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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. [ABSTRACT FROM AUTHOR]

The rapid increase in the antibiotic resistance of microorganisms, capable of causing diseases in humans as destroying cultural heritage sites, is a great challenge for modern science. In this regard, it is necessary to develop fundamentally novel and highly active compounds. In this study, a series of N4-alkylcytidines, including 5- and 6-methylcytidine derivatives, with extended alkyl substituents, were obtained in order to develop a new generation of antibacterial and antifungal biocides based on nucleoside derivatives. It has been shown that N4-alkyl 5- or 6-methylcytidines effectively inhibit the growth of molds, isolated from the paintings in the halls of the Ancient Russian Paintings of the State Tretyakov Gallery, Russia, Moscow. The novel compounds showed activity similar to antiseptics commonly used to protect works of art, such as benzalkonium chloride, to which a number of microorganisms have acquired resistance. It was also shown that the activity of N4-alkylcytidines is comparable to that of some antibiotics used in medicine to fight Gram-positive bacteria, including resistant strains of Staphylococcus aureus and Mycobacterium smegmatis. N4-dodecyl-5- and 6-methylcytidines turned out to be the best. This compound seems promising for expanding the palette of antiseptics used in painting, since quite often the destruction of painting materials is caused by joint fungi and bacteria infection. [ABSTRACT FROM AUTHOR]

Two forms were found in the NMR spectra of N6-substituted 2-chloroadenosines. The proportion of the mini-form was 11–32% of the main form. It was characterized by a separate set of signals in COSY, 15N-HMBC and other NMR spectra. We assumed that the mini-form arises due to the formation of an intramolecular hydrogen bond between the N7 atom of purine and the N6–CH proton of the substituent. The 1H,15N-HMBC spectrum confirmed the presence of a hydrogen bond in the mini-form of the nucleoside and its absence in the main form. Compounds incapable of forming such a hydrogen bond were synthesized. In these compounds, either the N7 atom of the purine or the N6–CH proton of the substituent was absent. The mini-form was not found in the NMR spectra of these nucleosides, confirming the importance of the intramolecular hydrogen bond in its formation. [ABSTRACT FROM AUTHOR]

Compounds containing benzimidazole fragment exhibit pronounced antiviral, antispasmodic, neuroleptic, and antihistamine activity. The biological activity profileof benzimidazole nucleosides can be changed by introducing a substituent into the benzimidazole ring and/or carbohydrate residue. A series of new modified substituted benzimidazole nucleosides (3'd-Rib-Bm) has been prepared by enzymatic method with yields from 8 to 72% and with purity morethan 95%. 3'-Deoxyinosine (3'd-Ino) and substituted benzimidazoles (Bm) have been used as substrates in the transglycosylation reaction in the presence of recombinant purine nucleoside phosphorylase (PNP). At the first step, each reaction was optimized for substrate ratio and amount of PNP. Conditions: 50 ℃, 2 mM potassium-phosphate buffer, pH 7.0. The next step was preparative synthesis followed by isolation of enzymatic reaction products. Thestructures of 3'd-Rib-Bm were confirmed by NMR and mass-spectrometry. In the case of asymmetrically substituted benzimidazoles N1 and N3 regio-isomers were formed. [ABSTRACT FROM AUTHOR]

Nucleoside analogues (or modified nucleosides) are heterocyclic nitrogenous bases of natural or synthetic origin, structurally similar to naturally occurring nucleosides, containing cyclic pentoses - ribose, deoxyribose and arabinose. Modified nucleosidesare used as antiviral, anticancer and antibacterial drugs [1-3]. Recently, the role of nucleoside analogues of 1,2,4-triazole as antiviral agents has increased. Synthesis of such compounds is possible either by methods of chemical synthesis or with biocatalysis. A long multi-stage chemical synthesis has a number of significant drawbacks. "One-pot" enzymatic reactions using recombinant nucleoside phosphorylases (NP) provide an alternative way of making some nucleoside analogues and has proved to be highly effective [4]. NP catalyzes the reversible phosphorolysis of ribo- or 2'-deoxyribonucleosides to form a free heterocyclic base and ribose or 2-deoxyribose-1-phosphate (transglycosylation reaction). In this work, we studied the substrate specificity of recombinant E. coli purine nucleoside phosphorylase (EcPNP) to 3,5-substituted-1,2,4-triazoles and evaluated the antiviral effect of the obtained nucleosides on two strains of Herpes simplex virus type-1 (HSV-1), including a strain resistant to the antiherpetic drug acyclovir. The main result of study was that for the first time the new 3,5-alkyl/aryl-substituted-1,2,4-triazole nucleosides have been synthesized using enzymatic transglycosylation. The surprising ability of EcPNP to synthesize ribo- and 2'-deoxyribonucleosides having structurally diverse hydrophobic substituents at the 3 and 5 position of 1,2,4-triazole has been discovered. 3,5-Alkylsubstituted-1,2,4-triazole nucleosides showed remarkable anti-herpes viral effect, which is expressed in the inhibition of the development of a virus-induced cytopathic effect in a culture of Vero E6 cells infected with various strains of HSV-1. [ABSTRACT FROM AUTHOR]

