Modified nucleosides constitute a large group of synthetic drugs. Cladribine, nelarabine, fludarabine, cytarabine are widely used for the treatment of oncohematological diseases; for viral infections - azidothymidine, maribavir, ribavirin, vidarabine, sofosbuvir. Their production is facilitated by the development of an integrated chemico-enzymatic synthesis approach using recombinant nucleic acid exchange enzymes. Within the framework of this project, it is planned to synthesize new potential anticancer drugs: N6-purine-substituted analogues of Clofarabine, an inhibitor of ribonucleotide reductase and DNA polymerase, which is used in the treatment of acute lymphoblastic leukemia, as well as to carry out glycosylation of a purine conjugate with benzoxazine, which has a high cytotoxic activity, where as a linker the 11-aminoundecanoic acid residue is used. It is planned to expand the series of 3-substituted analogues of ribavirin, a synthetic nucleoside used in the treatment of hepatitis C. Using biosynthesis, a series of new ribo- and 2'-deoxyribosides of 1,2,4-triazole with hydrophobic substituents in the C3 position will be obtained. We have shown that 2-fluorocordycepine has antiproliferative activity, but the biosynthesis of this drug using recombinant E. coli purine nucleoside phosphorylase (PNP) occurs with low yield. This project proposes to consider two potential solutions: a) studying the possibility of using 3'-deoxyuridine in biosynthesis; b) rational design of the active site of nucleoside phosphorylases (NPs), aimed at increasing the affinity for the enzyme. The substrate specificity of PNP does not allow the production of antiviral (phospho)ribosylated derivatives of pyrazine-2-carboxamide, in particular, the substance T-705, an antiviral purine mimetic prodrug known as “favipiravir” (“avifavir”), which is used in the treatment of various viral infections, including COVID-19. The active substance is T-705-riboside-5'-triphosphate, which is formed in vivo, but the low efficiency of drug activation by phosphoribosylation requires a very high dose load. The experience of our research (including those supported by the Russian Science Foundation) has shown that the problem of phosphoribosylated derivatives of the prodrug T-705 synthesis can be solved using rational enzyme design. We plan to utilize and expand this approach to design mutant forms of recombinant nucleoside phosphorylases. Using the engineered mutants, we plan to carry out the biosynthesis of antiviral favipiravir derivatives. Previously, our group has developed mono- and polyenzymatic cascades intended for the production of antiviral and antitumor drugs. Our planned studies are aimed at optimizing these systems through the rational design of enzyme active sites. The results of such studies will make it possible to overcome the limitations of the natural substrate specificity of enzymes and simplify technologies for the production of pharmaceutically significant compounds. The goal of this project is to develop at a new level a chemical-enzymatic approach to the synthesis of new modified nucleosides and nucleotides to obtain a series of compounds with unnatural heterocyclic bases and carbohydrate fragments and to study their antitumor and antiviral activity in in vitro experiments. Due to the wide variety of targets that the new compound can act on, this project involves screening for antiviral and antitumor activity of all obtained substances. The results obtained within the framework of the project will include not only new active modified nucleosides and nucleotides, but also preparations of recombinant enzymes with modified substrate characteristics, which contributes to the fulfillment of the global goal of the project - the targeted creation of new drugs for the treatment of socially significant human diseases in accordance with the approaches “green chemistry”.