher production and excretion of red pigments into the medium and yelJl which produced yellow pigments rather than red. Thus it appears to lack the gene responsible for the late-stage oxidation at C-5 of f~sarubin.~~ MeOfypMe J,W. \ I1 0 OH OH 0 '1 (34) R = H COzH (35) R = OH (36) Gene cloning methods are beginning to make a major impact in secondary metabolite production and biosynthesis. One area where there has been particularly significant work relates to the antibiotic actinorhodin and related octaketide-derived isochromanequinone antibiotics.Actinorhodin (36) is produced by Streptomyces coelicolor. All of the genes for the biosynthesis of actinorhodin are clustered on one contiguous region of the bacterial genome which has been cloned in a low-copy-number vector (pIJ922) as one large (32.5 kilobase) segment of DNA.37 The resultant plasmid (pIJ2303) has been used to transform other Streptomyces e.g. S. paruulus thereby conferring on them the ability to produce a~tinorhodin.~~ In related work it was shown that introduction of a plasmid vector carrying the afsB gene of S. coelicolor into S. liuidans or strains of S. coelicolor which had lost the ability to produce actinorhodin caused high production of actinorhodin in both.3Y The afsB gene appears to consist of a ca.2 kilobase segment of DNA4' which controls biosynthe~is~' of the regulatory substance known as A factor which in turn controls expression of genes controlling cell differentiation and antibiotic production.This work has been taken further to induce the formation of new or hybrid antibiotic^,^^ by making use of the close structural relationships between actinor- hodin (36) and medermycin (37) a metabolite of Streptomyces uiolacember and granaticin (38) produced by Streptomyces sp. AM-7161. Transformation of Strep romyces sp. AM-7161 with the whole act gene cluster carried on vector pIJ2303 resulted in production of both actinorhodin (36) and granaticin (38). However when transformed with a partial act clone a new metabolite dehydrogranitirhodin (39) was produced as a result of cooperation between gene products in the pathway 35 A.K. Demetriadou E. D. Laue and J. Staunton J. Chem. SOC.,Chem. Commun. 1985 764. 36 D. Parisot M. Deuys and M. Barbier Phytochemistry 1985 24 1977. 37 D. J. Lydiate F. Malpardita and D. A. Hopwood Gene 1985 35 223. 38 F. Malpardita and D. A. Hopwood Nature (London) 1984 309,462. 39 S. Horinuchi and T. Beppu Agr. Biof. Chem. 1984 48 2131. S. Horinuchi 0.Hara and T. Beppu J. Bacteriol 1983 155 1238. 41 S. Horinuchi Y. Kumada and T. Beppu J. Bacteriol 1984 158 481. 42 D. A. Hopwood F. Malpardita H. M. Kieser H. Ikeda J. Duncan I. Fujii B. A. M. Rudd H. G. Floss and S. Omura Nature (London) 1985 314 642. Biosynthesis 353 NMe OH OH 0 Me Me (38) (37) '0 NMe I to actinorhodin and granaticin.In contrast when S. violuceoruber was transformed with vector pIJ2303 normal antibiotic production was switched off and a new compound mederrhodin A (40) was produced instead. Mevinolin (41) is representative of a group of compounds containing the decalin ring system which have provoked considerable interest due to their ability to block cholesterol biosynthesis and to act as antibiotics or show other biological activities. The incorporation of 13C- 'H- and "0-labelled acetates into mevinolin in Aspergillus terreus give results consistent with biogenesis of (41) by intramolecular cycloaddition of a C18-polyunsaturated acid or intramolecular anionic condensations of a partially reduced p~lyketide.~~ illicicolin Similar studies on nargenicin Al ,44 nodu~micin,~~ A,46the beta en one^,^^ tetrocarcin A,48and chl~rothricin~~ have been reported.43 R. N. Moore G. Bigam J. K. Chan A. M. Hogg T. T. Nakashima and J. C. Vederas J. Am. Chem. SOC.,1985 107 3694. 44 D. E. Cane and C.-C. Yang J. Anribior. 1985 28 423. 45 W. C. Snyder and K. L. Rinehart jun. J. Am. Chem. Soc. 1984 106 287. 46 M. Tanabe and S. Urano Tetrahedron 1983 39 3569. 47 H. Ockawa A. Ichihara and S. Sakamura J. Chem. SOC.,Chem. Cornmun. 1984 814. 48 T. Tamaoki and F. Tomita J. Antibiot. 1983 36,595. 49 0.Mascaretti C. Chang D. Hook H. Otsuka E. F. Kreutzer and H. G. Floss Biochemistry 1981 20 919. 354 T. J. Simpson Incorporation of 13C-labelled acetates and malonate into oxytetracycline (42) in Streptomyces rimosus have established the mode of folding of the precursor chain5’ and the involvement of an intact malonate as the starter unit.5’ The mycotoxin viridicatumtoxin (43) has a similar structure but a quite different mode of assembly.52 0’ NMe2 N’2 HO HO 0 0 0 OH (42 1 (43) The mycotoxin asteltoxin (44) is derived from a nonaketide precursor.Surprisingly the first three carbons in the precursor chain can be derived either from acetate and a methionine-derived methyl or from pr~pionate.’