Predominant allylic hydroxylation at carbons 6 and 7 of 4 and 5-ene functionalized steroids by the thermophilic fungus Rhizomucor tauricus IMI23312

An unusual ring—A opening and other reactions in steroid transformation by the thermophilic fungus Myceliophthora thermophila

“…The transformation initiated with hydrolysis of testosterone acetate (2), generating testosterone (4) which was then oxidised to androst-4-en-3,17-dione (5) and then hydroxylated at C-14. This result further supports the notion that the progestogens (1,7,8) have a role in switching on the enzymatic attack observed on ring-A resulting in (6). In support of this, single functional groups on steroids transformed by fungi, have already been shown to control specific enzyme induction and therefore metabolic fate [2,20].…”

“…There were no significant differences observed in the methyl resonance signals indicating that this substitution was at distance from these centres. The 13 C NMR spectrum demonstrated ␤-carbon downfield shifts for C-6 (8.89 ppm) and C-8 (3.64 ppm) and ␥-carbon upfield shifts for C-9 (7.99) and C-14 (4.75 ppm) confirming hydroxylation at C-7, as did comparison with an authentic sample [6]. …”

“…A significant downfield shift was observed for the 19-methyl (0.19 ppm) in the 1 H NMR spectrum indicating a hydroxyl group in close proximity to this group. The 13 C NMR spectrum of (13) revealed downfield ␤-carbon shifts for C-4 (2.86 ppm), C-7 (7.22 ppm) and C-19 (2.64 ppm) and an upfield shift for C-8 (5.30 ppm) confirmed hydroxylation at C-6 as did comparison to an authentic sample [6].…”

“…trehalase, invertase, ␤-glycosidase) [3][4][5] but as of yet, very little is known about their ability to transform steroids. The only study [6] in this area to date determined that Rhizomucor tauricus IMI 23312 had patterns of metabolism similar to mesophilic fungi, with hydroxylation reactions predominating.…”

“…This organism is known to generate exogenous enzymes such as laccase [7], xylanases [8,9] and cellulases [10] which, have found practical application in the food industry [11], as biocatalysts [12] in spectroscopic science [13] and biosensor technologies [14]. In order to determine the pattern of metabolic handling of steroids by this fungus, a range of structurally diverse steroidal compounds namely progesterone Transformation products 5␣-Androstan-3␣,17␤-diol (6) 0.73 0.79 3.63 (1H, t, J = 8.6 Hz, 17␣-H), 4.04 (1H, t, J = 2.6 Hz, 3␤-H) 11␤-Hydroxy-progesterone (7) 5.82 0.80 1.38 2.29 4.37 (1H, t, J = 2.5 Hz, 11␣-H) 11␣-Hydroxy-progesterone (8) 5.73 0.70 1.32 2.13 4.03 (1H, td, J = 5 Hz, J = 10 Hz, 11␤-H) 14␣-Hydroxy-androst-4-en-3,17-dione (9) 5.75 1.05 1.22 17␤-Hydroxy-5␣-androstan-3-one (10) 0.76 1.02 3.64 (1H, t, J = 8.6 Hz, 17␣-H) 4-Hydroxy-3,4-seco-pregn-20-one-3-oic acid (11) 0 of sterile media and were incubated for 72 h at 40 • C. The cultures were shaken at 180 rpm on an orbital shaker. Aliquots (5 mL) from the seed flask were transferred aseptically to 10 flasks and grown for a further 72 h as above, at the end of which the fungus is in log phase growth.…”