Abstract

α-Arabinosyl-8-azaadenine (α-ara-8-azaA) and α-arabinosyladenine (α-araA) were toxic to H.Ep.2 cells in culture, whereas the corresponding β anomers were not toxic at the highest concentrations (375 µM) assayed. α-Ara-8-azaA was more cytotoxic than α-araA and therefore was selected for more detailed study. Cell lines deficient in adenosine kinase (EC 2.7.1.20) were resistant to inhibition by α-ara-8-azaA. Cultured H.Ep.2 cells grown in the presence of [2-¹⁴C]α-ara-8-azaA contained compounds migrating on paper chromatograms like mono-, and di-, and triphosphates. Characterization of these compounds by paper chromatography and by high-pressure liquid chromatography showed them to be phosphates of α-ara-8-azaA; there were no detectable amounts of ribonucleotides and thus, presumably, no cleavage of α-ara-8-azaA to 8-azaA. There was also no detectable radioactivity in polynucleotides isolated by extraction with hot NaCl solution. α-Ara-8-azaA was a substrate for adenosine kinase partially purified from H.Ep.2 cells; the K_m was 110 µM and the V_max was about 15% of that of adenosine. α-Ara-8-azaA was not a substrate for adenosine deaminase; several other α-nucleosides assayed also had little or no activity as substrates for this enzyme. These results, which show that an analogue of adenosine in the "unnatural" α configuration has biological activity and differs markedly from the β anomer in biological activity and in activity as a substrate for the principal enzymes acting on adenosine, are of importance for understanding the modes of action of adenosine analogues and should also find application in the design of new nucleoside analogues with biological activity. In the course of characterizing the metabolites, a rapid and convenient method was developed for the separation, by high-pressure liquid chromatography, of the α and β anomers of ara-8-azaA; this method should also be applicable to the separation of anomers of other nucleosides.