Amidation of methyl-esterified oligogalacturonides: examination of the reaction products using MALDI-TOF MS

Amidation of methyl-esterified oligogalacturonides (oligoGalA) was studied to produce partly and fully amidated oligoGalA to be used as substrates and/or inhibitors for the characterization of pectolytic enzymes acting on the homogalacturonan backbone. The reactions were performed with varying concentrations of ammonia or methylamine and monitored in time using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) that allows sensitive monitoring of the reactions. MALDI-TOF MS reveals the degree of amidation (DAm) and extent of hydrolysis of methyl-esters. Using this technique the conditions for each of the reactions was optimized. Amidation was performed best under anhydrous conditions at a concentration of 4 M ammonia or methylamine at ambient temperature. Amidation using methylamine reached almost completeness (DAm 95) without hardly any hydrolysis of methyl-esters while amidation with ammonia reached a DAm of 70 on average. After an initial fast amidation, precipitation of the partly amidated oligoGalA reduced the reaction rate enormously. The use of ammonia in aqueous solutions instead off anhydrous ammonia resulted in 6–10% lower DAm values due to the hydrolysis of methyl-esters. Therefore, anhydrous conditions are preferred during amidation. Furthermore, methylamine is a better reagent for amidation of oligoGalA and pectins then ammonia, but also results in totally different products with other properties.     

Reaction of methylamine with human alpha 2-macroglobulin. Mechanism of inactivation

The human protease inhibitor alpha 2-macroglobulin (alpha 2 M) is inactivated by reaction with methylamine. The site of reaction is a protein functional group having the properties of a thiol ester. To ascertain the relationship between thiol ester cleavage and protein inactivation, the rates of methylamine incorporation and thiol release were measured. As expected for a concerted reaction of a nucleophile with a thiol ester, the rates were identical. Furthermore, both rates were first order with respect to methylamine and second order overall. The methylamine inactivation of alpha 2M was determined by measuring the loss of total protease-binding capacity. This rate was slower than the thiol ester cleavage and had a substantial initial lag. However, the inactivation followed the same time course as a conformational change in alpha 2M that was measured by fluorescent dye binding, ultraviolet difference spectroscopy, and limited proteolysis. Thus, the methylamine inactivation of alpha 2M is a sequential two-step process where thiol ester cleavage is followed by a protein conformational change. It is the latter that results in the loss of total protease-binding capacity. A second assay was used to monitor the effect of methylamine on alpha 2M. The assay measures the fraction of alpha 2M-bound protease (less than 50%) that is resistant to inactivation by 100 microM soybean trypsin inhibitor. In contrast to the total protease-binding capacity, this subclass disappeared with a rate coincident with methylamine cleavage of the thiol ester. alpha 2M-bound protease that is resistant to a high soybean trypsin inhibitor concentration may reflect the fraction of the protease randomly cross-linked to alpha 2M. Both the thiol ester cleavage and the protein conformational change rates were dependent on methylamine concentration. However, the thiol ester cleavage depended on methylamine acting as a nucleophile, while the conformational change was accelerated by the ionic strength of methylamine. Other salts and buffers that do not cleave the thiol ester increased the rate of the conformational change. A detailed kinetic analysis and model of the methylamine reaction with alpha 2M is presented. The methylamine reaction was exploited to study the mechanism of protease binding by alpha 2M. At low ionic strength, the protein conformational change was considerably slower than thiol ester cleavage by methylamine. Thus, at some time points, a substantial fraction of the alpha 2M had all four thiol esters cleaved, yet had not undergone the conformational change. This fraction (approximately 50%) retained full protease-binding capacity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Production of Pyridine Synthons by Biotransformations of Benzene Precursors and Their Cyclization with Nitrogen Nucleophiles

