Studies in terpenoid biosynthesis. Part VII. The biosynthesis of helicobasidin

摘要

586 J.C.S. Perkin IStudies in Terpenoid Biosynthesis. Part V1I.l The Biosynthesis ofHelicobasidinBy Miss P. M.BN1 9QJAdams and J. R. Hanson,' School of Molecular Sciences, University of Sussex, BrightonHelicobasidin has been shown to incorporate four tritium atoms from 2,2-3H, mevalonate, two from 4R-4-aH-mevalonate, and two from 5,5-SH,mevalonate. Feeding with 2-3H,2-14Cgeranyl pyrophosphate has shownthat the 4R-mevalonoid tritium atom from the second prenyl unit is retained in the biosynthesis.THE fungal pigment helicobasidin (l), isolated from cuprenenes (4). In view of the close relationship ofHelicobasidiwn mompa,2 has been shown to be ses- this scheme to that which has been proposed for the tri-quiterpen~id.~,~ On biogenetic grounds it has been cothecanes (cf.ref. 7), we have examined the origin of thesuggested that helicobasidin belongs to the cuparane skeletal hydrogen atoms of helicobasidin. If either theclass, whose carbon skeleton is thought to arise by bisabolene route or the ' direct ' theory of Bentleyprotonation of y-bisabolene (2) followed by cyclization and Chen is correct then helicobasidin should incorpor-to the tertiary cation (3) which could then afford the ate up to four tritium atoms from the 2,2-3H,mevalon-1 Part VI, R. Evans, J. R. Hanson, and A. F. White, J .2 S. Natori, H. Nishikawa, and H. Ogawa, Chem. and Pharm.3 S . Natori, Y. Inouye, and H. Nishikawa, Chem. and Phamz.Chem. SOC. (C), 1970, 2601.Bull. (Japan), 1974, 12, 236.Bull. (Japan), 1967, 15, 380.R. Bentley and D.Chen, Phytochemistry, 1969, 8, 2171.W. Parker, J. S. Roberts, and R. Ramage, Quart. Rev.,C. Enzell and H. Erdtman, Tetrahedron, 1958, 4, 361.7 P. M. Adams and J. R. Hanson, Chem. Comm., 1970, 1569.1967, 21, 3311972 587ate progenitor, one from 4R-4-3Hmevalonate, and twofrom 5,5-3H2mevalonate. While this work was inprogress, a note appeared8 which, on the basis of theincorporation of two 4(R)-mevalonoid protons, excluded' -*(51 bsol; bsol;( 6 ) ( 7 1* Label from 2-position in mevalonic acid; 0 , from 4-posi-tion; and 0, from 6-position. PP = Pyrophosphate.a y-bisabolene intermediate, and proposed a directcyclization to a cuparenyl cation. Our results alsoexclude the bisabolene pathway.2,2-3H2,2-14CMevalonic acid lactone (3H : 14C,7.56: 1) was fed to Helicobasidium mompa 70 daysafter inoculation. After a further 13 days the helico-basidin was isolated from the mycelium by sublimation.To overcome quenching problems, the helicobasidinwas converted into its leuco-acetzs d(5) by reductionwith zinc and acetic anhydride.US showed activity(3H : 14C, 4.92 : 1) corresponding to 3.9 tritium atoms.When 4R-4-3H,2-14Cmevalonic acid lactone (3H : 14C,9.33 : 1) was used, surprisingly, the helicobasidin leuco-acetate showed an activity (3H : 14C, 5.74 : 1) correspond-ing to the retention of two tritium atoms, in agreementwith the results of Nozoe.8 Finally when 5,5-3H2,2-14C-mevalonic acid, as its NN'-dibenzylethylenediaminesalt (3H : l4C,1O.4 : l), was used, the helicobasidinleucoacetate showed an activity (3H : 14C, 3.3 : 1)corresponding to the incorporation of 1.9 mevalonoidtritium atoms.The origin of the additional 4-$ro-R-mevalonoidproton from the central prenyl unit of farnesyl pyro-phosphate precursor (6) i.e., from C-6 of the pyro-* S.Nozoe, M. Morisaki, and H. Matsumoto, Chenz. Comm.,1970, 926.phosphate (6) was established by feeding experimentswith 2-3H,2-14Cgeranyl pyrophosphate (3H : l4C,7.06 : 1). The helicobasidin leuco-acetate showed therelative activity CH : 14C of 7.04: 1) and thus the4-$ro-R-mevalonoid proton from the central prenylunit has been retained in the biosynthesis.If we make the reasonable assumption that themevalonoid labels are not ' scrambled ' during the bis-synthesis, then these results exclude y-bisabolene as anintermediate in the fungal pathway.The locationof the labels in the related tricothecane case has beenestablished.'