~ 180-Labelling results show the involvement of a tris-epoxide (45) in the rearrangement and cyclization responsible for the formation of the bisfuranoid system.54 Analogous results are reported for ~itreoviridin’~ and aurovertin B.56 Me Me (44) 0 (45 1 The pathway to aflatoxin B (46) continues to be actively studied.The intermediacy of averufin (47) has been firmly e~tablished.~~ The other highlight in this area has been Townsend’s detailed studies58359 of the mechanism of conversion of averufin via versiconal acetate into versicolorin A (48) using synthetic intermediates specifically labelled with I3C 2H and l8O. The origins of all the oxygen atoms in monensin (49) have been and these are consistent with its formation uia a tris-epoxide itself formed from the 50 R. Thomas and D. J. Williams J. Chem. SOC.,Chem. Commun. 1983 128. 51 R. Thomas and D. J. Williams J. Chern. SOC.,Chem. Commun. 1983 677. 52 A. E. de Jesus W. E. Hull P. S. Steyn F. R. van Heerden and R.Vleggaar J. Chem. SOC.,Chem. Commun. 1982 902. 53 P. S. Steyn and R. Vleggaar J. Chem. SOC.,Chem. Comrnun. 1984 977. 54 A. E. de Jesus P. S. Steyn and R. Vleggaar J. Chem. SOC.,Chem. Commun. 1985 1633. 55 P. S. Steyn and R. Vleggaar J. Chern. SOC.,Chem. Comrnun. 1985 1531. 56 P. S. Steyn and R. Vleggaar J. Chem. SOC.,Chem. Commun. 1985 1791. 57 T. J. Simpson A. E de Jesus P. S. Steyn and R. Vleggaar J. Chem. SOC.,Chem. Commun. 1982 631. 58 C. A. Townsend and S. B. Christensen Tetrahedron 1983 39 3575. 59 C. A. Townsend and S. B. Christensen J. Am. Chem. Soc. 1985 107 270. D. E. Cane T.-C. Liang and H. Hasler J. Am. Chem. SOC., 60 1982 104 7274. ’‘ A. A. Ajaz and J. A. Robinson J. Chem. SOC.,Chem. Commun. 1983 679. Biosyn thesis 355 HO 0 (46’ (47) HO 0 Me OH Me 1 1 OMe \OH COzH (49) Me Me..Me )Me OH Me C02H (50) triene (50),the synthesis of which has been reported.62 An important theoretical paper presents a unified stereochemical model which attempts to correlate the structures and stereochemistries of more than 30 different polyether antibiotics and to show how they can be biosynthesized via polyepoxide prec~rsors.~~ Amino acids such as valine can act as efficient carbon sources in the biosynthesis of monensin and other antibiotics as can butyrate and i~obutyrate.~~ The results of a number of 62 F. van Middlesworth D V. Patel J. Conaubauer P. Gannett and C. J. Sih J. Am. Chem. Soc. 1985 107 2996. 63 D.E. Cane W. D. Celmer and J. W. Westley J. Am. Chem. Soc. 1983 105 4110. 64 S. PopKil P. Sedmera M. Havrhek V. Krumphanzl and Z. Vanek J. Antibiotics 1983 36 617. 356 T. J. Simpson studies notably by Robinson and co-workers in S. cinnarnonensi~~~ and the nonactin producer Streptomyces griseus,66 but also by others has led to much information on the stereochemical and mechanistic details of the interconversions of these com- pounds being elucidated. These need to be appreciated in interpreting many of the biosynthetic results in this area. 18 0-Labelling studies on erythromycin have been reported in full.67 These results are consistent with the view that the oxidation level that is observed in the aglycone (51) is established during elongation of the carbon chain and exclude alternative oxidation or dehydration-rehydration mechanisms.These and other aspects of the biosynthesis of macrolides and polyether antibiotics particularly their relationship to fatty acid biosynthesis are discussed in an excellent review article.28 3 Terpenoids and Steroids Mevinolin (see above) is believed to act by inhibiting hydroxymethylglutaryl-CoA reductase the key enzyme in regulation of terpenoid biosynthesis. Full length cDNA clones have been obtained that revealed the complete amino acid sequence of the reductase (M.W. ca. 95K)from hamster cells.68 The cyclases responsible for the formation of several mono- and sesquiterpenoids in plants have been isolated and studied in detail. Enzyme preparations which mediate the formation of d -bornyl pyrophosphate (52) and I-bornyl pyrophosphate from geranyl pyrophosphate (GPP)have been isolated from SaIvia ~ficinalis~~ and 65 C.Gani D. O'Hagan K. Reynolds and J. A. Robinson J. Chem. SOC.,Chem. Commun. 1985 1002; K. Reynolds and J. A. Robinson ibid. 1985 1831. 66 C. A. Clark and J. A. Robinson J. Chem. SOC.,Chem. Commun. 1985 1568. 67 D. E. Cane H. Hasler P. B. Taylor and T.-C. Liang Tetrahedron 1983 39 3449. 