Biotransformation of benzene and derivatives has been used to accumulate meta-ring fission products of several catechols, including catechol, 3- and 4- methylcatechols, and 3- (trifluoromethyl) catechol. Dilute solutions of these yellow highly absorbing products were used to examine reaction conditions that affect their facile cyclizations with nitrogen nucleophiles, principally NH₃. The effects of NH₄⁺/NH₃ concentration, pH and temperature on the non-enzymic cyclizations of 2-hydroxymuconic semialdehyde to 2-picolinate and its substituted derivatives have been characterised. As expected, the enzymic product from 3-(trifluoromethyl) catechol cyclised 20 times more slowly than that from 3-methylcatechol under identical reaction conditions. Most of the biological and chemical transformations were studied in very dilute solutions for the convenience of optical absorbance measurements. Because of the known sensitivity of the spectral shifts and extinction coefficients to minor changes in pH between 4 and 8 both of the pyridine synthons (vinylogous β-dicarbonyls) generated by ring cleavage of the catechols and of the 2-picolinates, the pK_a values were determined for the pH-dependent tautomeric shifts of the reactants and products so that reasonably accurate assessments of the stoichiometries of the cyclizations could be made. The yield of pyridines from the ring cleavage synthons was usually 71 to 95 per cent. Other nitrogen nucleophiles (methylamine, hydroxylamine, hydrazine and phenylhydrazine) also reacted with the ring-fission products but the putative N-substituted 2-picolinates were not characterised.     

Kinetics of appearance of sulfhydryl groups in alpha 2-macroglobulin on reaction of the inhibitor with amines

The mechanism of the appearance of sulfhydryl groups in alpha 2-macroglobulin in the reaction with amines was characterized by analyses of the kinetics with ammonia and methylamine. All reactions occurred under pseudo-first-order conditions in the range of pH (7.0-8.6) and amine concentration (10-600 mM) investigated. The logarithm of the pseudo-first-order rate constant increased linearly as a function of pH with a slope of unity, indicating that the unprotonated amine is the active species in the reaction. Plots of the observed pseudo-first-order rate constants vs. concentration of unprotonated amine at constant pH were also linear and gave second-order-rate constants of 0.32 and 13.8 M-1 s-1 for ammonia and methylamine, respectively, at pH 8.0; similar values were obtained at pH 8.6. Activation energies of 85 and 100 kJ mol-1 and activation entropies of 10 and 95 J K-1 mol-1 for ammonia and methylamine, respectively, were estimated from Arrhenius plots, suggesting that the higher reaction rate for methylamine is due primarily to a higher activation entropy. These results are consistent with the release of sulfhydryl groups being caused by a nucleophilic attack of the uncharged amine on a thio ester bond of alpha 2-macroglobulin in a bimolecular reaction occurring under pseudo-first-order conditions. The characteristics of the reaction suggest that the thio ester in each alpha 2-macroglobulin subunit reacts independently and equivalently with the amine and also that the thio ester bond cleavage initiates the reaction sequence leading to inactivation of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optimization of Protease-Catalyzed Acyl Transfer Reactions. Determination of the Partition Constant Characterizing Nucleophile Efficiency

The partitioning of the acyl-enzyme between aminolysis by an added nucleophile and hydrolysis plays a key-role in protease-catalyzed acyl transfer reactions. It can be characterized by the partition constant, which is equal to the nucleophile concentration for which aminolysis and hydrolysis proceed at the same velocity. We describe a method for calculation of the partition constant from the product ratio which is based on the integrated rate equation. Therefore, it can be applied to reactions performed under synthesis-like conditions, i.e. a high degree of nucleophile consumption during the reaction. In principle, the dependence of the partition constant on nucleophile concentration can be determined from a single reaction. V8-protease-catalyzed acyl transfer reactions using Z-Glu-OMe as acyl donor and amino acid amides as nucleophiles were investigated as an application of the method. The central role of the partition constant in optimization of preparative protease-catalyzed acyl transfer reactions is discussed.     

