A new high affinity, iodinated adenosine receptor antagonist as a radioligand/photoaffinity crosslinking probe.

A new high affinity antagonist photoaffinity crosslinking radioligand has been synthesized for use in studying adenosine receptors. This compound, PAPAXAC (8-[-4-[[[[[2-(4-aminophenylacetylamino)ethyl]amino]carbonyl]- methyl]oxy]phenyl]-1,3-dipropylxanthine), has been labeled with 125I by a chloramine T method. The radioligand [125I]PAPAXAC binds to A1 adenosine receptors from bovine cerebral cortex with high affinity (KD = 0.1 nM), appropriate stereoselectivity, and A1 adenosine receptor specificity. Binding is not perturbed by guanine nucleotides. Adenylate cyclase assays document that PAPAXAC is an antagonist capable of completely blocking the ability of N6-R-phenyl-2-propyladenosine (R-PIA) to inhibit adenylate cyclase activity via A1 adenosine receptors. [125I]PAPAXAC can be incorporated covalently into a peptide of Mr = 40,000 using the heterobifunctional crosslinking agent N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate. Covalent labeling can be inhibited with adenosine receptor ligands to demonstrate a potency series of R-PIA greater than S-PIA greater than NECA much greater than IBMX. Guanine nucleotides do not decrease covalent incorporation. These results suggest that antagonists such as [125I]PAPAXAC recognize the same A1 adenosine receptor-binding subunit as agonists, such as [125I]AZPNEA, which labels a similar Mr peptide with the same pharmacological potency series. This new antagonist photoaffinity crosslinking probe/radioligand should be of great utility in the molecular characterization of A1 adenosine receptors.