17β-Hydroxy-17α-(hydroxymethyl)estr-4-en-3-one

The title compound, C19H28O3, the fungal-transformed metabolite of the steroid methyloestrenol contains four fused rings A, B, C and D. Ring A adopts a half-chair and the trans-fused rings B and C adopt chair confirmations; the five-membered D ring is folded like an envelope. In the crystal, adjacent molecules are linked by O—H⋯Ocarbonyl and O—H⋯Ohydroxy hydrogen bonds into a layer structure.

The title compound, C 19 H 28 O 3 , the fungal-transformed metabolite of the steroid methyloestrenol contains four fused rings A, B, C and D. Ring A adopts a half-chair and the transfused rings B and C adopt chair confirmations; the fivemembered D ring is folded like an envelope. In the crystal, adjacent molecules are linked by O-HÁ Á ÁO carbonyl and O-HÁ Á ÁO hydroxy hydrogen bonds into a layer structure.

Culture preparation
In 4 L water were dissolved glucose (40 g), peptone (20 g), yeast extract (12 g), potassium dihydrogen phosphate (20 g), sodium chloride (20 g) and glycerol (40 ml). The solution was distributed among 40 conical flasks (100 ml each); the mouths of the flasks were covered with cotton wool. The flasks were then heated at 374 K for 15 minutes. The spores of Aspergillus niger were transferred from slants grown on saboraud dextrose agar. The flasks were left on rotary shaker until there was sufficient growth of the spores. Methyloestrenolone (1 g) was distributed equally among the flasks in the form of its solution in acetone (20 ml, 0.5 ml per flask).

Fermentation of methyloestrenolone
Methyloestrenolone was also incubated with a liquid phase culture of Aspergillus niger (4 L) for 14 days. The biomass was separated by filtration and the filtrate extracted with dichloromethane The extract was dried with sodium sulfate; the solvent was evaporated to leave about 3 g of a brown gummy material. This was subjected to fractionation on a silica gel column with petroleum ether-ethyl acetate gradient solvent system. The fractions were subjected to size exclusion HPLC (GS-320, methanol, 35 minute retention time). Evaporation of the solvent gave the title compound as colorless prisms of (I).

Refinement
Carbon-bound H-atoms were placed in calculated positions (C-H 0.93 to 0.98 Å) and were included in the refinement in the riding model approximation, with U(H) set to 1.2 to 1.5U(C).
The hydroxy H-atoms were located in a difference Fourier map, and were refined with a distance restraint of O-H 0.84±0.01 Å; their temperature factors were freely refined.