Inhibitory effect of methyl esters of arginine-containing oligopeptides on thrombin and trypsin

Stereoisomeric oligopeptides were studied for their inhibitory effect on the hydrolysis of benzoyl-L-arginine methyl ester catalyzed by thrombin and trypsin, as well as on the thrombin-fibrinogen reaction. Comparison of the peptide structures, their conformational flexibility and inhibitory effects on thrombin and trypsin shows the availability of the essential differences in organization and functioning of the subsites S3, S2 and S'1 of these enzymes. In contrast to trypsin, thrombin is shown to be characterized by more pronounced secondary stereospecificity. This manifests in the more vigorous dropping of the catalytic constants of thrombin-catalyzed esterolysis than those of trypsin-catalyzed hydrolysis of the substrates, containing D-amino acids at the subsite P2. It is revealed that the peptide Tos-D-Val-D-Ala-D-Agr-D-Phe-OCH3 is the most powerful inhibitor among studied compounds. It is noteworthy that its antithrombin effect is almost an order of magnitude higher than its antitrypsin effect.     

Production of L-lysine by immobilized trypsin. Study of DL-lysine methyl ester resolution

The possibility of producing L-lysine from chemically synthesized DL-lysine has been investigated. Optical resolution of racemic DK-lysine may be achieved by using the stereospecific esterasic activity of trypsin on DL-lysine methyl ester, which gives L-lysine and unchanged D-lysine methyl ester. SL-lysine methyl ester spontaneous hydrolysis may be neglected when operating at pH 5.5 and 30 degrees C. Effect of pH and substrate concentration on hydrolysis rate has been investigated when using as a catalyst either soluble or immobilized trypsin. For this purpose, trypsin was coupled onto an amine porous silica, Spherosil, activated with glutaraldehyde. The optimal pH is 5.8 for soluble trypsin and 6.0 for immobilized trypsin. It was yet possible to lower the parent optimal pH of immobilized trypsin, and thus increase its activity at 5.5, by co-grafting onto Spherosil an aminosilane, for enzyme coupling via glutaraldehyde activation and a positively charged diethyl amino ethyl (DEAE) silane, for decreasing the pH of trypsin microenvironment.     

Comparative study of hydrolysis of methyl esters of N-arylsulfonyl-valyl-arginine by thrombin and trypsin]

The esterase action of thrombin and trypsin on N-arylsulfonyl-valyl-arginine methyl esters was studied. The values of Km and kcat under steady-state conditions at pH 8,5 were determined. It was shown that the nature of the arylsulfonyl group does not affect the kinetic parameters of the reactions under study. The Michaelis constants of the thrombin-catalyzed reactions appeared to be one order of magnitude lower than the Km values of the corresponding TAME analogs.     

The interaction in human plasma of antiplasmin, the fast-reacting plasmin inhibitor, with plasmin, thrombin, trypsin and chymotrypsin.

The inhibition of plasmin, (EC 3.4.21.7), thrombin (EC 3.4.21.5), trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) by antiplasmin, the recently described fast-reacting plasmin inhibitor of human plasma, was studied. To determine the quantitative importance of antiplasmin relative to the other plasma protease inhibitors, enzyme inhibition assays were performed on whole plasma and on plasma specifically depleted in antiplasmin, after addition of excess enzyme. Plasmin was the only enzyme for which the inhibitory capacity of antiplasmin-depleted plasma was lower than that of normal plasma. To determine the affinity of the enzymes for antiplasmin, as compared to the other inhibitors, various amounts of enzymes were added to normal plasma and the formation of enzyme-antiplasmin complexes studied by crossed immunoelectrophoresis using specific antisera against antiplasmin. Plasmin and trypsin, but not thrombin or chymotrypsin formed complexes with antiplasmin. It is concluded that antiplasmin is the only fast-reacting plasmin inhibitor of human plasma. It is also a fast-reacting inhibitor of trypsin but only accounts for a very small part of the fast-reacting trypsin-inhibitory activity of plasma. This can be explained by the low concentration of antiplasmin (1 muM) in normal plasma, compared to the other inhibitors (e.g. alpha1-antitrypsin: 40-80 muM).     

Antithrombic activity of N(alpha)-lauroyl-L-arginine ethyl ester (LAE) and 9-fluorenylmethoxycarbonyl-L-arginine methyl ester (Fmoc-L-Arg-OMe)

Simple synthetic compounds of lauroyl-arginine ethyl ester (LAE) and 9-fluorenylmethoxycarbonyl-L-agrinine methyl ester (Fmoc-Arg-OMe) were studied for their inhibitory effect on the hydrolysis of chromogenic substrate Tos-Gly-Pro-Arg-pNA (Chromozym TH) by thrombin with K(i) for LAE 1.92 microM and 77 microM for Fmoc Arg-OMe. It was shown that LAE inhibits thrombin activity almost 20 times more strongly than trypsin (K(i) = 18.9 microM). At the same time LAE preserves the ability to be hydrolyzed by thrombin at pH 8.5 (k(cat) = 3.6 c(-1)) and trypsin (k(cat) = 56 c(-1)). It is suggested, that LAE ability to suppress growth of some microorganisms is conditioned to some extent by its ability to inhibit the activity of trypsin-like serine proteases, participating in the infection process.     

