title: Direct chemical synthesis of 1 alpha,25-dihydroxy[26,27-3H]vitamin D3 with high specific activity: its use in receptor studies.
abstract: The first 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 six 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 dissociation constant measurements with the high specific activity radiohormone indicate a Kd of 8.2 x 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) x 10(-9) M previously reported.
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D002117:Calcitriol; D002645:Chickens; D003600:Cytosol / Q000378:metabolism; D004100:Dihydroxycholecalciferols / Q000138:chemical synthesis* / Q000378:metabolism; D004386:Duodenum / Q000378:metabolism; D006887:Hydroxycholecalciferols / Q000138:chemical synthesis*; D007413:Intestinal Mucosa / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D013058:Mass Spectrometry; D011955:Receptors, Drug / Q000378:metabolism*; D014316:Tritium
chemical_list: D004100:Dihydroxycholecalciferols; D006887:Hydroxycholecalciferols; D011955:Receptors, Drug; D014316:Tritium; D002117:Calcitriol