To elucidate the mechanism for oxidative attack in ring A resulting in the production of estrogens, the stereochemistry and cofactor requirements for aromatization of estr-4-ene-3,l7-dione (I) were determined and compared with the aromatization characteristics of androst-4-ene-3,17-dione (II). Incubations of [4-I4C]II stereoselectively tritiated at C-l/3 or C-la with the lO.OOOg supernatant of human term placenta and a reduced nicotinamide-adenine dinucleotide phosphate generating system showed that the 13hydrogen was lost in the conversion to estrone. Incubation of [4-14C-l-3H (83% 3)]I under similar conditions yielded estrone in which 82% of the tritium T A he conversion of androstenedione1 or testosterone to estrogens by human placental preparations requires two general steps: (1) hydroxylation and eventual elimination of the C-10 methyl group, and (2) oxidative attack resulting in double-bond introduction in ring A (Talalay, 1965). The available evidence suggests step 2 occurs after C-19 hydroxylation and before or during cleavage of the C-10,19 bond. No potential intermediate beyond 19-oxoandrostenedione has been isolated, and it is not known how desaturation occurs in ring A to produce estrogen.' In a study to elucidate the events occurring in ring A, leading to aromatization, we found evidence, presented in a preliminary communication, that the 13-equatorial hydrogen is removed. This suggested enzymatic participation or steric interaction of the C-19 group at C-13 (Morato et al., 1962). 19-Nor steroids also serve as estrogen precursors in placental and ovarian preparations

The incorporation of C5-amino-modified 2′-deoxyuridine analogues into DNA have found application in nucleic acid labelling, the stabilization of nucleic acid structures, functionalization of nucleic acid aptamers and catalysts, and the investigation of sequence-specific DNA bending. In this study, we describe the physicochemical properties of four different C5-amino-modified 2′-deoxyuridines in which the amino group is tethered to the base via a 3-carbon alkyl, Z- or E-alkenyl or alkynyl linker. Conformational parameters of the nucleosides and their pKa values were deduced using 1H NMR. All of them display the expected anti-conformation of the nucleoside with 2′-endo sugar puckers for the deoxyribose ring. A preference for the cisoid conformation for the Z-alkenyl analogue is found, while the E-alkenyl analogue exists exclusively as its transoid conformation. The pKa values range from 10.0 for the analogue with an aliphatic propyl linker to 8.5 for the propargylamino analogue. The analogues have been used for the synthesis of triple-helix forming oligonucleotides (TFOs) in which they replace thymidine in the natural sequence. Oligonucleotides containing the propargylamino analogue display the highest stability especially at low pH, while those containing analogues with propyl and especially Z-alkenyl linkers are destabilized to a great extent. TFOs containing the analogue with the E-alkenyl linker have stability similar to the unmodified structures. The chemical synthesis of TFOs containing the analogue, 5-(3-hydroxyprop-1-ynyl)-2′-deoxyuridine that possesses a neutral but polar side chain show a remarkable stability, which is higher than that of all TFOs containing the alkylamino or alkenylamino analogues and only slightly lower than that of TFOs containing the propargylamino analogue. Both the hydroxyl and propargylamino substitutions impart enhanced triple-helix stability relative to the analogous sequences containing C5-propynyl-2′-deoxyuridine. Furthermore, a similar dependence of stability on pH is found between TFOs containing the hydroxypropynyl modifications and those containing the propargylamino side chains. This suggests that the major factor responsible for stabilizing such triple helices is due to the presence of the alkyne with an attached electronegative group.

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We have proposed that inhibition of placental steroid 3-sulphatase by endogenous steroids may regulate oestrogen synthesis during human pregnancy. The possibility that an additional regulatory mechanism, involving the placental 3\g=b\-hydroxy-steroiddehydrogenase (SDH), may also be operative has now been examined. Inhibitory effects of naturally occurring steroids on SDH activity were determined from the reduction in initial rate of conversion of 3H-dehydroepiandrosterone to non-digitonin precipitable products by 10 000 \m=x\ g supernatant from homogenates of human term placentae. The apparent Km for dehydroepiandrosterone was 0.33 \m=x\ 1 0 \ m = -\ 6 m. \g=D\4-3-Oxoproducts of SDH action (4-androstene-3,17-dione, app. Ki = 0.60 \m=x\ 10\m=-\6 m; progesterone, app. Ki = 1.5 \m=x\10\m=-\6 m) were the most potent inhibitors and appeared to act non-competitively. \g=D\5-3\g=b\-Hydroxy alternative substrates were less inhibitory and in the case of pregnenolone (app. Ki = 4.5 \m=x\ 10\ m=-\ 6 m) behaved competitively. 11\g=b\-,16\g=a\-, 17\g=a\-or 21\ x=r eq-\ hydroxylation and epimerization of 3\g=b\or 17\g=b\-hydroxyl functions of inhibitors decreased their activity. It is concluded that inhibition of both sulphatase and SDH by endogenous steroids may provide complementary methods of regulating placental oestrogen synthesis in vivo. The SDH mechanism may regulate oestrogen synthesis from unconjugated precursors, either formed within the placenta or derived from the circulation. The major potential inhibitors appear to be \g=D\4-3-ketones, acting non\x=req-\