Доступность пиперидина, широко применяемого для деблокирования α-аминогрупп в твердофазном синтезе пептидов с использованием Fmoc-методологии, в настоящее время существенно ограничена, так как этот реагент имеет статус контролируемого вещества. Поэтому поиск доступных реагентов для удаления Fmoc-защиты, альтернативных пиперидину, представляется актуальным. В работе проведена сравнительная оценка пригодности трех деблокирующих реагентов на основе вторичных аминов: 4-метилпиперидина, пирролидина и пиперазина для твердофазного синтеза пептидов различной структуры — аналога нейрогипофизарного гормона окситоцина атозибана, агониста рецептора апелина (APJ) — метилина и фрагмента 11 – 19 аминокислотной последовательности регуляторной легкой цепи миозина. Показано, что перечисленные реагенты являются подходящей альтернативой пиперидину при получении исследованных нами физиологически активных пептидов.

The availability of piperidine, which is widely used for deblocking α-amine groups in solid-phase peptide synthesis using Fmoc methodology, is now significantly limited because this reagent is classified as a controlled substance. Therefore, a search for other available reagents capable of removing Fmoc-protection seems relevant. The suitability of three deblocking reagents based on secondary amines (4-methylpiperidine, pyrrolidine, and piperazine) for the solid-phase synthesis of peptides of various structures, i.e., atosiban (an analog of the neurohypophysial hormone oxytocin), metilin [an agonist of the apelin receptor (APJ)], and a fragment of the 11-19 amino-acid segment of the regulatory myosin light chain was comparatively assessed. These reagents were shown to be suitable alternatives to piperidine for the preparation of the physiologically active peptides being studied.

Type 1 diabetes mellitus (T1DM) is the most severe form of diabetes, which is characterized by absolute insulin deficiency induced by the destruction of pancreatic beta cells. The aim of this study was to evaluate the effect of a structural analogue of apelin-12 ((NαMe)Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Nle-Pro-Phe-OH, metilin) on hyperglycemia, mitochondrial (MCh) respiration in permeabilized cardiac left ventricular (LV) fibers, the myocardial energy state, and cardiomyocyte membranes damage in a model of streptozotocin (STZ) diabetes in rats. Metilin was prepared by solid-phase synthesis using the Fmoc strategy and purified using HPLC. Four groups of animals were used: initial state (IS); control (C), diabetic control (D) and diabetic animals additionally treated with metilin (DM). The following parameters have been studied: blood glucose, MCh respiration in LV fibers, the content of cardiac ATP, ADP, AMP, phosphocreatine (PCr) and creatine (Cr), the activity of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) in blood plasma. Administration of metilin to STZ-treated rats decreased blood glucose, increased state 3 oxygen consumption, the respiratory control ratio in MCh of permeabilized LV fibers, and increased the functional coupling of mitochondrial CK (mt-CK) to oxidative phosphorylation compared with these parameters in group D. In STZ-treated animals metilin administration caused an increase in the PCr content and prevention of the loss of total creatine (ΣCr=PCr+Cr) in the diabetic hearts, as well as restoration of the PCr/ATP ratio in the myocardium and a decrease in the activity of CK-MB and LDH in plasma to initial values. Thus, metilin prevented energy disorders disturbances in cardiomyocytes of animals with experimental T1DM.

An industry-ready approach for the synthesis of terlipressin is developed: the entire peptide chain assembly and subsequent disulfide bond closure are carried out on a polymeric carrier with a specific precaution for the protection of the N-terminal α-amino group. The effect of various factors on the purity of crude disulfide was studied, and the solid-phase cyclization process was optimized. The designed approach has high fidelity and reproducibility and is applicable for a large-scale peptide synthesis (we show it up to 15 g of the pure product). Using molecular modeling, we have found that the state of the N-terminal amino group (free or protected) has a significant influence on the ability of two cysteine side chains to reach each other, paving the way to a rational choice of protecting groups in peptide synthesis.

