Microbial transformation of steroids: contribution to 14 alpha-hydroxylations

The regioselective and stereoselective hydroxylation of steroids by fungal strains previously known for their hydroxylation capabilities, such as Thamnostylum (= Helicostylum) piriforme ATCC 8992, Mucor griseocyanus ATCC 1207a, Actinomucor elegans (= Mucor parasiticus) MMP 3122 (Mucorales), and Zygodesmus sp. ATCC 14716, was investigated with special interest for the 14 alpha-hydroxylation reaction. A preliminary screening had shown that some of these microorganisms were adequate for the production of 14 alpha-hydroxylated derivatives of the following steroids: progesterone, 5 beta-pregnane-3,20-dione, 3 beta-hydroxy-5 beta-pregnane-20-one, 3 beta-hydroxy-5 beta-17 (alpha H)-etianic acid methyl ester, androst-4-ene-3,17-dione, and testosterone. About 20 metabolites have been isolated and purified by silicagel chromatography and semi-preparative reverse-phase HPLC. These metabolites have been fully characterized by 1H, 13C NMR and mass spectrometry. All the identified metabolites were hydroxylated at some distinct positions, such as 6 beta-, 7 alpha-, 9 alpha-, 14 alpha-, 15 beta-, or dihydroxylated at 6 beta,14 alpha-,7 alpha,14 alpha-, 9 alpha,14 alpha-, 14 alpha,15 alpha-, 14 alpha,15 beta-positions; nine of these metabolites have not been reported previously. The relationship between the structural features of the investigated steroids and the site-specific hydroxylation has been delineated, and progesterone was found to be the best substrate for the production of 14 alpha-hydroxylated derivative, using T. piriforme.