Bis(o-trifluoromethylphenyl)dithiophosphinic methyl ester

When bis(o-trifluoromethylphenyl)dithiophosphinic acid (1) is dissolved in methanol, crystals of bis(o-trifluoromethylphenyl)dithiophosphinic methyl ester (2) are formed. The structure of dithiophosphinic methyl ester (2) has been characterized via single-crystal X-ray diffraction and multinuclear NMR studies. Compound 2 is remarkable in that the dithiophosphinic (PS₂) core is preserved during this transformation. The transformation to 2 suggests that the o-trifluoromethylphenyl groups on phosphorus assist in retaining the PS₂ core, possibly by steric hindrance.     

Benzimidazole-based fXa inhibitors with improved thrombin and trypsin selectivity

Optimization of the benzimidazole-based fXa inhibitors for selectivity versus thrombin and trypsin was achieved by substitution on the benzimidazole ring and replacement of the naphthylamidine group. Substitution of a nitro group at the 4-position on the benzimidazole improves both potency against fXa and selectivity versus thrombin. Alternatively, replacement of the naphthylamidine with either a biphenylamidine or propenylbenzamidine not only improves fXa potency and selectivity versus thrombin, but selectivity versus trypsin as well.     

Regioselective nitration of N(alpha),N(1)-bis(trifluoroacetyl)-L-tryptophan methyl ester: efficient synthesis of 2-nitro and 6-nitro-N-trifluoroacetyl-L-tryptophan methyl ester

Nitration of N(alpha),N(1)-bis(trifluoroacetyl)-l-tryptophan methyl ester with HNO(3) in acetic anhydride at 0 degrees C provides N(alpha)-trifluoroacetyl-2-nitro-l-tryptophan methyl ester in 67% yield, whereas nitration in trifluoroacetic acid at 0 degrees C gives N(alpha)-trifluoroacetyl-6-nitro-l-tryptophan methyl ester in 69% yield.     

The kinetics and mechanisms of the reaction of iron(III) with gallic acid, gallic acid methyl ester and catechin.

The kinetics and mechanisms of the reactions of a number of pyrogallol-based ligands with iron(III) have been investigated in aqueous solution at 25 degrees C and ionic strength 0.5 M NaClO(4). Mechanisms have been proposed which account satisfactorily for the kinetic data. These are generally consistent with a mechanism in which the 1:1 complex that is formed initially when the metal reacts with the ligand subsequently decays through an electron transfer reaction. There was also some evidence for the formation of a 1:2 ligand-to-metal complex at higher pH values. The kinetics of complex formation were investigated with either the ligand or metal in pseudo-first-order excess. Rate constants for k(1) of 2.83(+/-0.09)x10(3), 1.75(+/-0.045)x10(3) and 3300(+/-200) M(-1) s(-1) and k(-1) of 20(+/-6.0), 35(+/-13) and 25+/-7.6 M(-1) s(-1) have been evaluated for the reaction of Fe(OH)(2+) with gallic acid, gallic acid methyl ester and catechin, respectively. The stability constant of each [Fe(L)](+) complex has been calculated from the kinetic data. The iron(III) assisted decomposition of the initial iron(III) complex formed was investigated. Analysis of the kinetic data yielded both the equilibrium constants for protonation of the iron(III) complexes initially formed together with the rate constants for the intramolecular electron transfers for gallic acid and gallic acid methyl ester. All of the suggested mechanisms and calculated rate constants are supported by calculations carried out using global analysis of time-dependent spectra.     

The interaction of thrombin with fibrinogen. A structural basis for its specificity.

The structure of the ternary complex of human alpha-thrombin with a covalently bound analogue of fibrinopeptide A and a C-terminal hirudin peptide has been determined by X-ray diffraction methods at 0.25 nm resolution. Fibrinopeptide A folds in a compact manner, bringing together hydrophobic residues that slot into the apolar binding site of human alpha-thrombin. Fibrinogen residue Phe8 occupies the aryl-binding site of thrombin, adjacent to fibrinogen residues Leu9 and Val15 in the S2 subsite. The species diversity of fibrinopeptide A is analysed with respect to its conformation and its interaction with thrombin. The non-covalently attached peptide fragment hirudin(54-65) exhibits an identical conformation to that observed in the hirudin-thrombin complex. The occupancy of the secondary fibrinogen-recognition exosite by this peptide imposes restrictions on the manner of fibrinogen binding. The surface topology of the thrombin molecule indicates positions P1'-P3', differ from those of the canonical serine-proteinase inhibitors, suggesting a mechanical model for the switching of thrombin activity from fibrinogen cleavage to protein-C activation on thrombomodulin complex formation. The multiple interactions between thrombin and fibrinogen provide an explanation for the narrow specificity of thrombin. Structural grounds can be put forward for certain congenital clotting disorders.