Synthesis of [3α- 3H]cholesta-5,8-dien-3β-ol and tritium-labeled forms of other sterols of potential importance in the Smith-Lemli-Optiz syndrome☆

Abstract

Five unsaturated sterols relevant to the Smith-Lemli-Opitz syndrome have been prepared in high radiochemical purity with a tritium label at the 3α position. Swern oxidation of cholesta-5,8-dien-3β-ol and other unlabeled C 27 sterols afforded the corresponding 3-ketosteroids, and reduction with tritiated NaBH 4 gave the desired 3α- 3H sterols, with double bonds at the Δ 5,8, Δ 5,8(14), Δ 6,8, Δ 6,8(14), and Δ 8 positions. High radiochemical purity of the tritiated sterols was demonstrated by normal phase, reversed phase, and silver-ion (Ag +) high-performance liquid chromatography (HPLC). In the course of this work, we developed a medium-pressure variant of Ag +-HPLC for purifying radiolabeled samples, documented significant isotopic fractionation of the 3α-tritiated sterols and their acetates on Ag +-HPLC, and discovered unexpected effects of a Δ 8(14) bond on the conformation of 3-keto-Δ 5-steroids. The synthetic and analytical methodologies described herein should provide a sound basis for investigating the origin and metabolism of sterols involved in the Smith-Lemli-Opitz syndrome and in late stages of cholesterol biosynthesis.