Chemical Glycosylation Research Articles

Synthesis and immunological study of tetrasaccharide repeating units of capsular polysaccharide of Group B Streptococcus serotype VIII and their protein conjugates

This study reports the efficient synthesis of three tetrasaccharide derivatives of Group B Streptococcus (GBS) serotype VIII capsular polysaccharide with alternative glycan sequences and spacer chains through a convergent chemical glycosylation manner or a one-pot three-enzyme assembly protocol. Using bifunctional glutarate ester as the linker, these synthesized oligosaccharides were efficiently conjugated with recombinant ScpA193 protein, generating the resultant oligosaccharide-ScpA193 conjugates with desirable carbohydrate loading. ELISA analysis of the antisera obtained from mice inoculated with the synthetic glycoconjugates showed the elicitation of strong oligosaccharide-specific immunoglobulin G (IgG) antibodies, suggesting the potential of the synthesized oligosaccharides for use in the development of GBS vaccines.

One-pot synthesis of stable vitamin C analogue using trans-glycosylating enzyme from Aspergillus carbonarius BTCF 5

Biocatalysis using transglycosidases for one-pot glycosylation of small molecules is a greener alternative to chemical glycosylation. The transglycosylation potential of the crude culture filtrate of Aspergillus carbonarius BTCF 5 was evaluated based on the biotransformation of the target molecule, ascorbic acid (Vitamin-C), to its more stable glycosylated analogue -2-O-α-D-glucopyranosyl ascorbic acid (AA2G), using maltose as the glycosyl donor. Media engineering increased the transglycosylation yield by 64%, whereas the use of concentrated culture filtrate increased the transglycosylation yield by 70%. Protein purification studies identified the candidate transglycosylating enzyme to be a GH 31 family CAZyme (α-transglucosidase). A minimally purified enzyme could enhance the transglycosylation yield by 77% with a total AA2G yield of 91.5 mM (10.32 g/L) and improved the overall conversion rate of ascorbic acid and maltose in the reaction by 16% and 6.3% respectively from the base level. A two-step column chromatography helped in product purification with 46% recovery. The structural characterization of purified AA2G provided a detailed insight into the regioselectivity of the enzyme to glycosylate C2 of ascorbic acid and its ability to form AA2G as the predominant isomer, which is also the most preferred one for end applications.

Direct radical functionalization of native sugars

Naturally occurring (native) sugars and carbohydrates contain numerous hydroxyl groups of similar reactivity1,2. Chemists, therefore, rely typically on laborious, multi-step protecting-group strategies3 to convert these renewable feedstocks into reagents (glycosyl donors) to make glycans. The direct transformation of native sugars to complex saccharides remains a notable challenge. Here we describe a photoinduced approach to achieve site- and stereoselective chemical glycosylation from widely available native sugar building blocks, which through homolytic (one-electron) chemistry bypasses unnecessary hydroxyl group masking and manipulation. This process is reminiscent of nature in its regiocontrolled generation of a transient glycosyl donor, followed by radical-based cross-coupling with electrophiles on activation with light. Through selective anomeric functionalization of mono- and oligosaccharides, this protecting-group-free ‘cap and glycosylate’ approach offers straightforward access to a wide array of metabolically robust glycosyl compounds. Owing to its biocompatibility, the method was extended to the direct post-translational glycosylation of proteins.

Chemical and Chemoenzymatic Synthesis of Peptide and Protein Therapeutics Conjugated with Human N-Glycans.

Glycosylation is one of the most ubiquitous post-translational modifications. It affects the structure and function of peptides/proteins and consequently has a significant impact on various biological events. However, the structural complexity and heterogeneity of glycopeptides/proteins caused by the diversity of glycan structures and glycosylation sites complicates the detailed elucidation of glycan function and hampers their clinical applications. To address these challenges, chemical and/or enzyme-assisted synthesis methods have been developed to realize glycopeptides/proteins with well-defined glycan morphologies. In particular, N-glycans are expected to be useful for improving the solubility, in vivo half-life and aggregation of bioactive peptides/proteins that have had limited clinical applications so far due to their short duration of action in the blood and unsuitable physicochemical properties. Chemical glycosylation performed in a post-synthetic procedure can be used to facilitate the development of glycopeptide/protein analogues or mimetics that are superior to the original molecules in terms of physicochemical and pharmacokinetic properties. N-glycans are used to modify targets because they are highly biodegradable and biocompatible and have structures that already exist in the human body. On the practical side, from a quality control perspective, close attention should be paid to their structural homogeneity when they are to be applied to pharmaceuticals.