Optimization of Protease-Catalyzed Acyl Transfer Reactions. Determination of the Partition Constant Characterizing Nucleophile Efficiency

The partitioning of the acyl-enzyme between aminolysis by an added nucleophile and hydrolysis plays a key-role in protease-catalyzed acyl transfer reactions. It can be characterized by the partition constant, which is equal to the nucleophile concentration for which aminolysis and hydrolysis proceed at the same velocity. We describe a method for calculation of the partition constant from the product ratio which is based on the integrated rate equation. Therefore, it can be applied to reactions performed under synthesis-like conditions, i.e. a high degree of nucleophile consumption during the reaction. In principle, the dependence of the partition constant on nucleophile concentration can be determined from a single reaction. V8-protease-catalyzed acyl transfer reactions using Z-Glu-OMe as acyl donor and amino acid amides as nucleophiles were investigated as an application of the method. The central role of the partition constant in optimization of preparative protease-catalyzed acyl transfer reactions is discussed.     

Deodorizing effects of tea catechins on amines and ammonia

Deodorizing effects of tea catechins on amines were examined under alkaline conditions to eliminate the neutralization reaction. They showed deodorizing activity on ethylamine, but none on dimethylamine or trimethylamine. Deodorizing activity on ethylamine was found to be in the order of (-)-epigallocatechin gallate > gallic acid > (-)-epigallocatechin (EGC) > (-)-epicatechin gallate > ethyl gallate > (+)-catechin = (-)-epicatechin. Further, reaction products of EGC with methylamine, ethylamine, and ammonia were detected by HPLC, indicating that a deodorizing reaction other than neutralization occurs. From structural analysis of the reaction product with the methylamine isolated as a peracetylated derivative, the product was presumed to be methylamine substituted EGC, in which the hydroxyl group of EGC at the 4' position is replaced by the methylamino group. The same replacement reaction took place in the case of ethylamine and ammonia.     

The covalent-binding reaction of complement component C3.

The complement protein C3, when activated by limited proteolysis, forms a short-lived reactive intermediate fragment, 'nascent' C3b, which is known to bind covalently to certain surfaces. The characteristics of the covalent binding reaction have been studied by using Sepharose-trypsin as a combined proteolytic activator and binding surface for C3. Binding of C3 to Sepharose-trypsin is saturable, with a maximum of 25-26 molecules of C3b bound per molecule of trypsin. A minimum life-time of about 60 microseconds for the reactive intermediate has been calculated from binding of C3 at saturation. Initial binding efficiencies of over 30% can be obtained at physiological pH and ionic strength. The efficiency of C3 binding to Sepharose-trypsin decreases as pH increases and also shows a slight decline at high ionic strength. The covalent binding of C3 to Sepharose-trypsin can be inhibited by a range of oxygen and nitrogen nucleophiles. Activation of C3 in the presence of radioactive forms of four such nucleophiles, phenylhydrazine, methylamine, glycerol and glucosamine results in apparent covalent incorporation of the nucleophile into the C3d fragment of C3. The quantity of radioactive nucleophile bound can be predicted from the observed potency of the nucleophile as an inhibitor of the binding of C3 to Sepharose-trypsin. The radioactive nucleophiles may be considered as 'active-site' labels for C3.     

Nucleophilic Substitution Reactions of 5-Bromo-6-methyluridines

Abstract

The reactions of the 5-bromo-6-methyl-2′,3′-O-isopropylideneuridines 9 and 10 with a number of nucleophiles in hot DMF have been investigated. With acetate ion as the nucleophile, either the 5-acetoxy- (11,12) or the 6-acetoxymethyl- (15) products can be obtained in modest yield depending upon the exact reaction conditions. With nitrogen nucleophiles (aniline or p-methoxybenzylamine) reaction takes place at the 6-methyl carbon, whereas with sulfur nucleophiles (thiophenol, thioacetate) only the 5-substituted products are obtained.     

Papain-catalysed synthesis of dipeptides: a novel approach using free amino acids as nucleophiles.

