19 Biosynthesis By E. McDONALD University Chemical Laboratory Lensfield Road Cambridge CB2 1EW 1 Monoterpenoids Chrysanthemic acid (1) bears a close structural relationship to presqualene pyrophosphate and prephytoene pyrophosphate the important biosynthetic precursors of the steroids and carotenoids.' Each of the three compounds has a pair of isoprene units which are not linked in a head-to-tail manner and this irregularity gives a special interest to studies of their biosynthesis. Doubly labelled [(4R)-4-3H]mevalonic acid (MVA) (2) was incorporated' (0.4 %) into (1) (2) chrysanthemic acid (1) by Chrysanthemum cinerariaefolium without a change in the ratio of 3H to 14C. The isolated monoterpenoid was purified as a crystalline amide and a straightforward degradation showed that all of the tritium was located at C(4).Clearly MVA in this experiment is providing only half of the carbon skeleton of chrysanthemic acid a result often found in the monoterpenoid area.lb In a parallel experiment [(4S)-4-3H]MVA lost its tritium during bio- synthesis of chrysanthemic acid and so the stereospecificity of hydrogen removal in this study parallels that found in other system^.^ In the same plant ''C-labelled chrysanthemic acid (1) was shown to be specifically incorporated4 (0.08%) into pyrethrin I1 (3). Me02CII ' (a)E. McDonald Ann. Reports (E) 1971 68 395; (b)E. McDonald Ann. Reports (E) 1972 69 467. ' G. Pattenden and R. Storer Tetrahedron Letters 1973 3473. J. W. Cornforth Chem. SOC.Rev. 1973 2 1. S.A. Abou Donia C. F. Doherty and G. Pattenden Tetrahedron Letters 1973 3477. 597 598 E. McDonald 2 Sesquiterpenoids Farnesol(4) is the parent of the sesquiterpene family but its conversion into the 2-cis-isomer (5) is frequently postulated in biogenetic schemes. The isomers of farnesol had identical labelling ratios when they were biosynthesized’ from (4) (5) [(4R)-or (4S)-4-3H,2-I4C]MVA by an enzyme system from Andrographis pani- culata. However when the incubation was carried out using [S3H, 2-I4C]MVA (6),all-trans-farnesol was formed without a change in the 3H 14C ratio but the 2-cis-isomer had lost one sixth of its tritium. Essentially the same result has been obtained by another research group who incubated6 labelled all-trans-farnesol with a cell-free system from Tricothecium roseurn.Their farnesol (prepared biosynthetically using pig liver) was tritiated randomly at C(1) C(5) and C(9) and it lost one sixth of its tritium during isomerization to 2-cis,6-trans-farnesol. It seems therefore that the 2-cis-isomer is formed from 2-trans-farnesol with obligatory loss of a hydrogen atom from C(1). The aldehydes corresponding to (4)and (5)are probably intermediates as suggested last yearlb for the isomeri- zation in Helminthosporium sativum. Although the insect juvenile hormones (7)and (8) are closely related to farnesol (4),they are not derived from it by direct methylation. A cell-free extract from the corpus allatum of Manduca sexta on incubation with S-adenosylmethionine tritiated in the S-methyl group does give labelled juvenile hormone (7),but all the tritium activity is confined to the ester methyl group.7 This result is in full K.H. Overton and F. M. Roberts J.C.S. Chem. Comm. 1973 378. R. Evans A. M. Holtom and J. R. Hanson J.C.S. Chem. Comm. 1973 465. ’ D. Reibstein and J. H. Law Biochem. Biophys. Res. Comm. 1973.55 266. Biosynthesis 599 (7) R' = R2 = Me (8) R' = Me R2 = H (9)R' = R2 = H agreement with earlier worklb using intact Hyalophora cecropia. The reason for these negative results has become clear from the outstanding experimental study of the Zoecon group8 [2-I4C]MVA was converted by gland cultures of Manduca sexta into juvenile hormones I1 (8) and I11 (9). After purification the hormones were each degraded and the carbons of the epoxide terminus were isolated and counted in the form of the crystalline derivatives (10)and (11).The NH (10) R' = Me (11) R' = H derivative (11)carried one third of the activity of hormone I11 (9),but derivative (10) from hormone I1 (8) was completely inactive. This clearly indicates that hormone I11 is a regular sesquiterpenoid but that hormone I1 is biosynthesized from only two MVA molecules and a C unit possibly homomevalonic acid (12). HOHZC COZH (12) Further experiments showing that hormone I11 incorporates nine acetate mole- cules but hormone I1 incorporates eight acetates and one propionate are con- sistent with the homo-MVA proposal. A regular farnesyl unit can be discerned as part of the skeleton of cochlio- quinone (13)and it has now been showng that [2-14C]MVA is incorporated only * D.A. Schooley K. J. Judy B. J. Berget M. S.Hall and J. B. Siddall Proc. Nut. Acud. Sci. U.S.A. 1973 70 2921. L. Canonica B. M. Ranzi B. Rindone A. Scala and C. Scolastico J.C.S. Chem. Comm. 1973 213. 