P-nitrophenyl Alkanoates Research Articles

P-Nitrophenyl esters provide new insights and applications for the thiolase enzyme OleA

The OleA enzyme is distinct amongst thiolase enzymes in binding two long (≥C8) acyl chains into structurally-opposed hydrophobic channels, denoted A and B, to carry out a non-decarboxylative Claisen condensation reaction and initiate the biosynthesis of membrane hydrocarbons and β-lactone natural products. OleA has now been identified in hundreds of diverse bacteria via bioinformatics and high-throughput screening using p-nitrophenyl alkanoate esters as surrogate substrates. In the present study, p-nitrophenyl esters were used to probe the reaction mechanism of OleA and shown to be incorporated into Claisen condensation products for the first time. p-Nitrophenyl alkanoate substrates alone were shown not to undergo Claisen condensation, but co-incubation of p-nitrophenyl esters and CoA thioesters produced mixed Claisen products. Mixed product reactions were shown to initiate via acyl group transfer from a p-nitrophenyl carrier to the enzyme active site cysteine, C143. Acyl chains esterified to p-nitrophenol were synthesized and shown to undergo Claisen condensation with an acyl-CoA substrate, showing potential to greatly expand the range of possible Claisen products. Using p-nitrophenyl 1-13C-decanoate, the Channel A bound thioester chain was shown to act as the Claisen nucleophile, representing the first direct evidence for the directionality of the Claisen reaction in any OleA enzyme. These results both provide new insights into OleA catalysis and open a path for making unnatural hydrocarbon and β-lactone natural products for biotechnological applications using cheap and easily synthesized p-nitrophenyl esters.

Purification and biochemical characterization of FrsA protein from Vibrio vulnificus as an esterase.

Fermentation-respiration switch protein (FrsA) was thought to play an important role in controlling the metabolic flux between respiration and fermentation pathways, whereas the biochemical function of FrsA was unclear yet. A gene coding for FrsA protein from Vibrio vulnificus was chemically synthesized. The recombinant VvFrsA was expressed as a soluble protein and purified by Ni-NTA affinity chromatography. The protein had a subunit molecular weight of ca. 45 kDa by SDS-PAGE and preferred short-chain esters when p-nitrophenyl alkanoate esters were used as substrates. Optimum condition for VvFrsA was found to be at pH 9.0 and 50 °C. The protein retained high esterase activity at alkaline condition and would denature slowly at over 50 °C. With p-nitrophenyl acetate as the substrate, the Km and kcat were determined to be 18.6 mM and 0.67 s-1, respectively, by steady-state kinetic assay. Molecular dynamics simulation and docking model structure revealed that p-nitrophenyl acetate could be the substrate of VvFrsA. In conclusion our results demonstrated that the protein was able to catalyze the hydrolysis of esters, especially p-nitrophenyl acetate, for the first time.

Characterization of a novel immobilized biocatalyst obtained by matrix-assisted refolding of recombinant polyhydroxyoctanoate depolymerase from Pseudomonas putida KT2442 isolated from inclusion bodies

Purification and matrix-assisted refolding of recombinant His-tagged polyhydroxyalkanoate (PhaZ) depolymerase from Pseudomonas putida KT2442 was carried out. His-tagged enzyme was overproduced as inclusion bodies in recombinant E. coli M15 (pREP4, pPAZ3), which were denatured by 8 M urea, immobilized on Ni(2+)-nitrilotriacetate-agarose matrix, and refolded by gradual removal of the chaotropic agent. The refolded enzyme could not be eluted with 1 M imidazole buffer, leading to an immobilized biocatalyst where PhaZ depolymerase was homogeneously distributed in the agarose support as shown by confocal scanning microscopy. Polyhydroxyoctanoate could not be hydrolyzed by this novel immobilized biocatalyst, whereas the attached enzyme was active in the hydrolysis of p-nitrophenyl alkanoate esters, which differed in their alkyl chain length. Taking advantage of the observed esterase activity on p-nitrophenylacetate, functional characterization of immobilized PhaZ depolymerase was carried out. The immobilized enzyme was more stable than its soluble counterpart and showed optimal hydrolytic activity at 37°C and 50 mM phosphate buffer pH 8.0. Kinetic parameters were obtained with both p-nitrophenylacetate and p-nitrophenyloctanoate, which had not been described so far for the soluble enzyme, representing an attractive and alternative chromogenic assay for the study of this paradigmatic enzyme.