Synthesis and unexpected binding of monofluorinated N,Nʹ-diacetylchitobiose and LacdiNAc to wheat germ agglutinin

Fluorination of carbohydrate ligands of lectins is a useful approach to examine their binding profile, improve their metabolic stability and lipophilicity, and convert them into 19F NMR-active probes. However, monofluorination of monovalent carbohydrate ligands often leads to a decreased or completely lost affinity. By chemical glycosylation, we synthesized the full series of methyl β-glycosides of N,N′-diacetylchitobiose (GlcNAcβ(1–4)GlcNAcβ1-OMe) and LacdiNAc (GalNAcβ(1–4)GlcNAcβ1-OMe) systematically monofluorinated at all hydroxyl positions. A competitive enzyme-linked lectin assay revealed that the fluorination at the 6′-position of chitobioside resulted in an unprecedented increase in affinity to wheat germ agglutinin (WGA) by one order of magnitude. For the first time, we have characterized the binding profile of a previously underexplored WGA ligand LacdiNAc. Surprisingly, 4′-fluoro-LacdiNAc bound WGA even stronger than unmodified LacdiNAc. These observations were interpreted using molecular dynamic calculations along with STD and transferred NOESY NMR techniques, which gave evidence for the strengthening of CH/π interactions after deoxyfluorination of the side chain of the non-reducing GlcNAc. These results highlight the potential of fluorinated glycomimetics as high-affinity ligands of lectins and 19F NMR-active probes.

Unraveling chemical glycosylation: DFT insights into factors imparting stereoselectivity

Stereoselective chemical glycosylation reactions are pivotal for preparing manifold biologically and medically important compounds, while mechanisms of chemical glycosylation reactions remain obscure and largely speculative. Herein, we performed DFT calculations to delve into the multifaceted mechanistic details of glycosylation reactions, including the equilibria among reactive glycosyl triflate intermediates in solution, the stereoselectivity imparting protecting groups, solvent effects, base, and the anomeric effect. Our results provided theoretical corroboration to 2-OAc neighbouring group participation (NGP), the arming/disarming effect, the coordination theory of solvent effect on glycosylation stereochemistry, and the influence of solvent polarity on the reaction kinetics spanning the SN1-SN2 continuum. For the first time, the existence of putative contact-ion-pairs (CIP) of glycosyl oxocarbenium and triflate anion in organic solutions was theoretically confirmed with the identification of multiple ground state structures employing an implicit Solvation Model based on Density (SMD). Kinetics of nucleophilic attack of model glucosyl triflates by simple alcohol acceptors ethanol (EtOH) and trifluoroethanol (TFE), complexed with 2,4,6-tri-tert-butylpyrimidine (TTBP) were explored, revealing the essential role of the close accompanying base for rendering glycosidic bond formation thermodynamically favourable. Our work deepens the comprehension of the glycosylation mechanism, paving the way for the rational design and future advancement of efficient and environmentally friendly stereoselective glycosylation reactions.

VaporLIFT: On‐Chip Chemical Synthesis of Glycan Microarrays

AbstractLaser‐induced forward transfer (LIFT) of polymers is a versatile printing method for parallel in situ synthesis of peptides on microarrays. Chemical building blocks embedded in a polymer matrix are transferred and coupled in a desired pattern to a surface, generating peptides on microarrays by repetitive in situ solid‐phase synthesis steps. To date, the approach is limited to simple, heat induced chemical reactions. The VaporLIFT method, disclosed here, combines LIFT with chemical vapor glycosylation to rapidly generate glycans on microarray surfaces while maintaining inert, low temperature conditions required for glycosylations. Process design and parameter optimization enables the synthesis of a collection of glycans at defined positions on a glass surface. The synthetic structures are detected by mass spectrometry, fluorescently labeled glycan‐binding proteins, and covalent staining with fluorescent dyes. VaporLIFT is ideal for parallel screening of other chemical reactions, that require inert and well‐defined reaction conditions.