~~ Furthermore the retention of the centralprenyl olefinic proton, which in the tricothecane casehas been shown to occupy the Z-po~ition,~ suggests thata direct cyclization of farnesyl pyrophosphate (6) occurs.The primary enzyme-bound intermediate (7) may bedisplaced from the enzyme surface by a hydride shiftand elimination which leads to the cuprenenes (4) andthence by oxidation to deoxyhelicobasidin and helico-basidin.EXPERIMENTALGeneral details have been described previously.1deg; Count-ing was carried out with a Beckman LS 100 liquid scintil-lation counter.Helicobasidium mompa was grown as astill culture in Roux bottles (250 ml) and in Thompsonbottles (750 ml) on a medium containing (per litre) sucrose(20-0 g), potassium dihydrogen phosphate (0.5 g), di-potassium hydrogen phosphate (0.5 g) , magnesium sulphate(0.2 g), manganese sulphate (0.01 g), ferrous sulphate(0.01 g ) , sodium chloride (0.01 g), and L-asparagine(3.0 g). The ferments were harvested 80-90 days afterinoculation. The helicobasidin was recovered simplyfrom the dried mycelium by sublimation. The mycelium(95 g) from 10 1 afforded helicobasidin (250 mg) as orangeneedles, m.p. 194" (from ethanol), aID2o -125" (c 0.1 inCHC1,) (lit.,293 190-192", aD25 - 123" (CHCl,)} (Found:C, 68.1; H, 7.5. Calc. for C,,H,,O,: C, 68.2; H, 7 ~ 6 ) ~z 9-17 (3H, s), 8.93 (3H, s), 8.67 (3H, s), and 8.08 (3H, s).The fermentations also yielded mompain and deoxy-helicobasidin.Incorporation of 2, 2-,H,, 2-14C Mevalonic Acid Lactone.-70 Days after inoculation, the mevalonate (3H : 14C, 7.56 : 1)in ethanol (1 5 ml) was distributed in Helicobasidizcrn mompa(3.5 1).The ferment was harvested after a further 14 daysand the active helicobasidin (25 mg), m.p. 194", wasisolated and diluted with inactive material (35 mg). It washeated in acetic anhydride (4 ml) under reflux with zincpowder (400 mg) for 2 h. The hot filtrate was poured intowater and neutralized with sodium carbonate. Extractionwith ether led to helicobasidin leuco-acetate, white prisms(30 mg), m.p. 156-158" (from ethyl acetate-light petrol-eum), -9.8" (c 0.3 in CHCl,) (lit.,, 152-154", ~,,25-9.5" (CHCl,)) (Found: C, 63-9; H, 7.15.Calc. forC~~H~OO,: C, 63-6; H, 7*0), 7 9.23 (3H, s), 8.98 (3H, s),8-72 (3H, s), 8.06 (3H, s), and 7.74 (12H, s), relative activityIncorporation of 4R-4-3H,2-14C Mevalonic Acid Lactone.-70 Days after inoculation, the (3H :lac, 9-33 : 1) inlo B. Achilladelis and J. R. Hanson, Phytochemistry, 1968, 7,: 14C, 4.92 : 1).P. M. Adams and J. R. Hanson, unpublished work.589J.C.S. Perkin Iaqueous ethanol (1.75 ml) was distributed in Helicobasidiurnmompa (4 1). The ferment was harvested after a further14 days and helicobasidin (44 mg), m.p. 193-194", wasrecovered by sublimation. This was converted into theleuco-acetate (36 mg) (3H : 14C, 5.74 : l), m.p.156-158',as before.Incorporation of 5, 5-3H,, 2-14C Mevalonic Acid.-Themevalonate, as its NN'-dibenzylethylenediamine salt(3H : 14C, 10.4 : l), in ethanol (2 ml) was distributed inHelicobasidium mornpa (5 1) 74 days after inoculation.The ferment was harvested after a further 13 days andhelicobasidin (10 mg), m.p. 193", was isolated by sublim-ation. It was diluted with inactive helicobasidin (17 mg)and converted into its leuco-acetate (13 mg) (3H: 14C,3.3 : l), m.p. 158", as before.Incorporation of 2-3H,2-14CGeranyZ Pyyophosphate.-The pyrophosphate (3H : 14C, 7.06 : 1) in aqueous ethanol(12 ml) was distributed in Helicobasidium momfia (8 1) 70days after inoculation. After a further 14 days the mycel-ium was filtered off, dried, and extracted with light petrol-eum. The extract (0.186 g) was purified by p.1.c. on silicagel which had been treated with 3 oxalic acid in water.Benzene-light petroleum (1 : 1) was used as the mobilephase. This gave helicobasidin (34 mg) (RP 0.9), m.p.188-191", which was converted into the leuco-acetate(16 mg) (3H : 14C, 7.04 : l), m.p. 157-159", as before.for financial support.We thank the M.R.C. for a studentship and the S.R.C.1/1775 Received, 28th September, 1971