Chemoenzymatic preparation of nucleoside triphosphates

Area of Science:

• Biochemistry
• Synthetic Chemistry
• Molecular Biology

Background:

• Nucleoside triphosphates are essential substrates for DNA and RNA polymerases.
• Developing modified nucleoside triphosphates is crucial for creating biochemical probes and nucleic acid mimics.
• Azole carboxamide nucleotides are of particular interest due to their conformational flexibility during DNA replication.

Purpose of the Study:

• To present an efficient chemoenzymatic preparation of azole carboxamide deoxyribo- and ribonucleoside triphosphates.
• To establish a reliable method for synthesizing these novel nucleotide analogs for further research.

Main Methods:

• Nucleoside diphosphates were synthesized from nucleoside 5'-O-tosylate via displacement with tris(tetra-n-butylammonium) pyrophosphate.
• Enzymatic phosphorylation of deoxyribonucleoside diphosphates utilized nucleoside diphosphate kinase with ATP as a phosphate donor, coupled with an ATP regeneration system (phospho(enol)pyruvate and pyruvate kinase).
• Ribonucleoside diphosphates were phosphorylated using phospho(enol)pyruvate and pyruvate kinase.

Main Results:

• An efficient chemoenzymatic route for preparing azole carboxamide deoxyribo- and ribonucleoside triphosphates was successfully established.
• Optimized purification using boronate affinity gel yielded highly purified nucleoside triphosphates.
• The synthesized compounds are suitable for use as biochemical probes and precursors for nucleic acid mimics.

Conclusions:

• The developed chemoenzymatic method provides a robust and efficient way to synthesize azole carboxamide nucleoside triphosphates.
• These novel nucleotide analogs are valuable tools for advancing research in DNA replication, biochemical probing, and the synthesis of nucleic acid mimics.
• The accessibility of these modified nucleotides will facilitate further investigations into their properties and applications.