68 D. J. Chin G. Gil D. W. Russell L. Liscum K. L. Luskey S. K. Basu H. Okayama P. Berg J. L. Goldstein and M. S. Brown Nature (London) 1984 308 613. 69 H. Gambliel and R.Croteau J. Biol. Chem. 1982 257 2335. Biosyn thesis 357 Tanacetum ~ulgare~~ respectively. Inter alia these have been used with [3H,32P]- and ['4C,'80]-labelled GPP as substrates to show that the migration of the pyrophos- phate anion during cyclization involves formation of a very tight ion-pair (53).In contrast a cell-free preparation from Foeniculum uulgare (fennel) yielded (-)-endo-fenchol (54) from GPP by direct attack of water on the presumed bicycloterpinyl cation pyrophosphate anion non-pair. Consistent with this hypothesis no label was found in (54) if [1-180]GPP was used in the sub~trate.~' All of the plant phosphatases that have been studied catalyse P-0 bond cleavage so fenchyl pyrophosphate cannot be an intermediate. Cell-free extracts from Streptomyces spp. have been used to study the cyclization of farnesyl pyrophosphate to pentalene (55) the parent hydrocarbon of the pentalenolactone antibiotic^.^^ Retention of both hydrogens from C-9 of FPP suggested that cyclization occurred at a single enzyme active-site with no kinetically free intermediates.The cyclization of FPP to humulene (56) and caryophyllene (57) in a cell-free system from Saluia oficinalis represents the first Me Me I (55) (56) (57) example of a soluble Cls-cyclase from a higher plant.73 Plant tissue cultures provide a good source of cell-free systems for biosynthetic studies as has been demonstrated by the use of extracts from callus cultures of Andrographis panicul~ta~~ to show inter alia that (2E,6E)-FPP and not the (22,6Z)-isomer is the precursor of 7-bisabolene (58). The detailed stereochemistry and mechanisms of enzymic cycliz- ations to form several classes of sesquiterpenes and monoterpenes have been excel- lently re~iewed.~ *H N.m.r.and 'H isotope-induced shifts in 13C n.m.r. have also been used to provide details of sequiterpenoid diterpenoid and triterpenoid biosynthesis. Incor- poration experiments with labelled acetates and mevalonates revealed the occurrence of hydrogen migrations to C-1 and from C-5 to C-6 during biosynthesis of alliaciolide 70 D. E. Cane A. Saito R. Croteau J. Shaskuo and M. Felton J. Am. Chem. SOC.,1982 104 5831. 71 R. Croteau S. Shaskus D. E. Cane A. Saito and C. Chang J. Am. Chem. SOC.,1984 106 1142. 72 D. E. Cane and A. M. Tillman J. Am. Chem. SOC.,1983 105 122. 73 R. Croteau and A. Gundy Arch. Biochem. Biophys.1984 233 838. 74 P. Anastasis I. Freer C. Gilmore H. Mackie K. H. Overton D. Picken and S. Swanson Can. J. Chem. 1984 62 2079. 358 T.J. Simpson (59) in Marasmius allia~eus;~~ and the retention of label at C-6 of illudin M (60) enriched from [S2H2 ,5-'3C]mevalonate in Clitocybe illudens indicated that there is no hydride rearrangement involving this centre as had been previously reported.76 The appearance of a 2H-'3C coupling in the 2H n.m.r. spectrum of the antiviral and antitumour metabolite aphidicolin (61) when enriched from [4-'H2 ,3-'3C]meval- onate in Cephalosporium aphidicola established that a P-hydrogen at C-9 migrated to C-8 during the bio~ynthesis.'~ Further studies using advanced intermediates have shown that biosynthesis may proceed via aphidicolan-16-01 (62).78 R (61) R = OH (62) R = H A number of important studies in relation to the biosynthesis of the gibberellins have appeared.A microsomal enzyme preparation from Marah macrocarpus removes the 19-( pro-R) hydrogen during the stereospecific oxidation of ent-kauren- 19-01 (63)79to the aldehyde. A cell-free system which converts kaurene into the 19-aldehyde has also been obtained from maize seedlings.80 The kaurenolides e.g. (64) are co-metabolites of gibberellins in Giberella fujikuroi. Both oxygen atoms from C-19 of ent-kaur-16-en-19-oic are retained in their formation.81 The diene (65) has been (63) R = CH,OH 0 (65) R = C0,H; 6,7-dehydro (64) implicated in this step which presumably proceeds via the 6p,7P-epoxide which is opened in a trans diaxial manner by the carboxyl group.Resuspended cultures of G. fujikuroi metabolized [3-'3C]mevalonate to 13C-enriched gibberellins plus I3CO2 which results from the loss of C-20.82 The formation of gibberellic acid (GA,) could 75 A. P. W. Bradshaw J. R. Hanson and I. H. Sadler J. Chem. SOC.,Chem. Commun.,1982. 292. 76 A. P. W. Bradshaw J. R. Hanson and I. H. Sadler J. Chem. SOC.,Perkin Trans. I 1982 2445. 77 M. J. Ackland J. R. Hanson A. H. Ratcliffe and I. H. Sadler J. Chem. Soc. Chem. Commun. 