A critical review on the stability of natural food pigments and stabilization techniques

This comprehensive review article delves into the complex world of natural edible pigments, with a primary focus on their stability and the factors that influence them. The study primarily explores four classes of pigments: anthocyanins, betalains, chlorophylls and carotenoids by investigating both their intrinsic and extrinsic stability factors. The review examines factors affecting the stability of anthocyanins which act as intrinsic factors like their structure, intermolecular and intramolecular interactions, copigmentation, and self-association as well as extrinsic factors such as temperature, light exposure, metal ions, and enzymatic activities. The scrutiny extends to betalains which are nitrogen-based pigments, and delves into intrinsic factors like chemical composition and glycosylation, as well as extrinsic factors like temperature, light exposure, and oxygen levels affecting for their stability. Carotenoids are analyzed concerning their intrinsic and extrinsic stability factors. The article emphasizes the role of chemical structure, isomerization, and copigmentation as intrinsic factors and discusses how light, temperature, oxygen, and moisture levels influence carotenoid stability. The impacts of food processing methods on carotenoid preservation are explored by offering guidance on maximizing retention and nutritional value. Chlorophyll is examined for its sensitivity to external factors like light, temperature, oxygen exposure, pH, metal ions, enzymatic actions, and the food matrix composition. In conclusion, this review article provides a comprehensive exploration of the stability of natural edible pigments, highlighting the intricate interplay of intrinsic and extrinsic factors. In addition, it is important to note that all the references cited in this review article are within the past five years, ensuring the most up-to-date and relevant sources have been considered in the analysis.

SFox imidates as versatile glycosyl donors for chemical glycosylation.

Described herein is a continuation of our studies dedicated to the development of novel classes of leaving groups based on O- and S-imidates. The main focus of the study presented herein is the synthesis of novel 3,3-difluoro-3H-indol-2-ylthio (SFox) imidates and their application as glycosyl donors in chemical glycosylation. Being thioimidates, these compounds are more stable than O-imidates albeit much more reactive than conventional alkyl/arylthio glycosides. This study demonstrates that SFox imidates can be activated either with soft thiophilic reagents (N-iodosuccinimide or transition metal salts), typical for the activation of thioglycosides or thioimidates, or hard electrophilic reagents (protic or Lewis acids) common for the activation of O-imidates. Expectedly, complete β-selectivity was obtained from SFox donors equipped with 2-O-benzoyl group. Surprisingly, complete α-selectivity was obtained from 2-O-benzylated SFox imidates in all investigated cases.

Zinc(ii)-mediated stereoselective construction of 1,2-cis 2-azido-2-deoxy glycosidic linkage: assembly of Acinetobacter baumannii K48 capsular pentasaccharide derivative.

The capsular polysaccharide (CPS) is a major virulence factor of the pathogenic Acinetobacter baumannii and a promising target for vaccine development. However, the synthesis of the 1,2-cis-2-amino-2-deoxyglycoside core of CPS remains challenging to date. Here we develop a highly α-selective ZnI2-mediated 1,2-cis 2-azido-2-deoxy chemical glycosylation strategy using 2-azido-2-deoxy glucosyl donors equipped with various 4,6-O-tethered groups. Among them the tetraisopropyldisiloxane (TIPDS)-protected 2-azido-2-deoxy-d-glucosyl donor afforded predominantly α-glycoside (α : β = >20 : 1) in maximum yield. This novel approach applies to a wide acceptor substrate scope, including various aliphatic alcohols, sugar alcohols, and natural products. We demonstrated the versatility and effectiveness of this strategy by the synthesis of A. baumannii K48 capsular pentasaccharide repeating fragments, employing the developed reaction as the key step for constructing the 1,2-cis 2-azido-2-deoxy glycosidic linkage. The reaction mechanism was explored with combined experimental variable-temperature NMR (VT-NMR) studies and mass spectroscopy (MS) analysis, and theoretical density functional theory calculations, which suggested the formation of covalent α-C1GlcN-iodide intermediate in equilibrium with separated oxocarbenium-counter ion pair, followed by an SN1-like α-nucleophilic attack most likely from separated ion pairs by the ZnI2-activated acceptor complex under the influence of the 2-azido gauche effect.

