4-Ethenylidene steroids as mechanism-based inactivators of 3 beta-hydroxysteroid dehydrogenases.

The synthesis of 4-ethenylidene-5 alpha-androstane-3 beta, 17 beta-diol (5) and of 4-ethenylidene-5 alpha-androstane-3, 17-dione (4) is described. Compound 5 is a competitive inhibitor of solubilized bovine microsomal adrenal delta-5-3 beta-hydroxysteroid dehydrogenase, with Ki = 2.7 microM, and is converted by the enzyme to the corresponding 3-ketone. Compound 4 is shown to irreversibly inactivate the enzyme in a time-dependent manner (t 1/2 = 31 min; 55 microM; pH = 7.0). The substrate, dehydroepiandrosterone, protects against inactivation by compound 4. In contrast, compound 5 is not oxidized at the 3-position by the 3 beta-(and 17 beta)-hydroxysteroid dehydrogenase from P. testosteroni, but is oxidized at the 17-position. Nevertheless, the 4-ethenylidene-3,17-diketone (4) causes irreversible time-dependent inactivation (t 1/2 = 28 min; 64 microM; pH = 7.0) when incubated directly with this bacterial enzyme, acting as an affinity label.