Direct chemical synthesis of 1.alpha.,25-dihydroxy[26,27-3H]vitamin D3 with high specific activity: its use in receptor studies

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Abstract
The 1st direct chemical synthesis of radiolabeled 1.alpha.,25-dihydroxyvitamin D3 is reported. Unlike all previous syntheses, the new approach does not rely on enzymatic 1.alpha.-hydroxylation of radiolabeled precursors. Rather, isotope is introduced in the last synthetic step by reaction of [3H]-methylmagnesium bromide with methyl 1.alpha.-hydroxy-26,27-dinorvitamin D3-25-carboxylate to give 1.alpha.,25-dihydroxy-[26,27-3H]vitamin D3 with a specific activity of 160 Ci/mmol. Mass spectroscopy confirmed that the radiohormone consists of a single isomer with 6 tritium atoms bound to carbons 26 and 27. Synthetically produced 1.alpha.,25-dihydroxy[26,27-3H]vitamin D3 is indistinguishable from 1.alpha.,25-dihydroxy-[26,27-3H]vitamin D3 obtained from the enzymatic 1.alpha.-hydroxylation of 25-hydroxy[26,27-3H]vitamin D3 (160 Ci/mmol) by high-pressure liquid chromatography analysis and in the competitive binding assay using chick intestinal cytosol as the receptor source. Equilibrium Kd measurements with the high specific activity radiohormone indicate a Kd of 8.2 .times. 10-11 M for the chick intestinal cytosol 1.alpha.,25-dihydroxyvitamin D3 receptor, a value considerably lower than the constants in the range of (1-5) .times. 10-9 M previously reported.