Overcoming Challenges in Chemical Glycosylation to Achieve Innovative Vaccine Adjuvants Possessing Enhanced TLR4 Activity.

Lipopolysaccharide (LPS) mimicry leading to toll-like receptor 4 (TLR4) active compounds has been so far based mainly on reproducing the lipid A portion of LPS. Our work led to a series of structurally simplified synthetic TLR4 agonists in preclinical development as vaccine adjuvants called FPs. FPs bind MD2/TLR4 similarly to lipid A, inserting the lipid chains in the MD2 lipophilic cavity. A strategy to improve FPs' target affinity is introducing a monosaccharide unit in C6, mimicking the first sugar of the LPS core. We therefore designed a panel of FP derivatives bearing different monosaccharides in C6. We report here the synthesis and optimization of FPs' C6 glycosylation, which presented unique challenges and limitations. The biological activity of glycosylated FP compounds was preliminarily assessed in vitro in HEK-Blue cells. The new molecules showed a higher potency in stimulating TLR4 activation when compared to the parent molecule while maintaining TLR4 selectivity.

Iodine-triphenylphosphine triggers an easy one-pot alpha stereoselective dehydrative glycosylation on hemiacetalic benzylated glycosyl donors

The discovery of new glycosylation reactions is still a major challenge in carbohydrate chemistry. Traditional glycosylation reactions require the preparation of sugar donors with anomeric active or latent leaving groups. Dehydrative glycosylation is a fascinating alternative that enables the direct formation of the glycosidic bond from the hemiacetal, eliminating the need for (sometimes unstable) leaving groups, and allowing to reduce reaction, work-up, and purification times. Although some interesting methods of dehydrative glycosylation have been reported, in order to compete with conventional chemical glycosylation, a greater number of efficient and stereoselective methods need to be developed. Herein, a dehydrative procedure that uses a combination of iodine, triphenylphosphine, and a base (DMAP or imidazole) is described. This methodology allows for the preparation of sugar derivatives from commercially available 1-hydroxy glycosyl donors. The reaction takes place under mild conditions through the in situ-formation of an anomeric iodide intermediate, which, upon reaction with an alcohol, gives the corresponding glycosides up to quantitative yields and with high α-stereoselectivity.

Radical Pathway Glycosylation Empowered by Bench-Stable Glycosyl Donors.

ConspectusThe study of carbohydrates has emerged as a crucial research area in various disciplines due to their pivotal roles in cellular processes. To facilitate in-depth exploration of their biological functions, chemical glycosylation reactions that allow facile access of glycoconjugates to a broad research community are highly needed. In classical glycosylation reactions, a glycosyl donor is activated by an acid to generate a reactive electrophilic intermediate, which subsequently forms a glycosidic bond upon reaction with a nucleophilic acceptor. Such an ionic pathway glycosylation has been the mainstay technique for glycoconjugate synthesis and allowed the synthesis of numerous intricate structures. Nevertheless, limitations still exist. For instance, when labile glycosyl donors or harsh activating conditions are required, these methods show limited tolerance to hydroxyl groups that are abundant on sugar rings. In addition, achieving good stereocontrol represents another longstanding obstacle. In recent years, new modes of donor activation have been sought to tackle the above challenges.We noted that glycosylation methods passing through the intermediacy of glycosyl radicals via a cascade of single-electron transfer steps possess significant but underexplored potential. Progress in this area has been slow due in large part to a dearth of handy methods to generate and maneuver glycosyl radicals. Most existing methods call for either forcing conditions or unstable/inconvenient starting materials. In order to better exploit the power of the radical pathway glycosylation, we have developed a range of glycosyl donors─namely, glycosyl sulfoxides, glycosyl sulfones, and glycosyl sulfinates─that are bench stable and can be readily prepared from simple starting materials. These donors can be activated to form glycosyl radicals under mild conditions. Enabled by the use of these donors, we have developed a series of glycosylation methods that could be used for making O-, S-, or C-glycosides, some of which were previously difficult to access. In many cases, no protecting group on glycosyl donors is required. As an illustration of their potential utility, our methods have been adopted in the preparation of sugar-drug conjugates, sugar-DNA conjugates, glycopeptides, and even glycoproteins. While in most cases the intrinsic reactivity of glycosyl radical intermediates can be explored to access axially configured products, some of the methods also allow the utilization of external, delicate reagents, or catalysts to override such innate preference and achieve catalyst-controlled stereoselectivity.We believe that radical pathway glycosylation has enormous potential and can inspire the development of novel methods for glycoside synthesis. In this Account, we highlight the design principles for the development of our glycosyl donors, summarize our recent advancements in radical pathway glycosylation enabled by their use, and provide an outlook on the future directions of this field.

