14 Biological Chemistry Part (v) Biosynthesis By R.B. BOAR Department of Chemistry Chelsea College London SW3 6LX and D. A. WIDDOWSON Department of Chemistry Imperial College London SW7 2AY 1 Introduction This year's literature on biosynthesis has been dominated by applications of "C n.m.r. spectroscopy. Experiments using precursors in which two adjacent atoms are labelled with carbon- 13 have proved to be particularly informative. The conditions under which carbon-13 studies are most likely to be successful were defined last year,' and have since been further reviewed.2 The decline in more classical investiga- tions employing radiochemically-labelled precursors is exemplified by the area of alkaloid biosynthesis which no longer warrants a separate section.2 Terpenoids The incorporations of [14C]carbon dioxide and [14C]acetate into a representative range of monoterpenes have been ~tudied.~ Comprehensive degradations have established that not only does the half of the molecule derived from isopentenyl pyrophosphate contain the majority of the radioactivity but that labelling within this unit itself is uneven. Thus a sample of (+)-isothujone (1) isolated from l7zujaplicata after administration of [2-14C]acetate was labelled as shown. Such results are understandable if one accepts the concept of compartmentalized pools of particular intermediates. R. B. Boar and D. A. Widdowson Ann. Reports (B) 1974,71,455. T.J. Simpson Chem.Soc. Rev. 1975,4497; G.A. McInnes and J. L. C. Wright Accounts Chem.Res.1975,8,313;R.E.London V. H. Kollman and N. A. Matwiyoff J.Amer. am.Soc. 1975,97,3565. 3 D. V. Banthorpe 0.Ekundayo J. Mann and K.W. Turnbull Phytochernistry 1975,14,707. 415 R. B. Boarand D.A. Widdowson The additional carbon atoms of the homosesquiterpenoid insect juvenile hor- mones (3) and (4) are supplied by propionate units. [l-*4C]Propionate gave JH I (3) in which the radioactivity was shown to be equally divided between C-7 and C- 11.4 In an independent study a mixture of [Me-14C]methionine and [5-3H]homomevalonic acid (2) was administered to an in vitro culture from Manduca sexta.’ The derived homosesquiterpenoid JH I1 (4) and the unmodified sesquiter- pene JH 111 (5)were each labelled with carbon-14 in the methoxy.-group but only the former was labelled with tritium (at C-9).R‘ R2 (3) R’= R2 = Me (4) R’= Me; R2 = H (5) R’ = R2 = H In contrast it is well established that the additional carbon atom(s) present at C-24 of many triterpenoids originate from the S-methyl group of methionine. The exact mechanism of the alkylation varies from one system to an~ther.~” Feeding experi- ments with [Me-2H3]methionine and [2-I4C 4R -3H] mevalonic acid (MVA) sup-port the biosynthesis of the sitosterol side-chain in the higher plant Hordeurn vulgare by the pathway shown in Scheme 1. Scheme 1 Work defining the occurrence of stereospecific 173-hydride shifts in the biosyn- thesis of certain classes of sesquiterpenes has been When initial cyclization of the farnesol precursor involves bond formation bztween C- 1and C- 11 M.G. Peter and K. H. Dahm Helv. Chim. Acta 1975,58,1037. R. C. Jennings K. J. Judy and D. A. Schooley J.C.S. Chem. Comm. 1975,21. E. I. Mercer and W. B. Harries Phytochemistry 1975,14,439; E. I. Mercer and S. M. Russell ibid. pp. 445 451. J. R. Lenton L. J. Goad and T. W. Goodwin Phytochemistry 1975,14 1523. D. Arigoni Pure Appl. Chem. 1975,41 219. 9 F. Dorn P. Bernasconi and D. Arigoni Chimia (Switz.) 1975 29 24. lo J. R. Hanson and R. Nyfeler J.C.S. Chem. Comm. 1975 824. l1 A. Corbella P. Gariboldi G. Jommi and M. Sisti J.C.S. Chem. Comm. 1975 288. Biological Chemistry -Part (v) Biosynthesis there follows migration of a pro-S-hydrogen from C-1 to C-10 whereas bonding between C- 1and C-10 results in a pro -R-hydrogen migrating from C-1to C-11.8-11 The isomeric hydrocarbons (-)-longifolene (6)and (-)-sativene (7)are thus biosyn- thesized in Helminthosporium species according to Scheme 2.