A series of 5-alkyl/aryl-oxymethyl-ribavirin analogs was synthesized. Surprisingly, ability of E. coli purine nucleoside phosphorylase (PNP) to synthesize ribonucleosides and 2′-deoxyribonucleosides having structurally diverse alkyl(aryl)oxymethyl substituents at the 5 position of 1,2,4-triazol-3-carboxamide has been discovered. It has been shown that the enzymatic synthesis of 2′-deoxyribonucleosides proceeds with better conversion and is faster (up to 50 h) than the synthesis of ribonucleosides (more than 300 h). Therefore, ribonucleosides were synthesized by chemical glycosylation of base triazoles by 2,3,4,5-tetra-O-acetyl-D-ribose in the presence of bis(4-nitrophenyl) phosphate. 2′-Deoxyribonucleosides were synthesized by the transglycosylation reaction with E. coli PNP. For the first time, based on the kinetic parameters of the 2′-deoxyribonucleosides synthesis process, the positive cooperativity effect of wild type PNP active sites was discovered. The antiviral activity of nucleosides has been tested against herpes simplex virus type 1 (HSV-1) and influenza virus H5N1. The cytotoxicity and antiviral activity of the synthesized nucleosides differ, and it is impossible to draw an unambiguous conclusion about the structure-activity correlation.