[Synthesis and cardioprotective properties of apelin-12 and its structural analogs].

The apelin-12 and a number of its analogs, resistant to degradation of proteases, were synthesized by Fmoc- method of SPPS. By-products of synthesis were examined. It was found that serine hydroxyl group was sulfating during the final deprotection of apelin-12 (I) and its analogs. Sulfate moiety of Arg-protecting group transfer into hydroxyl group of Ser. Amount of by-product depends on presence of water in cleavage mixture. Furthermore, the final deprotection of amide analogs of apelin-12 (III, IV) is closed with formation of by-product--4-hydroxybenzylamide, its amount range on 20-8% on reaction mixture accordance HPLC data and also depend on composition of cleavage mixture. Effects of the synthesized peptides on recovery of cardiac function after ischemia were examined in a model of isolated perfused rat heart. Infusions of any of the peptides (I-V) before ischemia resulted in a significant improvement of contractile and pump function recovery compared to the control. Cardioptotective efficacy of the peptides increased in the following rank (I) < (II) = (III) < (IV) = (V).

[Peptide inhibitors of myosin light chain kinase. Development of peptidase resistant analogues].

Myosin light chain kinase (MLCK) is the key regulator of various forms of cell motility including endothelial and epithelial permeability in particular. One of the potential MLCK inhibitors to be used in humans is a membrane permeable peptide H-RKKYKYRRK-NH2 (L-PIK). In present work we used solid phase peptide synthesis and Fmoc-technology to produce five modifications of L-PIK. Based on (1)H NMR analysis revealed that these peptides demonstrated improved resistance to degradation in blood plasma. One of de novo synthesized peptides, L-[MeArg(1)]PIK inhibited MLCK activity in vitro with the same efficiency as L-PIK whereas other modified peptides showed reduced inhibitory activity. D-amino acid analog of PIK was the least active inhibitor. Thus, we have demonstrated the possibility to produce an effective MLCK peptide inhibitor with increased resistance to biodegradation that is suitable for further pharmacological development.

[Novel peptide inhibitors of the myosin light chain kinase suppress hyperpermeability of vascular endothelium].

The ability of novel cell-permeating peptide molecules derived from the peptide inhibitor of the myosin light chain kinase (MLCK) L-PIK (Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys) to inhibit this kinase in vitro and attenuate the thrombin-induced hyperpermeability of endothelial cell monolayer in culture has been studied. It was found that the compounds [NalphaMeArg1]-L-PIK and [Cit1]-L-PIK possess the inhibitory activity towards MLCK comparable to that of L-PIK and the ability to suppress the hyperpermeability of endothelium, whereas other modifications of L-PIK were less effective. Thus, among de novo synthesized peptides, [NalphaMeArg1]-L-PIK and [Cit1]-L-PIK demonstrate the inhibitory properties of the original peptide L-PIK and additionally surpass it by stability in blood plasma. These peptides may be used in the design of novel antiedemic drugs.

[Penetrating peptide inhibitor of the myosin light chain kinase suppresses hyperpermeability of vascular endothelium].

Nonapeptide H-Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH2 corresponding to a modified sequence of autoinhibitory region of myosin light chain kinase (MLCK) was synthesized from L-amino acids and from D-amino acids. Using nuclear magnetic resonance spectroscopy it has been demonstrated that D-peptide is significantly more stable in human blood plasma than its L-enantiomer. D-peptide accumulated in cultured human umbilical vein endothelial cells suppressed development of hyperpermeability in endothelial monolayer induced by thrombin addition. Following intravenous administration D-peptide decreased the extent of lung oedema in rats induced by infusion of oleic acid in bloodstream. Thus, the peptide molecules based on an autoinhibitory peptide of MLCK may serve as a prototype for development of a novel antioedematous drugs that directly affect the MLCK-dependent motile processes in vascular endothelium.

[The synthesis of immunomodulating peptide alloferon, the active component of the antiviral drug allokine-alpha].