1982 165. 78 M. J. Ackland J. R. Hanson B. L. Yeoh and A. H. Ratcliffe J. Chem. SOC.,Perkin Trans. 1. 1985 2705. 79 P. F. Sherwin and R. M. Coates J. Chem. SOC.,Chem. Commun. 1982 1013. 80 E.S. Wurtele P. Hedden and B. 0.Phinney J. Plant Growth Regul. 1982 1 15. 81 M. H. Beale J. R. Bearder G.H. Down,M. Hutchison J. MacMillan and B. P. Phinney Fhytochemistry 1982 21 1279. 82 P. Lewer and J. MacMillan Phytochemistry 1984 23 2803. Biosyn thesis 3 59 be observed in vivo as well as 13C02 if the filtered medium was concentrated. The metabolism of gibberellins has been reviewed.83 The aromatase-catalysed oxidation of androst-4-ene-3,17-dione(66) to oestrone has been the subject of particularly interesting work. Using samples of (66) with chiral 19-methyl labelling Caspi et ul. have demonstrated that the initial C-19 hydroxylation occurs with retention of ~tereochemistry.~~ 2H and l80doubly labelled alcohol (67) and aldehyde (68) also have been converted into oestrone by human placental microsomes to give the results shown.85 Me ,o 0 HO \ =* 0 HCOOH (68) The formation of oleanane and urs- 12-ene pentacyclic triterpenes has been exten- sively studied using 13C and 2H labelled acetates and mevalonate in tissue cultures of Isodon juponicus;86 and the incorporation of [1,2-13C2]acetate into the side chains of a number of sterols has been studied using cell-tissue cultures of several higher plants.87 A number of compounds of mixed terpenoid-polyketide origins (meroterpenoids) have been studied.The phthalide (69) is converted into mycophenolic acid (70) by two separate pathways.88 13C-Labelling studies demonstrate that the mycotoxin austalide D (71) is also derived via (69).89 Austin (72) is formed by alkylation of 3,5-dimethylorsellinic acid by farnesyl pyrophosphate followed by extensive oxida- tive modification^.'^ Andibenin B and terretonin have been shown to be products of the same path~ay.~',~~ 83 J.MacMillan in 'Analysis of Plant Hormones and Metabolism of Gibberellins' ed. A. Crozier and J. R. Hillman Society for Experimental Biology Cambridge 1984 Vol. 23 p. 9. 84 E. Caspi T. Arunachalam and P. A. Nelson J. Am. Chem. Soc. 1983 105 6987. 85 M. Akhtar M. R. Calder D. L. Corina and J. N. Wright Biochem. J. 1982 201 569. 86 S. Seo Y. Tomita and K. Tori J. Am. Chern. SOC.,1981 103 2075. 87 S. Seo A. Uomori Y. Yoshimura and K. Takeda J. Am. Chern. SOC.,1983 105 6343. 88 L. Colombo C. Gennari D.Potenza C. Scolastico F. Aragozzini and R. Gualandris J. Chem. SOC. Perkin Trans. 1 1982 365. 89 A. E. de Jesus R. M. Horak P. S. Steyn and R. Vleggaar J. Chem. SOC.,Chem. Commun. 1983 716. 90 T. J. Simpson D. J. Stenzel R. N. Moore L. A. Trimble and J. C. Vederas J. Chem. SOC.,Chem. Commun. 1986 1242. 91 C. R. McIntyre T. J. Simpson R. N. Moore L. A. Trimble and J. C. Vederas J. Chem. SOC.,Chem. Commun. 1984 1498. 92 C. R. McIntyre T. J. Simpson D. J. Stenzel A. J. Bartlett E. O'Brien and J. S. E. Holker J. Chem. Soc. Chem. Commun. 1982 781. 360 T. J. Simpson Me0 (71) (72) 4 Shikimate Metabolism Studies of the mechanisms of the individual steps in the shikimic acid pathway have been greatly facilitated by the availability of the individual enzymes in large quantities from gene cloning methodology.For example purification of dehydroquinate syn- thase from E. coli K-122 has been reported93 and a plasmid-bearing strain that overproduces the enzyme 1000 fold has been constructed by subcloning the aroB gene into E. coli RB791 resulting in production of the enzyme as 5% of the total water-soluble protein.94 Studies of the steric course of the 5-enolpyruvyl-shikimic acid-3-phosphate (EPSP) synthase reaction have shown that (2E)-[3-2H ,3- 3H,]phospho-enol pyruvate is incorporated into chorismate (73) with the double bond configuration retained so that in the EPSP synthase reaction the addition step must have the opposite steric course to that of the elimination The subsequent rearrangement of chorismate to prephenate (74) catalysed by chorismate mutase has been shown using (E)-and (Z)-[9-’H1 ,9-3H,]chorismates to proceed via a chair-like transition as does the corresponding uncatalysed thermal Claisen I I OH OH (73) (74) 93 J.W. Trost J. L. Bender J. T. Kadonaga and J. R. Knowles Biochemistry 1984 23 4470. 94 K. Duncan and J. R. Coggins Biochern. Soc. Trans. 1984 12 275. 95 C. E. Grimshaw S. G. Sogo S. D. Copley and J. R. Knowles J. Am. Chem. Soc. 1984 106 1699; J. J. Lee Y. Asano T.-L. Shieh F. Speafico K. Lee and H. G. Floss J. Am. Chem. Soc. 1984 106 3367. 