Objectives: The aim of this work was to elucidate the role of GalR2 receptor activation in protecting the rat heart in vivo from ischemia/reperfusion (I/R) damage by a pharmacological peptide agonist WTLNSAGYLLGP beta AH-OH (G1) and full-length rat galanin GWTLNSAGYLLGPHAIDNHRSFSDKHGLT-NH2 (G2) using M871, a selective inhibitor of GalR2. Methods: The peptides were prepared by the automatic solid-phase synthesis using the Fmoc-strategy and purified by high-performance liquid chromatography (HPLC). A 40-min left anterior descending (LAD) coronary artery occlusion followed by a 60-min reperfusion was performed. The criteria for damage/protection of the heart were the infarct size (IS) and plasma activity of creatine kinase-MB (CK-MB) at the end of reperfusion. Results: Intravenous injection of G1 or G2 at an optimal dose of 1 mg/kg at the fifth minute of reperfusion significantly reduced the IS (by 35% and 32%, respectively) and activity of CK-MB at the end of reperfusion (by 43% and 38%, respectively) compared with the control. Administration of M871 (8 mg/kg) 5 min before the onset of reperfusion abolished the effects of G1 on IS and CK-MB activity, returning them to control values. Co-administration of M871 (8 mg/kg) with G2 attenuated protective effect of G2 on both IS and plasma CK-MB activity. However, differences in these parameters between the M871+G2 and G2 groups did not reach statistical significance (P = 0.139 and P = 0.121, respectively). Conclusion: Thus, GalR2 is the principal receptor subtype that transduces the protective effects of galanin and ligand G1 in myocardial I/R injury. This suggests that GalR2-specific peptide agonists could be used as drug candidates for treating ischemic heart disease.

The neuropeptide galanin (G) and its N-terminal fragments reduce the formation of reactive oxygen species and normalize myocardial metabolic and antioxidant state in experimental cardiomyopathy and ischemia/reperfusion injury. This study intends to elucidate the cardioprotective effect of G in rats treated with streptozotocin (STZ). The rat galanin (GWTLNSAGYLLGPHAIDNHRSFSDKHGLT-NH2) was prepared by the solid phase peptide synthesis using the Fmoc-strategy and purified by preparative HPLC. Its chemical structure was identified by 1H-NMR spectroscopy and MALDI-TOF mass spectrometry. The animals were divided in four groups: C—normal control; S—STZ control, a single i.p. injection of STZ (35 mg/kg); SG—a single i.p. injection of STZ (35 mg/kg) and i.p. injections of G (5 nmol/kg/day for 4 weeks); and G—normal animals treated with G (5 nmol/kg/day for 4 weeks). Treatment with G prevented hyperglycemia in STZ rats. G significantly improved maximal ADP-stimulated respiration and respiratory control ratio in saponin-skinned myocardial fibers in SG group compared to S group. G enhanced myocardial metabolic state in animals treated with STZ by reducing ATP, phosphocreatine (PCr) and total creatine (ΣCr = PCr + Cr) losses, and decreasing lactate accumulation in parallel with elevation of glucose level. Administration of G reduced the increased activity of creatine kinase-MB and lactate dehydrogenase in blood plasma of STZ-treated rats. G also prevented the formation of STZ-induced lipid peroxidation products, thiobarbituric acid reactive substances, in blood plasma to a value not different from baseline. The results suggest that G may be a promising tool for reducing myocardial metabolic disorders in diabetes mellitus.

The aim of this work was to design and characterize peptides based on the α-helices h1 and h2 of the ACE2 receptor, forming the interaction interface between the receptor-binding domain (RBD) of the SARS-CoV-2 S protein and the cellular ACE2 receptor. Monomeric and heterodimeric peptides connected by disulfide bonds at different positions were synthesized. Solubility, RBD-binding affinity, and peptide helicity were experimentally measured, and molecular dynamics simulation was performed in various solvents. It was established that the preservation of the helical conformation is a necessary condition for the binding of peptides to RBD. The peptides have a low degree of helicity and low affinity for RBD in water. Dimeric peptides have a higher degree of helicity than monomeric ones, probably due to the mutual influence of helices. The degree of helicity of the peptides in trifluoroethanol is the highest; however, for in vitro studies, the most suitable solvent is a water-ethanol mixture.

Разработана новая высокотехнологичная методика получения дезаминоаналогов нейрогипофизарных гормонов, которая представляет полностью твердофазный вариант, включая стадию замыкания дисульфидной связи. Все сырые продукты получены с чистотой выше 92 % и минимальным количеством примесей, что является привлекательным для крупномасштабного синтеза S–S-пептидов.

