New indazole and pyrazole nucleoside analogues were synthesized by enzymatic transglycosylation using E. coli purine nucleoside phosphorylase (PNP). Indazoles substituted at position 5 or 6 with bromine, pyrazole or pyrimidine fragments were used as heterocyclic bases. In the case of indazole-pyrazole hybrids, the selectivity of PNP in glycosylation of the pyrazole fragment, rather than the indazole one, was established. Molecular modeling methods allowed the identification of the preferred orientations of substrates in the active site of the enzyme, leading to the formation of N1- and N2-regioisomers of indazole. Unique substrate specificity ensured the possibility of glycosylation of the bases with conversion rates of 65–100% (according to HPLC analysis of the reaction mixtures) and isolated yields of 19–94%. It was shown that the synthesized nucleoside analogues are not inhibitors of E. coli adenosine deaminase (ADA). They do not exhibit cytotoxicity towards cancer cell lines (SH-SY5Y neuroblastoma cell lines, K-562 lymphoblastic cells, and HL-60 promyeloblastic cells). The activity of indazole derivatives against Mycobacterium tuberculosis (strain H37Rv) was determined by the microdilution method. 1-(β-D-2′deoxyribofuranosyl)-5-bromo-indazole and 5-(pyrimidin-5-yl)-1H-indazole at a concentration of 50 μg/mL were able to inhibit 50% and 90% of the culture growth, correspondingly.

In this study, the analgesic activity of the Cl-Ala-OH-AR compound was evaluated in various models of somatogenic somatic pain. The results showed that the Cl-Ala-OH-AR compound significantly increases the latency of the reaction in tests of the formation of thermal somatic pain, such as "Hot plate" and "Tail Twitching", however, its effectiveness was lower than that of tramadol and adenosine. In the algogen-induced somatic pain model evaluated in the formalin test, Cl-Ala-OH-AR demonstrated more pronounced analgesic properties, especially in the inflammatory phase, where the reduction in pain reactions was 3.4 times greater than the effect of adenosine. These results indicate a significant analgesic potential of Cl-Ala-OH-AR in the context of somatogenic somatic pain caused by algogens, which opens up prospects for further study.

A series of new 3′-deoxyribosides of substituted benzimidazoles was obtained by the chemo-enzymatic method using genetically engineered E. coli purine nucleoside phosphorylase (PNP). In the case of asymmetrically substituted benzimidazole derivatives, a mixture of N1- and N3-regioisomers was formed (confirmed by NMR). The antiviral activity of the obtained compounds against herpes simplex virus 1 of reference strain L2 and a strain deeply resistant to acyclovir in Vero E6 cell culture was studied. 4,6-Difluoro-1-(β-D-3′-deoxyribofuranosyl)benzimidazole (IC50 = 250.92 µM, SI = 12.00) and 4,5,6-trifluoro-1-(β-D-3′-deoxyribofuranosyl)benzimidazole (IC50 = 249.96 µM, SI = 16.00) showed significant selective activity against both viral models in comparison to ribavirin (IC50 = 511.88 µM, SI > 8.00).

The investigation of the structure–function relationship in hypoxanthine-guanine phosphoribosyltransferases (HGPRT) is a direction that is relevant for the development of drugs and approaches of enzymatic synthesis of modified nucleosides and nucleotides. This research paper is dedicated to the investigation of binding of sulphonate molecules, such as HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) in the active sites of HGPRT and similar proteins. We report the crystal structure of HGPRT from Escherichia coli (EcoHGPRT) in a complex with HEPES. In the obtained X-ray structure, a HEPES molecule binds to the active site in a position that mimics one of the HGPRT substrates, namely phosphoribosylpyrophosphate (PRPP). Enzymological study has shown that HEPES is an inhibitor of EcoHGPRT, along with two structurally similar molecules, namely MES and PIPES. Comparison of the observed EcoHGPRT/HEPES complex to other reported structures in the context of inhibition study results provides an opportunity to explore the variety of binding modes of HEPES and similar molecules and to discuss the structure–function relationship in this enzyme and similar proteins.

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.

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-(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.

A new approach to the synthesis of Anti-Reverse Cap Analog (ARCA) of the classical structure – dimethylated guanosine dinucleotide 2mGpppG is proposed. The classical approach to obtaining consists in condensation of activated 5′-diphosphate of 7,3′-O-dimethylguanosine with 5′-guanosine monophosphate. We suggest to use a commercially available 3′-O-methylguanosine for the synthesis of 5′-monophosphate of dimethylated guanosine only, and to use an activated 5′-guanosine diphosphate in the condensation. The conditions of 3′-O-methylguanosine phosphorylation were determined to avoid the formation of a side product 3′-O-methyl-5′-deoxy-5′-chloroguanosine-3′-phosphate. ARCA was synthesized with a high yield (89% at the condensation stage). Our approach to the synthesis is easily scaled and can be used for a preparative synthesis of ARCA (several grams).