96 S. G. Sogo T. S. Widlanski J. H. Hoare C. E. Grimshaw G. A. Berchtold and J. R. Knowles J. Am. Chem. Soc. 1984 106 2071; Y. Asano J. J. Lee T.L. Shieh F. Speafico C. Kowal and H. G. Floss ibid. 1985 107 4314. Biosynthesis 361 rearrangement.97 The mechanism of the prephenate dehydrogenase reaction has been studied making elegant use of I3C isotope effects on the decarboxylation of both deuteriated and non-deuteriated substrate^.^^ It was concluded that the reaction proceeds by a concerted mechanism rather than a stepwise one in which dehydroge- nation precedes decarboxylation. Studies of the substrate specificity of chalcone synthase from Pteroselinum hortense (parsley) showed that malonyl CoA could not be replaced by malonyl acyl carrier protein but that butyryl- hexanoyl- and benzoyl-CoA could function efficiently as chain-initiating units whereas acetyl- and octanoyl-CoA were relatively poor sub- strate~.~~ An isoflavone synthase has been detected for the first time in a microsomal preparation from cell-suspension cultures of soybean (Glycine max) treated with a glucan phytoalexin elicitor from Phytophthoramegasperma.'OOCofactor requirements were consistent with a monooxygenase so a pathway to isoflavone (75) via epoxida-tion of the enol form of the flavanone leading to the spiro-cyclopropyl-cyclohexadienone (76) has been proposed..OH H HO H+ (76) (75) 'H and 13C labelled precursors have been used to obtain useful biosynthetic information on a number of flavonoid metabolites including phaseolin,"' psilotin,"' formon~netin,'~~ and pisatin.Io4 5 Alkaloids and other Amino Acid Derived Metabolites The mechanism whereby phenylalanine is converted into tropic acid (77) has been much studied.Incorporation of the four possible stereoisomers of [carboxyl-97 S. D. Copley and J. R. Knowles J. Am. Chem. Soc. 1985 107 5306. 98 J. D. Hermes P. A. Tipton M. A. Fisher M. H. O'Leary J. F. Morrison and W. W. Cleland Biochemistry 1984 23 6263. 99 R. Schiizm W. Heller and K. Hahlbrock J. Bid. Chem. 1983 258 6730. I00 M. Hagmann and H. Grisebach FEBS Letf. 1984 175 199. ini P. M. Dewick and M. J. Steele Phytochemistry 1982 21 1599. 102 E. Leete A. Muir and G. H. N. Towers Tetrahedron Lett. 1982 23 2635. 103 H. A. M. Al-hi and P. M. Dewick J. Chem. Soc. Perkin Trans. 1 1984 2831. 104 S. W. Banks and P. M. Dewick. 2. Naturforsch. Sect. C 1983 38 185.362 T. J. Simpson C,P3Hl]alanine into hyoscyamine in Datura stramonium indicated that the rearrangement involves a 1,2-migration of both hydrogen and the ~arboxyl."~ A highlight of the period has been the complementary work of two research groups on elucidating the details of the biosynthesis of the pyrrolizidine and olizidine alkaloids from putrescine and cadaverine respectively. Extensive use has been made of 2H- 13C- and "N-labelled precursors and n.m.r. analysis of enriched metabolites. For example on incorporation of [1,9-13C2]homospermidine (78) into retronecine (79) the 13C n.m.r. spectrum showed doublets for C-8 and C-9 to confirm that homospermidine is an intact precursor.lo6 [l-13C,1-amino-15N]cadaverine (80) has been incorporated inter alia into lupinine (81) and lupanine (82).Observation NH2 HO of 13C-"N couplings indicated the mode of incorporation of cadaverine into these precurs~rs.'~~~'~~ Incorporations of (R) and (S) [l-2Hl]cadaverine have also been rep~rted.'"".'~ Aspects of this work have been recently reviewed."' Observation of two sets of I3C-l3C couplings on incorporation of [4,5-13C,]lysine (83) into vertine (84) and lythrine (84; 10PH) confirms that lysine is incorporated via a symmetrical intermediate cadaverine."' Detailed and valuable information on alkaloid biosynthesis has been obtained using enzymes from cell-tissue cultures. Inter alia an O-methyl-transferase which converts scoulerine (85) into tetrahydrocolumbamine (86) has been isolated from 105 E.Leete 1. Am. Chem. Soc. 1984 106 7271. I06 J. Rana and D. J. Robins J. Chem. Rex (S) 1983 146. 107 J. Rana and D. J. Robins J. Chem. Soc. Chem. Commun. 1983 1325. I08 W. M. Golebiewski and I. D. Spencer J. Am. Chem. SOC.,1984 106 7925. 109 A. M. Fraser and D. J. Robins J. Chem. Soc. Chem. Commun. 1984 1477. 110 I. D. Spenser Pure Appl. Chem. 1985 57 453. 111 S. H. Hedges R. B. Herbert and P. C. Wormald J. Chem. Soc. Chem. Commun. 1983 145. Biosyn thesis 363 OH (86) R = Me suspension cultures of Berberis wilsoniae.112 Vinorine .synthase which catalyses the conversion of 16-epi-vellosimine into vinorine (87) in the presence of acetyl CoA has been isolated from cell cultures of RauwolJa serpent in^."