Incubation of 8a,9ot-epoxytetrahydrolanosterol [3] with Mycobacterium sp. gave 20(5)-hydroxymethyl-12a-hydroxypregn-4-en-3-one [4] and 20(5)hydroxymethyl-4,4,14a-trimethylpregna-7,9( 11 )-dien-3ß-ol [5] in 5.0% and 1.6% yields, respectively. Incubation of lanosta-7,9(l l)-dien-3P-ol [6] resulted in the isolation of two C-19 steroids as the major metabolites that have been isolated previously from the incubation of lanosterol, and methyl 12a-hydroxybisnorchola-4,17(20)-dien-22-oate [91 in low yield. These results indicated that this microorganism is capable of carrying out saturation of a C=C bond, methylation of a carboxyl function, and 12a-hydroxylation in the steroid nucleus, besides earlier reported transformations.In previous reports we have disclosed that cycloartenol and 24-methylenecycloartanol are transformed by Mycobacterium sp. (NRRL B-3805) to give androsta-4,8( 14)-diene-3,17-dione {1} as the major metabolite, in 34 and 35% yields, respectively (1,2). It was also shown that when lanosterol [2] was incubated with this microorganism 1 was isolated as the major product in 30% yield. During this transformation, a series of complicated chemical changes occurred including demethylation at the 4 ,4ß-and 14apositions, formation of a 8<14> double bond accompanied by the disappearance of the Am> double bond, and cleavage of the C-17 side-chain. Among these changes, we were particularly interested in the disappearance of the 8*" double bond accompanied by the formation of the 8<14> double bond in 1. Therefore, lanosterol derivatives with substituent modification at C-8 and C-9 were prepared and subjected to transformation with this microorganism. RESULTS AND DISCUSSIONAfter unsuccessful attempts in hydrogenating the Am) double bond, 2 was converted smoothly with catalytic hydrogenation (H2-Pd) and peroxidation (wz-CPBA) to 8a,9a-epoxy-tetrahydrolanosterol [31 (3). Compound 3 showed a molecular ion at m!z 444 in its eims corresponding to a formula of C30H52O2. The presence of two oxygenated quaternary carbon signals at 70.6 and 68.2, along with the disappearance of two olefinic carbon signals (C-8 and C-9, overlapping at 134.5 in dihydrolanosterol) confirmed its structure. The favored -side attack by w-CPBA was confirmed by an nOe nmr study, in which a critical observation was the interaction of H-18 and H-19, which is only possible for an 8a,9a-epoxy structure in 3.From the incubation mixture of 3 with Mycobacterium sp. (NRRL B-3805), compounds 4 and 5 were isolated in relatively low yields of 5.0% and 1.6%, respectively (Scheme 1).The hreims of 4 showed a molecular ion at mlz 346.2510, consistent with an elemental formula of C^H^O^T he *H-nmr spectrum showed an olefinic proton signal at 5.70 (br s), typical for H-4 in a 3-one-4-ene sterol. The presence of this functionality was confirmed by an ir absorption at 1659 cm-1 and the uv absorption maximum at 242 nm. The ir spectrum also indicated the presence of hydroxy groups (3425 cm '). Three methyl signals in the 'H-nmr spectrum of 4 including two singlets ( 0.7...