The photoreactivity of a number of photosensitive estrogen derivatives with the estrogen binding protein of rat uterus can be ascertained using a cytosol exchange assay. The
Fourteen estrogen derivatives bearing a photosensitive functional group have been synthesized for use as photoaffinity labels for estrogen binding proteins: diazoacetate derivatives of 173-estradiol and estrone, 16-diazoestrone, and 3-diazo-2-ketopropyl ether and ortho azide derivatives of estradiol, estrone, and hexestrol. All of these compounds are reasonably stable and easily purifiable. Attempts to introduce a diazo function at B-ring positions 6 and 7 of the estrogens have been unsuccessful: the 6-diazo derivative is thermally labile and the 6keto-7-diazo derivative fails to form. This is presumably due to the hindrance at position 7, as the corresponding position (2) in 7-hydroxy-l-tetralone is easily functionalized.
The ability of two radiolabeled, photoreactive estrogen analogues, [3H]hexestrol diazoketopropyl ether ([3H]Hex-DKP) and [3H]hexestrol azide ([3H]Hex-N3), to covalently label the uterine estrogen receptor is studied. Lamb uterine estrogen receptor preparations that have been partially purified (ammonium sulfate precipitation, Sephadex G-200 chromatography) and disaggregated by limited trypsinization can be electrophoresed on polyacrylamide gels under conditions where binding activity is preserved. This electrophoretic procedure was used to fractionate the proteins labeled by the two photoreactive estrogen analogues. Prior to photolysis, peaks of radioactivity indicating estrogen specific binding of [3H]-Hex-N3 and [3H]Hex-DKP are evident on the gels, although dissociation of the latter compound is extensive. When preparations of uterine estrogen receptor that contain the photoreactive derivatives are irradiated and then electrophoresed, reversibly labeled proteins can be distinguished from irreversibly labeled ones (covalently bonded), by extraction of the individual gel slices with organic solvents. While no irreversible binding to receptor appears to result from irradiation with [3H]Hex-DKP, irradiation with [3H]Hex-N3 does covalently label the estrogen receptor. The receptor covalently labeled with [3H]Hex-N3 has the same electrophoretic mobility as the unlabeled receptor; the covalent labeling process is estrogen-site specific, and the efficiency of labeling (15-20%) is consistent with the inactivation efficiency of Hex-N3, previously measured by an indirect assay. This is the first example of the labeling of a steroid hormone receptor by photoaffinity labeling.
Two photosensitive estrogen derivatives, hexestrol diazoketopropyl ether (5) and hexestrol azide (8a), have been synthesized in radiolabeled form, and their photochemical behavior in solution has been studied. The radiolabeled compounds were prepared in good yields according to improved synthetic procedures; they are stable and were obtained with specific activities in the range of 50-100 Ci per mmol and radiochemical purities in excess of 95%. A simpler model system, phenyl diazoketoprophyl ether, was used to study the photochemical behavior of the diazoketopropyl ether group. Direct irradiation of this compound at 254 nm in methanol led to 33% insertion product (methoxyketone) and 67% Wolff rearrangement product (ester). Irradiation of [3H]hexestrol diazoketopropyl ether (5) in methanol gives mainly nonpolar photoproducts (presumed to be the methoxy ketone and ester); however, irradiation in aqueous medium leads to large amounts of free hexestrol (52%). Photolysis of hexestrol azide (8a) in either methanol or water gives the corresponding amine in low yield as the only identifiable photoproduct.
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