600 E. McDonald HO.. HOpo into this C unit by CochlioboIus miyabeunus. The methyl groups marked a are derived from [Me-14C]methionine and the quinone ring and its attached side- chain are probably of acetate origin. The labelling pattern illustrated for dendrobine (14) was obtained" from feeding [5-3H,, 2-14C]MVA (6) to Dendrobium nobile the location of tritium at C(5)and C(8)being obtained by specific degradations to (15) and (16),respectively.T (14) The tritium at C(8) must have migrated from either C(3) or from C(5) and the authors assume that this happened at an early stage of the biosynthesis during interconversion of cations (17)and (18). If so then the isomerization of 2-trans- to 2-cis-farnesol must have occurred without loss of tritium a suggestion in conflict with results now discovered in three different organisms (uide supra). The alter- native possibility that tritium migrates from C(3) to C(8) later in the pathway lo A. Corbella P. Gariboldi and G. Jommi J.C.S. Chem. Comm. 1973 729. Biosyn thesis 601 nicely accounts for the presence of oxygen at C(3) and a feeding of specifically labelled farnesol should make the necessary distinction. Labelled illudin M (19) was obtained" after incubating the mushroom Clitocybe illudens with [2-14C]MVA.Further studies show that [(4R)-4-3H]MVA provides one tritium at C(1) or C(11) while [2-3H,]MVA gives illudin M with three tritium atoms only one of which is located in the cyclopropane ring. This shows that one of the CH groups of the cyclopropane changed its oxi- dation level during the biosynthesis and (20)is suggested as an intermediate. Incubation of [2-' 3C]MVA with Helicobasidium mompa gave' * a 3C-enriched sample of helicobasidin (21). This was converted into the diacetate before running the 13C n.m.r. spectrum in the presence of the relaxation reagent chromium trisacetylacetonate. Comparison of the spectrum with that of a natural-abundance sample revealed the enhancement of three carbon resonances assigned as C(4) C(12) and either one of the equivalent atoms C(8) or C(10).No justification is given for the distinction which is made between the pairs C(8),C(10) and C(7) C(11) (both remain singlets during off-resonance decoupling) or between C(2) and C(4) (both triplets) or between C(12) C( 13) and C(14)(all quartets). In all cases the differences in 13C chemical shift seem too small to be significant. J. R. Hanson and T. Marten J.C.S. Chem. Comm. 1973 171. l2 M. Tanabe K. T. Suzuki. and W. C. Jankowski Tetrahedron Letters 1973 4723. 602 E. McDonald 3 Diterpenoids (-)-Kaurene (22),biosynthesized from [(SS)-S-3H,2-14C]MVA,is converted by Gibberellufujikuroi (1.4%)into gibberellic acid (23) without loss of 3H.In the present work13 the stereochemistry of the 3H at C(11) was assigned from the rate of base-catalysed exchange of tritium from the rearranged ketone (24). T9'-*T HO W 11 H H T' C0,H (23) H py I Me0,C OH Preliminary experiments with D,O had shown that the exo-9-H exchanges faster than endo-9-H in (24). [17-' 4C]Kaurene (22) is poorly incorporated' (0.003-0.004%) by leaves from lsodon juponicus into the highly oxygenated diterpenoid enmein (25) and Sugar R CH,OH (26) R = CH,OH (27) R = CH oridonin but degradation of these compounds showed that all of their radio- activity was located in their vinyl methylene group. Both fusiococcin (26) and fusiococcin H (27) have a skeleton of twenty carbon atoms and are structurally l3 R.Evans J. R. Hanson and L. J. Mulheirn J.C.S. Perkin I 1973 753. l4 T. Fujita S. Takao and E. Fujita J.C.S. Chem. Comm. 1973 434. Biosynthesis 603 related to the CZ5 sesterterpenoids. It seemed possible that oxidative cleavage of the side-chain of a sesterterpene might give rise to the CH,OH residue in fusiococcin. Fusiococcin H might then be formed by reduction of fusiococcin. In fact such a reduction does not occur1’ in Fusiococcurn amygddi but rather fusiococcin H is oxidatively transformed (2%) into fusiococcin. This result strongly supports the classification of these compounds as true diterpenoids rather than degraded sesterterpenoids. The dilactone (28) with a c16 skeleton is very probably a degraded diterpene.The evidence for this comes from feeding experiments’ with Acrostalagrnus. 