^ The conversion of anhydro-vinblastine into vinblastine by a cell-free homogenate of a cell-line of Catharanthus roseus that did not produce dimeric indole alkaloids has been repor- ted,Il4 and a review of one group's studies on terpenoid alkaloid biosynthesis in different cell-lines of C.roseus has appeared."' Incorporation of [Me-2H3]mevalonic acid into echinulin (88) in Aspergillus arnstelodarni followed by degradation and *H n.m.r. analysis of the enantiotopic methyl of the resultant 2,2-dimethylbutan- 1-01 using a chiral shift reagent revealed HN,!,,Me I12 S. Muemmler M. Ruffer N. Nagakura and M. H. Zenk Plant Cell Rep. 1984 4 36. I13 A. Pfitzner and J. Stockigt Tetrahedron Lett. 1983 24 5197. I14 W. R. McLauchlin M. Hasan R. L. Baxter and A. I. Scott Tetrahedron 1983 39 3777.11s J. P. Kutney B. Aweryn L. S. L. Choi T. Honda P. Kolodziejczyk N. G. Lewis T. Sato S. K. Leigh K. L. Stuart B. R. Worth W. G. W. Kurz. K. B. Chatson and F. Constabel Tetrahedron 1983,39.3781. 364 T. J. Simpson Ph '1 that most of the label was present in the 2-pro-S-methyl group."6 [ indole-2-13C,2-15 NITryptophan has been incorporated into roquefortine (89) in Penicillium roquefor- tii and into oxaline (90) in P. o~alicum."~ Incorporation was confirmed by observa- tion of 13C-'5N coupling in both metabolites. The cyclodipeptide (91) labelled with 35S has been shown to be a precursor for the aranotin derivative (92) in Aspergillus terreus.' l8 Analysis of the 13C n.m.r. spectrum of streptonigrin (93) isolated from feeding [ U-'3C]-glucose to Streptomyces Jocculus showed that the metabolite was formed from the intact units indicated."' Further evidence has come from feeding [ l-'3C]erythr~~e'20 resulting in enrichment(*) of C-8 C-6 and (2-9'.Detailed studies with 13C- and "N-labelled arginines have unambiguously estab- lishedI2l the way in which the molecule is used to construct the streptolidine moiety of streptothricin F (94). The mechanism for the conversion of a-lysine into the P-lysine component of (94) has been studied'22 in both Clostridium and Streptomyces species and is in general agreement with results for P-lysine formation in tissue cultures of Andrographilis pani~ulata.'~~ A huge body of work has appeared on the biosynthesis of the p-lactams.A useful general review'24 and a more detailed one on the enzymology of penicillin biosyn- thesis have appeared.'25 The enzyme which catalyses the cyclization of the tripeptide 116 D. M. Harrison and P. Quinn J. Chem. SOC.,Chem. Commun. 1983 879. I I7 P. S. Steyn and R. Vleggaar J. Chem. SOC.,Chem. Commun. 1983 560. 118 G. W. Kirby D. J. Robins and W. M. Stark J. Chern. SOC., Chem. Commun.,1983 812. 119 S. J. Gould and D. E. Cane J. Am. Chem. SOC., 1982 104 343. I20 W. J. Gerwick S. J. Gould and H. Fonouni Tetrahedron Lett. 1983 24 5445. 121 K. J. Martinkus C.-H. Tann and S. J. Gould Tetrahedron 1983 39 3493. 122 T. K. Thiruvengadam S. J. Gould D. J. Aberhart and H.-J. Lin J. Am. Chem. SOC.,1983 105 5470. 123 J. Freer G. Pedrocchi-Fantoni D.J. Picken and K. H. Overton J. Chem. SOC.,Chem. Commun. 1981 80. 124 R. Southgate and S. Elson Fortschr. Chem. Org. Naturst. 1985 47 1. 125 J. A. Robinson and D. Gani Nat. Prod. Rep. 1985 2 293. Biosynthesis 365 0 Me0&c02H / H2N H2N Me Me0 HoQ9' 0LNH2 OMe (93) (94) L,L,D-a-aminoadipylcysteinylvahe isopenicillin-N synthetase has been purified to homogeneity by several groups.'26 The gene from Cephalosporium acremoniurn that codes for isopenicillin-N synthetase has been identified using a synthetic oligonucleotide probe corresponding to a portion of the N-terminal amino-acid sequence of the enzyme and has been cloned into E. c~li.'~~ The substrate specificity of the enzyme has been studied extensively using synthetic tripeptide analogues and this has led to the formation of many novel P-lactams.'28 Use of suitably deuteriated samples of LLD-ACV in competitive mixed-label experiments with isopenicillin-N synthetase and detailed analysis of the deuterium isotope effects has provided evidence that the p-lactam ring forms before the thiazolidine ring.129 Model studies provided good support for a free radical mechanism for thiazolidine ring forma- tion,13' and a penicillin synthesis has been reported which begins with a monocyclic P-lactam and forms the thiazolidine ring oxidatively using Fe2+ and ascorbic acid which are the same cofactors used in the enzymic ~eacti0n.l~' Other noteworthy work concerns the incorporation of '3C,'5N-labelled LLD-ACV into isopenicillin N;'32 the synthesis of valine with chiral methyl groups and incorporation into cephalosporin C;133the biosynthetic origins of nocardicin A;134and the origins of the C3 and C5 units of clavulanic acid.'35 126 C.