Conversion of 19-oxo[2β-²H]androgens into oestrogens by human placental aromatase. An unexpected stereochemical outcome

Aromatase is a cytochrome P-450 enzyme that catalyzes the conversion of androgens into oestrogens via sequential oxidations at the 19-methyl group. Despite intensive investigation, the mechanism of the third step, conversion of the 19-aldehydes into oestrogens, has remained unsolved. We have previously found that a pre-enolized 19-al derivative undergoes smooth aromatization in non-enzymic model studies, but the role of enolization by the enzyme in transformations of 19-oxoandrogens has not been previously investigated. The compounds 19-oxo[2β-²H]testosterone and 19-oxo[2β-²H]androstenedione have now been synthesized. Exposure of either of these compounds to microsomal aromatase, in the absence of NADPH, for an extended period led to no significant ²H loss or epimerization at C-2, leaving open the importance of an active-site base. However, in the presence of NADPH there was an unexpected substrate-dependent difference in the stereoselectivity of H loss at C-2 in the enzyme-induced aromatization of 19-oxo[2β-²H]testosterone versus 19-oxo[2β-²H]androstenedione. The aromatization results for 17β-ol derivative 19-oxo[2β-²H]testosterone correspond to about 1.2:1 2β-H/2α-H loss from unlabelled 19-oxotestosterone. In contrast, aromatization results from 19-oxo[2β-²H]androstenedione correspond to at least 11:1 2β-H/2α-h loss from unlabelled 19-oxoandrostenedione. This substrate-dependent stereoselectivity implies a direct role for an enzyme active-site base in 2-H removal. Furthermore, these results argue against the proposal that 2β-hydroxylation is the obligatory third step in aromatase action.