0 [2-13C]Acetate gave a sample of dilactone enriched as shown. The enrichment at C(12) and C(15) particularly precludes the possibility that the compound might be a methylated sesquiterpenoid. Furthermore the results obtained from feeding doubly labelled MVA were consistent with the incorporation of four rather than three molecules of MVA. 4 Triterpenoids The text of Professor Cornforth’s Robert Robinson lecture3 should not be missed by anyone interested in mechanistic studies of biosynthetic processes and it provides a background for many of the stereochemical results reported in these pages. For example the mechanism of the biosynthesis of squalene is such that [(5S)-5-3H 2-I4C]MVA (29) should lead to the labelling pattern illustrated in (30).The unsymmetrical labelling at the central pair of carbons C(12) and C(13) might in principle be maintained if squalene oxidase were to operate on only one of the terminal double bonds but because of the symmetry of the molecule it would be necessary for newly biosynthesized squalene to be transferred from one enzyme to another without release into the medium. To test this hypothesis the appro- priate MVA (29) was fed” to intact growing Fusidiurncoccineum,and [14C6,3H4]-fusidic acid (31) was isolated. Careful degradation established that one of the K. D. Barrow D. H. R. Barton Sir E. Chain U. F. W. Ohnsorge and R.P. Sharma J.C.S. Perkin I 1973 1590.l6 H. Kakisawa M. Sato T. Ruo,and T. Hayashi J.C.S.Chem. Comm. 1973 802. R. C. Ebersole W. 0. Gotfredsen S. Vangedal and E. Caspi J. Amer. Chem. SOC. 1973,95 8 133. 604 E. McDonald four tritium atoms is equally distributed between C(11) and C(12) so that the asymmetry of squalene is not transferred to later biosynthetic stages. The absence of tritium at C(16) suggests that hydroxylation took place with retention of configuration as expected. T i several steps HO (33) Biosynthesis 605 P-Amyrin (32) is formed in peas (Pisumsatiuurn)in high yield (13 %)from squalene oxide. The ion (33) (which is similar to that implicated in lanosterol biosynthesis) might be cyclized by the pea enzyme and transformed into (34).Support for the intermediacy of the tetracyclic ion (34) comes'* from the transformation of the synthetic compound (35) by the pea enzyme into p-amyrin (32) (0.006%). Oxidation of the P-amyrin to the 1 1-oxo-derivative established the specificity of labelling in the product. Oleanolic acid (36) and maslinic acid (37) were isolated from lsodon japonicus tissue culture after inc~bation'~ with [(4R)-4-3H 2-14C]MVA (2). The labelling ratio in the two acids was the same as that of the precursor and the pattern illus- trated can be deduced from earlier studies. In contrast 3-epi-maslinic acid iso- lated from the same experiment had retained five rather than six tritium atoms i (36) R = OH (37) R = H and degradation showed that the discrepancy is entirely at C(3).Thus 3-epi- maslinic acid must be biosynthesized via a 3-ox0 precursor. After feeding [2-I4C]MVA to Convallaria majalis degradation of convallamarogenin (38) established" that the exocyclic methylene group carries one sixth of the radio- activity and that C(26) is radioinactive. The same result has been observed in other sapogenins. H. Horan J. P. McCormick and D. Arigoni J.C.S. Chem. Comm. 1973 73. l9 Y. Tomita and S. Seo J.C.S. Chem. Comm. 1973 707. *O F. Ronchetti and G. Russo J.C.S. Chem. Comm. 1973 184. 606 E. McDonald HO (38) From two independent studies of the biosynthesis of the ecdysone hormones the sequence cholesterol (39)+ (40)-B (41) has been established. 6,7&0xido- cholestanol was reduced by borotritide and the product converted into cholesterol (39) (41) R = H ; a-ecdysone R = OH ; P-ecdysone (ecdysterone) stereospecifically labelled at C(7).In the insect Calliphora erythrocephala the tree Tuxus baccata and the fern Polypodium vulgare ecdysone and ecdysterone were formed from cholesterol and in every case the 7/3-hydrogen was lost.” Furthermore [3-3H]-(40) was incorporated22 by larvae of Calliphora stygia into ’’ I. F.Cook J. G. Lloyd-Jones H. H. Rees and T. W. Goodwin Biochem. J. 1973 136 135. M. N. Galbraith D. H. S.Horn E. J. Middleton and J. A. Thomson J.C.S. Chem. Comm. 1973 203. Biosynrhesis 607 a- and P-ecdysone (41),and degradation showed that the tritium of each hormone was exclusively located at C(3).Several pathways for ergosterol biosynthesis seem to operate in yeast and the complexities have been described by two different research gro~ps.~~,~~ The biosynthesis from [(4R)-4-3H 2-14C]MVA (2) of the plant sterols which carry an ethyl group at C(24) proceeds via cholesterol (39) and the ion (42) having tritium at C(25). It has now been shown that in four different plants stigmasterol (43)and a-spinasterol (44),produced in this way do not carry 3H at C(25),and ...TAT ..T& Jw 25 Jw. (43) cholesterol nucleus (42) (44) 5-a A8.9-cholestene nucleus therefore a 24-eth~l-A~~.~~-intermediate is likely as discussed earlier for p-sitosterol biosynthesis. The Liverpool studied a-spinasterol biosynthesis in leaves from Spinacea oleracea and Medicago sativa while the Osaka group26 used tissue cultures of Nicotiana tabacurn and Dioscorea tokoro and isolated stigmasterol.The biosynthetic origin of the highly complex Daphniphyllurn alkaloids has been a fascinating problem for some years and it may not surprise others who have given it some thought that squalene is in~orporated~~ into daphniphylline (45) by Daphniphyllurn rnacropodurn. Further support for the suggestion that this alkaloid is a triterpenoid comes from feeding [2-14C]-and [5-14C]-MVA and degrading the radioactive products to (46)and (47)having a specific activity ratio of 2.0 and 5.0 respectively. Daphnilactone (48) carried one quarter of its radio- activity at the site marked 0 after feeding” [2-I4C]MVA to Daphniphyllurn D.H. R. Barton J. E. T. Corrie P. J. Marshall and D. A. Widdowson Bio-org. Chem. 1973,2 363. 