**' A similar rationale applies to the biosynthesis of culmorin (8)" and dendrobine (9).8*11 Scheme 2 The labelling patterns of the fungal metabolite fusicoccin (10) when biosyn- thesized from [l-13C]acetate (@) and [2-13C]acetate (A)are fully consistent with an unexceptional formation via geranylgeranyl pyrophosphate.l2 Precursors doubly labelled with carbon-13 at adjacent sites can provide rapid solutions to otherwise complicated problems. Feeding experiments using [1,2-13CJacetate and [2-14C,4R -3H]MVArequire that the biosynthesis of the diterpene l2 K.D. Barrow R. B. Jones P. W. Pemberton and L. Phillips J.C.S.Perkin 1 1975 1405. R. B.Boar and D.A. Widdowson OAc Ho -A 0H Ho\ /Q A' A ACH,OMe (10) antibiotic aphidicolin (11) proceeds according to Scheme 3.13The usual observation that it is the 4a-methyl group of 4,4-dimethyl-di- and -tri-terpenoids which is derived from C-2 of MVA is confirmed by this13 and elated'^*'^ studies. J" 1 T+> H &:T\oHk' HO** + f' OH (1 1) Scheme 3 l3 M. R. Adams and J. D. Bu'Lock J.C.S. Chem. Comm. 1975,389. l4 J. Polonsky G. Lukacs N. Cagnoli-Bellavita and P. Ceccherelli TetruhedronLetters 1975,481. S. Seo Y. Tomita and K. Tori J.C.S. Chern. Comrn. 1975 270 954. Biological Chemistry -Part (v)Biosynthesis 419 The use of [1,2-13C2]acetate as a precursor permits an unambiguous definition of the rearrangement leading from (3s)-squalene 2,3-epoxide to triterpenoids of the a-amyrin (urs-12-ene) group [for example (l2)]." The absence in the 'I3Cn.m.r.spectrum of coupling between carbon atoms 20 and 21 indicates that this bond has not survived intact from acetate and therefore rules out the possible intermediacy of the carbonium ion (13) (Scheme 4). +l CH,-CO; -+ HO H t Scheme 4 H The chirally labelled MVA (14) has been used to show that in the rearrangement leading from (3s)-squalene 2,3-epoxide to lanosterol the 1,2-shift of a methyl group from C-14 to C-13 proceeds with retention of configuration.16 l6 K. H.Clifford and G.T.Phillips J.C.S.Chem. Comm. 1975,419. R. B.Boar and D.A. Widdowson 3 Polyketides The use of acetate that is doubly labelled with 13Chas become dominant in this area of biosynthesis. In addition to the vicinal coupling which shows the incorporation of intact acetate units long-range (1,3)coupling has been observed in the product of a 1,2-alkyl shift.17 The method promises to be useful in the general area of 1,2-alkyl migrations. Terrein (15) a metabolite of Aspergillus terreus is now known to be derived from 3,4-dihydro-6,8-dihydroxy-3-methylisocoumarin(16). The proposed pathway including the novel extrusion of one carbon atom from the aromatic ring is given in Scheme 5.'* A pyrone metabolite (17) of Aspergillus melleus has been studied by using ['3C-Jacetate.Simpson suggested" a tropolone intermediate with a contraction of the side-chain as the method of generation of the unusual juxtaposition of two H02C Scheme 6 T. J. Simpson and J. S. E. Holker Tetrahedron Letters 1975,4693. 1s R. A. Will R. H. Carter and J. Staunton J.C.S. Gem. Comm. 1975,380. Biological Chemistry -Part (v) Biosynthesis 42 1 acetate methyl carbons (Scheme 6). In a subsequent collaboration with Holker” a simpler process (Scheme 7) was proposed. From the [13C2]acetate feeding they used the 1,3-coupling of 6.2 Hz between C-1 and C-7 in the metabolite to show the origin I Scheme 7 of these carbons to be as indicated. This result rules out the earlier Simpson hypothesis although a modified form would fit the later results.The necessity for formate involvement (Scheme 6) was not established. A similar juxtaposition of two acetate methyl carbons is found in the aflatoxins. [13C]Acetate feedings to Aspergillus flavus and A.parasiticus confirmed2’ the earlier results using 14C-labelled compounds and the double-labelled work21 gave the pattern shown for aflatoxin B1(18). A related metabolite sterigmatocystin (19) has previously been shown22 to be an efficient precursor of aflatoxin B1 (18) in A. parasiticus and a reassignment of the 13C spectrum of (19) has led23 to a new hypothesis for aflatoxin B1 biosynthesis (Scheme 8). The earlier hypothesis24 is no longer compatible with the newly established acetate-labelling pattern. The new scheme is based on a suggestion by Thomas2’ and encompasses the related metabo- lites averufin (20) and versicolorin A (21).The six-carbon side-chain would seem superfluous however for the aflatoxin route beyond (22) and a four-carbon analogue could be The labelling pattern in the furanoid system could be explained by a process related to the pyrone (17) pathway. l9 T. J. Simpson Tetrahedron Letters 1975 175. 