Steroidal 21-diazo ketones: photogenerated corticosteroid receptor labels.
The 21-diazo derivatives of 9 alpha-fluoro- and 9 alpha-bromo-21 deoxycorticosterone, 21-deoxycorticosterone, and progesterone were synthesized for use as photoaffinity labels for corticosteroid receptors. In the isolated toad bladder system, 9 alpha-bromo-21-diazo-21-deoxycorticosterone was as active as d-aldosterone and more active than 9 alpha-fluoro-cortisol in augmenting active Na+ transport. The activities of 21-diazoprogesterone and progesterone were equal; both were much less potent than d-aldosterone, however. These results indicate that the 21-diazo derivatives had significant functional activity in the toad bladder system. The rat kidney slice system was used to estimate the relative affinities of the diazo steroids for aldosterone receptor sites by competition experiments. At 100-fold excess of competitor to [3-H]aldosterone, the order of affinities was 9 alpha-fluoro-21-diazo-21-deoxycorticosterone greater than 9 alpha-bromo-21-diazo-21-deoxycorticosterone greater than 21-diazoprogesterone. Moreover, 9 alpha-bromo-21-diazo-21-deoxycorticosterone reduced binding of [3-H]aldosterone to cytoplasmic and nuclear forms of the receptor proportionately. On the basis of competition for [3-H]corticosterone binding, presumably to corticosteroid-binding globulin (CBG), the order of affinities was 21-diazo-21-deoxycorticosterone greater than 21-diazoprogesterone greater than 9 alpha-bromo-21-diazo-21-deoxycorticosterone. These findings indicate that 21-diazo steroids may be suitable as photogenerated affinity labels for mineralocorticoid receptors. The tritiated derivative, [1,2-3-H]-9 alpha-bromo-21-diazo-21-deoxycorticosterone (specific activity 25 Ci/mol) was synthesized and used in model experiments on photogenerated covalent binding to rat plasma proteins. Irradiation with uv light resulted in binding of [1,2-3-H]-9 alpha-bromo-21-diazo-21-deoxycorticosterone to plasma proteins, that was resistant to extraction with methylene dichloride and did not exchange with unlabeled corticosterone. The diazocorticosteroids, therefore, may have the requisite functional and selectivity properties for photoaffinity labeling of corticosteroid-binding proteins. Further studies are needed, however, to assure that photogenerated labeling with these steroids was site specific.
D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D001966:Bromine; D002024:Bufo marinus; D003593:Cytoplasm / Q000378:metabolism; D003900:Desoxycorticosterone / Q000032:analysis* / Q000494:pharmacology; D003979:Diazonium Compounds; D005461:Fluorine; D007659:Ketones; D007668:Kidney / Q000378:metabolism; D011374:Progesterone / Q000032:analysis* / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats; D011956:Receptors, Cell Surface; D012964:Sodium / Q000378:metabolism; D013329:Structure-Activity Relationship; D001743:Urinary Bladder / Q000187:drug effects / Q000378:metabolism
D003979:Diazonium Compounds; D007659:Ketones; D011956:Receptors, Cell Surface; D005461:Fluorine; D003900:Desoxycorticosterone; D011374:Progesterone; D012964:Sodium; D001966:Bromine