24 M. Fryber A. C. Oehlschlager and A. M. Unrau J. Amer. Chem. Soc. 1973,95 5747. 25 W. L. F. Armarego L. J. Goad and T. W. Goodwin Phyrochemistry 1973 12 2181. ” Y. Tomita and A. Uomori J.C.S. Perkin I 1973 2656. ’’ K. T. Suzuki S. Okada H. Niwa M. Toda Y. Hirata and S. Yamamura Tetrahedron Letters 1973 799. 28 H. Niwa Y. Hirata K. T. Suzuki and S. Yamamura Tetrahedrorr Letters 1973 2129. 608 E. McDonald & &co2H -N Me (47) teijsrnanii,and this suggests that four molecules of MVA have been incorporated. The alkaloid is presumably a degraded relative of daphniphylline (49 and a possible biosynthetic pathway is outlined in Scheme 1.Scheme I 5 Polyketides More detailst9 of the biosynthesis of the highly unsaturated fatty acids found in the Cornpositae have appeared one example*'" being the conversion (3%) by Anthernis austriaca of synthetic (49) into pyrone (50). 29 (a) F. Bohlmann and P.-D. Hopf Chem. Ber. 1973 106 3772; (6) F. Bohlmann and D. Weber ibid. p. 3020. Biosynthesis 609 Samples of [l-"C]-and [2-' 3C]-acetate have been incorporated into several metabolites and the labelling patterns deduced by '3C n.m.r. are summarized in the accompanying formulae (51)-(58). The carbon skeleton of nigrifactin (51) is formed3' entirely from acetate by Streptomyces nigrifaciens whereas several related compounds in plants are derived from lysine and acetate.2 G3C0 *aH fiO a 15 11 a (51) 0 (52) (58) A notable feature observed3' in the 13C n.m.r. spectrum of avenaciolide (52) was the 75 Hz '3C(1 lk' 3C(15) coupling which demonstrates clearly a deviation from the alternating pattern of enrichment in the remainder of the molecule. The 30 T. Terashima E. Idaka Y. Kishi andT. Goto J.C.S. Chem. Comm. 1973 75. 31 M. Tanabe T. Hamasaki Y. Suzuki and L. F. Johnson J.C.S. Chem. Comm. 1973 212. 610 E. McDonald authors conclude that the three-carbon unit is formed from acetate via succinate. The absence of I3C-enrichment in the three-carbon unit [C(l) C(2) C(21)] of thermozymocidin (53) and the incorporati~n~~ of [I4C]serine together reveal the nature of its biosynthesis. In addition to the ['3C]acetate incorporations [14C]-(54) is in~orporated~~ very well (25%) by Phyllosticta into epoxydon (59,and the same hydroquinone (54) may well be involved34 in the biosynthesis of patulin (56) by Penicilliurn patulurn.Nybomycin (57) an antibiotic produced by a strain of Streptornyces incorporates four acetate units as shown and the atoms marked were labelled after feeding ['4CH3]methionine.35 The central aromatic ring is not labelled by acetate thus demonstrating that nybomycin is a compound of mixed biosynthetic origin. This is true also for the ansa antibiotics rifamycin S (58) and strepto- varicin D (59). Prelog has shown36 by feeding various radioactive propionate samples to Streptomyces rneditterranei that 23 of the 37 carbon atoms are derived from propionate.The results from feeding Nocardia rnediterranei with [' 3C]-acetate and ['3C]propionate now make it clear3' that the polyketide chain is built up in a clockwise manner since [l-13C]acetate provides enrichment at C(17) rather than C(19) in rifamycin S (58). The assignment of the I3C resonances for C(17) and C(19) has been established by specific single proton decoupling. Rinehart's group has found3' the same clockwise growth for the polyketide chain of streptovaricin D (59)in Streptornyces spectabilis. The crucial observation was "bH Oe -rut c-CH ,CH,CO,H n"Y II the enrichment of C(15) and C(19) rather than C(17) when the antibiotic was biosynthesized from [l-'3C]propionate. Ring B in these antibiotics is not of polyketide origin.32 F. Aragozzini M. G. Beretta G. S. Ricca C. Scolastico and F. W. Wehrli J.C.S. Chem. Comm. 1973 788. 33 K. Nabeta A. Ichihara and S. Sakamura J.C.S. Chem. Comm. 1973 814. 34 A. I. Scott L. Zamir G. T. Phillips and M. Yalpani Bio-org. Chem. 1973 2 124. 35 W. M. J. Knoll R. J. Huxtable and K. L. Rinehart jun. J. Amer. Chem. Sac. 1973 95 2703. 36 M. Brufain D. Kluepfel G. C. Lancini J. Leitich A. S. Mesentsev V. Prelog F. P. Schmook and P. Sensi Helv. Chim. Acra 1973 56 2315. 37 R. J. White E. Martinelli G. G. Gallo G. Lancini and P. Beynon. Nature 1973 243 273. 38 B. Milavetz K. Kakimuna K. L. Rinehart jun. J. P. Rolls and W. J. Haak. J. Amer. Chem. Soc. 1973,9S 5793. Biosynthesis 61 1 The final 13C biosynthetic experiment in this section is rather different.