Synthesis and Use of 3′-(Azidoiodosalicyl) Derivatives of 2′,5′-Dideoxyadenosine as Photoaffinity Ligands for Adenylyl Cyclase

Abstract

3′-[(4-Azidosalicyl)glycyl]-2′,5′-dideoxyadenosine (1), 3′- [(4-azidosalicyl)-γ-aminobutyryl]-2′,5′-dideoxyadenosine (2), and the 125I-labeled mono- and diiodinated analogs of 1 were synthesized and tested as photoaffinity probes for adenylyl cyclases. Kinetics for inhibition of purified type I enzyme 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 membrane-bound and purified forms of recombinant type I bovine adenylyl cyclase expressed in ovarian cells of either the fall armyworm (Sf9) or Trichoplasia ni (High Five). Irreversible inactivation was independent of 5′-ATP and was prevented by 2′,5′-dideoxyadenosine. Adenylyl cyclase, whether purified from bovine brain or in membranes from High Five cells expressing type I enzyme, when subjected to UV irradiation in the presence of 125I-labeled 1 resulted in radioactive incorporation into protein migrating at ∼116 kDa. The cross-linking of 1 and its iodinated derivative with adenylyl cyclase suggests potential for such compounds to be useful in structural studies of adenylyl cyclases or of other proteins for which adenine nucleosides are substrates or allosteric regulators.