Nitrophenyl Esters Research Articles

29] Human plasminogen

Publisher Summary Human plasminogen is known to possess multiple isoelectric forms, which can be isolated by isoelectric-focusing techniques. Glu-plasminogen 1 possesses multiple forms of pI values of 6.2, 6.3, 6.4, and 6.6. Glu-plasminogen possesses two isoelectric forms with pI values of 6.4 and 6.6. On the other hand, Lys77-plasminogen 1 contains three major isoelectric forms of pI 6.7, 7.2, and 7.5; whereas LysrT-plasminogen 2 contains three major forms of pI values 7.5, 7.8, and 8.1. Assays of plasminogen first require its conversion to plasmin by common activators. The conversion of human plasminogen to human plasmin is accomplished as a result of cleavage of the Arg-Val­-sl peptide bond. The enzyme is then assayed based on its ability to hydrolyze a-N-tosyl-Larginine methyl ester (TAME), utilizing a recording pH-stat to titrate the amount of acid, a-N-tosyl-L-arginine, liberated with time. Assays that rely on the spectrophotometric determination of the plasmin-catalyzed release of p -nitroanilide from the substrate H-D-valyl-L-leucyl-L-lysine-pnitroanilide dihydrochloride and on the determination of the plasmin-catalyzed release of p-nitrophenolate from N-Cbz-L-lysine-p - nitrophenyl ester 3° have also been fruitfully employed. Other assay methods for plasmin are available, such as the casein, azocasein, and fibrin plate assay.

42] Cathepsin G

This chapter presents the procedure for purification and assaying of cathepsin G. Cathepsin G is purified from human neutrophil leukocytes. Cathepsin G is eluted from the carboxymethyl-cellulose column at a higher salt concentration than the elastase. The cathepsin G tends to be contaminated by traces of elastase, which are readily removed by immunoadsorption, or by rechromatography on carboxymethyl-cellulose. Cathepsin G is well stored by lyophilization from solution in a 0.5 M pyridine acetate buffer, pH 5.5. Solutions in 0.5 M NaC1 are also stable at 20°C for long periods. The activity of cathepsin G can be detected in electrophoresis gels by use of a naphthol ester substrate such as Bz-Phe-ONap or the acetate or chloroacetate of naphthol AS-D, liberated naphthol being coupled with a diazonium salt. Moreover, it is quite possible that a burst-assay with a nitrophenyl ester could be adapted for cathepsin G.

Mechanistic alteration in micellar peptide esterolysis reactions

Buffer or CTACl-catalyzed basic cleavages of Z - D - or L -AA- L -Pro p -nitrophenyl esters proceed mainly via intramolecular cyclization to diketopiperazines. These reactions are more facile with DL than with LL substrates.

The effect of ultrasound on the alkaline hydrolysis of nitrophenyl esters

Ultrasound was found to increase the rate of hydrolysis of a series of esters by up to 15%. No effect of molecular structure upon this enhancement was observed.

Unusual kinetic behaviour of plasmin (fibrinolysin) digestion of fibrin and N-benzyloxycarbonyltyrosine nitrophenyl ester.

Unusual kinetic behaviour of plasmin (fibrinolysin) digestion of fibrin and N-benzyloxycarbonyltyrosine nitrophenyl ester.

ChemInform Abstract: SYNTHESIS OF 4‐NITROPHENYL ESTERS OF THYMIDINE 3′‐PHOSPHATE AND 3′‐PHOSPHOROTHIOATE USING A NEW PHOSPHORYLATING AGENT

AbstractDie durch Substitution des entsprechenden Dichlorids mit Anilin zugängliche Verbindung (II) reagiert mit dem Thymidin (I) zu den Diastereomeren (III), die durch Säulenchromatographie getrennt werden können.

Interaction of trypsin with immobilized p-aminobenzamidine derivatives studied by means of affinity electrophoresis

The strength of the interaction of trypsin with immobilized p-aminobenzamidine derivatives was studied by affinity electrophoresis on polyacrylamide gel in Trisdiethylbarbituric acid buffer (pH 8). p-Aminobenzamidine was coupled to two kinds of soluble macromolecular carrier: (i) periodate-oxidized Dextran T-500 and (ii) a synthetic copolymer of N-(2-hydroxypropyl)methacrylamide and 4-nitrophenyl esters of 6-(methacroyl)aminohexanoic acid. The enzyme inhibitor was attached to the periodate oxidized dextran either directly or through glycine, 6-aminohexanoic acid or 12-aminododecanoic acid spacer. The strength of the interaction of trypsin with immobilized p-aminobenzamidine did not depend on the type of macromolecular carrier that the ligand was bound to but it did depend on the length of the spacer arm. The dissociation constants of the trypsin-immobilized p-aminobenzamidine complexes decreased with increasing length of the spacer arm.