[1,2-13C,]Acetate was converted3' by Mollisia caesia into mollisin (60). In the 3C n.m.r. spectrum of the enriched mollisin (60)the resonances assigned to C(3) C(6) C(12) and C(14) appeared as doublets (J = 45 52 61 and 47 Hz respec-tively) whereas that for C(11) was a clean singlet. The remaining carbon reson- ances could not be observed because they have long relaxation times. c1 ,\,.-J F''.0 go 0 P'...(.../ 00 (60) (604 The high enrichment of the precursor (90% at each carbon so that 81 % of acetate molecules have two ad.jacent 3C atoms) together with the dilution of the precursor with endogenous ['2C,Jacetate has ensured that only the carbon atoms of C units which have remained intact throughout the biosynthesis will appear as doublets a singlet methyl reveals a site at which decarboxylation has occurred.So on the 13C assignments made by the authors C(12) and C(14) represent the methyl ends of two separate chains while C( 11) is the degraded end of the chain. The illustrated arrangement (60a) of two acetate chains then accounts for the results and requires that C(1) should be a singlet. The use of Cr(acac) might reveal the fully substituted carbon resonances and allow this point to be checked. [There are alternative schemes in which C(8) or C(l0) but not C(l) would appear as singlets]. 6 Shikimate Metabolites In Mycobacterium phlei [7-14C]shikimate (61) was incorporated4' into mena- quinone (62) and a subtle degradation gave the quinoxalines (63) (radioactive) and (64) (inactive) proving that the I4C was located specifically at C(4).Clearly any naphthoquinone precursors of menaquinone have to be unsymmetrically substituted. ~-[6-'~C]Shikimic acid was in~orporated~~ very efficiently (36 %) by Pseudomonas aureofaciens into phenazine-1 -carboxylic acid (65). Oxidative degradation served only to restrict the label to four possible sites leaving a num- ber of possibilities for the biosynthesis. '3C-Techniques would surely help to solve this problem. The labelling pattern found42 in chloramphenicol (66) after '' H. Seto L. W. Cary and M. Tanabe J.C.S. Chem. Comm. 1973 867. 40 R. M. Baldwin C. D. Snyder and H. Rapoport J. Amer. Chem. Soc. 1973,95 276. 41 K. Hollstein and D.A. McCamey J. Org. Chem. 1973. 38 3415. 42 W. P. O'Neill R. F. Nystrom K. L. Rinehart jun. and D. Gottlieb Biochemisiry 1973,12,4775. 612 E. McDonald OH 0 N0 feeding [6-4C]glucose to Streptomyces venezuelae suggests that the glucose is first converted into [2,6-'4C,]shikimate and thence into triply labelled prephenate (67). C02H C02H The radioactive oxime (68)can be isolated43 from Sorghum vulgare after feeding [U-'4C]tyrosine. Further the oxime is converted in high yield (44%)into the cyanogenic glucoside dhurrin (69). Since p-hydroxyphenylacetonitrileis 6-7 times less effective as a precursor of dhurrin it is concluded that hydroxylation occurs at the oxime stage. Using tyrosine stereospecifically tritiated at C(3),it has been shown44 that the biosynthesis of mycelianamide (70) in Penicilliurn 43 K.J. F. Farnden M. A. Rosen and D. R. Liljegren Phytochemistry 1973 12 2673. 44 G. W. Kirby and S. Narayanaswami J.C.S. Chem. Comm. 1973 322. Biosynthesis 613 griseojiilvum involves loss of the 3-pro-S-hydrogen from the (2S)-amino-acid a formal cis-dehydrogenation. OGlucose 0 0 (70) Stereospecifically deuteriated tyramine (71) was converted45 by Williaanornala into tyrosol (72) whose stereochemistry was established by comparison with a sample synthesized by deuterioboration of the monodeuteriostyrene (73). Clearly BzO the transformation involves subsequent oxidation-reduction at C(l) and the reductive step (perhaps catalysed by an alcohol dehydrogenase) is stereospecific.The oxidative step was next studied by measuring the tritium retention in tyrosol formed from various tyramine derivatives randomly tritiated at C(1). In each case a high (70-96 %) retention of tritium was observed and this result is inter-preted to mean that the oxidative step (monoamine oxidase) is non-stereospecific which seems rather unlikely. When [4-3H]phenylalanine is hydroxylated to tyrosine (in this case by Pseudo-monas) appreciable amounts of 3H are retained having migrated to the adjacent 45 C. Fuganti D. Ghiringhelli P. Grasselli and A. Santopietro-Amisano,J.C.S. Chem. Comm. 1973 862. 614 E. McDonald ring position (so-called NIH shift). In principle the isotope effect k,/kT (or kJkD) can be calculated from the percentage retention of 3H(or 2H) at completion of the reaction but in practice the calculation is very sensitive to error.It is now reported46 that (74) is converted into tyrosine with 74% retention of 3H. This D (74) corresponds to a value of kD/b= 2.8 k0.1 and from this k,/k can be calculated as 10 & I. The value is so close to that observed in studies of purely chemical enolization reactions that it is apparent that the final step of the NIH shift mechanism (75) -P (76) is not under enzymatic control. 7 Aliphatic Amino-acid Metabolism Miscellaneous.