Design of Glycoengineered IL-4 Antagonists Employing Chemical and Biosynthetic Glycosylation.

Interleukin-4 (IL-4) plays a key role in atopic diseases. It coordinates T-helper cell differentiation to subtype 2, thereby directing defense toward humoral immunity. Together with Interleukin-13, IL-4 further induces immunoglobulin class switch to IgE. Antibodies of this type activate mast cells and basophilic and eosinophilic granulocytes, which release pro-inflammatory mediators accounting for the typical symptoms of atopic diseases. IL-4 and IL-13 are thus major targets for pharmaceutical intervention strategies to treat atopic diseases. Besides neutralizing antibodies against IL-4, IL-13, or its receptors, IL-4 antagonists can present valuable alternatives. Pitrakinra, an Escherichia coli-derived IL-4 antagonist, has been evaluated in clinical trials for asthma treatment in the past; however, deficits such as short serum lifetime and potential immunogenicity among others stopped further development. To overcome such deficits, PEGylation of therapeutically important proteins has been used to increase the lifetime and proteolytic stability. As an alternative, glycoengineering is an emerging strategy used to improve pharmacokinetics of protein therapeutics. In this study, we have established different strategies to attach glycan moieties to defined positions in IL-4. Different chemical attachment strategies employing thiol chemistry were used to attach a glucose molecule at amino acid position 121, thereby converting IL-4 into a highly effective antagonist. To enhance the proteolytic stability of this IL-4 antagonist, additional glycan structures were introduced by glycoengineering utilizing eucaryotic expression. IL-4 antagonists with a combination of chemical and biosynthetic glycoengineering could be useful as therapeutic alternatives to IL-4 neutralizing antibodies already used to treat atopic diseases.

Computational study of chemical phenol glycosylation mechanism in the gas phase for modeling direct glycoconjugate formation in raw plant material

Glycosylation in a broad sense is commonly considered a chemical reaction between a carbohydrate and another molecule with any functional group which can act as a glycosyl acceptor forming covalent bond. Such modification of small natural biologically active molecules (e.g., flavonols) is of significant importance for drug delivery and dietary supplements due to the increase in their bioavailability. Nevertheless, classical chemical synthesis is complicated by the oxidation of flavonols and requires multiple stages to prevent its degradation. Thus, the idea of direct solid-state chemical synthesis in raw plant material arises. To evaluate such a possibility, we provide a computational gas phase study of this process based on the model system of reaction between glucose and phenol. This investigation is based on the assumption that water and flavonol (here phenol as a model) molecules are deficient in raw plant material and do not form liquid phase, whereas dielectric constant (Ɛ = 2–5) is close to 1 in vacuo, and absence of regular-crystals structure. The reaction mechanism and particular path of phenol direct chemical glycosylation are calculated as well as all intermediate products, transition states, and energy barriers tacking into account molecular symmetry. This work proves a concept of the possibility of such reaction in the raw plant material, including flavonol direct glycosylation using mechanochemistry.

Chemical Glycosylation with p-Methoxyphenyl (PMP) Glycosides via Oxidative Activation

A novel persulfate-mediated oxidative glycosylation system using p-methoxyphenyl (PMP) glycosides as bench-stable glycosyl donors is developed. This study shows that both K2S2O8 as an oxidant and Hf(OTf)4 as a Lewis acid catalyst play important roles in the oxidative activation of the PMP group into a potential leaving group. This convenient glycosylation protocol proceeds under mild conditions and delivers a wide range of biologically and synthetically valuable glycoconjugates, including glycosyl fluorides.