Two variants of the synthesis of tridecapeptide alloferon, the active principle of antiviral preparation allokine-alpha, were developed on the basis of fragment condensation in solution or on the Merrifield resin. The solid phase variant of the synthesis was shown to be more technological; it allows the preparation of the product at a higher total yield (40% vs. 17% for conventional synthesis in solution from the starting derivatives of the C-terminal dipeptide). The by-products formed during the synthesis of alloferon were identified.

[Peptide fragment 66-77 of monocyte chemoattractant protein 1 and its retro-enantio analogue inhibits the migration of cells in vitro and in vivo].

The retro-enantio analogue of peptide 66-77 of the chemokine MCP-1 and two hexapeptide fragments 66-71 and 72-77 of the C-terminal sequence of this protein were synthesized using the Fmoc strategy of solid phase peptide synthesis. The effect of the synthetic peptides upon the MCP-1-stimulated migration of THP-1 mononuclear cells was studied in vitro. The activity of the retro-enantio analogue was found to be comparable with that of the initial peptide 66-77: both peptides inhibit the migration of monocytes and granulocytes into inflammation zones of experimental animals.

[Peptide fragments and structural analogues of chemokine MPC-1: synthesis and effect on the MPC-1-induced migration of monocytes]. / Peptidnye fragmenty khemokina MCP-1, ikh strukturnye analogi i ikh vliianie na MCP-1-oposredovannuiu migratsiiu monotsitarnykh kletok.

Fourteen fragments and structural analogues of chemokine MCP-1 were synthesized using the Fmoc strategy of solid phase peptide synthesis. The effect of synthesized peptides on the MCP-1-stimulated migration of mononuclear cells was examined. Both in vitro stimulants and inhibitors of the monocyte migration were found among the peptides. A possible participation of the C-terminal part of the MCP-1 molecule in the inhibition of the MCP-1-stimulated cell migration was found for the first time. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.

[Solid-phase synthesis of peptides containing arginine with an unprotected guanidine group]. / Tverdofaznyi sintez peptidov, soderzhashchikh arginin s nezashchishchennoi guanidinovoi gruppoi.

A new variant of the solid phase synthesis of arginine-containing peptides was proposed. The conditions for the attachment to the Wang polymer of N alpha-Fmoc-arginine containing a protonated guanidine group were found. We demonstrated that this attachment is accompanied by neither racemization nor the attachment of the second Arg residue. Side reactions involving the guanidine group of arginine were studied, and methods for their prevention were proposed. The comparison of the carbodiimide method with a 1-hydroxybenzotriazole additive and a modified method with the use of Kastro's reagent for the introduction of N alpha-Fmoc-Arg residue with the unprotected guanidine group into the growing peptide chain demonstrated the advantages of the second method. Bradykinin and a peptide corresponding to the 584-591 sequence of the transmembrane gp41 from HIV-1 were synthesized by the method proposed here.

[Solid phase synthesis of beta amyloid (1-42)]. / Tverdofaznyi sintez beta-amiloida-(1-42).

A solid phase synthesis of the polypeptide corresponding to the 1-42 sequence of beta-amyloid protein that accumulated in brain cells during Alzheimer's disease was performed using Fmoc strategy. Two alternative approaches to the synthesis, stepwise elongation of the peptide chain and fragment coupling, were compared, and the advantage of the latter approach was shown. Effects of various factors (solvents, reagents for deprotection of alpha-amino functions, and substitution level of polymer carrier) on the synthesis was studied. The appropriate conditions of HPLC for an analysis of the homogeneity of the beta A4(1-42) peptide, as well as the conditions of its gel chromatography on Sephadex G-50 providing the preparation of the end product of 90-95% purity according to HPLC were found.

Comparative evaluation of different methods for disulfide bond formation in synthesis of the HIV-2 antigenic determinant.