Regio-selective acylation of biologically important iridoid glycosides by Candida antarctica lipase

Lipase catalyzed regio-selective acylation of five iridoid glycosides viz., picroside I&II, catalpol, agnuside and negundoside in the presence of various acyl donors such as vinyl acetate and p-nitrophenyl alkanoates was studied. The regio-selectivity of enzymatic acylation and yields were found to vary amongst different substrates. Monoacylated products were isolated with all the substrates under scrutiny indicating high regio-selective nature of such transformations. A series of acyl esters of picroside-I, picroside-II, catalpol, agnuside and negundoside have been synthesized by this enzymatic trans-esterification methodology.

Biomimetic Hydrolysis of p-Nitrophenyl Alkanoates with Functionalized Mesoporous Silicas

Abstract A biomimetic catalysis of p-nitrophenyl alkanoate esters as a function of their alkyl chain lengths through multifunctionalization of molecular self-assembly onto ordered silica nanopores has been investigated in Tris-HCl buffer, and their kinetic parameters were calculated including a Michaelis–Menten kinetics.

Cloning, purification and properties of a hyperthermophilic esterase from archaeon Aeropyrum pernix K1

The gene APE1547 of the aerobic thermophilic Aeropyrum pernix K1 encoding 582 amino acid residues was cloned into Escherichia coli. BL21 (DE3) by using vector pET11a with a T7 promoter. An alignment of similarity analysis of APE1547 with protein sequences from A. pernix K1 databank revealed that it showed a lipase motif and low homology with the known thermophilic esterases. However, it had a high degree homology with several acyl amino acid-releasing enzymes. After purified by ion exchange chromatography and gel filtration chromatography, the recombinant protein showed both esterase activity and acylamino acid-releasing enzyme (AARE) activities. The optimum of temperature and pH of the esterase activity are 90 °C and 8.0, respectively. The recombinant protein showed the hydrolytic activity for a wide range of substrates, such as p-nitrophenyl alkanoate esters of varying alkyl chain lengths, pNA-labelled amino acid and peptide. The highest activity was observed for the substrate p-nitrophenyl caprylate. The recombinant enzyme was extremely stable and protein concentration-dependent. Its half-life at 90 °C was over 160 h. at the concentration of 2.14 mg/ml, which renders this new esterase very attractive for biotechnological applications.

Catalysis by colloidal polymers in aqueous media

Crosslinked polymer latexes were prepared by emulsion copolymerization of either styrene or one of 14 different alkyl methacrylates and vinylbenzyl chloride and converted to quaternary ammonium ion latexes by reaction with trimethylamine or tributylamine. As catalytic colloidal media the cation exchange latexes increased the rate decarboxylation of 6-nitrobenzisoxazole-3-carboxylate up to 10 000 times and the rate of basic hydrolysis of p-nitrophenyl alkanoates up to 16 times in aqueous dispersions at room temperature. Latexes containing either 2-ethylhexyl methacrylate as the inert component of the polymer or tributylammonium ion active sites were most active. Catalytic activity for the decarboxylation did not depend on particle size when the diameter was <1 μm. Polyampholyte latexes containing 29/18 and 25/23 molar ratios of styrylmethyl(trimethyl)ammonium cations and methacrylate anions were colloidally stable in 4 M NaCl. Catalytic activity decreased with increasing concentrations of NaCl in the dispersions, and all of the latexes except the polyampholytes coagulated at>0.1 M NaCl. Dendrimers having one hydrophobic octyl group and one hydrophilic tri(ethylene glycol) methyl ether group on each tertiary amine end were prepared from commercial poly(propylene imine) dendrimers having eight and 32 primary amine end groups and were converted to quaternary ammonium dendrimers by methyl iodide. After ion-exchange for chloride, the polycationic dendrimers increased the rate of the decarboxylation by 200–500 times the rate in pure water.

