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

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 C₂₇ sterols afforded the corresponding 3-ketosteroids, and reduction with tritiated NaBH₄ 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 Δ⁸ 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-Δ⁵-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. Copyright (C) 2000 Elsevier Science Inc.