The peptide H-Asn-Ser-Trp-Gly-Cys-Ala-Phe-Arg-Gln-Val-Cys-NHEt corresponding to the 593-603 sequence of gp41 protein of the HIV-2 was used to evaluate different methods for the removal of Acm-protection and subsequent disulfide bond formation. The studied methods involved the treatment by salts of heavy metals (silver and mercury) and subsequent cyclization by oxygen, potassium ferricyanide or hydrogen peroxide. The direct oxidative conversion of Acm-peptide to the corresponding cyclic disulfide by iodine under acidic and neutral conditions was investigated, and the structure of by-products was also studied. The best results were obtained using mercuric acetate followed by oxidation with hydrogen peroxide.

[Direct transition of S-acetamidomethyl-protected peptide 593-603 of HIV-2 gp41 into a cyclic disulfide using iodine. Study of side reactions]. / Priamoe prevashchenie S-atsetamidometilzashchimshennogo peptida 593-603 gp-41 VICh-2 v tsiklicheskii disul'fid pri pomoshchi ioda. Izuchenie pobochnykh reaktsii.

Removal of Acm-protecting group from thiol functional groups of Cys residues with simultaneous disulfide bridge formation by iodine in acetic acid was studied in the course of the synthesis of a peptide fragment corresponding to 593-603 sequence of HIV-2 gp41 glycoprotein. The excess iodine influence on the cyclization process was investigated. By-products of the oxidative disulfide formation were isolated, and their structures were elucidated by means of amino acid and elemental analyses, mass spectrometry, NMR, and UV-spectroscopy.

The flexible region of protein L12 from bacterial ribosomes studied by proton nuclear magnetic resonance.

The dimeric protein L7/L12 from bacterial ribosomes has a highly elongated and flexible structure. We have, using 1H NMR methods, analyzed the extent of the flexible region and also the size of the organized structures of the molecule. A number of mutants of the protein as well as monomeric and dimeric forms of the protein and a COOH-terminal fragment have been used for the identification of certain resonances. Thus, residues 37-50 were found to be highly mobile whereas the amino-terminal and COOH-terminal regions are organized into folded domains. The flexibility between the domains and its relation to functional properties of the protein are discussed.

Neutral glycolipids of atherosclerotic plaques and unaffected human aorta tissue.

The composition, structure and localization of neutral glycosphingolipids of human aorta taken from subjects who had died after myocardial infarction were studied. Individual glycosphingolipids were purified by high-performance liquid chromatography and were characterized on the basis of their chromatographic mobility, carbohydrate composition, methylation analysis and by 1H-NMR spectroscopy. The main aortic glycosphingolipids were identified as glucosylceramide, lactosylceramide, globotriaosylceramide and globotetraosylceramide. Significant differences in the neutral glycosphingolipid composition of intima and media were detected. The neutral glycosphingolipid profile of medial plaques resembled that of unaffected media; however, significant differences were detected between intimal plaques and unaffected intima. Whereas the latter contained trihexosylceramide and globoside as the only neutral glycolipids, the intimal plaque glycolipids consisted mainly of glucosylceramide and also contained appreciable amounts of lactosylceramide which were completely absent in the unaffected intima. In comparison to intimal plaques, unaffected intima is characterized by a much higher content of cerebrosides terminating by beta-galactosyl residues which are known to interact with growth factors and other external stimuli. It thus seems possible that the proliferative activity of smooth muscle cells in atherosclerotic diseases is to some extent associated with their neutral glycolipid profile.

C-H ... O hydrogen bonding in solutions of methylated nucleic acid base analogs as revealed by NMR.

