Synthesis and biological properties of 5-azido-2'-deoxyuridine-5'-triphosphate: a photoaffinity labelling reagent and a tool for the enzymatic synthesis of photoactive DNA

To produce light sensitive DNA for the study of protein-DNA interactions, 5-azido-2'-deoxyuridine-5'-triphosphate (5-N3dUTP), was synthesized to serve as a substrate for the enzymatic incorporation of a photoactive nucleotide analog into DNA. 5-N-dUTP was synthesized from dUMP in five steps with the key reaction being the nitration of dUMP to 5-NO2dUMP in 98% yield using an excess of nitrosonium tetrafluoroborate in anhydrous dimethylformamide. The triphosphate form of the nucleotide (5-N3dUTP), formed through a chemical coupling of pyrophosphate to 5-N3dUMP, was found to support DNA synthesis by E. coli DNA polymerase I at a rate indistinguishable to that supported by dTTP. Using UMP as the starting compound 5-N3UTP was formed in an analogous fashion with similar yields and produced a photoactive nucleotide which is a substrate for E. coli RNA polymerase. To prepare [γ32P] 5-N3dUTP for use as an active site directed photoaffinity labeling reagent, a simple method of preparing [γ32P] labeled pyrimidine nucleotides was developed. [γ32P] 5-N3dUTP is an effective photoaffinity labeling reagent for DNA polymerase I which binds to the active site of the enzyme with a two fold higher affinity than dTTP