A method was developed for the synthesis of high-specific-activity 21-diazo-21[6,7-3Hdeoxycorticosterone, an analog of corticosterone. This analog was used as a photoaffinity label of a high affinity steroid-binding protein, human corticosteroid-binding globulin. Based on direct binding studies and crosscompetition experiments, this diazo derivative exhibited the requisite affinity (within a factor of 1.5 times that of corticosterone) and site specificity to qualify as an affinity labeling ligand. Purification and definition of the properties of the corticosteroid receptors have been frustrated by their low abundance in cytoplasm and irreversible loss of binding activity during isolation (1-4). In principle, these problems could be circumvented by site-specific covalent labeling of the receptor (5, 6). In a previous study, 21-diazo derivatives of corticosteroids exhibited reasonable affinities for intracellular receptors and functional activity in a toad bladder assay (5). In addition, covalent labeling of plasma proteins was obtained with 9a-bromo-21-diazo-21-[1,2-3H]deoxycorticosterone. The utility of this compound was limited, however, by the low specific activity (25 Ci/mol). We now report on the preparation of high-specific-activity 21-diazo-21- [6,7-3H] (5) with the following solvent systems: system A, benzene-ethyl acetate (1:2 vol/vol); system B, benzene-ethanol (6:1 vol/vol); system C, chloroform-ethyl acetate (1:3 vol/vol). Melting points, microanalyses, and. UV spectroscopy were performed as described (5). Catalytic tritiation was performed with the assistance of Dr. Chin-Tzu Peng and Mr. Ray Aune at the Lawrence Radiation Laboratory (Berkeley, Calif.). The reaction sequence used to prepare 21-diazo-21-[6,7-3H]deoxycorticosterone (compound V) is shown in Fig. 1. Compound II was obtained by treatment of compound I (2.0 g) with chloranil (6.5 g) in t-butanol (120 ml). The filtered mixture was evaporated, taken up in ether, washed with dilute NaOH and water, and reevaporated. The residue was crystallized from aqueous methanol; mp 171-174°[lit. mp 155-177° (8)], Xmax 286 nm. Compound-III was prepared by reaction of compound II (0.9 g in 10 ml of dioxane + 1.5 ml of H20) with sodium hypobromite (prepared from 0.3 ml of Br2 and 0.5 g of NaOH in 8 ml of H20) at 00. The reaction mixture was diluted with dioxane (4 ml), stirred for 2.5 hr at 00, treated with NaHSO3 (50 mg) for 15 min, extracted with ether, and acidified with HCL. The precipitate was washed with H20 and crystallized from aqueous acetone (mp 270-275°, Xmax 286 nm) (Anal. calcd. for C20H2604: C, 72.70; H, 7.93. Found: C, 72.54; H, 7.83). Compound IV [mp 251-255°, Xmax 242 nm, (E 1.55 X 104)] was prepared from compound I as described (5). Compound V [mp 1750 (dec), Xmax 247 nm (E 2.3 X 104)], with a shoulder at 280 nm, was prepared from compound IV as described (5). Tritium-labeled compound IV (at C6-C7) was obtained by catalytic tritiation of compound III in benzene-tetrahydrofuran solvent using 10% palladium on charcoal and carrier-fre...