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.

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.

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.

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.

The full-length rat galanin (GWTLNSAGYLLGPHAIDNHRSFSDKHGLT-NH2, G29) was prepared by the solid phase peptide synthesis using the Fmoc-strategy. The peptide chain was elongated both by one amino acid and by a fragment condensation. Fragments with the C-terminal glycine residue were synthesized by the solid phase method on the 2-chlorotrityl chloride resin or by the method of conventional peptide synthesis in solution. After the reversed phase HPLC, galanin had the correct molecular weight and 98% purity. We studied the G29 cardioprotective properties on a model of acute myocardial infarction in rats. The G29 administration reduced the infarct size by 40% and decreased the activity of necrosis markers CK-MB (Creatine Kinase-MB) and LDH (Lactate Dehydrogenase) in the blood plasma. The peptide improved metabolic state of the infarcted heart, increased the ATP content, the total adenine nucleotide pool, phosphocreatine, and total creatine, and decreased the lactate level compared to a control. These results indicated the possibility of the G29 use as a drug for reducing the myocardial reperfusion injury. In addition, the mechanisms of the G29 action should be studied.

— The solid phase synthesis (SPS) of the ingramon peptide antagonist of the human monocyte chemoattractant protein-1 (H-Asp-His-Leu-Asp-Lys-Gln-Thr-Gln-Thr-Pro-Lys-Thr-OH), which exhibited the anti-inflammatory activity, was optimized. Side products of the Fmoc-SPS of this peptide were studied. Their structures were determined by the 1 H NMR spectroscopy and confirmed by the synthesis. Methodological ways to minimize of a formation of impurities in the course of the ingramon SPS were found. The reproducible SPS procedure of the ingramon preparation that resulted in the formation of the minimum amount of such side products as [Asi 4 ]-, [D-Asp 4 ]-, and [βAsp 4 ]-peptides was elaborated.

The use of the anticancer drug doxorubicin (Dox) is limited due to its cardiotoxic effect. Using the method of automatic solid-phase peptide synthesis, we obtained a synthetic agonist of galanin receptors GalR1-3 [RAla14, His15]-galanine (2-15) (G), exhibiting cardioprotective properties. It was purified by high performance liquid chromatography (HPLC). The homogeneity and structure of the peptide was confirmed by HPLC, 1H-NMR spectroscopy and mass spectroscopy. The purpose of this study was to study the effect of G on the metabolism and cardiac function of rats with chronic heart failure (CHF) caused by Dox. Experiments were performed using male Wistar rats weighing 280-300 g. The control group of animals (C) was intraperitoneally treated with saline for 8 weeks; the doxorubicin group (D) of rats was intraperitoneally treated with Doх; the group of Doх + peptide G (D+G) received intraperitoneally injections of Doх and subcutaneously injections of peptide G; the peptide G group (G) was subcutaneously treated with G. At the beginning and at the end of the study, the concentration of thiobarbituric acid reactive substances (TBARS) and the activity of creatine kinase-MB (CK-MB) were determined in blood plasma; the animals were weighed, and cardiac function was assessed using echocardiography. At the end of the experiments, the hearts were used for determination of metabolites and assessment of oxidative phosphorylation in mitochondria. After 8-week treatment, animals of group D were characterized by severe heart failure, the lack of weight gain and an increase in plasma TBARS concentration and CK-MB activity. These disorders were accompanied by a decrease in the content of myocardial high-energy phosphates, a reduction inmitochondrial respiratory parameters, accumulation of lactate and glucose in the heart, and disturbances in the metabolism of alanine and glutamic and aspartic acids. Coadministration of G and Dox prevented the increase in plasma CK-MB activity and significantly reduced the plasma TBARS concentration. At the end of the experiments animals of group D+G had higher myocardial energy state and the respiratory control index of mitochondria than animals of group D, there was a decrease in anaerobic glycolysis and no changes in the amino acid content compared to the control. The peptide G significantly improved the parameters of cardiac function and caused weight gain in animals of group D+G in comparison with these parameters in group D. The obtained results demonstrate the ability of a novel agonist of galanin receptors GalR1-3 to attenuate Dox-indiced cardiotoxicity.