For the first time, papain-catalysed synthesis of peptide bonds was successfully carried out using free amino acids as nucleophiles. In kinetically controlled experiments employing pH-Stat-mode, the ester substrates Z-Ala-OMe and Z-Gly-OMe were coupled with alanine, glutamine, and Cys(Acm)-OH, respectively. Under optimized reaction conditions (pH 9.2, high ratio nucleophile/carboxyl component, 10 mumol substrate mg-1 papain), the peptide yields ranged from 17% to 79%, depending on the structure of the amino and/or carboxyl component. The peptides formed were not hydrolysed under the chosen reaction conditions. With Z-Gly-OMe as the ester substrate, formation of the dipeptide was both rapid and high yielding. Papain-catalysed formation of peptide bonds applying free amino acids as nucleophiles might serve as an economic and easily manageable approach for the synthesis of short-chain peptides to be used in clinical nutrition.     

Nucleophilic cleavage of the complex between human alpha-1-antitrypsin and porcine pancreatic elastase

Several nucleophilic amines were examined to determine their ability to split the alpha-1-antitrypsin-elastase complex. Hydrazine and hydroxylamine, their methyl derivatives, and methylamine were tested in the pH range of 8 to 10.6. Only hydrazine and methylamine produced complete and clean cleavage of the complex into its inhibitor and enzyme components. When [¹⁴C]-methylamine at pH 10.6 was used about 0.3 mol of the nucleophile was specifically bound per mol of the inhibitor component. An interfering reaction between methylamine and the native inhibitor was controlled by preincubation with unlabeled methylamine.     

Mg2+和Na+对多核酶系统体外切割反应的影响

为了进一步了解2价Mg2+和1价Na+存在与否的情况下,多核酶系统对底物RNA的切割效率,构建了pGEM-Coat'A,pGEM-Coat'A196Rz质粒和pGEM-MDR1靶质粒,通过用SP6/T7转录试剂盒在体外转录RNA,在无细胞系统进行切割反应,反应产物通过6%变性聚丙烯酰胺凝胶电泳,干胶、x光片曝光自显影,利用Image J生物图像分析软件分析,结果表明,多核酶系统的切割效率依赖于二价Mg2+的浓度,切割产物随Mg2+浓度的增加而增加,而且具有反应时间的依赖性,在Na+浓度低于200 mmol/L且单独存在时,没有切割产物生成,相反,在Na+和Mg2+共存时,表现出Na+抑制Mg2+诱导的切割活性,切割效率明显低于Mg2+单独存在时的结果.这些结果提示,在生理环境下,Mg2+对于多核酶系统对底物的切割反应是必需的,而Na+则不是.     

Further characterization of the covalent linking reaction of alpha 2-macroglobulin.

It is shown that non-proteolytic proteins can become covalently linked to alpha 2M (alpha 2-macroglobulin) during its reaction with proteinases, and that this probably occurs by the mechanism that leads to the covalent linking of proteinases described previously [Salvesen & Barrett (1980) Biochem. J. 187, 695-701]. The covalent linking of trypsin was at least partly dependent on the presence of unblocked lysine side chains on the protein. The covalent linking of proteinases was inhibited by nucleophiles of low Mr, and these compounds were themselves linked to alpha 2M in a molar ratio approaching one per quarter subunit. Peptide "mapping" indicated that the site of proteinase-mediated incorporation of the amines was the same as that at which methylamine is incorporated in the absence of a proteinase. The nucleophile-reactive site revealed in alpha 2M after reaction with a proteinase was shown to decay with a t1/2 of 112 s, at pH 7.5. After the reaction with a proteinase or with methylamine, a free thiol group was detectable on each subunit of alpha 2M. We propose that the site for incorporation of methylamine in each subunit is a thiol ester, which in S-alpha 2M (the electrophoretically "slow" form) is sterically shielded from reaction with large nucleophiles, but is revealed as a highly reactive group, free from steric hindrance, after the proteolytic cleavage. We have designated the activated species of the molecule "alpha 2M".     

