Synthesis and polymerase incorporation of 5′‐amino‐2′,5′‐dideoxy‐5′‐N‐triphosphate nucleotides

Owing to the markedly increased reactivity of amino functional groups versus hydroxyls, the 5′‐amino‐5′‐deoxy nucleoside and nucleotide analogs have proven widely useful in biological, pharmaceutical and genomic applications. However, synthetic procedures leading to these analogs have not been fully...

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Bibliographic Details
Published in:Nucleic acids research 2002-09, Vol.30 (17), p.3739-3747
Main Authors: Wolfe, Jia Liu, Kawate, Tomohiko, Belenky, Alexei, Stanton Jr, Vincent
Format: Article
Language:English
Subjects:
Adenosine - analogs & derivatives
Adenosine - metabolism
Base Sequence
Chromatography, High Pressure Liquid
Cytidine - analogs & derivatives
Cytidine - metabolism
Dideoxynucleosides - chemical synthesis
Dideoxynucleosides - metabolism
DNA - chemistry
DNA - genetics
DNA - metabolism
DNA Polymerase I - metabolism
Guanosine - analogs & derivatives
Guanosine - metabolism
Inosine - analogs & derivatives
Inosine - metabolism
Magnetic Resonance Spectroscopy
Molecular Sequence Data
Sequence Analysis, DNA
Thymidine - analogs & derivatives
Thymidine - metabolism
Uridine - analogs & derivatives
Uridine - metabolism
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Summary:Owing to the markedly increased reactivity of amino functional groups versus hydroxyls, the 5′‐amino‐5′‐deoxy nucleoside and nucleotide analogs have proven widely useful in biological, pharmaceutical and genomic applications. However, synthetic procedures leading to these analogs have not been fully explored, which may possibly have limited the scope of their utility. Here we describe the synthesis of the 5′‐amino‐2′,5′‐dideoxy analogs of adenosine, cytidine, guanosine, inosine and uridine from their respective naturally occurring nucleosides via the reduction of 5′‐azido‐2′,5′‐dideoxy intermediates using the Staudinger reaction, and the high yield conversion of these modified nucleosides and 5′‐amino‐5′‐deoxythymidine to the corresponding 5′‐N‐triphosphates through reaction with trisodium trimetaphosphate in the presence of tris(hydroxymethyl)aminomethane (Tris). We also show that each of these nucleotide analogs can be efficiently incorporated into DNA by the Klenow fragment of Escherichia coli DNA polymerase I when individually substituted for its naturally occurring counterpart. Mild acid treatment of the resulting DNA generates polynucleotide fragments that arise from specific cleavage at each modified nucleotide, providing a sequence ladder for each base. Because the ladders are generated after the extension, the corresponding products may be manipulated by enzymatic and/or purification processes. The potential utility of this extension–cleavage procedure in genomic sequence analysis is discussed.
ISSN:0305-1048
1362-4962
1362-4962
DOI:10.1093/nar/gkf502