Synthesis and biological properties of 5-azido-2'-deoxyuridine 5'-triphosphate, a photoactive nucleotide suitable for making light-sensitive DNA

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Abstract
A photoactive nucleotide analogue of dUTP, 5-azido-2''-deoxyuridine 5''-triphosphate (5-N3dUTP), was synthesized from dUMP in five steps. The key reaction in the synthesis of 5-N3dUTP is the nitration of dUMP in 98% yield in 5 min at 25.degree. C using an excess of nitrosonium tetrafluoroborate in anhydrous dimethylformamide. Reduction of the resulting 5-nitro compound with zinc and 20 mM HCl gave 5-aminodeoxyuridine monophosphate (5-NH2dUMP). Diazotization of 5-NH2dUMP with HNO2 followed by the addition of NaN3 to the acidic diazonium salt solution gave a photoactive nucleotide derivative in 80-90% yield. The monophosphate product was identified as 5-N3dUMP by proton NMR, UV, IR, and chromatographic analysis as well as by the mode of synthesis and its photosensitivity. After formation of 5-N3dUTP through a chemical coupling of pyrophosphate to 5-N3dUMP, the triphosphate form of the nucleotide was found to support DNA synthesis by Escherichia coli DNA polymerase I at a rate indistinguishable from that supported by dTTP. When UMP was used 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 [.gamma.-32P]-5-N3dUTP for use as an active-site-directed photoaffinity labelling reagent, a simple method of preparing .gamma.-32P-labeled pyrimidine nucleotides was developed. [.gamma.-32P]-5-N3dUTP is an effective photoaffinity labeling reagent for DNA polymerase I and was found to bind to the active site with a 2-fold higher affinity than dTTP. The photoactivity of 5-N3dUMP is stable to extremes of pH, and [.gamma.-32P]-5-N3dUTP was an effective photolabeling reagent even in the presence of 10 mM dithiothreitol. 5-Azidouracil-containing nucleotides have potential applications as active-site-directed photoaffinity labeling reagents and as tools for generating photoactive DNA and RNA to study nucleic acid binding proteins.

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