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J. Org. Chem. 1986,51, 2906-2910
way as product 6, by using 17 U 3&hydroxysteroiddehydrogenase and 20 U formate dehydrcgenase. The reaction was complete after 25 h. The turnover number for the coenzyme was 140. The product was recovered as was product 3, and the yield was 0.49 g (86%). The product was c r y s m from ethyl acetateacetone: mp 270-272 "C; Rf 0.51 (system 2); HPLC t , 18 min; [a]D+22"; 'H NMR 6 0.98 (3 H, s, C-18 Me), 1.26 (3 H, s, (2-19 Me), 0.74 (3 H, d, J = 6.3 Hz,C-21 Me). Synthesisof 7a-Hydroxy-3,12-dioso-5~-cholan-24-oic Acid (S)?' Sodium dehydrocholate (0.7 g) was reduced in the same way as product 6, by using 0.1 M glucose instead of 0.1 M formate, 5 U 7a-hydroxysteroid dehydrogenase, and 10 U glucose dehydrogenase. The reaction was complete after 9 h. The turnover number for the coenzyme was 160. The product was recovered in the same way as product 3, and the yield was 0.61 g (91%). The product was crystallized from ethyl acetate: mp 193-195 "C; R,0.48 (system 2); HPLC t, 36 min; [ a ]+73"; ~ 'H NMR 6 1.00 (3 H, s, C-18 Me), 1.05 (3 H, s, C-19 Me), 0.77 (3 H, d, J = 6.3 Hz, C-21 Me). Synthesis of 78-Hydro.y-3,12-dioxo-SB-cholan-249ic Acid (9). Sodium dehydrocholate (0.6 g) was reduced in the same way as product 8, by using NADPH instead of NADH, 8 U 78hydroxysteroid dehydrogenase,and 15 U glucose dehydrogenase. The reaction was complete after 30 h. The turnover number for the coenzyme was 140. The product was recovered in the same way as product 3,and the yield was 0.49 g (86%). The product was crystallized from ethyl acetak mp 187-189°C; Rf 0.47 (system (36) Compound 7 waa previously prepared from cholic acid by a fivestep synthesis,which ale0 involved a chromatographic purification of an intermdata, with a yield lower than 30% (lit.n and: Chaug, F. C. J. Org. Chem. 1979, 44, 4567. Blickenstaff, R. T.; Atkineon, K.; Breaux, D.; Foster, E.; Kim,Y.; Wolf, G. C. J. Org. Chem. 1971,36, 1271). (37) Compound 8 waa previously prepared from cholic acid by a four-step synthesis (lit." and Nicotra, F.; Ranzi, B. M. Ann. Microbiol. 1978, 28, 11). The yield was not given.
2); HPLC t, 14 min; [aID+90°; 'H NMR 6 1.01 (3 H, s, (2-18 Me), 1.05 (3 H, s, C-19 Me),0.77 (3 H, d, J = 6.3 Hz, C-21 Me); mass spectrum (positive FAB), mlz (relativeintensity) 405 ((M + H)', 100), 387 (98),369 (30), 353 (14), 351 (17),341 (21),323 (12), 263 (12),219 (9), 145 (18),121 (23),105 (35). Anal. Calcd for CaHS05: C, 71.26; H, 8.97. Found: C, 70.97; H, 8.78. Synthesis of 12~-Hydroxy-3,7-dioxo-5~-cholan-24-oic Acid (lo).% Sodium dehydrocholate (0.6 g) was reduced in the same way as product 9, by using 12 U 12a-hydroxysteroid dehydrogenase coimmobilizedwith 8 U glucose dehydrogenase. The reaction was complete after 26 h. The turnover number for the coenzyme was 140. The product was recovered as was product 2, and the yield was 0.50 g (87%). The product was crystallized from ethyl acetate: mp 168-170 "C (lit.18mp 168-169 "C, from ethyl acetate); R 0.44 (system 2); HPLC t, 31 min; ["ID -9"; 'H NMR 6 0.63 (3 E, C-18 Me), 1.22 (3 H, E, C-19 Me), 0.93 (3 H, d, J = 6.3 Hz,C-21 Me).
