Chemoenzymatic preparation of nucleoside triphosphates

Current Protocols in Nucleic Acid Chemistry
Weidong WuV Jo Davisson

Abstract

The design and synthesis of alternative nucleoside triphosphate substrates for DNA and RNA polymerases holds continued promise to create biochemical probes and precursors for synthesis of nucleic acid mimics. The azole carboxamide nucleotides are of particular interest, as they display multiple conformations in the context of DNA replication. An efficient chemoenzymatic preparation of azole carboxamide deoxyribo- and ribonucleoside triphosphates is presented. Nucleoside diphosphate is prepared from nucleoside 5'-O-tosylate by displacement with tris(tetra-n-butylammonium) pyrophosphate. Enzymatic phosphorylation of the azole carboxamide deoxyribonucleoside diphosphate to its triphosphate is based on ATP as the phosphate donor and nucleoside diphosphate kinase as the catalyst, coupled with phospho(enol)pyruvate (PEP) and pyruvate kinase as an ATP regeneration system. Enzymatic phosphorylation of the azole carboxamide ribonucleoside diphosphate requires PEP as the phosphate donor and pyruvate kinase as the catalyst. The optimized purification uses boronate affinity gel to yield highly purified nucleoside triphosphate.

References

Jul 1, 1975·Nucleic Acids Research·A Simoncsits, J Tomasz
Dec 1, 1967·Tetrahedron Letters·M YoshikawaT Takenishi
Feb 28, 1998·Nucleic Acids Research·G C HoopsV J Davisson
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Mar 27, 2003·Pediatric Dermatology·Khalid Al HawsawiAhmad Al Githami
Apr 20, 1965·Journal of the American Chemical Society·D E HOARD, D G OTT

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Citations

Apr 14, 2012·Nucleosides, Nucleotides & Nucleic Acids·Anilkumar R KoreBalasubramanian Srinivasan
Dec 3, 2016·Current Protocols in Nucleic Acid Chemistry·Muthian ShanmugasundaramAnilkumar R Kore
Jan 1, 2010·Current Protocols in Chemical Biology·Robert D Kuchta

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