‘Inverse’ substrates for butyrylcholinesterase

1. 1. ‘Inverse’-type substrates for butyrylcholinesterase (acylcholine acylhydrolase, EC 3.1.1.8), i.e., p- and o- nitrophenyl esters of (3-carboxypropyl)-trimethylammonium iodide, (4-carboxybutyl)trimethylammonium iodide and (5-carboxypentyl)trimethylammonium iodide were prepared, and their kinetic parameters for butyrylcholinesterase-catalyzed hydrolysis were determined. 2. 2. The hydrolysis of these ‘inverse’-type substrates were found to proceed through specific binding with the enzyme and efficient production of acyl enzyme intermediates, −a pathway essentially identical with that followed by choline esters, normal type substrates.

Preparation of derivatives of inosine 5'-phosphorothioate for use in affinity chromatography

In the paper the known methods of preparation of ribonucleoside 5'-phosphorothioates II are evaluated and an improved method of their synthesis is described, based on the reaction of 2',3'-O-ethoxymethyleneribonucleosides III with thiophosphoryl chloride in the presence of one equivalent of pyridine, and subsequent hydrolysis. Inosine 5'-phosphate and inosine 5'-phosphorothioate(IIc) were converted to 2',3'-O-cyclic ketals of levulinic acid Vb, VIb on reaction with ethyl levulinate and ethyl orthoformate and subsequent alkaline hydrolysis. O-(4-Aminophenyl)inosine 5'-phosphorothioate (IX) was prepared from 2',3'-O-isopropylideneinosine (VII) with O-(4-nitrophenyl)thiophosphoryldiimidazolide and subsequent catalytic hydrogenation of the O-(4-nitrophenyl)ester VIII formed.

Synthesis of 4-nitrophenyl esters of thymidine 3′-phosphate and 3′-phosphorothioate using a new phosphorylating agent

Download options Please wait... Article information DOI https://doi.org/10.1039/C39800000524 Article type Paper Download Citation J. Chem. Soc., Chem. Commun., 1980, 524-525 BibTex EndNote MEDLINE ProCite ReferenceManager RefWorks RIS Permissions Request permissions Synthesis of 4-nitrophenyl esters of thymidine 3′-phosphate and 3′-phosphorothioate using a new phosphorylating agent W. Niewiarowski, W. J. Stec and W. S. Zielinski, J. Chem. Soc., Chem. Commun., 1980, 524 DOI: 10.1039/C39800000524 To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page. If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given. If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page. Read more about how to correctly acknowledge RSC content. Search articles by author Wojcieh Niewiarowski Wojcieh J. Stec Wojcieh S. Zielinski

Syntheses and configurational assignments of the diastereomers of the 4-nitrophenyl esters of thymidine 3′-(N-phenyl phosphoramidate) and thymidine 5′-(N-phenyl phosphoramidate)

The separated diastereomers of 3′-(4-nitrophenyl N-phenyl phosphoramidate)-5′-monomethoxytrityl thymidine and 3′-monomethoxytrityl-5′-(4-nitrophenyl N-phenyl phosphoramidate) thymidine have been prepared and their absolute configurations at phosphorus have been determined.

Mechanism-controlled stereospecificity. Acylation of subtilisin with enantiomeric alkyl and nitrophenyl ester substrates.