Neuropeptide galanin and its N-terminal fragments reduce the generation of reactive oxygen species and normalize metabolic and antioxidant states of myocardium in experimental cardiomyopathy and ischemia/reperfusion injury. The aim of this study was to elucidate the effect of WTLNSAGYLLGPβAH-OH (peptide G), a pharmacological agonist of the galanin receptor GalR2, on the cardiac injury induced by administration of streptozotocin (STZ) in rats. Peptide G was prepared by solid phase peptide synthesis using the Fmoc strategy and purified by preparative HPLC; its structure was confirmed by 1H-NMR spectroscopy and MALDI-TOF mass spectrometry. Experimental animals were randomly distributed into five groups: C, control; S, STZ-treated; SG10, STZ + peptide G (10 nmol/kg/day); SG50, STZ + peptide G (50 nmol/kg/day); G, peptide G (50 nmol/kg/day). Administration of peptide G prevented hyperglycemia in SG50 rats. By the end of the experiment, the ATP content, total pool of adenine nucleotides, phosphocreatine (PCr) content, and PCr/ATP ratio in the myocardium of animals of the SG50 group were significantly higher than in rats of the S group. In the SG50 and SG10 groups, the content of lactate and lactate/pyruvate ratio in the myocardium were reduced, while the glucose content was increased vs. the S group. Both doses of peptide G reduced the activation of creatine kinase-MB and lactate dehydrogenase, as well as the concentration of thiobarbituric acid reactive products in the blood plasma of STZ-treated rats to the control values. Taken together, these results suggest that peptide G has cardioprotective properties in type 1 diabetes mellitus. Possible mechanisms of peptide G action in the STZ-induced diabetes are discussed.

Anew high-tech methodology for the synthesis of desamino-analogs of neurohypophyseal hormones has been developed. The procedure is a completely solid-phase variant including the disulfide bond closure step. All crude products are obtained with 92

The dose-dependent action of the M871 synthetic peptide antagonist of the GalR2 galanin receptor (H-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Glu-His-Pro-Pro-Pro-Ala-Leu-Ala-Leu-Ala-NH2) and the pharmacological agonist G (H-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-βAla-His-OH) on sizes of the myocardial infarction (MI) and an activity of creatine kinase (CK-MB) in the blood plasma was studied on the in vivo model of regional ischemia and reperfusion of the rat heart. The peptides were prepared by the automatic solid phase synthesis using the Fmoc-strategy and purified by HPLC. The peptides had the appropriate molecular mass and the homogeneity of 97–98%. A blockage of the GalR2 receptors by the intravenous injection of M871 in doses of 3, 6, and 8 mg/kg before the beginning of the reperfusion had no influence on the MI sizes and the CK-MB activity in comparison with the control. The intravenous injection of the agonist G in a dose of 1 mg/kg at the beginning of the reperfusion significantly decreased the MI sizes and the CK-MB activity in the blood plasma in comparison with the control by 38 and 40%, respectively. Peptides M871 and G did not significantly affect the hemodynamic parameters of the heart. The preliminary intravenous injection of increasing doses of peptide M871 before an administration of agonist G resulted in a gradual increase in the MI sizes and the CK-MB activity. An application of compound M871 in doses of 6 or 8 mg/kg completely abrogated the cardioprotective effects of agonist G. These results suggested the participation of the GalR2 receptor in the protective action of the chimeric agonist G on the heart that was subjected to ischemia and reperfusion and pointed to the considerable promise of the molecular engineering of the peptide agonists of the GalR2 receptor for a design of therapeutic agents for a treatment of cardiovascular diseases.

Computer simulation has been used to identify peptides that mimic the natural target of the SARS-CoV-2 coronavirus spike (S) protein, the angiotensin converting enzyme type 2 (ACE2) cell receptor. Based on the structure of the complex of the protein S receptor-binding domain (RBD) and ACE2, the design of chimeric molecules consisting of two 22-23-mer peptides linked to each other by disulfide bonds was carried out. The chimeric molecule X1 was a disulfide dimer, in which edge cysteine residues in the precursor molecules h1 and h2 were connected by the S-S bond. In the chimeric molecule X2, the disulfide bond was located in the middle of the molecule of each of the precursor peptides. The precursors h1 and h2 modelled amino acid sequences of α1- and α2-helices of the extracellular peptidase domain of ACE2, respectively, keeping intact most of the amino acid residues involved in the interaction with RBD. The aim of the work was to evaluate the binding efficiency of chimeric molecules and their RBD-peptides (particularly in dependence of the middle and edge methods of fixing the initial peptides h1 and h2). The proposed polypeptides and chimeric molecules were synthesized by chemical methods, purified (to 95-97% purity), and characterized by HPLC and MALDI-TOF mass spectrometry. The binding of the peptides to the SARS-CoV-2 RBD was evaluated by microthermophoresis with recombinant domains corresponding in sequence to the original Chinese (GenBank ID NC_045512.2) and the British (B. 1.1.7, GISAID EPI_ISL_683466) variants. Binding to the original RBD of the Chinese variant was detected in three synthesized peptides: linear h2 and both chimeric variants. Chimeric peptides were also bound to the RBD of the British variant with micromolar constants. The antiviral activity of the proposed peptides in Vero cell culture was also evaluated.