Catalysis of ester aminolysis by cyclodextrins. The reaction of alkylamines with p-nitrophenyl alkanoates

The effects of four cyclodextrins (alpha-CD, beta-CD, hydroxypropyl-beta-CD, and gamma-CD) on the aminolysis of p-nitrophenyl alkanoates (acetate to heptanoate) by primary amines (n-propyl to n-octyl, isobutyl, isopentyl, cyclopentyl, cyclohexyl, benzyl) in aqueous solution have been investigated. Rate constants for amine attack on the free and CD-bound esters (k(N) and k(cN)) have ratios (k(cN)/k(N)) varying from 0.08 (retardation) to 180 (catalysis). For the kinetically equivalent process of free ester reacting with CD-bound amine (k(Nc)), the ratios k(Nc)/k(N) vary from 0.2 to 28. Either way, there is evidence of catalysis in some cases and retardation in others. Changes in reactivity parameters with structure indicate more than one mode of transition state binding to the CDs. Short esters react with short alkylamines by attack of free amine on the ester bound by its aryl group, but for longer amines, free ester reacts with CD-bound amine. Reaction of long esters with long amines, which is catalyzed by beta-CD and gamma-CD, involves inclusion of the alkylamino group and possibly the ester acyl group. The larger cavity of gamma-CD may allow the inclusion of the ester aryl group, as well as the alkylamino group, in the transition state. Reaction between an ester bound to the CD by its acyl group and free amine appears not to be important.

Molecular mechanics and molecular dynamics calculations of the β-cyclodextrin inclusion complexes with m-, and p-nitrophenyl alkanoates

The inclusion complexes of a series of m- and p-nitrophenyl alkanoates have been studied using molecular mechanics and molecular dynamics computations. Inclusion complexes are likely to exist for the m-series while external complexation seems to be important in the p-series. Solvation effects depend on the force field used. While amber ∗ stabilizes more the separated molecules, mm3 ∗ favors the inclusion complex; computed dissociation constants change accordingly.

Syntheses of imidazolium‐bridged cyclodextrin dimers and their catalytic properties in the hydrolytic cleavage of p‐nitrophenyl alkanoates

AbstractTwo imidazolium‐bridged cyclodextrin dimers 3a and 3b were prepared by reacting 6‐deoxy‐6‐N‐imidazolyl‐β‐CD (2) with bis(bromomethyl) benzene. The catalytic properties of 2, 3a and 3b in the hydrolytic cleavage of p‐nitrophenyl alkanoates, in the form of acetate (PNPA), butanoate (PNPB), hexanoate (WH) and octanoate ( PNPO), were examined. CD dimers showed middling rate enhancements around neutrality. Catalytic rate constants ( kc) in the presence of 3a or 3b did not vary much with chain length of esters. In contrast, dissociation constants (Kd) and selectivity factors (kc/Kd) for “long‐chain” esters were much smaller and significantly larger than those for “short‐chain” ones respectively, indicating CD dimers 3a and 3b have good dimensional recognition ability and substrate selectivity in the hydrolytic cleavage of p‐nitrophenyl alkanoate. Their kinetic consequences are briefly interpreted.