The activation parameters of acylation of subtilisin with alkyl and p-nitrophenyl esters of N-acylamino acid enantiomers were determined. It was found that (1) the activation entropy is much higher with the nitrophenyl esters than with the corresponding methyl esters, (2) the difference in rate constants between enantiomers is 10(4)--10(5) with methyl esters whereas it is only of the order of 10 with nitrophenyl esters. The results indicate that the catalytic mechanism is simpler for nitrophenyl esters than for alkyl esters. The simple mechanism requires only general base catalysis, and thus permits more freedom of motion in the transition state, whereas the complex mechanism involves both general base and general acid catalysis. Furthermore, the strikingly low enantiomeric specificity with nitrophenyl esters indicates that not only binding but also the catalytic mechanism is an important factor in determining the stereospecificity of an enzyme. The activation parameters for enantiomeric nitrophenyl ester reactions suggest that structurally related substrates can be transformed by the enzyme in different conformations which may be energetically similar or not. The energetically different conformations may account for the activation enthalpy-entropy compensation.

Catalytic Hydrolysis of p-Nitrophenyl Esters in the Presence of Representative Ammonium Aggregates. Specific Activation of a Cholesteryl Nucleophile Bound to a Dialkylammonium Bilayer Membrane

Abstract Catalytic hydrolysis of p-nitrophenyl esters (acetate and nonanoate) by hydroxamate and imidazole nucleophiles was studied at 30 °C in the presence of aqueous aggregates of single-chain (hexadecyl), double-chain (didodecyl and dioctadecyl) and triple-chain (trioctyl) ammonium amphiphiles. These three types of ammonium salts give rise to very different aggregate morphologies. The rate constant of ester cleavage by the nucleophiles was enhanced several hundred folds in the presence of these hydrophobic aggregates. Simple long-chain nucleophiles possessed esterolytic reactivities which reflect the hydrophobic microenvironment of the respective aggregates: single-chain<double-chain<triple-chain. On the other hand, the cholesteryl ester of imidazolecarboxylic acid showed an especially high reactivity when bound to the didodecylammonium bilayer. Cholic acid-derived nucleophiles showed normal reactivity patterns. Apparently, specific binding of the cholesterol derivative to the bilayer is responsible for the unusual catalytic behavior.

Characteristics of human splenic proteinases active in a neutral medium

Fractionation of proteins of aqueous and salt (1 M KCl) extracts of human spleen on Sephadex G-100 revealed several proteinases hydrolyzing histone and casein and active in a neutral medium. The enzymes were found in the extracts in a relatively inactive form, due to the presence of an inhibitor, chiefly in the aqueous extract. Proteinases active in a neutral medium were found in two protein fractions. “High-molecular-weight” proteinases are inhibited by di-isopropylfluorophosphate (DFP), so that they can be classed in the group of serine proteinases. The fraction of “low-molecular-weight” proteinases contains neutral SH-dependent proteinase (proteinases) and enzymes inhibited by DFP. Kininogenase activity and activity hydrolyzing N-Boc-L-alanine nitrophenyl ester, N-benzoyl-L-tyrosine ethyl ester, and N-benzyl-DL-arginine-p-nitroanilide also were found in this fraction.

Effects of metal ions on the hydrolysis of p‐nitrophenyl caproate by modified poly(ethylenimine)s

AbstractHydrolysis of nitrophenyl caproate by modified poly(ethylenimine)s containing imidazole moieties is markedly enhanced in the presence of divalent metal ions. The order of effectiveness is Cu(II) > Co(II) > Zn(II) > Ni(II) > Mn(II), with Cu increasing the rate about 20‐fold. The acceleration by the metal ion is much greater in the presence of CH3COO− or Cl− as counterions than in the presence of ClO4−. Turnover experiments, in which moles of substrate cleaved were in substantial excess of moles of metal present, established the catalytic nature of the effects of the metals. The extent of binding of Cu(II) by the polymers was measured. Maximum accentuation in rate of hydrolysis of nitrophenyl ester was observed for the imidazole‐containing polymer when the ratio of imidazole:Cu(II) was 2.75. Some possible mechanisms for the rate enhancement by metal ions are described.

Multifunctional hydrolytic catalyses x. Catalytic hydrolysis of p-Nitrophenyl acetate by a polyethylenimine derivative containing hydroxamate (Zwitterionic) group

Abstract A water-soluble polyethylenimine derivatives was prepared containing the zwittenionic hydroxamate group, 11 mol% dococyl side chain, and a tertiary amino group. This polymer hydrolyzes p -nitrophenyl esters very effeciently (30°C, 3 v/v% EtOHH 2 O), according to the Michaelis-Menten kinetics. The catalysis involves substrate binding ( K m = (2–5) × 10 −4 M in the case of p -nitrophenyl acetate) due to hydrophobic interaction, and facile acylation and de-acylation at the hydroxamate site. The zwitterionic nature and the hydrophobic microenvironment contribute to the enchanced nucleophilicity to the hydroxamate group. It is suggested that the de-acylation process involves general-base catalysis by the tert-amino group. The overall catalytic effeciency for p -nitrophenyl acetate exceeded that of α-chymotrypsin by a factor of ca. 100 under a comparable condition. Polymers which do not contain the hydroxamate group or contain the imidazole group in its place were much less effective.

