Synthesis and use of 3 '-(azidoiodosalicyl) derivatives of 2 ',5 '-dideoxyadenosine as photoaffinity ligands for adenylyl cyclase

3'-[(4-Azidosalicyl)glycyl]-2',5'-dideoxyadenosine (1) 3'-[(4-azidosalicyl) -gamma-aminobutyryl]-2',5'-dideoxyadenosine (2), and the I-125-labeled mono - and diiodinated analogs of 1 were synthesized and tested as photoaffinity probes for adenylyl cyclases, Kinetics for inhibition of purified type I e nzyme by 1 was noncompetitive with respect to Mn(.)5'-ATP in the absence of light, implying a P-site mechanism of inhibition. In a UV-dependent manner both 1 and 2 and the iodinated derivative of 1 irreversibly inactivated me mbrane-bound and purified forms of recombinant type I bovine adenylyl cycla se expressed in ovarian cells of either the fall armyworm (Sf9) or Trichopl asia ni (High Five). Irreversible inactivation was independent of 5'-ATP an d was prevented by 2',5'-dideoxyadenosine, Adenylyl cyclase, whether purifi ed from bovine brain or in membranes from High Five cells expressing type I enzyme, when subjected to UV irradiation in the presence of I-125-labeled 1 resulted in radioactive incorporation into protein migrating at similar t o 116 kDa. The cross-linking of 1 and its iodinated derivative with adenyly l cyclase suggests potential for such compounds to be useful in structural studies of adenylyl cyclases or of other proteins for which adenine nucleos ides are substrates or allosteric regulators, (C) 2000 Academic Press.