2o D. P. H. Hsieh J. N. Sieber C. A. Reece D. L. Fitzell S. L. Yang J. I. Dalezios G.N. LaMar D. L. Budd and E. Motell Tetrahedron,1975,31,661. 21 P. Steyn R. Vleggaar and P. L. Wessels J.C.S. Chem. Comm. 1975 193. 22 M. T. Lin and D. P. H. Hsieh J. Amer. Chem. Soc. 1973,95 1668. 23 K. G. R. Pachler P. Steyn R. Vleggaar and P. L. Wessels J.C.S. Chem. Comm. 1975 355. 24 M.Bollaz G. Buchi and G. Milne J. Amer. Chem. SOC.,1970 92 1035. 25 R. Thomas personal communication to M. 0.Moss in ‘Phytochemical Ecology’ ed. J. B. Harborne Academic Press London 1972 p. 140. z6 (a)J. G. Heathcote M. F. Dutton an3 J. R. Hibbert Chem. andZnd. 1973,1021; (b)J. S. E. Holker and L. J. Mulheirn J.C.S. Chem. Comm. 1968 1576. R. B. Boar and D.A. Widdowson 0 -H,C-C02-J 0 0 (22) 1 0 0 0 (18) Scheme 8 The polyketide antibiotics continue to attract attention. The aglycone of the macrolide tylosin (23) is derived27 from acetate propionate and butyrate (2-ethylmalonate) as indicated. The structure of piericidin A has been revised to (24).28 The biosynthesis in Streptomyces mobaraensis had been shown to be from four acetate units and five propionate This was confirmed by the I3C-feedings.The isolation of erythromycin E (25) from Streptomyces erythreus and its forma- tion from erythromycin A (25) throws further light on the later stages of biosynthesis of this class of complex macrolide~.~~ 27 S. Omura A. Nakagawa H. Takeshima J. Miyazawa C. Kitao F. Piriou and G. Lukacs Tetrahedron Letters 1975,4503. S. Yoshida S. Shiraishi K. Fujita and N. Takahashi Tetrahedron Letters 1975 1863. 29 Y. Kimura N. Takahashi and S. Tamura Agric. and Bid. Chern. (Japan) 1969,33 1507. 30 J. R. Martin R. S. Egan A. W. Goldstein and P. Collum Tetrahedron 1975,31 1985. Biological Chemistry -Part (v)Biosynthesis mycinose I m ycaminose I mycarose (23) \ acetate; A Propionate; butyrate NMe, 1 Me H0.-12 HO..Me :e$o,J$Me . 0 Me 0 Me .VOH Me Me Me -Me 4 Shikimic Acid Metabolites Some interesting new routes to modified shikimic acid metabolites have appeared. Thus echinatin (27) a retro-chalcone is formed in Glycyrrhiza echinata by an apparent reversal of the substitution patterns of the aryl rings. Feeding experi- ment~~’ (Scheme 9) showed a 1,3-transposition of the unsaturated ketone function after formation of the ‘normal’ intermediate (28). 31 T. Saitoh S. Shibata and U. Sankawa Tetrahedron Letters 1975,4463. R. B. Boar and D.A. Widdowson (27) Scheme 9 Eucomin (29) one of a group of 3-benzylchroman-4-ones from Eucornis and Scilla spp. is formed by an oxidative cyclization of a methoxy-group onto the chalcone system.Scheme 10 shows a suggested pathway.32 The sequence of the OH (29; R = Me) Scheme 10 biosynthesis of the pterocarpan demethylhomopterocarpin (30) from an isoflavone in red clover (Trifoliurn pratense) has been determined to be as shown in Scheme 11-33.34 5 Nitrogen Metabolites The preparation from Catharanthus rmew of cell-free systems which can for example convert tryptamine and secologanin into alkaloids of the Corynanthe group with considerable efficiency promises to revolutionize the study of the 32 P. W. Dewick Phytochemistry 1975,14 983. 33 P. W. Dewick Phytochemistry 1975 14 979. 