Mechanistic studies on C-19 demethylation in oestrogen biosynthesis

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Abstract
Mechanistic aspects of the biosynthesis estrogen were studied with a microsomal preparation from full-term human placenta. The overall transformation, termed the aromatization process, involves 3 steps using O2 and NADPH, in which the C-19 methyl group of an androgen is oxidized to formic acid with concomitant production of the aromatic ring of estrogen: .**GRAPHIC**. To study the mechanism of this process in terms of the involvement of the oxygen atoms, a number of labeled precursors were synthesized. Notable among these were 19-hydroxy-4-androstene-3,17-dione (II) and 19-oxo-4-androstene-3,17-dione (IV) in which the C-19 was labeled with 2H in addition to 18O. In order to follow the fate of the labeled atoms at C-19 of (II) and (IV) during the aromatization, the formic acid released from C-19 was benzylated and analyzed by mass spectrometry. Experimental procedures were devised to minimize the exchange of O2 atoms in substrates and product with oxygens of the medium. In the conversion of the 19-[18O] compounds of types (II) and (IV) into 3-hydroxy-1,3,5-(10)-estratriene-17-one (V, estrone), the formic acid from C-19 retained the original substrate oxygen. When the equivalent 16O substrates were aromatized under 18O2, the formic acid from both substrates contained 1 atom of 18O. It is argued that in the conversion of the 19-hydroxy compound (II) into the 19-oxo compound (IV), the C-19 oxygen of the former remains intact and that 1 atom of oxygen from O2 is incorporated into formic acid during the conversion of the 19-oxo compound (IV) into estrogen. This conclusion was further substantiated by demonstrating that, in the aromatization of 4-androstene-3,17-dione (I), both the oxygen atoms in the formic acid originated from molecular oxygen. 10.beta.-Hydroxy-4-estrene-3,17-dione formate, a possible intermediate in the aromatization, was synthesized and shown not to be converted into estrogen. In the light of the cumulative evidence available to date, stereochemical aspects of the conversion of the 19-hydroxy compound (II) into the 19-oxo compound (IV), and mechanistic features of the C-10.sbd.C-19 bond cleavage step during the conversion of the 19-oxo compound (IV) into estrogen are discussed.