Control of Substrate Specificity in Polymer-Catalyzed Solvolysis Reactions of p-Nitrophenyl Alkanoates by Changing the Buffer System

The substrate specificity in solvolysis reactions of p-nitrophenyl alkanoates 2 (n = 2−18) catalyzed by 4-(dialkylamino)pyridine-functionalized polymer 1 was examined in buffered aqueous methanol solution at pH 8.0 and 30 °C. The chemical reactivity and substrate specificity in this catalytic system were found to be controlled by changing the buffer system. In 1:1 (v/v) methanol−aqueous phosphate buffer solution, macromolecule 1 exhibits substrate specificity for 2 (n = 14) below 1.0 × 10-5 unit mol L-1, and the substrate specificity changes from 2 (n = 14) to 2 (n = 12) as the concentration of 1 increases to 2.5 × 10-5 unit mol L-1 and changes again from 2 (n = 12) to 2 (n = 10) when the concentration of 1 increases further to 7.5 × 10-5 unit mol L-1. However, in 1:1 (v/v) methanol−aqueous Tris buffer solution, macromolecule 1 was found to demonstrate the same substrate specificity for 2 (n = 14) when the concentration of 1 is increased from 5.0 × 10-6 to 1.0 × 10-4 unit mol L-1. The control of substrate specificity by the change of the buffer system is believed to be unprecedented for catalysis of ester solvolysis.

Polymer Concentration-Controlled Substrate Specificity in Solvolysis of p-Nitrophenyl Alkanoates Catalyzed by 4-(Dialkylamino)pyridine-Functionalized Polymer in Aqueous Methanol Solution

The substrate specificity in solvolysis reactions of p-nitrophenyl alkanoates 2 (n = 2−18) catalyzed by 4-(dialkylamino)pyridine-functionalized polymer 1 can be controlled by the concentration of 1 in 50:50 (v/v) methanol−aqueous phosphate buffer solution at pH 8.0 and 30 °C. Below 1.0 × 10-5 unit mol L-1, macromolecule 1 exhibits substrate specificity for 2 (n =14). As the concentration of 1 increases to 2.5 × 10-5 unit mol L-1, the substrate preference changes from 2 (n = 14) to 2 (n = 12). The substrate specificity changes again from 2 (n = 12) to 2 (n = 10) when the concentration of 1 increases further to 7.5 × 10-5 unit mol L-1. The control of substrate specificity by polymer catalyst concentration is believed to be unprecedented for catalysis of ester solvolysis.

Transition state stabilization by micelles: the hydrolysis of p-nitrophenyl alkanoates in cetyltrimethylammonium bromide micelles

The cleavage of p-nitrophenyl alkanoates (acetate to octanoate) at high pH is modestly catalyzed by micelles formed from cetyltrimethylammonium bromide (CTAB) in aqueous solution. Rate constants exhibit saturation behaviour with respect to [CTAB], consistent with substrate binding in the micelles. The strength of substrate binding and transition state binding to the micelles increases monotonically with the acyl chain length, and with exactly the same sensitivity. As a result, the extent of acceleration (or catalytic ratio) is independent of the ester chain. These and earlier results are consistent with the reaction centre being located in the Stern layer of the micelle, with the acyl chain of the ester being directed into the hydrophobic micellar interior. The chain length dependence of kinetic parameters found in this work is comparable to that found previously for ester cleavage by cyclodextrins and by various enzymes with hydrophobic binding sites, as well as to that observed for other phenomena involving hydrophobic effects. Keywords: catalysis, ester hydrolysis, micelles, transition state.