Formation and thermodynamic characteristics of C-H ... O hydrogen bonding of methylated uracils and caffeine have been studied by nmr along two lines. 1. The concentration and temperature dependencies of the PMR spectra of 1,3-dimethyluracil (m2 1,3Ura), 1,3-dimethylthymine (m2 1,3Thy), and 1,3,6-trimethyluracil (m3 1,3,6Ura) in chloroform at high concentrations of base analogs indicated the self-association of m2 1,3Ura and m2 1,3Thy via C(6)H ... O hydrogen bonding and the competitive formation of C-H ... O bonds between carbonyl oxygens and chloroform. The intermolecular interaction energy and the arrangement of molecules in the local minima of various m2 1,3Ura dimers were calculated by the method of atom-atom potentials. The deepest minimum for the m2 1,3Ura coplanar dimer corresponds to a C(6)-H ... O hydrogen-bond formation. 2. At low concentration of m2 1,3Ura and caffeine in CCl4, C(6)-H ... O bonding for m2 1,3Ura and C(8)-H ... O bonding for caffeine with oxygens of dimethyl sulfoxide (DMSO) and acetone were observed. The association constants of these complexes were obtained at different temperatures. The enthalpies delta H, of the m2 1,3Ura-DMSO, m2 1,3Ura-accetone, caffeine-DMSO, and caffeine-acetone complexes were -2 +/- 0.1 kcal/mol. The calculations showed that the deepest minimum of the caffeine-acetone coplanar complex corresponds to C(8)-H ... O bonding with energy of -3.5 kcal/mol and that of the m2 1,3Ura-acetone complexes corresponds to C(6)-H ... O bonding with energy of -3.4 kcal/mol. The approximate correction for the solvent effect provides good agreement of the experimental data with the calculations.

[1H-NMR study of protein L7/L12 from bacterial ribosomes]. / Issledovanie belka L7/L12 iz bakterial'nykh ribosom metodom 1H-IaMR.

The 500 MHz 1H-NMR spectra of dimeric protein L12 from ribosomes shows a limited number of unusually sharp signals at room temperature. This is interpreted as evidence for substantial segmental flexibility of the region in the protein molecule. We have analysed the extent of the flexible region and also the size of the organized structures of the molecule. Thus residues 37-50 were found to be highly mobile whereas the N-terminal and C-terminal region are organized into folded domains.

[Structural analysis of translating ribosomes. Proton magnetic resonance method]. / Strukturnye issledovaniia transliruiushchikh ribosom. III. Metod protonnogo magnitnogo rezonansa.

Comparison has been made of the proton magnetic resonance (PMR) spectra of translating ribosomes in the pre-translocation and post-translocation states as well as of the complexes of translating ribosomes with elongation factors Tu (EF-Tu) or G (EF-G) in the presence of the uncleavable analogue of GTP--guanylyl-imidodiphosphate (GMP-PNP). It is shown that proteins L7/L12 within the translating ribosomes possess a high intramolecular mobility both in the pre-translocation and in the post-translocation states. The interaction of EF-G with translating ribosomes results in a decrease of the mobility of the L7/L12 proteins. The interaction of EF-Tu with translating ribosomes leads to slight changes in the PMR spectra different from the changes caused by EF-G.

Nuclear magnetic resonance techniques for studying structure and function of ribosomes.

The following conclusions can be drawn from the use of NMR techniques for studies of ribosomes: 1. The majority of ribosomal proteins are rigidly fixed within the particles, and the most mobile components in the isolated ribosome are L7/L12 proteins from the large subunit. 2. Interaction of EF-G with ribosomes results in some changes in ribosomal domains, and, particularly, immobilization of L7/L12 proteins takes place. The changes may pertain to the translocation reaction, since complexes with ribosomes, EF-G, and GTP are functional. The results of these studies using 1H NMR show that structural studies with this technique are limited as only a few proteins express their resonances in the 1H NMR spectra (S1, L7/L12). At the same time such studies are not exhaustive, since only the simplest samples were studied (ribosomes, the ribosomal complex with EF-G). Complexes with other ligands (tRNA, EF-Tu) have not yet been studied. It is also possible to enhance the resolution of 1H NMR techniques with the help of deuterated factors, ribosomes, and proteins, and to adapt the use of NMR to other nuclei (e.g., the use of fluorinated labels or incorporation of fluoroamino acids into the proteins). Many other approaches using NMR in biology have still to be explored. Therefore it is hoped that the use of NMR techniques will prove to be very useful in studies of the different functional steps of protein biosynthesis.