A CONVENIENT SYNTHESIS OF 7-ALPHA-HYDROXYCHOLEST-4-EN-3-ONE BY THE HYDROXYPROPYL-BETA-CYCLODEXTRIN-FACILITATED CHOLESTEROL OXIDASE OXIDATION OF 3-BETA,7-ALPHA-CHOLEST-5-ENE-3,7-DIOL

The initial biosynthetic conversions of cholesterol to the bile acids involve sequential 7 alpha-hydroxylation (catalyzed by cholesterol 7 alpha-hydroxylase) followed by C-3 oxidation and concomittant double bond migration (to a Delta(4)-configuration, catalyzed by 3 beta-Delta(5)-C-27-steroid oxidoreductase) to provide 7 alpha-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 3 beta-(benzoyloxy)-cholest-5-en-7-one with LiB(sec-butyl)(3)H provided a 4:1 mixture, respectively, of the 7 alpha- and 7 beta-hydroxy diastereomers, which were separated chromatographically. Solvolytic removal of the C-3 benzoyl group gave 3 beta,7 alpha-cholest-5-ene-3,7-diol. A suspension of the 1:1 (nu/nu) complex (formed by mutual dissolution in MeOH, followed by evaporation of the solvent) of this diol with hydroxypropyl-beta-cyclodextrin, at a concentration of 1 mg mL(-1) (in neutral phosphate bl(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 O-2 from the H2O2 produced by the enzymatic oxidation) to a suspension of 7 alpha-hydroxycholest-4-en-3-one and the hydroxypropyl-beta-cyclodextrin. The yield for the enzymatic conversion was in excess of 90%. A much poorer and less reproducible yield (<20%) was seen in the absence of the hydroxypropyl-beta-cyclodextrin. Routine extraction of this aqueous suspension, and chromatographic purification (85:15 CHCl3/acetone nu/nu on silica) of the residue, gave pure 7 alpha-hydroxycholest-4-en-3-one in 68% 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.