Effects of Surfactants on Solvolysis of p-Nitrophenyl Alkanoates Catalyzed by 4-(Dialkylamino)pyridine-Functionalized Polymer in Aqueous Methanol Solution

Catalysis of the solvolysis of p-nitrophenyl alkanoates 2 (n = 2-18) by 4-(dialkylamino)pyridine-functionalized polymer 1 in the presence of sodium n-dodecyl sulfate (SDS), n-dodecyltrimethylammonium bromide (DTAB), and heptaethylene glycol n-dodecyl ether (HEGDE) was investigated in 50:50 (v/v) methanol-water buffer (0.05 M H 2 PO 4 - /HP0 4 2- , pH 8.0) solution. Strikingly, a large rate enhancement is observed in the presence of anionic surfactant SDS for the solvolysis of 2 (n = 10-18) as compared with the corresponding reaction catalyzed by 1 or SDS alone. SDS is believed to bind both substrate and catalyst within micellar aggregates and influence reactivity by a combination of catalyst-substrate proximity and micellar microenvironment. In the presence of HEGDE, no significant effects on the 1-catalyzed solvolysis of 2 (n < 8) were observed. At (2.5-7.5) x 10 -5 unit mol L -l 1, the solvolysis rate exhibits a modest decrease for 2 (n = 10 and 12) and a modest increase for 2 (n = 14, 16, and 18) with added nonionic surfactant HEGDE. The solvolysis rate for 1-catalyzed solvolysis of 2 (n = 2 -18) was found to be unchanged by cationic surfactant DTAB in aqueous methanol solution.

Esterolytic Reactivities of (Dialkylamino)pyridine Amphiphiles Solubilized in Different Pseudo-Three-Component Cationic Microemulsions

Hydrolysis reactions at pH 8.7 of p-nitrophenyl alkanoates of varying chain lengths mediated by four (dialkylamino)pyridine (DAAP)-functionalized amphiphiles were examined in three different microemulsion (ME) media. All the ME systems studied herein were stabilized by a cationic surfactant, cetyltrimethylammonium bromide (CTABr), and a cosurfactant, 1-butanol. The three MEs could also be described as (a) an oil-in-water, (b) a water-in-oil, and (c) a bicontinuous oil−water ME recipe. The DAAP surfactants employed in this study differ in their electrostatic character at the level of headgroup. In order to examine their esterolytic activities, individual DAAP surfactants were separately doped into each ME and the resulting kinetic data for esterolyses were compared with that of the nonamphilphilic (dimethylamino)pyridine. The ester-cleaving abilities of each DAAP catalyst in three different MEs were also compared. Hydrophilic substrates such as 3-acetoxy-4-nitrobenzoic acid or short chain esters like p-nitrophenyl acetate were found to be especially susceptible to cleavage compared to the alkanoate esters having long alkyl chain residues under these conditions in any of the MEs. Importantly these results are markedly different from what we found in CTABr micelles with the same DAAP catalysts, although the crucial catalytic turnover behavior of the DAAPs was maintained even in MEs.

Effect of β-cyclodextrin on the reaction of α-amino acid anions with p-nitrophenyl acetate and p-nitrophenyl hexanoate

The effects of β-cyclodextrin (β-CD) on the kinetics of the reaction of α-amino acid anions with p-nitrophenyl acetate (pNPA) and p-nitrophenyl hexanoate (pNPH) have been investigated. Pseudo-first-order rate constants obtained with various concentrations of nucleophile and β-CD, in aqueous solution at pH 9.88, were analyzed to yield rate constants for reaction of the nucleophiles with free ester (kN) and with ester bound to β-CD (kcN). For pNPA reacting with amino acids that bind weakly to β-CD, if at all, the ratio kcN/kN is very close to one, but for amino acids that bind appreciably to β-CD this ratio is greater than one (up to 3.5). Generally similar behaviour is observed for reactions with pNPH, but the rate ratios are smaller and all less than one. These findings are comparable to those obtained in this laboratory for the CD-mediated reactions of p-nitrophenyl alkanoate esters with other nucleophiles, including simple alkylamines that bind quite strongly to CDs, but they differ from those reported in an earlier study of the reaction of pNPA with amino acid anions (Can. J. Chem. 69, 1124 (1991)) where the data analysis suggested more substantial catalysis by cyclodextrins. Key words: amino acids, cyclodextrin, ester cleavage, kinetics, catalysis.