New peptide analogs of galanin corresponding to fragments of the N -terminal sequence were synthesized by an automatic solid-phase method using Fmoc-technology. Incomplete cleavage of the Boc-protection from the Trp indole ring was found during postsynthetic procedures and a method for solving this problem was proposed. Physicochemical properties of a number of natural and synthetic N -terminal fragments of galanin were been studied. A comparative evaluation of cardioprotective action of peptide agonists of the galanin GalR2 receptor was carried out on the model of the regional ischemia and reperfusion of the heart in rats in vivo. The analog that contained the carnosine sequence, H-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-βAla-His-OH, was found to most effectively protect the heart from ischemic and reperfusion stress.

A method for the solid-phase synthesis of the apelin-12 analog has been developed using the Fmoc methodology in combination with the temporary protection of the guanidine function of the arginine residues by protonation (salt formation) during the formation of the amide bond. Proton magnetic resonance spectroscopy has been used to compare the proteolytic stability of apelin-12 and its structural analog in human blood plasma. The half-life of the analog in plasma has been shown to be about three times longer than that of the natural peptide.

The P26 peptide corresponding to the 197–222 sequence of the second extracellular loop of the β 1 -adrenoreceptor (β 1 -AR) was synthesized by solid-phase fragment condensation on the Wang polymer. Pentapeptide fragments were prepared on the 2-chlorotrityl resin. The racemization degree of the C-terminal alanine residue of the pentapeptide was experimentally evaluated for the synthetic H-Glu-Ser-Asp-Glu-Ala-Arg-OH hexapeptide β 1 -АR-(202–207) which was prepared by the 5 + 1 fragment condensation with the use of various condensing agents. A content of the diastereoisomeric peptide in the products of the fragment condensation was determined by HPLC on a reversed phase. The D-alanine-containing hexapeptide was specially synthesized and used for a comparison. The minimum racemization degree of the C -terminal alanine residue was observed if complex F was applied to the synthesis of the hexapeptide.

The automated Fmoc solid-phase technique was used to synthesize Cys-containing linear peptide fragments of monocyte chemoattractant protein-1, chemokine domain of fractalkine, and their analogues, with the Cys residue being either modified or replaced with Ser. Chimeric symmetric and asymmetric disulfides were also prepared from the linear precursors. A SAR study of a set of the newly synthesized peptides showed that the capacity to stimulate migration of monocytes and influence cell motility in vitro critically depends, in general, on the presence of free Cys thiol group in the molecule. Notably, all analogues, including chimeric disulfides, containing no SH groups demonstrated the lack of chemokinetic properties.

Automated Fmoc solid-phase technique was used to synthesize Cys-containing linear peptide fragments of monocyte chemoattractant protein-1 and chemokine domain of fractalkine along with their analogues with Cys residue being either modified or replaced with Ser. Chimeric symmetric and asymmetric disulfides were also prepared from the former linear precursors. A SAR study on a set of the newly synthesized peptides revealed that capacity to stimulate migration of monocytes and to influence cell motility in vitro, in general, critically depends on the presence of Cys free thiol group in the molecule. Notably, all analogs lacking this feature, including chimeric disulfides, demonstrated lack of effect on monocyte migration.

Fragments corresponding to the 83–98 sequence of the first extracellular loop and to the 168–192 and 171–182 sequences of the second extracellular loop of the M2-muscarinic receptor (antibodies to this receptor could be markers of early symptoms of heart disorders) were synthesized by solid phase method using the Fmoc-SPPS strategy. A new conformational antigen with the natural location of the disulfide bridge was prepared by selective formation of disulfide bond between the corresponding cysteine residues in the synthe-sized peptides and characterized. The comparative analysis of reactivity of the synthesized peptides towards sera from patients which had no organic heart disease was performed. A new conformational antigen was effectively bound to the sera from patients with idiopathic arrhythmias, but without symptoms of organic heart disease.

Рассмотрены теоретические и экспериментальные методы определения антигенных детерминант белков с известной аминокислотной последовательностью. Проведена систематизация методов на основе используемых теоретических подходов. Приводятся данные по сравнительной оценке эффективности различных предсказательных методов, а также примеры нахождения эпитопов экспериментальным путем для ряда белков.Theoretical and experimental methods for locating antigenic determinants of proteins with known amino acid sequences are discussed. These methods are systematized on the basis of the theoretical approaches applied, and the efficiency of various predictive methods is compared. Some examples of experimental epitope determination for a number of proteins are given.