-P.Pang B. Chakravarti R. M. Adlington H.-H. Ting R. L. White G. S. Jayatilake J. E. Baldwin and E. P. Abraham Biochem. J. 1984 222 789; J. Kupka J.-Q. Shen S. Wolfe and A. L. Demain Can. J. Microbiol. 1983 29 488; F. R. Ramos M. J. L6pez-Nieto and J. F. Martin Antimicrob. Agents Chemother. 1985 27 381. 127 S. M. Samson R. Belagaje D. T. Blankenship J. L. Chapman D. Perry P. L. Skatrud R. M. van Frank E. P. Abraham J. E. Baldwin S. W. Queener and T. D. Ingolia Nature (London) 1985,318,191. 128 See for example J. E.Baldwin R. M. Adlington A. E. Derome H.-H. Ting and N. J. Turner 1.Chem. SOC.,Chem. Commun.,1984 1211. 129 J. E. Baldwin R. M. Adlington S. E. Moroney L. D. Field and H.-H. Ting J. Chem. Soc. Chem. Commun. 1984 984. 130 C. J. Easton J. Chem. Soc. Chem. Commun. 1983 1349; C. J. Easton and N. J. Bowman ibid. p. 1193. 131 J. E. Baldwin R. M. Adlington and R. Bohlmann J. Chem. SOC.,Chem. Commun. 1985 357. 132 R. L. Baxter C. J. McGregor G. A. Thomson and A. 1. Scott J. Chem. SOC.,Perkin Trans. 1 1985 369. 133 C.-P. Pang R. L. White E. P. Abraham D. H.G. Crout M. Lustorf P. J. Morgan and A. E. Derome Biochem. J. 1984 222 777. 134 C. A. Townsend and A. M. Brown J. Am. Chem. SOC.,1983 105 913; C. A. Townsend and G. M. Salituro J. Chem. SOC.Chem. Commun. 1984 631. 135 S. W. Elson R. S. Oliver B. W. Bycroft and E. A. Faruk J. Antibiot. 1982 35 81; C. A. Townsend and M.-F. Ho J. Am. Chem. Soc. 1985 107 1065 1066. 366 T J. Simpson The cyclopentyl isocyanide (95) has been shown to be formed from tyr~sine.'~~ Two labelling patterns are observed (from 13C-enriched precursor) showing that ring-fission OCCUTS at both a and b. The lincomycins A (96) and B (97) contain respectively the propyl- and ethyl-hygric acid moieties (98) and (99). Incorporations of 2H-labelled tyrosine and dopa (100) indicate their formation from oxidative cleavage of dopa and methi~nine.'~~ (96) R = Pr" (97) R = Et (98) X = Me (99) X = H The 2-amino-3-hydroxycyclopentenonemoiety occurs in several antibiotics of disparate structure.Results of experiments on formation of asukamycin in Streptomy-ces nodosus indicate that this moiety is formed by intramolecular cyclization of 5-aminolaevulinic Interesting biosynthetic studies on showd~mycin,'~~ 136 J. E. Baldwin H. S. Bansal J. Chondrogianni L. D. Field A. A. Taka V. Thaller D. Brewer and A. Taylor Tetrahedron 1985 41 1931. 137 N. M. Brahme J. E. Gonzalez J. P. Rolls E. J. Hassler S. Mizsak and L. H. Hurley J. Am. Chem. Soc. 1984 106 7873. 138 A. Nakagawa T.-S. Wu P. J. Keller J. P. Lee S. Omura and H. G. Floss J. Chem. SOC.,Chem. Commun. 1985 519. 139 J. G. Buchanan A. Kumar R. H. Wightman S. J. Field and D. W. Young J. Chem. SOC.,Chem. Commun. 1984 1517. Biosynthesis 367 virginiamycin antibiotic^,'^' and naphthyrid~mycin'~~ elia~mycin,'~~ have appeared.[2-'3C,'5N]Aspartic acid is incorporated intact into 3-nitropropionic acid in Penicil-Zium atro~enetum.'~~ A large number of tremorgenic mycotoxins have structures based on substitution of indole with highly modified terpenoid residues e.g. penitrem A (101). Structural and biosynthetic work on these and other tremorgens has been reviewed.'@ Me 6 Porphyrins Hydroxymethylbilane (102) is now firmly established as the product of the enzyme deaminase which is cyclized by co-synthetase to uroporphyrinogen I11 (103). Kinetic pulse-labelling experiments with both I3C- and 14C-labelled porphobilinogen (PBG) (104) have shown that the order of binding of the four pyrrolic rings to deaminase is first ring A then B C and finally D.'45 Different forms of deaminase which differ in having 1 2 3 or 4 molecules of PBG bound per molecule of deaminase have been detected and ~eparated.'~~ In the presence of excess PBG these forms could complete the synthesis of hydroxymethylbilane.Battersby and co-workers have P A HOZC $-P A HOCH2 >-P A P P I4O R. J. Parry and J. V. Mueller J. Am. Chem. SOC.,1984 106 5614. 141 J. W. LeFevre and D. G. I. Kingston J. Org. Chem. 1984 49 2588. 142 M. J. Zmijewski jun. M. Mikolajczak V. Viswanatha and V. J. Hruby J. Am. Chem. SOC.,1982 104 4969. 143 R. L. Baxter E. M. Abbott S. L. Greenwood and I. J. McFarlane J. Chem. Soc. Chem. Commun. 1985 564. 144 P.S. Steyn and R. Vleggaar Fortschr. Chem. Org. Naturst. 1985 48 1. 145 A. R. Battersby C. J. R. Fookes G. W. J. Mateham E. McDonald and R. Hollenstein J. Chem. SOC. Perkin Trans. 1 1983 3031; J. S. Seehra and P. M. Jordan J. Am. Chem. SOC.,1980 102 6841. 