A comparative study of the possibilities of using ribokinase → phosphopentomutase → nucleoside phosphorylase cascades in the synthesis of modified nucleosides was carried out. Recombinant phosphopentomutase from Thermus thermophilus HB27 was obtained for the first time: a strain producing a soluble form of the enzyme was created, and a method for its isolation and chromatographic purification was developed. It was shown that cascade syntheses of modified nucleosides can be carried out both by the mesophilic and thermophilic routes from D-pentoses: ribose, 2-deoxyribose, arabinose, xylose, and 2-deoxy-2-fluoroarabinose. The efficiency of 2-chloradenine nucleoside synthesis decreases in the following order: Rib (92), dRib (74), Ara (66), F-Ara (8), and Xyl (2%) in 30 min for mesophilic enzymes. For thermophilic enzymes: Rib (76), dRib (62), Ara (32), F-Ara (<1), and Xyl (2%) in 30 min. Upon incubation of the reaction mixtures for a day, the amounts of 2-chloroadenine riboside (thermophilic cascade), 2-deoxyribosides (both cascades), and arabinoside (mesophilic cascade) decreased roughly by half. The conversion of the base to 2-fluoroarabinosides and xylosides continued to increase in both cases and reached 20-40%. Four nucleosides were quantitatively produced by a cascade of enzymes from D-ribose and D-arabinose. The ribosides of 8-azaguanine (thermophilic cascade) and allopurinol (mesophilic cascade) were synthesized. For the first time, D-arabinosides of 2-chloro-6-methoxypurine and 2-fluoro-6-methoxypurine were synthesized using the mesophilic cascade. Despite the relatively small difference in temperatures when performing the cascade reactions (50 and 80 °C), the rate of product formation in the reactions with Escherichia coli enzymes was significantly higher. E. coli enzymes also provided a higher content of the target products in the reaction mixture. Therefore, they are more appropriate for use in the polyenzymatic synthesis of modified nucleosides.

Objectives. The purpose of this analytical review is to evaluate the market for osteoplastic materials and surgical implants, as well as study the features of new-generation materials and the results of clinical applications.Methods. This review summarizes the volumes of research articles presented in the electronic database PubMed and eLIBRARY. A total of 129 scientific articles related to biological systems, calcium phosphate, polymer, and biocomposite matrices as carriers of pharmaceutical substances, primary recombinant protein osteoinductors, antibiotics, and biologically active chemical reagents were analyzed and summarized. The search depth was 10 years.Results. Demineralized bone matrix constitutes 26% of all types of osteoplastic matrices used globally in surgical osteology, which includes neurosurgery, traumatology and orthopedics, dentistry, and maxillofacial and pediatric surgery. Among the matrices, polymer and biocomposite matrices are outstanding. Special attention is paid to the possibility of immobilizing osteogenic factors and target pharmaceutical substances on the scaffold material to achieve controlled and prolonged release at the site of surgical implantation. Polymeric and biocomposite materials can retard the release of pharmaceutical substances at the implantation site, promoting a decrease in the toxicity and an improvement in the therapeutic effect. The use of composite scaffolds of different compositions in vivo results in high osteogenesis, promotes the initialization of biomineralization, and enables the tuning of the degradation rate of the material.Conclusions. Osteoplastic materials of various compositions in combination with drugs showed accelerated regeneration and mineralization of bone tissue in vivo, excluding systemic side reactions. Furthermore, although some materials have already been registered as commercial drugs, a plethora of unresolved problems remain. Due to the limited clinical studies of materials for use on humans, there is still an insufficient understanding of the toxicity of materials, time of their resorption, speed of drug delivery, and the possible long-term adverse effects of using implants of different compositions.

The success in treatment of venous thromboembolism and acute coronary syndromes using direct thrombin inhibitors has stimulated research aimed at finding a new anticoagulant from haematophagous organisms. This study deals with the comparison between hirudin-1 from Hirudomedicinalis(desirudin), being the first-known and most well-studied natural anticoagulant, along with recombinant analogs of haemadin from the leech Haemadipsa sylvestris, variegin from the tick Amblyomma variegatum, and anophelin from Anopheles albimanus. These polypeptides were chosen due to their high specificity and affinity for thrombin, as well as their distinctive inhibitory mechanisms. We have developed a universal scheme for the biotechnological production of these recombinant peptides as pharmaceutical substances. The anticoagulant activities of these peptides were compared using the thrombin amidolytic activity assay and prolongation of coagulation time (thrombin time, prothrombin time, and activated partial thromboplastin time) in mouse and human plasma. The preliminary results obtained suggest haemadin as the closest analog of recombinant hirudin-1, the active substance of the medicinal product Iprivask (Aventis Pharmaceuticals, USA) for the prevention of deep venous thrombosis in patients undergoing elective hip or knee replacement surgery. In contrast, variegin can be regarded as a natural analog of bivalirudin (Angiomax, The Medicines Company), a synthetic hirudin-1 derivative certified for the treatment of patients undergoing percutaneous coronary intervention and of patients with unstable angina pectoris after percutaneous transluminal coronary angioplasty.

Development of efficient approaches for the production of medically important nucleosides is a highly relevant challenge for biotechnology. In particular, cascade synthesis of arabinosides would allow relatively easy production of various cytostatic and antiviral drugs. However, the biocatalyst necessary for this approach, ribokinase from Escherichia coli (EcoRK), has a very low activity towards D-arabinose, making the synthesis using the state-of-art native enzyme technologically unfeasible. Here, we report the results of our enzyme design project, dedicated to engineering a mutant form of EcoRK with elevated activity towards arabinose. Analysis of the active site structure has allowed us to hypothesize the reasons behind the low EcoRK activity towards arabinose and select feasible mutations. Enzyme assay and kinetic studies have shown that the A98G mutation has caused a large 15-fold increase in k_cat and 1.5-fold decrease in K_M for arabinose phosphorylation. As a proof of concept, we have performed the cascade synthesis of 2-chloroadenine arabinoside utilizing the A98G mutant with 10-fold lower amount of enzyme compared to the wild type without any loss of synthesis efficiency. Our results are valuable both for the development of new technologies of synthesis of modified nucleosides and providing insight into the structural reasons behind EcoRK substrate specificity.

A comparative analysis of the transglycosylation conditions catalyzed by E. coli nucleoside phosphorylases, leading to the formation of 2′-deoxynucleosides, was performed. We demonstrated that maximal yields of 2′-deoxynucleosides, especially modified, can be achieved under small excess of glycosyl-donor (7-methyl-2′-deoxyguanosine, thymidine) and a 4-fold lack of phosphate. A phosphate concentration less than equimolar one allows using only a slight excess of the carbohydrate residue donor nucleoside to increase the reaction’s output. A three-step methodology was elaborated for the preparative synthesis of purine-modified 2′-deoxyribonucleosides, starting from the corresponding ribonucleosides.