Synthesis of a photoaffinity analog of 3′-azidothymidine, 5-azido-3′-azido-2′,3′-dideoxyuridine: Interactions with herpesvirus thymidine kinase and cellular enzymes

Long term administration of 3′-azidothymidine (AZT) for the treatment of AIDS has led to detrimental clinical side effects in some patients, the biochemical causes of which are still being delineated. Base-substituted, azido-nucleotide photoaffinity analogs have routinely proven to be effective tools for identifying and characterizing nucleotide-utilizing enzymes. Therefore, we have synthesized 5-azido-3′-azido-2′,3′-dideoxyuridine, which is a potential photoaffinity analog of two human immunodeficiency virus drugs, AZT and 3′azido-2′,3′-dideoxyuridine. A partially purified herpes simplex virus type 1 thymidine kinase and [γ-³²P]ATP were used to make an AZT monophosphate analog, [³²P]5-azido-3′-azido-2′,3′-dideoxyuridine monophosphate. The photoaffinity properties of this analog were initially tested with herpes simplex virus type 1 thymidine kinase. Photoaffinity labeling of this enzyme was saturable (half-maximal, 30 μM) and could be specifically inhibited by AZT, AZT monophosphate, thymidine, and thymidine monophosphate. Photolabeling of rat liver microsomal membranes was also done, and several membrane proteins that interact with AZT monophosphate were identified. The antiviral and cytotoxic activities of 5-azido-3′-azido-2′,3′-dideoxyuridine were determined using human immunodeficiency virus, type 1 strain IIIB and an AZT drug-resistant strain in human T lymphocyte H9 cells.