146 P. M. Anderson and R. J. Desnick J. Biol. Chem. 1980 255 1993; A. Berry P. M. Jordan and J. S. Seehra FEBS Lett. 1981. 129 220. 368 T. J. Simpson demonstrated that the active site of deaminase from Euglena gracilis contains an essential lysine residue which is the likely point of attachment of the first molecule of PBG.14' A different conclusion was reached by Scott and co-workers who studied the 3H n.m.r. of a PBG-deaminase complex formed using 3H-labelled PBG with an activity of 132 Ci mmol-' and the deaminase from Rhodopseudornonas spheroides.14* The 3H n.m.r. of this complex showed inter alia a broad peak centred at 6 3.28 ppm which was attributed to an S-CHT-pyrrole residue indicating that the PBG was attached to a cysteine group! The spiro-pyrrolenine (105) is a likely intermediate in the conversion of hydroxymethylbilane into uro'gen 111. Two model systems (106) and (107) containing the previously unknown macrocyclic system of (105) have been synthesi~ed.'~' In vitro evidence for this fragmentation-recombination process has been provided by synthesis of the model pyrrolo-pyrrolenine (108) and its demonstrated facile rearrangement to (109) on mild acid treatment.l5' P A P AM" CN (106) X = x ,(lo71 X = CN H Me ,Me Bu'02C H Me The conversion of uro'gen I11 into cobyrinic acid during the biosynthesis of vitamin B, proceeds via the C-methylated products Factors I 11 and 111 the dihydro forms of which are believed to be the actual enzyme-active intermediates.This has been confirmed for Factor I1 by an isolation experiment under rigorously anaerobic condition^.'^' Post Factor I11 (110)the pathway requires five methylations decarboxylation of the C-12 side-chain insertion of cobalt ring contraction and loss of C-20 via acetic acid. In a fascinating series of biomimetic synthetic studies Eschenmoser and co-workers have studied the methylation of dipyrrocorphins and 147 G. J. Hart F. J. Leeper and A. R. Battersby Biochem. J. 1984 222 93. 148 J. N.S. Evans P. E. Fagerness N. E. Mackenzie and A. I. Scott J. Am. Chem. SOC.,1984 106 5738. 149 W. M. Stark M. G. Baker P. R. Raithby F. J. Leeper and A. R. Battersby J. Chem. Soc. Chern. Comrnun. 1985 1294. 1so A. R. Battersby H. A. Broadbent and C. J. R. Fookes J. Chem. Soc. Chem Comrnun. 1983 1240. 151 A. R. Battersby F. Frobel F. Hammerschmidt and C. Jones J. Chem. Soc. Chem. Comrnun. 1982,455. Biosynthesis 369 pyrrocorphins to obtain information on the likely biogenetic sequence.152 The actual sequence has been demonstrated in separate studies using pulse-labelling experi- ments with l3C-labe1led S-adenosyl methionine and unlabelled Factor 111in cell-free preparations from Clostridium tetanornorphurn153 or R. ~pheroides'~~ to be C-17 12 1 15 and finally C-5.Eschenmoser has also provided excellent biomimetic evidence for the ring-contraction process by demonstrating the interconversions shown in the scheme starting from the nickel or cobalt complexes of 20-methyl-20-hydroxydihy-drocorphin (1 11). The previously puzzling requirement for the introduction of the 10-methyl is explained by the ready tautomerization of the nor-methyl analogue (112) to the corresponding ketone which does not undergo ring-c~ntraction.'~~ Recent 180-labelling studies on bacteriochlorophyll biosynthesis have shown that the phytyl ester is formed by attack of the ring D propionate carboxyl group on the w-HJ-N M'NJ (111) R = Me (112) R = H I52 R. Wadischatka and A. Eschenmoser Angew. Chem. Int. Ed. Engl.1983 22,630; R. Wadischatka E. Diener and A. Eschenmoser ibid. p. 631;C. Leumann K.Hilpert J. Schreiber and A. Eschenmoser J. Chem. SOC.,Chem. Commun. 1983 1404. 153 H.C.Uzar and A. R. Battersby J. Chem. SOC., Chem. Commun. 1985 585. 154 A. I. Scott N. E. Mackenzie P. J. Santander P. E. Fagerness G. Muller E. Schneider R. Sedlmeier and G. Worner Bioorg. Chem. 1984 12 356. 155 V. Rasetti A. Pfaltz C. Kratky and A. Eschenmoser Proc. Natl. Acad. Sci. USA 1981 78 11; V. Rasetti K.Helpert A. Fassler A. Pfaltz and A. Eschenmoser Angew. Chern Znt. Ed. Engl. 1981 20 1058. 370 T. J. Simpson isoprenyl pyrophosphate with retention of both carboxylate oxygen^.'^^ The stereochemistry of formation of the ethyl group in bacteriochlorophyll a has been shown to occur by overall trans redu~tion'~' of the corresponding vinyl group with addition of hydrogen from the si face at C-8l and the re face at C-8*.I56 V. C. Emery and M. Akhtar J. Chem. SOC.,Chem. Commun. 1985 600. V. C. Emery and M. Akhtar J. Chem. SOC.,Chem. Commun. 1985 1646; A. R. Battersby A. L. Gutman C. J. R. Fookes M. Gunther and H. Simon ibid. 1981 645.