Direct Kinetic Evidence for the Formation of an Acylpyridinium Intermediate in Synthetic p-Nitrophenyl Esterase-Catalyzed Hydrolysis Reactions

The kinetics of the hydrolysis of p-nitrophenyl alkanoates 2 catalyzed by 1 were investigated in aqueous Tris buffer solution. Direct evidence for the existence of an N-acylpyridinium intermediate 3 was obtained for 1-catalyzed hydrolysis of 2 (n = 2−10). Increase of the alkyl chain length leads to an increase in the acylation rate, which reaches a maximum for 2 (n = 6). The acylation rate then decreases progressively with further increases of alkyl chain length in substrate esters. The deacylation rate was also found to exhibit a maximum for the same substrate 2 (n = 6). These results are similar to those previously reported with cholesterol esterase as catalyst for the same hydrolysis reaction. The acylation reaction is first order in catalyst concentration and exhibits saturation kinetics at high substrate concentration in accordance with the Michaelis−Menten model for enzyme reaction kinetics.

Dialkylaminopyridine catalysed esterolysis of p-nitrophenyl alkanoates in different cationic microemulsions

The reactions of p-nitrophenyl alkanoate esters with dialkylaminopyridine (DAAP) and its related mono- and di-anionic water-soluble derivatives have been studied separately in three different microemulsion (ME) media. These were (a) oil-in-water ME (O/W), (b) water-in-oil ME (W/O) and (c) a bicontinuous ME, where oil and water are in nearly comparable amounts. All the ME systems were stabilized by cationic surfactant, cetyltrimethylammonium bromide (CTABr) and butanol as a cosurfactant. The second-order rate constants (k2) in the microemulsion media were also determined over a phase volume (φ) of approximately 0.13–0.46. In order to explain the contribution of effective concentration of the nucleophiles in the aqueous pseudophase, corrected rate constants k2φ=k2(1 –φ) were obtained. The rate constants of the corresponding hydrolytic reactions were also examined in CTABr micelles. While the DAAP catalysts were partitioned between the micellar and aqueous pseudophases in ME, the hydrophobic substrates were found to be mainly confined to oil-rich phases. Present results indicate that the main effect of ME media on the hydrolysis reaction is due to both electrostatic reasons and substrate partitioning.

Salt Effects on Solvolysis Reactions of p-Nitrophenyl Alkanoates Catalyzed by 4-(Dialkylamino)pyridine- Functionalized Polymer in Buffered Water and Aqueous Methanol Solutions

Specific salting-in effects that lead to striking substrate selectivity were observed for the hydrolysis of p-nitrophenyl alkanoates 2 (n = 2−16) catalyzed by 4-(dialkylamino)pyridine-functionalized polymer 1 in aqueous Tris buffer solution at pH 8.0 and 30 °C. Macromolecule 1 was found to exhibit clear substrate preference for 2 (n = 6) in 0.05 M aqueous Tris buffer solution, as contrasted with the corresponding reaction in 0.05 M aqueous phosphate or borate buffer solutions where the substrate selectivity is absent. The formation of a reactive catalyst−substrate complex, 1·2, appears to be promoted by the presence of tris(hydroxymethyl)methylammonium ion, an efficient salting-in agent, from the Tris buffer system. The salting-in effect on formation of 1·2 complex is presumed responsible for the substrate specificity. The salting-out effects of sodium chloride on the solvolysis of 2 catalyzed by 1 were also investigated in 1:1 (v/v) methanol−water solution at pH 8.0 and 30 °C. The rate of 1-catalyzed sol...

Catalysis of the Reaction of p-Nitrophenyl Alkanoates with Cyclodextrins by Potential Inhibitors: Simple Allosteric Activation

Catalysis of the Reaction of p-Nitrophenyl Alkanoates with Cyclodextrins by Potential Inhibitors: Simple Allosteric Activation