-Proline serine alanine and methionine each labelled with I3C were incorporated4' into prodigiosin (77) by Serratia marescens. All the remaining carbon atoms come from acetate as discussed in the 1971 Annual Report [where incidentally formula (82) is in error].Q H 4c W. R. Bowman W. R. Gretton and G. W. Kirby J.C.S. Perkin I 1973,218. 47 H. H. Wasserman R. J. Sykes P. Peverada C. K. Shaw R. J. Cushley and S. R. Lipsky J. Amer. Chem. SOC.,1973,95 6874. Biosynthesis 615 Va1ine.-Valine (78) has a prochiral centre at C(3) and during the year not less than four syntheses have appeared of valine derivatives having a true chiral centre at C(3)by virtue of isotopic substitution in one of the methyl substituents. In three of the syntheses the key intermediates have only one chiral centre which has been elaborated from an optically pure starting material. The labelled cyclopropanecarboxylic acid (79) was prepared48 from the corresponding Grignard reagent and 3C0, and was resolved by fractional crystallization of the diastereoisomeric quinine salts.The key intermediate (80)was then prepared as shown. several MeO,C&H phQ. steps ‘50,H p 3 Optically active isopropyl alcohol has been prepared earlier and a clean sN2 displacement on the benzenesulphonate gave4’ the required ester (81). Optically C0,Et active epoxymethacrylate (82) was opened cleanly in an sN2 displacement by methyl-lithium and gaves0 the diol(83). Possible racemization by enolization of the aldehyde (84) was avoided by converting it directly into the Strecker a-amino-nitrile. CD,LI CH3 OH H CH ’ &t-H -* YL.CHO H C02H H CH,OH CD3 CH20H CD3 48 J. E. Baldwin J.Loliger W. Rastetter N. Neuss L. L. Huckstep and N. De la Higuera J. Amer. Chem. SOC.,1973,95,3796. 49 R. K. Hill S. Yan and S. M. Arfin J. Amer. Chem. SOC.,1973,95,7857. D.J. Aberhart and L. J. Lin J. Amer. Chem. SOC.,1973,95,7859. 616 E. McDonald In the fourth synthesis (Scheme 2) both chiral centres of valine are set up5' in optically pure form by the action of the enzyme /?-methylaspartase on acid (85). C0,Me ox::;; LiCuGe 1 Ill I C0,Me /*eps enzyme H3?fC02H -H3?fCH2D H3Y02H H0,C ' H2N CO2H H2N A CO2H H (85) Scheme 2 Deuterium may be introduced during reduction of the carboxy-group to afford the (3-R)-valine derivative while 3C-labelled (3-S)-valine may be prepared from ['3C]methyl iodide by the route illustrated.The chiral valines (86) have been used by two group^^'*^^ to investigate the biosynthesis of penicillin (87) and cephalosporin (88) in Cephdosporiurn acre-rnoniurn. Their valine precursors were fortuitously enantiomeric at C(3) and the results were therefore complementary as illustrated. So in the overall conversion of valine into penicillin sulphur is introduced with retention of configuration,* 0 II RCNH H 0 3, yJ-2 C02H (87) 0 Rgp>&o*c C02H (88) H. Kluender C. H. Bradley C. J. Sih P. Fawcett and E. P.Abraham J. Amer. Chem. SOC.,1973 95 6149. '' N. Neuss C. H. Nash J. E. Baldwin P. A. Lemke and J. B. Grutzner J. Amr . Chem. SOC.,1973 95 3797. * The assignment of the appropriate 13C resonances to the a-and 8-methyl groups is ultimately based on the difference between the NOE's of H(3) observed during irradiation of the r-and fi-methyl 'Hresonances.Biosynthesis 617 while the biosynthesis of cephalosporin involves a formal cis-dehydrogenation of L-valine. The biosynthesis of valine proceeds from dihydroxyisovaleric acid (89) and the diastereotopic methyl groups of (89) have been distinguished4' by the OH OH H,C>-\-H CDj CO2H CH H (3R) CD,-CrC-CO,R L'CuMez P CD3 COlR cD3y-{ OH CO,H(3s) (89) synthesis of deuteriated derivatives as shown. Each compound was incubated with the specific dehydrase-transaminase system from E. coli and gave a sample of valine chirally substituted at C(3). These could be distinguished by 'H n.m.r.spectroscopy and their stereochemistry was assigned by comparison with the synthetic specimens described above showing that the replacement of OH by H at C(3)happened with overall retention of configuration and that the proto- nation of enol(90) was a stereospecific reaction. Me \_pH Me C02H Lysine.-Quite frequently racemic labelled compounds are used for biosynthetic studies and the results can be readily interpreted because one enantiomer is completely rejected by the enzyme system in question. Thus (3R)-MVA has never been found to be utilized for terpenoid biosynthesis. With a-amino-acids it is more difficult to ascertain that only one enantiomer is being used because the configuration may be inverted via the corresponding keto-acid. An elegant method has now been described53 for determining which enantiomer is meta-bolized even during studies in intact plants.The resolved [3H]-precursor should be fed mixed with racemic [''C]-precursor the technique is best under- stood by considering one of the examples rep~rted.