Glycovaccine Design: Optimization of Model and Antitubercular Carrier Glycosylation via Disuccinimidyl Homobifunctional Linker.

Conjugation via disuccinimidyl homobifunctional linkers is reported in the literature as a convenient approach for the synthesis of glycoconjugate vaccines. However, the high tendency for hydrolysis of disuccinimidyl linkers hampers their extensive purification, which unavoidably results in side-reactions and non-pure glycoconjugates. In this paper, conjugation of 3-aminopropyl saccharides via disuccinimidyl glutarate (DSG) was exploited for the synthesis of glycoconjugates. A model protein, ribonuclease A (RNase A), was first considered to set up the conjugation strategy with mono- to tri- mannose saccharides. Through a detailed characterization of synthetized glycoconjugates, purification protocols and conjugation conditions have been revised and optimized with a dual aim: ensure high sugar-loading and avoid the presence of side reaction products. An alternative purification approach based on hydrophilic interaction liquid chromatography (HILIC) allowed the formation of glutaric acid conjugates to be avoided, and a design of experiment (DoE) approach led to optimal glycan loading. Once its suitability was proven, the developed conjugation strategy was applied to the chemical glycosylation of two recombinant antigens, native Ag85B and its variant Ag85B-dm, that are candidate carriers for the development of a novel antitubercular vaccine. Pure glycoconjugates (≥99.5%) were obtained. Altogether, the results suggest that, with an adequate protocol, conjugation via disuccinimidyl linkers can be a valuable approach to produce high sugar-loaded and well-defined glycovaccines.

Better Peptides via Chemical Glycosylation: Somatostatin Analogues Having a Human Complex-Type N-Glycan with Improved Drug Properties.

Somatostatin (somatotropin release-inhibiting factor, SRIF) is a growth hormone inhibitory factor in the form of a 14- or 28-amino acid peptide. SRIF affects several physiological functions through its action on five distinct SRIF receptor subtypes (sst1-5). Native SRIF has only limited clinical applications due to its rapid degradation in plasma. To overcome this obstacle, we have developed glycosylated SRIF analogues that possess not only metabolic stability but also high affinity to all five receptor subtypes by attaching human complex-type oligosaccharides. Such glycosylated SRIF analogues with improved pharmacokinetic profiles could be potent and novel therapeutic drugs for SRIF-related diseases in which several SRIF receptor subtypes are closely involved, and also shed light on new indications. Our results show that chemical glycosylation can be a powerful tool for the development of peptide and protein analogues superior to the original molecules with enhanced drug properties.

Developments in Plant Proteins Production for Meat and Fish Analogues

In recent years, there have been significant developments in plant proteins production for meat and fish analogues. Some of the key developments include the use of new plant protein sources such as soy, legumes, grains, potatoes, and seaweed, as well as insect proteins, leaf proteins, mushrooms, and microbial proteins. Furthermore, to improve the technological and functional properties of plant proteins, they can be subjected to traditional and unconventional treatments such as chemical (glycosylation, deamidation, phosphorylation, and acylation), physical (pulsed electric fields, ultrasound, high hydrostatic pressure, dynamic high-pressure treatment, and cold plasma), and biological (fermentation and enzymatic modification). To obtain the high quality and the desired texture of the food product, other ingredients besides proteins, such as water, fat, flavors, binders, dyes, vitamins, minerals, and antioxidants, also have to be used. The final product can be significantly influenced by the matrix composition, variety of ingredients, and water content, with the type of ingredients playing a role in either enhancing or constraining the desired texture of the food. There are several types of technologies used for meat and fish analogues production, including extrusion, shear cell technology, spinning, 3D printing, and others. Overall, the technologies used for meat and fish analogues production are constantly evolving as new innovations are developed and existing methods are improved. These developments have led to the creation of plant-based products that have a similar texture, taste, and nutritional profile to meat and fish, making them more appealing to consumers seeking alternatives to animal-based products.

Chemo-enzymatic synthesis of 5-substituted ribavirin analogs: Unexpected cooperative effect in the interaction of 5-alkyloxymethyl 1,2,4-triazol-3-carboxamides with E. coli purine nucleoside phosphorylase active site

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.