Development of Novel Photoaffinity Ligands for the β-Adrenergic Receptor

Abstract The successful synthesis and use of carrier-free radioiodinated β 2 -adrenergic receptor competitive antagonist photoaffinity labels (±)-[ 125 I]IABP, (±)-[ 125 I]MAPIT, (−)-[ 125 I]IAPTA, and (±)-[ 125 I]IAPCGP-12177, are described. In addition, the synthesis and use of two carrier-free radioiodinated β-adrenergic receptor agonist photoaffinity labels (±)-[ 125 I]iodoazidoprenalterol ((±)-[ 125 I]IAPr) and (−)- N -( p -azido- m -[ 125 I]iodophenethylamidoisobutyl)norepinephrine ((−)-[ 125 I]NAIN), are described. All antagonist photolabels were capable of highly specific derivatization of the purified recombinant hamster lung β 2 -adrenergic receptor. Tryptic cleavage of the photolabeled receptor into a 30-kDa radiolabeled fragment (transmembrane 1-5) and an 8-kDa radiolabeled fragment (transmembrane 6,7) showed variable Insertion ratios between the two juxtaposed domains, depending on the structure of the photolabel. Unique synthetic strategies were used for the agonist photolabels. The phenolic hydroxyl of (±)-IAPr was protected as the glucoside and deprotected enzymatically in the final step. The final coupling step in the synthesis of (−)-[ 125 I]NAIN was accomplished by reductive alkylation without protection of the catechol hydroxyls of norepinephrine using sodium cyanoborohydride. (±)-IAPr was found to be a partial agonist for the turkey erythrocyte β-adrenergic receptor and an effective photoaffinity label for the avian β-adrenergic receptor. (−)-NAIN was found to be a full agonist for the β 2 -adrenergic receptor in guinea pig lung membranes and a highly effective agonist photoaffinity label for the β 2 -adrenergic receptor. These photolabels will be useful for probing the β-adrenergic receptor binding site In order to "map" this site under nonactivated (antagonist photolabels) or activated states (agonist photolabels).