Hydrolytic Reactions of p-Nitrophenyl Esters in Reversed Micellar Systems

Abstract Hydrolytic reactions of several p-nitrophenyl esters were studied with and without catalysts in reversed micellar systems consisting of sodium octanoate, 1-hexanol and water. Hydrolytic rate constants become very large as compared with those in aqueous solutions, increasing with decrease in the molar ratio of water to sodium octanoate (R-value). The results of kinetic measurements were discussed in terms of the behavior of water molecules and polar groups of sodium octanoate as revealed by means of IR and 1H, 13C and 23Na-NMR spectroscopy. It was concluded that the decrease in the R-value induces the increase in the mobility of water molecules and the decrease in the polarity of the water core, which affects the partition coefficient of p-nitrophenyl esters between the 1-hexanol phase and the water phase, enhancing hydrolytic reactions.

Activation of the frog sartorius acetylcholine receptor by a covalently attached group

The frog sartorius motor endplate was treated with the specific disulfide bond reducing agent dithiothreitol and subsequently exposed to a covalently reacting compound (the nitrophenyl ester of p-carboxyphenyltrimethylammonium iodide, NPTMB) known to activate the dithiothreitol-reduced acetylcholine receptor in Electrophorus electroplax. NPTMB causes a maximum depolarization of about 35 mV when applied to the dithiothreitol-treated sartorious motor endplate. It is ineffective on postjunctional membrane prior to disulfide bond reduction and on extrajunctional regions, reduced or unreduced. High concentrations of a competitive antagonist such as (+)-tubocurarine prevent reaction between NPTMB and the reduced receptor and cause a repolarization of the membrane when applied to the already-depolarized preparation. We conclude that in frog muscle, as in electroplax, the attached activator bridges the acetylcholine binding site of the reduced receptor between a sulfhydryl group, to which it is covalently bound, and a negative subsite, with which it forms a reversible ionic band.

Collagen helix stabilization by hydroxyproline in (Ala-Hyp-Gly) n

(Ala-Hyp-Gly) n was synthesized and fractionated to yield three fractions in the molecular weight range 10,000 to 2,500. Optical rotatory and circular dichroism measurements indicated the collagen-like properties of this polymer in contrast to those reported for (Ala-Pro-Gly) n. We conclude that hydroxyproline contributes to collagen-helix stability in general, independent of the presence of an adjacent proline residue as in previously studied synthetic polypeptides. (Pro-Hyp-Gly) n showed identical circular dichroism spectra whether polymerized via the nitrophenyl ester or tetraethylpyrophosphite.

Secondary deuterium isotope effects for certain acyl transfer reactions of phenyl formates

Kinetic ..cap alpha.. secondary deuterium isotope effects, k/sub D//k/sub H/, for formyl group transfer from either the 4-methoxyphenyl or 4-nitrophenyl esters, or both, of formic and deuterioformic acids to a variety of oxygen acceptors have been measured at 25/sup 0/C in aqueous solution: hydroperoxide ion, 1.12; 2-propynol anion, 1.13; hexafluoropropan-2-ol anion, 1.14; and water, 1.22. In addition, corresponding isotope effects have been obtained for general-base-catalyzed formyl group transfer from the same substrates to acetate, 1.21, formate, 1.23, and trimethylamine N-oxide, 1.20. The ..cap alpha.. secondary deuterium isotope effect for acid-catalyzed hydrolysis of both esters has been determined to be 1.24. These data are interpreted to reflect considerable, and perhaps complete, carbon--oxygen bond formation in the transition state for addition of oxygen nucleophiles to phenyl formates. Finally, corresponding isotope effects were determined for reaction of fluoride, 1.19, and azide, 1.14, which also suggest substantial covalent bond formation between ester and nucleophile in the transition state. 4 tables.