Mass spectrometric analysis of oxygenations in aromatization of androst-4-ene-3,6,17-trione, a suicide substrate of aromatase, by placental microsomes. Isotope effect and stereochemistry.
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.
Numazawa
Mitsuteru
M
Tohoku Pharmaceutical University, 4-1 Komatsushima-4-chome, Aoba-ku, Sendai 981-8558, Japan. numazawa@tohoku-pharm.ac.jp
Takahashi
Madoka
M
Nagaoka
Masao
M
Handa
Wakako
W
Yamashita
Kouwa
K
D000736:Androstenes / Q000378:metabolism*; D005260:Female; D006801:Humans; D007554:Isotopes; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry / Q000379:methods*; D008861:Microsomes / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D010920:Placenta / Q000378:metabolism*; D013237:Stereoisomerism
D000736:Androstenes; D007554:Isotopes; C018213:androst-4-ene-3,6,17-trione; D010100:Oxygen