34 P. W. Dewick J.C.S. Chem. Comm. 1975,656. Biological Chemistry -Part (u) Biosynthesis J J Scheme 11 biosynthesis of indole alkaloids.35 Not only are more indicative incorporations thus obtained but the way is opened to studies using carbon-13-labelled precursors.Full details of Battersby's work on the biosynthesis of tetrahydroprotoberberine-type alkaloids have been published.36 The ambiguity in the origin of the cytochalasan D (31) has been resolved by experiments using d~ubly-'~C-labelled acetate.38 The C- 18 methyl group is derived from methionine and C-18 C-19 are derived from acetate [see (31)]. The A0 CH,-CO,-Ay$yOH H CH,-methionine (31) related macrolide cytochalasan B (phomin) (32) is formed39 by oxygen insertion into a carbocyclic precursor (33)' in a Phoma sp. (S 298) (Scheme 12). After the determination of the chiral origins of the methyl groups in penicillin G (34) attention has been turned to the nuclear hydrogen atoms.The a-hydrogens of both D-and L-valine are lost in the formation of penicillin G in a commercial high-yielding strain of Penicilliurn chrysogen~m.~~ Both isomers are equally effective precursors. D-Valine is therefore not directly incorporated and the chirality at C-3 in penicillin G is generated at a later stage. 35 A. 1. Scott and S.-L. Lee J. Amer. Chem. Soc. 1975,97 6906. 36 A. R. Battersby J. Staunton,H. R. Wiltshire B. J. Bircher and C. Fuganti J.C.S. Perkin I 1975 1162 and references there cited. 37 R. B. Boar and D. A. Widdowson Ann. Reports (B),1974,71,464. 38 J. C. Vederas W. Graf L. David and Ch.Tamm Helv.Chim. Acfu 1975,58 1886. 39 J.-L. Robert and Ch. Tamm Helv. Chim. Acfa 1975,58 2501. 40 B. W. Bycroft C. M. Wels K. Corbett A. P. Maloney and D. A. Lowe J.C.S. Chern.Cornrn. 1975,923. R.B.Boar and D.A. Widdowson -+ 0 (33) (32) Scheme 12 (34) Two groups have shown independently that the hydrogen at C-5 is derived largely (60-70%) from the 3-pro-R hydrogen of cy~teine.~',~~ The formation of the N-C-5 bond occurs therefore with overall retention of configuration. Porphyrin and corrin biogenesis continue to attract considerable interest. Akhtar has by feeding chiral a! -ketoglutarate to a haemolysed erythrocyte prepara- tion that the decarboxylation of the acetic acid side-chains in haem biosynthesis occurs with overall retention of configuration (Scheme 13).Uroporphyrinogen 111 Coproporphyrinogen 111 Scheme 13 Chemical degradation of the haem gave acetic acid from the methyl groups attached to rings c and D that had S-chirality. In an analogous series of experiments using Euglena gracilis Battersby has showna that the vinyl side-chains of protoporphyrin IX (35) are generated by an apparent antiperiplanar elimination of H' and CO from the propionic acid groups (Scheme 14). The stereospecifically labelled porphobilinogen (36) with (S,S)-41 D. J. Morecornbe and D. W. Young J.C.S. Chem. Comm. 1975 198. 42 D. J. Aberhart L. J. Lin and J. Y.-R. Chu J.C.S. Perkin f 1975 2517. 43 G. F. Barnard and M. Akhtar J.C.S. Chem. Comm. 1975,494. A. R. Battersby E. McDonald H. K. W.Wurziger and K. J. James J.C.S. Chem. Comm. 1975,493. 427 Biological Chemistry -Part (0) Biosynthesis (36) CO,H C0,H Scheme 14 chirality in the side-chain gave the monodeuteriated vinyl group as shown in (35). The (R,R)-isomer gave the converse 1,2-dideuteriovinyl function. By means of a specifically 14C-labelled uroporphyrinogen I11 (37) feeding Bat- tersby has also demonstrated4’ that the pro-S-methyl group in ring cof the cobyrinic acid derivative is derived from the acetic acid side-chain of (37) in Propionibacterium shermanii (Scheme 15). This transformation is the first unambiguous proof of the intermediacy of (37) in corrin biosynthesis. C0,H C0,Me CO,H C0,H Me0,C C0,Me (37) (38) Scheme 15 As a result of new studies on the origin of the angular methyl group in ring A of vitamin BIZ,Scott has proposed46 a new theory for corrin biogenesis.This methyl group is not derived from porphobilinogen and the meso-carbon of the uropor- phyrinogen I11 (37) precursor but from the C pool. 45 A. R. Battersby,M. Ihara E. McDonald F. Satoh and D. C. Williams J.C.S. Chem. Comm. 1975,436. 46 A. I. Scott C. A. Townsend K. Okada R. J. Cushley and P. J. Whitman J. Amer. Chem. Soc. 1974,% 8069.