Erratum

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.

The development of technologies for the efficient synthesis of innovative antiviral compounds remains an important challenge for modern biotechnology, especially in the context of the recent COVID-19 pandemic. One of the drugs that is currently in the research spotlight for potential anti-SARS-CoV-2 activity is the purine mimetic prodrug compound T-705, also known as favipiravir. Along with a similar compound, T-1105, the activation of T-705 is limited by the low rate of phosphoribosylation, mediated by an enzyme named hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Therefore, the synthesis of phosphoribosylated/ribosylated derivatives of these prodrugs is a viable direction for the discovery and development of antiviral pharmaceuticals. However, the chemical synthesis of such compounds is a complex and laborious process, whereas enzymatic cascades are not feasible because of the narrow HGPRT substrate specificity. Here, we report the successful rational design of an efficient biocatalyst for T-705/T-1105 phosphoribosylation. With two rounds of Thermus thermophilus HB27 HGPRT active site optimization, we have achieved a 325-fold increase in kcat toward the compound T-705 and a 125-fold increase toward T-1105 accompanied by a multifold decrease in KM. The practical applicability of the designed mutant was illustrated through the preparative synthesis of T-705/T-1105 nucleotide derivatives. Our engineered biocatalyst can become a basis for the technologies of enzymatic and chemoenzymatic synthesis of various T-705/T-1105 derivatives with proven antiviral activity. Moreover, our results provide insight into the molecular mechanism of T-705/T-1105 phosphoribosylation, including the experimental evidence explaining the reasons behind the low activity of HGPRT toward these compounds.

Objective: A new approach to the synthesis of the cap structure analogue with the correct orientation (Anti-Reverse Cap Analog, ARCA) having a classical structure (dimethylated guanosine dinucleotide 2mGpppG) is proposed. The traditional approach to its preparation is the condensation of activated 7,3′-O-dimethylguanosine-5′diphosphate with guanosine-5′-monophosphate. We suggest to use 3′-O-methylguanosine only for the synthesis of dimethylated guanosine-5′-monophosphate, and to introduce the activated guanosine-5′-diphosphate into the condensation. Methods: Conditions for phosphorylation of 3′-O-methylguanosine were determined to avoid the formation of by-product 3′-O-methyl-5′-deoxy-5′-chloroguanosine-2′-phosphate. Results and Discussion: At the last step of the synthesis, we used activated guanosine-5′-diphosphate (IV) and 7,3′-O-dimethylguanosine-5′monophosphate (VI). Compound (IV) was prepared by a three-step synthesis starting with guanosine. Compound (VI) was prepared by a two-step synthesis starting with 3′-O-methylguanosine. During phosphorylation of 3′-Omethylguanosine an unexpected compound (3′-O-methyl-5′-deoxy-5′-chloroguanosine-2′-phosphate) was formed. The structure of this compound was confirmed by NMR and mass-spectroscopy data. Conclusions: A new approach to the synthesis of ARCA of the guanosine type, methylated at the N7 and 3′ positions of guanosine was proposed. A scheme for the condensation of 7,3′-O-dimethylguanosine-5′-monophosphate and guanosine-5′-diphosphate was implemented. Guanosine 5′-diphosphate was used as activated component. Conditions for phosphorylation of 3′-Omethylguanosine were determined to avoid the formation of by-product 3′-O-methyl-5′-deoxy-5′-chloroguanosine2′-phosphate. ARCA was synthesized in high yield (89