Two approaches to the chemoenzymatic synthesis of 2-fluorocordycepin and 2-chlorocordycepin were studied: (i) the use of 3'-deoxyadenosine (cordycepin) and 3'-deoxyinosine (3'dIno) as donors of 3-deoxy-D-ribofuranose in the transglycosylation of 2-fluoro-(2FAde) and 2-chloroadenine (2ClAde) catalyzed by the recombinant E. coli purine nucleoside phosphorylase (PNP), and (ii) the use of 2-fluoroadenosine and 3'-deoxyinosine as substrates of the cross-glycosylation and PNP as a biocatalyst. An efficient method for 3'-deoxyinosine synthesis starting from inosine was developed. However, the very poor solubility of 2ClAde and 2FAde is the limiting factor of the first approach. The second approach enables this problem to be overcome and it appears to be advantageous over the former approach from the viewpoint of practical synthesis of the title nucleosides. The 3-deoxy-a-D-ribofuranose-1-phosphate intermediary formed in the 3'dIno phosphorolysis by PNP was found to be the weak and marginal substrate of E. coli thymidine (TP) and uridine (UP) phosphorylases, respectively. Finally, one-pot cascade transformation of 3-deoxy-D-ribose in cordycepin in the presence of adenine and E. coli ribokinase, phosphopentomutase, and PNP was tested and cordycepin formation in ca. 3.4% yield was proved. [ABSTRACT FROM AUTHOR]

A series of 5,6-disubstituted benzimidazole nucleosides, obtained earlier, did not show any significant antiviral activity at relatively low cytotoxicity in vitro. In the course of our research we have succeeded in introducing an additional fluorine atom into the benzimidazole ring system. A new series of 4,6-difluorobenzimidazoles, bearing various groups (fluoro-, methoxy-, ethoxy-, morpholino-, and pyrrolidino-) in the 5-position of the benzene ring, have been synthesized. All these compounds proved to be substrates for recombinant E. coli purine nucleoside phosphorylase (PNP) in the transglycosylation reaction. Effective methods for the synthesis of ribo- and 2'-deoxyribonucleosides with high yields (60-90%) have been described, and the formation of regioisomeric N3-nucleosides of benzimidazoles have been detected. The biological activity of the nucleosides obtained against herpes simplex virus type 1 (HSV-1) has been elucidated. All compounds show a low cytotoxicity in the cell culture Vero E6. 4,5,6-Trifluoro-1-(β-D-ribofuranosyl) benzimidazole and 5-methoxy-4,6-difluoro-1-(β-D-2'-deoxyribofuranosyl) benzimidazole proved to inhibit completely the progression of the virus cytopathic effect (CPE) at a multiplicity of infection (MOI) of 0.01 PFU/cell. [ABSTRACT FROM AUTHOR]

A wide range of natural purine analogues was used as probe to assess the mechanism of recognition by the wild-type (WT) E. coli purine nucleoside phosphorylase (PNP) versus its Ser90Ala mutant. The results were analyzed from viewpoint of the role of the Ser90 residue and the structural features of the bases. It was found that the Ser90 residue of the PNP 1) plays an important role in the binding and activation of 8-aza-7-deazapurines in the synthesis of their nucleosides, 2) participates in the binding of α- D-pentofuranose-1-phosphates at the catalytic site of the PNP, and 3) catalyzes the dephosphorylation of intermediary formed 2-deoxy-α- D-ribofuranose-1-phosphate in the trans-2-deoxyribosylation reaction. 5-Aza-7-deazaguanine manifested excellent substrate activity for both enzymes, 8-amino-7-thiaguanine and 2-aminobenzothiazole showed no substrate activity for both enzymes. On the contrary, the 2-amino derivatives of benzimidazole and benzoxazole are substrates and are converted into the N1- and unusual N2-glycosides, respectively. 9-Deaza-5-iodoxanthine showed moderate inhibitory activity of the WT E. coli PNP, whereas 9-deazaxanthine and its 2′-deoxyriboside are weak inhibitors. [ABSTRACT FROM AUTHOR]

A number of new 5,6-disubstituted benzimidazoles have been prepared and their substrate properties for recombinant E. coli purine nucleoside phosphorylase (PNP; the product of the deoD gene) in the transglycosylation reaction were investigated. The heterocyclic bases showed good substrate activity for PNP and the ribo- and 2'-deoxyribonucleosides were synthesized. The predominant (OMe and OEt) or exclusive (Oi-Pr, morpholino, and N-methylpiperazino) formation of the 5-substituted 6-fluoro-1-(ß-Dribofuranosyl) benzimidazoles was observed. The formation of the regioisomeric 6- methoxy-, 6-ethoxy-, or 6-isopropoxy-substituted 1-(2-deoxy-ß-D-ribofuranosyl)-5-fluorobenzimidazoles was observed in the trans-2-deoxyribosylation reaction of the corresponding bases. The predominant or exclusive formation of the regioisomeric N1-nucleosides with bulky 5-substituents of 6-fluorobenzimidazole points to a large hydrophobic pocket in the E. coli PNP active site that can accommodate these groups. The biological activity of the synthesized nucleosides was studied and revealed no inhibitory activity against a broad variety of DNA and RNA viruses. The compounds also lacked significant cytotoxicity. [ABSTRACT FROM AUTHOR]

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/R ACV ) 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-(β- D -ribofuranosyl)-1,2,4-triazole and 5-butylthio-1-(2-deoxy-β- 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.