A,
Acknowledgment. We thank the Consiglio Nazionale delle Ricerche, Rome, Progetto Finalizzato "Chimica Fine e Secondaria", and the Biomolecular Engineering Programme of the Commission of the European Communities for financial support of this work. We also thank Dr. Lucia Zetta (CNR Milano) for obtaining the NMR spectra and Prof. Annamaria Ferrari (Agricultural University, Milano) for providing C. absonum cells. W s t W NO. i,81-25-4;2,2304-89-4;3,911-40-0;4 , 2 m o a 4 ; 5,81-23-2;6, 517-33-9;7,3615-35-8;8,2304-91-8;9, 102649-81-0; 10,2304-92-9;EC 1.1.1.50,9028-56-2; EC 1.1.1.159, 39361-64-3; EC 1.1.1.51,9015-81-0; EC 1.4.1.4,9029-11-2; EC 1.1.1.47,902853-9; EC 1.1.1.176,61642-40-8;EC 1.4.1.3,9029-12-3; EC 1.2.1.2, 9028-85-7. (38) Compound 10 waa previously prepared from cholic acid by a six-step synthesis (lit.18). The yield was not given.
Roritoxins, New Macrocyclic Triohothecenes from Myrothecium roridum Bruce B. Jarvis* and C. S. Yatawara Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742
Received February 3, 1986
Four new macrocyclictrichothecenea (roritoxins A-D) which contain a tetrahydropyranylring fused to a y-lactone or lactol ring in the macrolide side chain were isolated from a culture of Myrothecium roridum ATCC 20605 grown on rice substrate. Roritoxin C is unique in that it is the first reported fungal-produced trichothecene which possesses a 9,lO-epoxide group. In liquid media this fungal culture produces a new antibiotic, (+)-(lR,6R)-5(S)-hydroxy-4-methyl-7-oxabicyclo[ 4.1.O]hept-3-en-2-one. During the past few years we have been involved in screening Myrothecium verrucaria and M. roridum isolates for the production of trichothecenes, which are a family of sesquiterpenes important as mycotoxins' and anticancer agents.2 The research group a t Warner-Lambert has reported on an interesting isolate of M. roridum, CL-514 (ATCC 20605), which in a liquid medium produced a novel array (1) (a) Bamburg, J. R.; Strong, F. M. In Microbial Toxins;Kadis, S., Ciegler, A., Ajl, S. J., Eds.;Academic Press: New York, 1971; Vol. VI1 p 207. (b) Ueno, Y., Ed. Trichothecenes-Chemica~,Biological, and Torcicological Aspects; Halated-KodanshaScientific Press: New YorkTokyo, 1983. (c) Ueno, Y. Ado. Nutr. Sei. 1980,3,301. (d) Bamburg, J. R. Bog. Mol. Subcell. 1983,8,41. (e) McDougal, P. G.; Schmuff, N. R. Fortschr. Chem. Org. Naturst. 1986,47, 163. (2) Doyle, T. W.; Bradner, W. T. In Anticancer Agents Baaed on Natural Product Models; Caeaidy, J. M., Douros, J. D., Eds.;Academic Press: New York, 1980; p 43.
of trichoverroids (roridin L-23and tri~hoverritone~) as well as two unusual macrocyclic trichothecenes, 12'-hydroxyisoverrucarin J and isosatratoxin H.S We have studied a variety of fermentation conditions for this isolate and now report the isolation of a series of new antibiotics whose production depends strongly on the type of media used to grow this culture.
Results and Discussion Initially, when M. roridum isolate CL-514 was grown in a sucrose/glycerol based liquid medium, conditions we (3) Bloem, R. J.; Smitka, T. A.; Bunge, R. H.; French, J. C.; Mazzola, E. P. Tetrahedron. Lett. 1983,24, 249. (4) Jarvis, B. B.; Vrudhula, V. M.; Pavanasasivam, G. Tetrahedron Lett. 1983,24, 3539. ( 5 ) Smitka, T. A.; Bunge, R. H.; Bloem, R. J.; French, J. C. J . Antibiotics 1984, 37, 823.
0022-326318611951-2906$01.50/0 0 1986 American Chemical Society
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Ror itoxins have found to be well-suited for trichothecene production by Myrothecium species,6 we observed production of a trichothecene metabolite (ca. 10 mg/L)' whose spectral characteristics did not match any of those reported for known trichothecenes. However, numerous attempts to reproduce this result using a variety of conditions failed to produce this new toxin (roritoxin A, vide infra), and we observed production of only small quantities (