A variety of ribo-, 2′-deoxyribo-, and 5′-norcarbocyclic derivatives of the 8-aza-7-deazahypoxanthine fleximer scaffolds were designed, synthesized, and screened for antibacterial activity. Both chemical and chemoenzymatic methods of synthesis for the 8-aza-7-deazainosine fleximers were compared. In the case of the 8-aza-7-deazahypoxanthine fleximer, the transglycosylation reaction proceeded with the formation of side products. In the case of the protected fleximer base, 1-(4-benzyloxypyrimidin-5-yl)pyrazole, the reaction proceeded selectively with formation of only one product. However, both synthetic routes to realize the fleximer ribonucleoside (3) worked with equal efficiency. The new compounds, as well as some 8-aza-7-deazapurine nucleosides synthesized previously, were studied against Gram-positive and Gram-negative bacteria and M. tuberculosis. It was shown that 1-(β-D-ribofuranosyl)-4-(2-aminopyridin-3-yl)pyrazole (19) and 1-(2′,3′,4′-trihydroxycyclopent-1′-yl)-4-(pyrimidin-4(3H)-on-5-yl)pyrazole (9) were able to inhibit the growth of M. smegmatis mc2 155 by 99% at concentrations (MIC99) of 50 and 13 µg/mL, respectively. Antimycobacterial activities were revealed for 4-(4-aminopyridin-3-yl)-1H-pyrazol (10) and 1-(4′-hydroxy-2′-cyclopenten-1′-yl)-4-(4-benzyloxypyrimidin-5-yl)pyrazole (6). At concentrations (MIC99) of 40 and 20 µg/mL, respectively, the compounds resulted in 99% inhibition of M. tuberculosis growth.

The great interest in studying the structure of human purine nucleoside phosphorylase (hPNP) and the continued search for effective inhibitors is due to the importance of the enzyme as a target in the therapy of T-cell proliferative diseases. In addition, hPNP inhibitors are used in organ transplant surgeries to provide immunodeficiency during and after the procedure. Previously, we showed that members of the well-known fleximer class of nucleosides are substrates of E. coli PNP. Fleximers have great promise as they have exhibited significant biological activity against a number of viruses of pandemic concern. Herein, we describe the synthesis and inhibition studies of a series of new fleximer compounds against hPNP and discuss their possible binding mode with the enzyme. At a concentration of 2 mM for the flex-7-deazapurines 1–4, a decrease in enzymatic activity by more than 50% was observed. 4-Amino-5-(1H-pyrrol-3-yl)pyridine 2 was the best inhibitor, with a Ki = 0.70 mM. Docking experiments have shown that ligand 2 is localized in the selected binding pocket Glu201, Asn243 and Phe200. The ability of the pyridine and pyrrole fragments to undergo rotation around the C–C bond allows for multiple binding modes in the active site of hPNP, which could provide several plausible bioactive conformations.

A highly effective producer strain Escherichia coli C3030/pET23d + - Ec HGPRT, allowing production of recombinant hypoxanthine‒guanine phosphoribosyltransferase from E. coli ( Ec HGPRT) in a soluble form, has been created. A method for isolating and purifying the recombinant protein has been developed. The specific activity against the natural substrate and pyrazine-2-carboxamide derivatives has been determined. Crystals of the Ec HGPRT complexes with 3-hydroxypyrazine-2-carboxamide (T-1105) and 6-fluoro-3-hydroxypyrazine-2-carboxamide (T-705), suitable for X-ray diffraction analysis, have been grown by capillary counter diffusion. X-ray diffraction sets with a resolution of up to 2.4 and 2.5 Å have been collected at the ESRF synchrotron (France, station ID23-1) at a temperature of 100 K. The crystals belong to the sp. gr. P 3(1)21; the independent part of the cell contains two enzyme molecules.

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-B m ) 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 (B m ) 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 o С, 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-B m were confirmed by NMR and mass-spectrometry.In the case of asymmetrically substituted benzimidazoles N1 and N3 regio-isomers were formed.

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][2][3].Recently, the role of nucleoside analogues of 1,2,4triazole as antiviral agents has increased.Synthesis ofsuch 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-1,2,4triazole 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.

Molecular dynamics simulations were performed for wild-type purine nucleoside phosphorylase in complexes with two substrates (adenosine and guanosine). The MD simulations were also performed for the mutant form of the enzyme with the same substrates. The free energy changes upon the formation of the complexes were evaluated from the molecular dynamics trajectories by the MM-GBSA method.

A number of purine arabinosides containing chiral amino acid amides at the C6 position of the purine were synthesized using a transglycosylation reaction with recombinant E. coli nucleoside phosphorylases. Arsenolysis of 2-chloropurine ribosides with chiral amino acid amides at C6 was used for the enzymatic synthesis, and the reaction equilibrium shifted towards the synthesis of arabinonucleosides. The synthesized nucleosides were shown to be resistant to the action of E. coli adenosine deaminase. The antiproliferative activity of the synthesized nucleosides was studied on human acute myeloid leukemia cell line U937. Among all the compounds, the serine derivative exhibited an activity level (IC50 = 16 μM) close to that of Nelarabine (IC50 = 3 μM) and was evaluated as active.

Ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamid, Virazole) is a modified nucleoside effective against a wide spectrum of RNA and DNA viruses.