Stereochemistry of the Enzymatic Conversion of a Δ⁴‐3‐Oxosteroid into a 3‐Oxo‐5β‐Steroid

Abstract

The synthesis of [4-3H,4-14C]7.cr-hydroxycholest-4-en-3-one is described. The transformation of this compound into cholic acid in the bile fistula rat occurred with retention of the tritium label. The position of the tritium label in the isolated cholic acid was established by oxidation at the C-3 position of the methylated derivative followed by dehydrogenation with selenium dioxide yielding methyl 7a,12a-dihydroxy-3-oxo-chol-4-enoate. This compound retained most of the tritium label. Since selenium dioxide dehydrogenation of a 3-oxo-5p-steroid could be shown to involve a preferential loss of a 4a-hydrogen, it was concluded that the tritium label in the cholic acid isolated from bile was located in the 4/3-position. The enzymatic reduction of the A4 double bond in 7a-hydroxycholest-4-en-3-one in its conversion into cholic acid thus involves a trans diaxial addition of hydrogens.The conversion of cholesterol into bile acids entails the intermediary formation of 7a-hydroxycholest-4-en-3-one and 7a, 12a-dihydroxycholest-4-en%one and of the corresponding 3-oxo-5P-steroids [I].The saturation of the A4 double bond is catalyzed by a soluble A4-3-oxosteroid 5b-reductase(s) [2,3]. The reaction was shown recently to involve the transfer of a hydride ion from the A-side of NADPH to the 5P-position of the steroid and the addition of a proton to the 4-position [4]. The stereochemistry of the addition o€ the proton was not established. The present communication provides evidence that the proton is introduced into the 4a-position. The saturation of the A4 double bond in the biosynthesis of bile acids is thus a trans diaxial addition of hydrogens. Wettstein 161. The reaction mixture was acidified with 0.5M sulfuric acid and was extracted with ether. The ether extract was washed with 0.5M sulfuric acid, with a 5 O l 0 solution of sodium hydroxide (wlv), and with water until neutral. The ether extract was evaporated to dryness under reduced pressure and the residue was chromatographed on a column of 15 g of aluminum oxide, grade I11 (Woelm, Eschwege, Germany). The column was eluted with increasing concentrations of benzene in hexane. Benzene in hexane, 500/, (vlv), eluted 65 mg of [4-3H,4-14C]cholest-4,6-dien-3-one. The material was crystallized from a methanol-water mixture yielding 57 mg, m. p. 78", reported m. p. 80-81" [7]. The ratio of 3H to 14C was 1.8 (Table 1). R X P E R I M E N T A L PROCEDURE Materials(4-3H,4-14G]7a-Hydroxycholest-4-en-3-one. [4-3H, 4-14C]Cholest-4,6-dien-3-one, 56 mg, was dissolved in 3.5 ml of chloroform and 2 ml of a solution of monoperphthalic acid in ether (30mg/ml) were added [S, 91. The mixture was allowed to stand a t room temperature for 20hours. The reaction mixture was then poured into a 5 0 / / , solution of sodium bicarbonate in water (w/v) and the mixture was extracted with ether. The ether extract was washed with water until neutral and the solvent was evaporated. The residue of the ether extract was chromatographed on a column of l o g of aluminum oxide, gradeIII. The column was elute...

Mechanism of Microbial Transformation of Cholesterol into Coprostanol

“…Alkaline enolization of coprostanone was carried out as described previously [a]. Conversion of coprostanone into A4-cholestenone with selenium dioxide and subsequent purification of the product were performed as described previously [12]. The removal of isotope from C-6 in the A4-cholestenone by alkaline enolization was performed as described by Rosenfeld, Hellman and Gallagher [13].…”

Mechanism and Stereochemistry of the Enzymatic Conversion of a Delta4-3-Oxosteroid into a 3-Oxo-5alpha-Steroid

“…Dehydrogenation of 3-0x0-5a-steroids by reagents such as selenium dioxide and dichlorodicyanoquinone leads primarily to the formation of A1-3-oxosteroids, which in turn may be converted into A1~4-3-oxosteroids. Dehydrogenation with selenium dioxide and dichlorodicyanoquinone of 3-0x0-5a-steroids and 3-0x0-5B-steroids has been shown to be stereospecific [3,20,21] and it is probable that the formation of A1~4-3-oxosteroids from A'-3-oxosteroids also is stereospecific. However, considerable difficulties arose in the isolation of a A1~4-3-oxosteroid from the reaction mixture.…”

“…The present results indicate that the saturation of the A 4 double bond in the conversion of 7cc-hydroxycholest-4-en-3-one into 7u-hydroxy-5a-choles~n-3-one also involves an ionic mechanism with transfer of a hydride ion from NADPH to the 5u-position of the steroid and addition of a proton from the medium to the 4-position. A notable difference between the Conversion of a C2,-A4-3-oxosteroid into a 3-oxo-5p-steroid [2,3] and that into a 3-0x0-5a-steroid is that in the former case the hydride ion comes from the A-side of NADPH and the addition of hydrogens is trans, whereas in the latter case the B-side of NADPH is used and the addition is cis, a t least to 5O0lO.…”

“…7a-Hydroxycholest -4-en-3 -one, 7a -hydroxy -58-cholestan-3 -one,7a, 12a-dihydroxycholest-4-en- 3 -one, and 7a,i2a-dihydroxy-5~-cholestan-3-one have been shown to be intermediates in the major pathways for the conversion of cholesterol into the main primary 5B-cholanoic acids, chenodeoxycholic acid and cholic acid [l]. The reduction of the A 4 double bond is catalyzed by soluble A4-3-oxosteroid 5B-reductases and the mechanism and stereochemistry of this reaction have been the subject of two recent communications [Z, 31.…”

“…[4-311,4-14C]7a-lIydroxycholest -4-en-3-one. This compound was prepared as described previously [3]. The preparation used in the experiments in vitro had a specific radioactivity of 0.5 x lo6 countslminl pmole with respect to 3H and 0.2 x lo6 countslminl pmole with respect to 14C.…”