~ ~-[4,5-~H,]Lysinewas mixed with ~L-[6-'~C]lysine to give (91) with a 3H/'4C ratio of 6.8. Any products formed entirely from L-lysine should therefore have a 3H/'4C ratio of 13.6 whereas those formed only from D-lysine will carry only I4C radioactivity. Clearly the tritium label must not be located at the chiral centre and it must 53 E. Leistner R. N. Gupta and I. D. Spenser J. Amer. Chem. Soc. 1973 95 4040. 618 E. McDonald first be established that the 3H/'4C ratio remains unchanged after feeding DL material.Using the technique it seems that pipecolic acid (92) is formed only from D-lySine in three different plants whereas in the same three plants other alkaloids are formed exclusively from L-lysine. T C02H NH2 NH Another interesting feature of lysine metabolism is that C(2) and C(6)do not become equivalent during the bioconversion into sedamine (93) yet the sym- metrical molecule cadaverine (94) acts as a precursor for sedamine is present in ,n NH NH the plant and is biosynthesized from lysine in the plant. The authors conclude54 on the basis of these facts and the non-incorporation of alternative precursors that cadaverine is a normal intermediate but that it maintains the distinction between C(2)and C(5)by staying bound to the enzyme (cf.the randomization of squalene during fusidic acid biosynthesis). An enantiomeric pair of stereospecifically labelled [1 -3H]cadaverine (94) molecules was prepared from lysine (91) by the decarboxylase from Bacillus cadaveris. Each was then mixed with [l-14C]cadaverine and fed to Sedurn sarrnentosurn. The N-methylpelletierine (95) from sample A had retained all its s4 E. Leistner and I. D. Spenser J. Amer. Chem. Soc. 1973 95 4715. Biosynthesis 619 tritium while that from sample B had lost one half. These results show that conversion of cadaverine into N-methylpelletierine involves stereospecific enzymatic oxidation at C(l)[C(6)]. Also by comparison with the results obtained from lysine feedings it appears that the lysine decarboxylases of B.cadaveris and of S. sarrnentosurn are of opposite stereospecificity. 6-Aminolaevulinic Acid.-The review lecture55 by Neuberger provides a brief background to many of the following topics. 6-ALA synthetase operates on succinyl-coenzyme A and glycine as substrates. Glycine randomly tritiated at C(2) and both enantiomers of [2-3H]glycine have now been incubated’ with 600-fold purified enzyme from Rhodopseudo- rnonas spheroides and the results clearly show that the 2-pro-R-hydrogen of glycine is lost during the formation of 6-ALA (96). The enzyme is known to pyridoxal (96) require pyridoxal phosphate as cofactor and is deactivated by sodium boro- hydride only in the presence ofsubstrate. It seems reasonable therefore to consider the enzymatic mechanism illustrated.The nature of the intermediates in the biosynthesis of uroporphyrinogen I11 (97)from four molecules of PBG (98) is still uncertain. All four pyrromethanes (99),(loo),(101) and (102) have been ~ynthesized,’.’’,~~ and the chemical studies reported by two independent group^^',^' are in agreement in showing some surprising differences in the behaviour of isomers. Both groups also report enzymatic studies but in different biological systems and no clear conclusion can yet be drawn concerning the possible intermediacy of the pyrromethanes. In contrast the nature of the rearrangement which results in the ‘reverse’ substitu- tion pattern in ring D of the natural macrocycles is now quite clear the carbon skeletons of three PBG molecules remain intact and provide rings A B and c and the 6- a- and P-meso-carbons respectively.A fourth PBG molecule suffers a 55 R. C. Davies A. Gorchen A. Neuberger J. D. Sandy and G. H. Tait Nature 1973 245 15. 56 T. Zaman P. M. Jordan and M. Akhtar Biochem. J. 1973 135 257. ” A. R. Battersby D. A. Evans K. H. Gibson E. McDonald and L. Nixon J.C.S. Perkin I 1973 1546. ’* A. R. Battersby J. F. Beck and E. McDonald J.C.S. Perkin I 1974 160. ’’ R. B. Frydman A. Valasinas and B. Frydman Biochemistry 1973 12 80. ‘O A. R. Battersby K. H. Gibson E. McDonald L. N. Mander J. Moron and L. Nixon J.C.S. Chem. Comm. 1973 768. 620 E. McDonald rearrangement intramolecular with respect to itself and provides ring D and the y-meso-carbon.AP (97) A = CH2C02H P = CH2CH2C02H R' R2 R3 R4 R' R2 R3 R4 (99) A (100) A P P A P P A (101) P A A P (102) P A P A A = CH2C02H P = CH2CH2C02H This situation was revealed61 by using diluted doubly 13C-labelled PBG. [5-' 3C]6-ALA (90% enrichment) (96) was synthesized from sodium [13C]-cyanide by a new route,62 and then converted into PBG (103)by the dehydratase from ox liver. 81% of the molecules actually* carry two 13C atoms and the 3Cn.m.r. spectrum revealed a long-range 13C-13Ccoupling of -4 Hz. The doubly labelled material was diluted four-fold with PBG of natural abundance and incubated with an enzyme system- from chicken blood. The (103) A = CH2CO2H; P = CH2CH2C02H A. R. Battersby E. Hunt and E.McDonald J.C.S. Chem. Comm. 1973 442. 