Synthesis of Substituted 1,2,4-Triazole-3-Thione Nucleosides Using E. coli Purine Nucleoside Phosphorylase.

1,2,4-Triazole derivatives have a wide range of biological activities. The most well-known drug that contains 1,2,4-triazole as part of its structure is the nucleoside analogue ribavirin, an antiviral drug. Finding new nucleosides based on 1,2,4-triazole is a topical task. The aim of this study was to synthesize ribosides and deoxyribosides of 1,2,4-triazole-3-thione derivatives and test their antiviral activity against herpes simplex viruses. Three compounds from a series of synthesized mono- and disubstituted 1,2,4-triazole-3-thione derivatives were found to be substrates for E. coli purine nucleoside phosphorylase. Of six prepared nucleosides, the riboside and deoxyriboside of 3-phenacylthio-1,2,4-triazole were obtained at good yields. The yields of the disubstituted 1,2,4-triazol-3-thiones were low due to the effect of bulky substituents at the C3 and C5 positions on the selectivity of enzymatic glycosylation for one particular nitrogen atom in the triazole ring. The results of cytotoxic and antiviral studies on acyclovir-sensitive wild-type strain HSV-1/L2(TK+) and acyclovir-resistant strain (HSV-1/L2/RACV) in Vero E6 cell culture showed that the incorporation of a thiobutyl substituent into the C5 position of 3-phenyl-1,2,4-triazole results in a significant increase in the cytotoxicity of the base and antiviral activity. The highest antiviral activity was observed in the 3-phenacylthio-1-(ß-D-ribofuranosyl)-1,2,4-triazole and 5-butylthio-1-(2-deoxy-ß-D-ribofuranosyl)-3-phenyl-1,2,4-triazole nucleosides, with their selectivity indexes being significantly higher than that of ribavirin. It was also found that with the increasing lipophilicity of the nucleosides, the activity and toxicity of the tested compounds increased.

Enzymatic Transglycosylation Features in Synthesis of 8-Aza-7-Deazapurine Fleximer Nucleosides by Recombinant E. coli PNP: Synthesis and Structure Determination of Minor Products.

Enzymatic transglycosylation of the fleximer base 4-(4-aminopyridine-3-yl)-1H-pyrazole using recombinant E. coli purine nucleoside phosphorylase (PNP) resulted in the formation of "non-typical" minor products of the reaction. In addition to "typical" N1-pyrazole nucleosides, a 4-imino-pyridinium riboside and a N1-pyridinium-N1-pyrazole bis-ribose derivative were formed. N1-Pyrazole 2'-deoxyribonucleosides and a N1-pyridinium-N1-pyrazole bis-2'-deoxyriboside were formed. But 4-imino-pyridinium deoxyriboside was not formed in the reaction mixture. The role of thermodynamic parameters of key intermediates in the formation of reaction products was elucidated. To determine the mechanism of binding and activation of heterocyclic substrates in the E. coli PNP active site, molecular modeling of the fleximer base and reaction products in the enzyme active site was carried out. As for N1-pyridinium riboside, there are two possible locations for it in the PNP active site. The presence of a relatively large space in the area of amino acid residues Phe159, Val178, and Asp204 allows the ribose residue to fit into that space, and the heterocyclic base can occupy a position that is suitable for subsequent glycosylation. Perhaps it is this "upside down" arrangement that promotes secondary glycosylation and the formation of minor bis-riboside products.