| Abstract [eng] |
Nucleoside and nucleotide analogues are extensively used in biochemistry, molecular biology, and pharmacology. These compounds function as substrates or inhibitors in enzymatic reactions and have significant applications in nucleic acid research, anticancer and antiviral treatments, and RNA vaccines. In this study, a series of novel N4-amino acid-modified deoxycytidine nucleosides were synthesized, enriching the library of nucleoside analogues. The newly synthesized compounds, along with additional pyrimidine nucleoside analogues, were tested as substrates for Drosophila melanogaster deoxynucleoside kinase (DmdNK) and Bacillus subtilis deoxycytidine kinase (BsdCK). The findings revealed that wild-type DmdNK and BsdCK exhibited activity toward pyrimidine nucleosides with minor modifications at the N4/O4 and C5 positions. Nucleoside kinases, coupled with a phosphate donor regeneration system, were applied for a milligram-scale synthesis of nucleoside monophosphates, showcasing potential for scalable and eco-friendly nucleotide production. Additionally, successful application of site-directed mutagenesis demonstrated that specific mutations in the active sites of DmdNK and BsdCK enhance enzymatic activity towards N4-modified nucleosides, enabling the customization of these enzymes for tailored applications. |