62 A. R. Battersby E. Hunt E. McDonald and J. Moron J.C.S. Perkin I 1973 2917. * The reader should appreciate that structures drawn elsewhere e.g. (30) as multiply labelled molecules are in fact assemblies of singly labelled molecules and unlabelled molecules. Biosynthesis 621 3Cn.m.r. spectrum of the dimethyl ester of the isolated protoporphyrin-IX (104) had three doublets (J = 5 Hz) corresponding to the a- p- and &carbon atoms and one doublet (J = 72 Hz) corresponding to the y-carbon atom. Manipulation C0,Me C0,Me I C0,Me CO,Me I + C0,Me C0,Me C0,Me C0,Me of the spectrum by lanthanide shift reagents and by conversion of both vinyl side-chains into acetyl groups served to increase the separation of the carbon resonances which were unambiguously assigned63 by the total synthesis of protoporphyrin-IX specifically 3C-labelled at the p- y- and 6-meso-carbon atoms respectively.At least 19 proposals have been published concerning the mechanism of the rearrangement but the vast majority are inconsistent with the facts just described. The timing of the rearrangement remains to be ascertained and the pyrromethane results described above demonstrate that future experiments will need to be very carefully designed. Neuberger reports64 that the ‘intermediates’ which accumulate when por- phyrin macrocycle biosynthesis is inhibited by ammonia hydroxylamine and methoxylamine actually incorporates these inhibitor molecules into their structures.They appear to be ‘Type I’ tetrapyrroles (105) and each cyclizes 63 A. R. Battersby G. L. Hodgson M. Ihara E. McDonald and J. Saunders J.C.S. Perkin I 1973 2923. ‘‘ R. C. Davies and A. Neuberger Biochem. J. 1973,133,471. 622 E. McDonald chemically to uroporphyrinogen I releasing the appropriate RNH molecule. The significance of these findings for uroporphyrinogen 111 biosynthesis is rather uncertain. AP AP AP AP (105) A = CH2C02H; P = CH2CH2C02H Three independent research teams have obtained ' 3C-enriched vitamin B (106) after feeding [ ''Clrnethionine to Propionibacterium shermanii. All three teams agree that the same seven carbon resonances in the vitamin show enrich- ment and that these resonances are due to seven of the eight methyl groups in (106) A = CH2C02H; P = CH2CH2C02H vitamin B12.The methyl group which is not enriched is one of those at C(12) but there is some confusion about the distinction between the 12a- and 12B-methyl resonances. The Cambridge group6' degraded their corrin to the imide (107) whose optical activity demonstrates that the stereochemistry at C( 13) remains unaltered. P (107) P = CH2CHzCOzH In the 'H n.m.r. spectrum of this imide in both CDC1 and C6D6 the high-field CH resonance was diminished in intensity and had 13C satellite signals [J('H-13C) = 128 Hz]. The high-field signal has been unambiguously assigned to the a-methyl group by Eschenmoser through the synthetic reactions (108) -P (109) (Scheme 3). The other two groups of workers based their conclusions on 65 A.R. Battersby M. Ihara E. McDonald J. R. Stephenson and B. T. Golding J.C.S. Chem. Comm. 1973,404. Biosynthesis 623 the chemical-shift values of the enriched signals in the corrin spectrum. Scott argues66 that the methyl groups at C(2) C(7) C(12a) and C(17) are all in similar environments and should have similar chemical shifts owing to the y-effect of P = CH2CH2CO2H Reagent i K0Bu'-ButOD; ii (R,P),RhCI; iii 0 Scheme 3 the syn propionate side-chain while that at C(12B) should be shifted downfield. Good support for this analysis was obtained by inspection of the 13Cn.m.r. spectra of the enriched corrin after epimerisation at C( 13) when one of the enriched signals [presumably C12a))I moved downfield by -12 p.p.m.Shemin and Katz have reached the opposite concl~sion.~~ They have by careful single-frequency 'H-decoupling correlated each of the enriched '3C resonances with the corresponding 'H resonance and then based their assignment on 'H n.m.r. assignments including those of Brodie and Poe.68 Unfortunately these 'H assignments appear to the reviewer to be uncertain in respect of the crucial methyl signals. The reviewer has attempted to set out clearly the experimental evidence and he must declare his interest of being involved in the Cambridge effort before suggesting that the 'H n.m.r. assignments have probably led Shemin and Katz to the wrong conclusion. If so then it is the C(12a)methyl resonance which is derived from methionine and a consistent pattern of formal trans-addition of CH3-H can be seen in all four pyrrole rings of vitamin B, .bb A. I. Scott C. A. Townsend E. Lee and R. J. Cushley J. Amer. Chem. SOC. 1973,95 5159. 67 C. E. Brown D. Shemin and J. J. Katz J. Biol. Chem. 1973 248 8015. 60 J. D. Brodie and M. Poe Biochemistry 1971 10 914.