Pregnenolone-17a -3H was synthesized by reaction of1 7a-bromopregnenolone with tritiated acetic acid (CH3C02 3H) and zinc metal dust. The distribution of the label in the product was studied: about 98 % was at the 17-position and the remainder was randomly distributed on the steroids nucleus. It was also found that when pregnenolone was stirred with deuterated acetic acid (CH3C02 2H) and zinc metal dust ,for a longer period of time (22 hr) and in relatively high concentration, the protons of the CzI-methyl group (-57 %) were exchangedfor deuterium.
For metabolic studies involving the biosynthesis of testosterone from pregnane precursors, it was necessary to synthesize 3P-hydroxy-5-pregnen-20one-[l7dH] (pregnenolone-1 ~z -~H , 1). This was accomplished by the stereospecific reaction (l) of 17~-bromopregnenolone (2) with zinc metal dust and tritiated acetic acid (CH,C0,3H).
When 1 was used as substrate for incubations, some tritium was unexpectedly observed in certain isolated C,,-metabolites. To determine whether this tritium resulted from metabolic transformation or from labeling of the steroid nucleus during the preparation of 1, the distribution of its tritium label was investigated.
Initially 1 was equilibrated * with potassium hydroxide in methanolwater to eliminate tritium from the 17-and 21-positions.
The product of equilibration was a mixture of pregnenolone 3a and isopregnenolone (4a), which were separated by fractional crystallization of * Pregneno10ne-4-'~C was used as internal standard.