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

Journal Article · Tue Jan 13 04:00:00 UTC 1987 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00375a037· OSTI ID:6524066

Evans, R K; Haley, B E

A photoactive nucleotide analogue of dUTP, 5-azido-2'-deoxyuridine 5'-triphosphate (5-N/sub 3/dUTP), was synthesized from dUMP in five steps. The key reaction in the synthesis of 5-N/sub 3/dUTP is the nitration of dUMP in 98% yield in 5 min at 25 /sup 0/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-NH/sub 2/dUMP). Diazotization of 5-NH/sub 2/dUMP with HNO/sub 2/ followed by the addition NaN/sub 3/ to the acidic diazonium salt solution gave a photoactive nucleotide derivative in 80-90% yield. The monophosphate product was identified as 5-N/sub 3/dUMP by proton NMR, UV, IR, and chromatographic analysis as well as by the mode of synthesis and its photosensitivity. After formation of 5-N/sub 3/dUTP through a chemical coupling of pyrophosphate to 5-N/sub 3/dUMP, 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-N/sub 3/UTP 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..-/sup 32/P)-5-N/sub 3/dUTP for use as an active-site-directed photoaffinity labeling reagent, a simple method of preparing ..gamma..-/sup 32/P-labeled pyrimidine nucleotides was developed. (..gamma..-/sup 32/P)-5-N/sub 3/dUTP 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-N/sub 3/dUMP is stable to extremes of pH, and (..gamma..-/sup 32/P)-5-N/sub 3/dUTP was an effective photolabeling reagent even in the presence of 10 mM dithiothreitol.

• Research Organization: Univ. of Wyoming, Laramie
• OSTI ID: 6524066
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:1; ISSN BICHA
• Country of Publication: United States
• Language: English

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59 BASIC BIOLOGICAL SCIENCES
62 RADIOLOGY AND NUCLEAR MEDICINE
AZIDO COMPOUNDS
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMISTRY
BIOSYNTHESIS
CHEMISTRY
DAYS LIVING RADIOISOTOPES
DNA
DNA POLYMERASES
ENZYMES
ESCHERICHIA COLI
INFRARED SPECTRA
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
MAGNETIC RESONANCE
MICROORGANISMS
NITRATION
NUCLEAR MAGNETIC RESONANCE
NUCLEI
NUCLEIC ACIDS
NUCLEOTIDES
NUCLEOTIDYLTRANSFERASES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
RADIOISOTOPES
RESONANCE
SPECTRA
SYNTHESIS
TRANSFERASES
ULTRAVIOLET SPECTRA
UMP
UTP