[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.

[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.

Physical properties of ribosomal proteins isolated under different conditions from the Escherichia coli 50 S subunit.

Physical properties of ribosomal proteins obtained with or without denaturating agents were compared. CD measurements and NMR studies have shown that proteins L2, L19, L24 and L30 isolated under denaturing conditions have the same properties as those prepared avoiding denaturating agents. CD and NMR spectra of proteins L1, L6, L11, L23, L25 and L29 obtained by us under denaturating conditions practically coincide with the data for the same proteins reported under 'mild' conditions. These findings suggest that the differences of reported physical properties can be due to different procedures of protein renaturation rather than to the methods of their isolation.

A comparative study of structural properties of fibronectin and its 180 kDa fragment.

Fibronectin from human plasma and its 180 kDa fragment which retained collagen-binding, cell-attachment and heparin-binding activities, were studied by velocity centrifugation and 1H-NMR methods. The fibronectin hydrodynamic radius strongly increased at pH 11 while the hydrodynamic properties of the fragment did not change noticeably. 1H-NMR spectroscopy also showed differences in the molecular properties of fibronectin and its 180 kDa fragment. Under physiological conditions the structure of fibronectin differs from that of its 180 kDa fragment. At pH 11 and in 4 M urea no differences in their structures are observed. It is suggested that interdomain and intersubunit interactions play an important role in maintaining the native conformation of intact fibronectin.

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.

[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.

[Nuclear magnetic resonance study of C--H...O type hydrogen bonds in analogs of nucleic acid base]. / Issledovanie metodom iadernogo magnitnogo rezonansa vodorodnykh sviazei tipa C--H...O v analogakh osnovanii nukleinovykh kislot.

Concentration and temperature dependence of nuclear magnetic resonance spectra of 1,3-dimethyluracil (m2 1,3Ura) in organic solvents has been investigated. The results indicate m2 1,3Ura dimer formation via C(6)--H...O hydrogen bond. In addition to that the formation of C--H...O H-bonds between solvent molecules and m2 1,3Ura take place. delta H of C---H...O H-bond formation between chloroform and m2 1,3Ura dimers have the value of about -2.5 and -2.0 kcal/mol, respectively. Interaction energy calculations by atom-atom potential functions for different types of m2 1,3Ura dimers are in good agreement with experimental data. The possible role of C--H...O H-bonds in intermolecular interactions and biological functions of nucleic acids are discussed.

[Spatial structure of the cro-repressor in a solution. I. Identification of interaction in a hydrophobic cluster using the nuclear Overhauser effect]. / Prostranstvennaia struktura cro-repressora v rastvore. I. Identifikatsiia vzaimodeistviia v gidrofobnom klastere s pomoshch'iu iadernogo éffekta Overkhauzera.

The structure of a bacteriophage lambda cro repressor hydrophobic globule was studied by the technique of 1H NMR spectroscopy at 500 MHz. The analysis of NOE difference spectra and building of the molecular models for the most probable fragments of the secondary structure allowed us to assign many signals in the protein spectrum and to identify the intramolecular interactions which stabilized the hydrophobic globule and the tertiary structure of the molecule. The results suggest that the structure of the cro repressor hydrophobic globule in solution coincides with the crystal one, although there are some differences in the mutual arrangement of the alpha 1 helix and the three-fold beta-sheet. Resonance assignment has made it possible to study conformational changes and specific interactions of the cro repressor by using suitable reporter groups.

[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.

[Comparison of the physical properties of ribosomal proteins from Escherichia coli 50S subparticles isolated by different methods]. / Sravnenie fizicheskikh svoistv ribosomnykh belkov 50S subchastitsy Escherichia coli, poluchennykh razlichnymi metodami.

Physical properties of ribosomal proteins obtained with or without denaturing agents were compared. CD measurements and PMR studies have shown that proteins L2, L19, L24 and L30 isolated under denaturing conditions have the same properties as those prepared avoiding denaturing agents. CD and PMR data of L1, L6, L11, L23, L25 and L29 obtained by us under denaturing conditions practically coincide with the data for these proteins obtained in "mild" conditions and published in the literature. These findings indicate that the differences of physical properties reported in the literature can be due to different procedures of protein renaturation rather than to the methods of their isolation.

[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.

[Study of peptide degradation in the serum by 1H-NMR]. / Issledovanie degradatsii peptidov v syvorotke krovi metodom 1H-IaMR.

Enzymatic degradation of hexapeptide Tyr-D-Ala-Gly-Phe-Leu-Arg in human serum has been investigated by 1H-NMR spectroscopy, and its pathways are suggested. Its final degradation products, tetra- and pentapeptide, are shown to be stable in human serum for several hours. The NMR spectroscopy application for pharmacokinetic studies is substantiated.

[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.

[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.

[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.

[Study by the proton magnetic resonance method of complex formation between nucleosides and compounds modeling amino acid residues of proteins in dimethyl sulfoxide]. / Issledovanie metodom protonnogo magnitnogo rezonansa kompleksoobrazovaniia mezhdu nukleozidami i soedineniiami, modeliruiushchimi aminokislotnye ostatki beldov, v dimetilsul'fokside

Proton magnetic resonance was used to investigate in dimethyl-sulfoxide specificity of complex formation between nucleosides and a number of substances modelling of carboxyl, guanidine and amide side chain residues of aminoacids. It was observed downfield chemical shift of NH2 group of guanosine and disappearence. NH groups resonance for all nucleosides containing its in heterocycle in complex with carboxyl group. The next selectivity of interaction of nucleosides with carboxyl was observed: Ino greater than Guo greater than Urd ==Thd greater than Ado==Cyd. Obtained data indicates formation specific hydrogen bonds between carboxyl group and NH2 group of guanine and more strong hydrogen bonds with NH groups of all bases. In the case of guanidine group there are more weaker interaction and for amide group complex is not observed at all.

[Identification of signals in overlapping NMR spectra of proteins by the differential spin echo method]. / Vydelenie signalov v perekryvaiushchikhsia spektrakh IaMR belkov metodom differentsial'nogo spin-ékha.

A novel method for identification of the spin systems of Thr and Ala amino acid residues in H--NMR spectra of proteins is proposed.