Testosterone 1β‐hydroxylation by human cytochrome P450 3A4

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Abstract
Human cytochrome P450 3A4 forms a series of minor testosterone hydroxylation products in addition to 6β‐hydroxytestosterone, the major product. One of these, formed at the next highest rate after the 6β‐ and 2β‐hydroxy products, was identified as 1β‐hydroxytestosterone. This product was characterized from a mixture of testosterone oxidation products using an HPLC‐solid phase extraction‐cryoprobe NMR/time‐of‐flight mass spectrometry system, with an estimated total of ≈ 6 µg of this product. Mass spectrometry established the formula as C19H29O3 (MH+ 305.2080). The 1‐position of the added hydroxyl group was established by correlated spectroscopy and heteronuclear spin quantum correlation experiments, and the β‐stereochemistry of the added hydroxyl group was assigned with a nuclear Overhauser correlated spectroscopy experiment (1α‐H). Of several human P450s examined, only P450 3A4 formed this product. The product was also formed in human liver microsomes.