Steroid nitrogen compounds. 3. A general method for introduction of O, N, S and other functionality into position-5 of cholestanes. Conjugate addition of nucleophiles to 6-nitrosocholesteryl acetate generated in situ from the 5 -chloro-6-oxime

The conjugate addition of organic and inorganic nucleophiles to 6-nitrosocholesteryl acetate (II) generated in situ from 3β-acetoxy-5-chloro-6-hydroxyimino-5α-cholestane (I) has been shown to give high yields of 5α-substituted 6-oximes (IIIa-h) under mild reaction conditions. Nucleophiles include ethanethiol, ethanol, methylamine, ammonia, nitrite, cyanide, thiocyanate and azide. Validity of the proposal that the replacement of chlorine atom of α-chlorooximes by nucleophiles proceeds via an elimination-addition mechanism through nitrosoolefins has been confirmed by the isolation and conversion of II into methoxy oxime (III, X = OMe) by the treatment with methanol. Due to the steric hindrance exerted by α-hydrogen atoms at λ-positions, bulky nucleophiles, which have been reported to react with α-chlorooximes, failed to react with the chlorooxime (I). Effects of 5α-substituents upon the signal position of the 3α-proton are described.     

Identification of alternative products and optimization of 2-nitro-5-thiocyanatobenzoic acid cyanylation and cleavage at cysteine residues

The reagent 2-nitro-5-thiocyanatobenzoic acid (NTCB) is commonly used to cyanylate and cleave proteins at cysteine residues, but this two-step reaction requires lengthy incubations and produces highly incomplete cleavages. In previous reports, incomplete cleavage was attributed to a competing beta-elimination reaction that converts cyanylated cysteine to dehydroalanine. In this study, previously unidentified side reactions of the NTCB cleavage were discovered and beta-elimination was not the major reaction competing with peptide bond cleavage. A major side reaction was identified as carbamylation of lysine residues. Carbamylation could be minimized by desalting the cyanylation reaction before cleavage or by reducing the reactant concentrations, but both methods suffered from further reductions in cleavage efficiency. Based on model peptide studies, poor cleavage was primarily caused by a mass neutral rearrangement of the cyanylated cysteine which produced a cleavage-resistant, nonreducible product. The formation of this product could be minimized by using stronger nucleophiles for the cleavage reaction. We discovered that base-catalyzed nucleophilic cleavage could be achieved with many amino-containing compounds. Most notably, glycine is capable of promoting efficient cleavage. In addition, efficient NTCB cleavage can be performed in a simple one-step method without a prior cyanylation step, rather than the previously described two-step reaction.     

Hydrolysis of nerve agents by model nucleophiles: a computational study

Density functional theory calculations were employed to study the reaction of five nerve agents with model nucleophiles, including EtX(-) and EtXH (X=O, S, Se) for serine, cysteine and selenocysteine, respectively. Calculations at the B3LYP/6-311++G(2d,p) level of theory predict an exothermic reaction between ethoxide and all of the nerve agents studied. As compared to EtO(-) as a nucleophile, these reactions become approximately 30kcal/mol more endothermic for EtS(-), and by approximately 40kcal/mol for EtSe(-). The equivalent reactions with the neutral nucleophiles (EtXH) were more endothermic. The effect of solvation on the reaction thermochemistry was determined using a polarizable continuum model simulating the dielectric constant of chloroform. While there was a large exothermic shift for reactions involving charged nucleophiles with solvation modeling, the corresponding shift was minimal for the reaction with neutral nucleophiles.     

Peptide synthesis with a proteinase from the extremely thermophilic organism Thermus Rt41A

