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Aromatase catalyzes the conversion of androstenedione (AD) to estrone through three sequential oxygenations of the 19-methyl group. 6-OxoAD (1) is one of the typical suicide substrates of aromatase, which is converted by aromatase to 6-oxoestrone through 19-alcohol (19-ol) and 19-aldehyde (19-al) intermediates 2 and 3. To study the deuterium isotope effect on the conversion of 19-ol 2 to 19-al 3 as well as the stereochemistry of the 19-hydrogen removal in this conversion, we initially synthesized [19,19-(2)H(2)] and [19S- or 19R-(2)H] 19-ols 2, starting from the corresponding deuterium-labeled 19-hydroxyAD derivatives. In incubation of non-labeled and [19,19-(2)H(2)]-labeled 19-ol 2 or that of their 1:1 mixture with human placental microsomes in the presence of NADPH under air, there was no significant deuterium-isotope effect on the production of the aromatized product 6-oxoestrone or on the conversion of 19-ol 2 to 19-al 3, based on gas chromatography-mass spectrometric analysis of the estrogen product or liquid chromatography-mass spectrometric (LC-MS) analysis of the deuterium contents of the product 19-al 3 and the recovered 19-ol 2. Moreover, in the incubations of [19S-(2)H] 19-ol 2 and its 19R isomer, LC-MS analysis of the product 3 demonstrated that the 19-pro-R hydrogen atom was stereospecifically removed in the conversion of 19-ol 2 to 19-al 3. These findings indicate that the 19-oxygenation of 19-ol 2 would proceed in the same mechanism as that involved in the AD aromatization.

To gain insight into the catalytic function of aromatase, we studied 19-oxygenation of 19-methyl-substituted derivative of the natural substrate androstenedione (AD), compound 1, with human placental aromatase by use of gas chromatography-mass spectrometry (GC-MS). Incubation of the 19-methyl derivative 1 with human placental microsomes in the presence of NADPH under an aerobic condition did not yield a detectable amount of [19S]19-hydroxy product 2 or its [19R]-isomer 3 when the product was analyzed as the bis-methoxime-trimethylsilyl (TMS) derivative by GC-MS; moreover, the production of estrogen was not detected as the bis-TMS derivative of estradiol (detection limit: about 3 ng and 10 pg per injection for the 19-ol and estradiol, respectively). The results reveal that the 19-methyl steroid 1 does not serve as a substrate of aromatase, although it does serve as a powerful inhibitor of the enzyme.