Stereospecific hydrogen transfer from C-4 to C-6 during enzymatic transformation of cholesterol into cholestenone.

Abstract

To clarify the steric mechanism for biotransformation of .DELTA.5-3.beta.-hydroxysteroid into .DELTA.4-3-ketosteroid the metabolic fate of H at C-4 during conversion of cholesterol into cholestenone by 3.beta.-hydroxysteroid oxidase [EC 1.1.3.6 from Brevibacterium sterolicum] was investigated. Of 3 substrates required for this purpose, 4.alpha.-d1-cholesterol was prepared through 4-d1-cholest-4-en-3.beta.-ol tert-butyldimethylsilyl ether as a key intermediate. The substrate was incubated with the enzyme, and the content and locality of deuterium in resulting cholestenone were determined by inspection of the mass, NMR and IR spectra. The transformation products formed from 4.alpha.- and 6-deuterated cholesterols retained the label almost intact, while that derived from 4.beta.-d1-cholesterol showed approximately 50% retention of the heavy isotope at 6.beta.. Incubation studies using 6.beta.-d1-cholestenone as a substrate implied that a 50% loss of the label would be ascribable in part to the exchange with the incubation medium under the enzymatic control. Apparently, 4.beta.-H was transferred stereospecifically to the 6.beta.-position during biotransformation of cholesterol into cholestenone.