Computer simulation has been used to identify peptides that mimic the natural target of the SARS-CoV-2 coronavirus spike (S) protein, the angiotensin converting enzyme type 2 (ACE2) cell receptor. Based on the structure of the complex of the protein S receptor-binding domain (RBD) and ACE2, the design of chimeric molecules consisting of two 22-23-mer peptides linked to each other by disulfide bonds was carried out. The chimeric molecule X1 was a disulfide dimer, in which edge cysteine residues in the precursor molecules h1 and h2 were connected by the S-S bond. In the chimeric molecule X2, the disulfide bond was located in the middle of the molecule of each of the precursor peptides. The precursors h1 and h2 modelled amino acid sequences of α1- and α2-helices of the extracellular peptidase domain of ACE2, respectively, keeping intact most of the amino acid residues involved in the interaction with RBD. The aim of the work was to evaluate the binding efficiency of chimeric molecules and their RBD-peptides (particularly in dependence of the middle and edge methods of fixing the initial peptides h1 and h2). The proposed polypeptides and chimeric molecules were synthesized by chemical methods, purified (to 95-97% purity), and characterized by HPLC and MALDI-TOF mass spectrometry. The binding of the peptides to the SARS-CoV-2 RBD was evaluated by microthermophoresis with recombinant domains corresponding in sequence to the original Chinese (GenBank ID NC_045512.2) and the British (B. 1.1.7, GISAID EPI_ISL_683466) variants. Binding to the original RBD of the Chinese variant was detected in three synthesized peptides: linear h2 and both chimeric variants. Chimeric peptides were also bound to the RBD of the British variant with micromolar constants. The antiviral activity of the proposed peptides in Vero cell culture was also evaluated.

A correction to this paper has been published: https://doi.org/10.1007/s10989-021-10233-9

Antioxidant properties of rat galanin GWTLNSAGYLLGPHAIDNHRSFSDKHGLT-NH2 (Gal), N-terminal fragment of galanin (2-15 aa) WTLNSAGYLLGPHA (G1), and its modified analogue WTLNSAGYLLGPβAH (G2) were studied in vivo in the rat model of regional myocardial ischemia and reperfusion and in vitro in the process of Cu2+-induced free radical oxidation of human blood plasma low-density lipoproteins. Intravenous administration of G1, G2, and Gal to rats after ischemia induction reduced the infarction size and activities of the necrosis markers, creatine kinase-MB and lactate dehydrogenase, in blood plasma at the end of reperfusion. G1, G2, and Gal reduced formation of the spin adducts of hydroxyl radicals in the interstitium of the area at risk during reperfusion, moreover, G2 and Gal also reduced formation of the secondary products of lipid peroxidation in the reperfused myocardium. It was shown in the in vivo experiments and in the in vitro model system that the ability of galanin peptides to reduce formation of ROS and attenuate lipid peroxidation during myocardial reperfusion injury was not associated directly with their effects on activities of the antioxidant enzymes of the heart: Cu,Zn-superoxide dismutase, catalase, and glutathione peroxidase. The peptides G1, G2, and Gal at concentrations of 0.01 and 0.1 mM inhibited Cu2+-induced free radical oxidation of human low-density lipoproteins in vitro. The results of oxidative stress modeling demonstrated that the natural and synthetic agonists of galanin receptors reduced formation of the short-lived ROS in the reperfused myocardium, as well as of lipid radicals in blood plasma. Thus, galanin receptors could be a promising therapeutic target for cardiovascular diseases.

This work is devoted to the large-scale solid-phase synthesis (SPS) of Atosiban, Mpa 1 -D-Tyr(OEt)-Ile-Thr-Asn-Cys 6 -Pro-Orn-Gly-NH 2 cyclic 1,6 disulfide, the only clinically used oxytocin receptor antagonist. The conditions have been selected for the closure of the disulfide bond (S–S) in the Atosiban molecule both in the solution and solid phase with the minimal formation of by-products. A comparative assessment of the formation of the S–S bond was carried out under various conditions. The formation of by-products during the closure of the disulfide bond has been studied both in solution and on the polymer support. The developed technique allows for the synthesis of Atosiban on an enlarged scale (10–20 mmol) involving the cyclization of a protected intermediate with the formation of the S–S bond during solid-phase synthesis with the minimal formation of by-products.