UNIT 13.2 Chemoenzymatic Preparation of Nucleoside Triphosphates
Published Online: 1 MAY 2004
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Nucleic Acid Chemistry
How to Cite
Wu, W., Bergstrom, D. E. and Jo Davisson, V. 2004. Chemoenzymatic Preparation of Nucleoside Triphosphates. Current Protocols in Nucleic Acid Chemistry. 16:13.2:13.2.1–13.2.19.
- Published Online: 1 MAY 2004
- Published Print: MAR 2004
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.
- nucleoside diphosphate;
- nucleoside triphosphate;
- synthesis, kinase