A proteinase isolated from Thermus RT41a was immobilized to controlled pore glass beads and was used in the free and immobilized forms for peptide synthesis. The observed maximum yield was the same in both cases. a number of dipeptides were produced from amino acid esters and amides. The best acyl components, from those tested, were found to be Ac-Phe-OEt and Bz-Ala-OMe. Tur-NH(2), Trp-NH(2), Leu-pNA, and Val-pNA were all reactive nucleophiles.The kinetically controlled synthesis of Bz-ala-Tyr-NH(2) was optimized by studying the effect of pH, temperature, solvent concentration, ionic strength, and nucleophile and acyl donor concentration, ionic strength, and nucleophile and acyl donor concentration on the maximum yield. The initial conditions used were 25 mM Bz-ala-OMe, 25 mM Tyr-NH(2), 70 degrees C, pH 8.0, and 10% v/v dimethylformamide (DMF). The optimum conditions were 90% v/v DMF using 80 mM bz-Ala-OMe and 615 mM Tyr-NH(2) at 40 degrees C and pH 10. These conditions increased the maximum conversion from 0.75% to 26% (of the original ester concentration). In a number of other cosolvents, the best peptide yields were observed with acetonitrile and ethyl acetate. In 90% acetonitrile similar yields were observed to those in 90% DMF under optimized conditions except that the acyl donor and nucleophile concentrations could be reduced to 25 mM and 100mM, respectively. The effect of the blocking group on the nucleophile was also investigated; -betaNA and -pNA as blocking groups improved the yields markedly. The blocking and leaving groups of the acyldonor had no effect on the dipeptide yield. (c) 1994 John Wiley & Sons, Inc.     

Addition of amines and carbon nucleophiles to vinyl sulfone-modified 6-deoxy-hex-3-enopyranoside: a case of nucleophile dependent diastereoselectivity

Reactions of amines and carbon nucleophiles with 4-sulfonyl-hex-3-enopyranoside generate a range of C-3 amino- and C-3 branched-chain sugars, which are analogues of 3-amino-3,6-dideoxy sugars and 3-C-branched-chain-3,6-dideoxy sugars. The diastereoselectivity of addition reaction is nucleophile dependent; while both nitrogen and carbon nucleophiles added in cis-fashion, amines generated C3-C4 trans-diaxial products (gulo-derivatives), and carbon nucleophiles afforded C3-C4 trans-diequatorial products (gluco-analogues).     

Activities of the feline immunodeficiency virus integrase protein produced in Escherichia coli.

Retroviral DNA integration requires the activity of at least one viral protein, the integrase (IN) protein. We cloned and expressed the integrase gene of feline immunodeficiency virus (FIV) in Escherichia coli as a fusion to the malE gene and purified the IN fusion protein by affinity chromatography. The protein is active in site-specific cleavage of the viral DNA ends, DNA strand transfer, and disintegration. FIV IN has a relaxed viral DNA substrate requirement: it cleaves and integrates FIV DNA termini, human immunodeficiency virus DNA ends, and Moloney murine leukemia virus DNA ends with high efficiencies. In the cleavage reaction, IN exposes a specific phosphodiester bond near the viral DNA end to nucleophilic attack. In vitro, either H2O, glycerol, or the 3' OH group of the viral DNA terminus can serve as nucleophile in this reaction. We found that FIV IN preferentially uses the 3' OH ends of the viral DNA as nucleophile, whereas HIV IN protein preferentially uses H2O and glycerol as nucleophiles.     

Microwave‐assisted TFA cleavage of peptides from Merrifield resin

Microwave‐assisted (MW) reactions are of special interest to the chemical community due to faster reaction times, cleaner reactions and higher product yields. The adaptation of MW to solid phase peptide synthesis resulted in spectacular syntheses of difficult peptides. In the case of Merrifield support, used frequently in synthesis of special peptides, the conditions used in product cleavage are not compatible with off‐resin monitoring of the reaction progress. The application of MW irradiation in product removal from Merrifield resin using trifluoroacetic acid (TFA) was investigated using model tetrapeptides and the effects were compared with standard trifluoromethanesulphonic acid (TFMSA) cleavage using elemental analysis as well as chromatographic (HPLC) and spectroscopic (IR) methods. The deprotection of benzyloxycarbonyl and benzyl groups in synthetic bioactive peptides was analyzed using LC‐MS and MS/MS experiments. In a 5 min microwave‐assisted TFA reaction at low temperature, the majority of product is released from the resin, making the analytical scale MW‐assisted procedure a method of choice in monitoring the reactions carried out on Merrifield resin due to the short reaction time and compatibility with HPLC and ESI‐MS conditions. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.