Abstract
As a series of our studies on estrogen biosynthesis, stereochemistry of hydrogen loss from C-2 during placental and microbial aromatization of 19-norsteroid has been investigated. The substrates required for this purpose, epimeric (2-3H) estr-4-ene-3, 17-diones, were prepared along the way which had previously been developed in this laboratory. These tritiated compounds were mixed with an appropriate amount of (4-14C)-estr-4-ene-3, 17-dione and recrystallized repeatedly up to a constant isotope ratio.
The labeled substrates were incubated with human placental preparation according to the method of Ryan. The incubation mixture consisted of the steroid substrate, placental 10,000×g supernatant and NADPH-generating system suspended in 0.05M phosphate buffer, pH 7.0. After incubation at 37° for 1 hr, the mixture was extracted with ethyl acetate and the extract was submitted to thin-layer chromatography (TLC) on silica gel. The products and unchanged substrate were separated, diluted with the carrier and then recrystallized up to constant specific activity, respectively. Estrone formed from the substrate with the label in the 2 β-position exhibited a 79% loss of tritium. In contrast, the 2 α-labeled substrate lost only 20% isotope. These data unambiguously tell us that hydrogen loss from C-2 in the aromatization process is stereoselectively β and in consequence C-1, 2 hydrogen elimination is 1β, 2 β-cis. The 1 β-hydroxylated products obtained from both incubations showed almost the same tritium content with a slight decrease.
The double-isotope labeled substrates were also incubated with respiring cultures of Bacillus sphaericus for 48 hr. The incubation mixture was extracted with ethyl acetate and the extract was similarly purified by TLC. The desired estrone was eseparated, diluted with the carrier and then recrystallized until a constant isotope ratio has been attained. The aromatized product from the 2 β-tritiated substrate lost 80% of the label, while 2 α-epimer showed only an 11% loss of tritium. Elimination of hydrogen from C-2 is thus stereospecifically β; in other words loss of hydrogen at C-1, 2 is 1α, 2 β-trans. Stereochemistry of Δ1 dehydrogenation of 19-norsteroid is then the same as that of C19 steroid.
