A convenient synthesis of 7α-hydroxycholest-4-en-3-one by the hydroxypropyl-β-cyclodextrin-facilitated cholesterol oxidase oxidation of 3β,7α-cholest-5-ene-3,7-diol

Article abstract of DOI:10.1016/0039-128X(95)93851-O

The initial biosynthetic conversions of cholesterol to the bile acids involve sequential 7α-hydroxylation (catalyzed by cholesterol 7α-hydroxylase) followed by C-3 oxidation and concomittant double bond migration (to a Δ⁴-configuration, catalyzed by 3β-Δ⁵-C₂₇-steroid oxidoreductase) to provide 7α-hydroxycholest-4-en-3-one.A straightforward, and economical, preparation (on a 0.1 g scale) of this pivotal biosynthetic intermediate has been devised.Reduction of 3β-(benzoyloxy)-cholest-5-en-7-one with LiB(sec-butyl)3H provided a 4:1 mixture, respectively, of the 7α- and 7β-hydroxy diastereomers, which were separated chromatographically.Solvolytic removal of the C-3 benzoyl group gave 3β,7α-cholest-5-ene-3,7-diol.A suspension of the 1:1 (v/v) complex (formed by mutual dissolution in MeOH, followed by evaporation of the solvent) of this diol with hydroxypropyl-β-cyclodextrin, at a concentration of 1 mg mL^-1 (in neutral phosphate buffer), was converted by Brevibacterium sp cholesterol oxidase (0.25 U mg^-1 of substrate) and catalase (70 U mg^-1 of substrate, to recover O2 from the H2O2 produced by the enzymatic oxidation) to a suspension of 7α-hydroxycholest-4-en-3-one and the hydroxypropyl-β-cyclodextrin.The yield for the enzymatic conversion was in excess of 90percent.A much poorer and less reproducible yield (< 20percent) was seen in the absence of the hydroxypropyl-β-cyclodextrin.Routine extraction of this aqueous suspension, and chromatographic purification (85:15 CHCl3/acetone v/v on silica) of the residue, gave pure 7α-hydroxycholest-4-en-3-one in 68percent isolated yield.This route is a significant improvement, in terms of reaction scale and convenience, over the previous procedures for the preparation of this steroid. - Keywords: (7α)-hydroxycholesterol; hydroxypropyl-β-cyclodextrin; cholesterol oxidase; (7α)-hydroxycholest-4-en-3-one; bile acid biosynthesis; cholesterol 7α-hydroxylase