Thermodynamic non-ideality as an alternative source of the effect of sucrose on the thrombin-catalyzed hydrolysis of peptide p-nitroanalide substrates

The inhibitory effect of sucrose on the kinetics of thrombin-catalyzed hydrolysis of the chromogenic substrate S-2238 (D-phenylalanyl-pipecolyl-arginoyl-p-nitroanilide) is re-examined as a possible consequence of thermodynamic non-ideality-an inhibition originally attributed to the increased viscosity of reaction mixtures. However, those published results may also be rationalized in terms of the suppression of a substrate-induced isomerization of thrombin to a slightly more expanded (or more asymmetric) transition state prior to the irreversible kinetic steps that lead to substrate hydrolysis. This reinterpretation of the kinetic results solely in terms of molecular crowding does not signify the lack of an effect of viscosity on any reaction step(s) subject to diffusion control. Instead, it highlights the need for development of analytical procedures that can accommodate the concomitant operation of thermodynamic non-ideality and viscosity effects.

Experimental investigation of heterogeneously catalyzed hydrolysis of esters

Thermodynamics, equilibirum, reaction kinetics, heterogeneous, hydrolysis, ion-exchange resin, calorimetry

A kinetic model for the ultrasound catalyzed hydrolysis of solventless TEOS-water mixtures and the role of the initial additions of ethanol

The acid and ultrasound catalyzed hydrolysis of solventless TEOS-water mixtures are studied, as a function of the initial additions of ethanol to the mixtures, by means of flux calorimetry measurements. A device was specially designed for this purpose. Under acid conditions, our proposed method has been able to resolve hydrolysis from other condensation reactions, by detecting the exothermal hydrolysis reaction heat. The process has been explained by a dissolution and reaction mechanism. Ultrasound forces the dissolution process to start the reaction. The alcohol produced in the reaction helps the dissolution process to further enhance the hydrolysis. Initial amounts of pure ethanol added to the mixtures shorten the start time of the reaction, due to an additional effect of dissolution, and diminish the reaction rate, as a result of the solvent dilution effect. Our dissolution and reaction mechanism modeling describes the main points arising from the experimental data and yields k(H) = 0.24 M(-1) min(-1) for the second-order hydrolysis rate constant at 39 degrees C.

Ras-catalyzed hydrolysis of GTP: a new perspective from model studies.

Despite the biological and medical importance of signal transduction via Ras proteins and despite considerable kinetic and structural studies of wild-type and mutant Ras proteins, the mechanism of Ras-catalyzed GTP hydrolysis remains controversial. We take a different approach to this problem: the uncatalyzed hydrolysis of GTP is analyzed, and the understanding derived is applied to the Ras-catalyzed reaction. Evaluation of previous mechanistic proposals from this chemical perspective suggests that proton abstraction from the attacking water by a general base and stabilization of charge development on the gamma-phosphoryl oxygen atoms would not be catalytic. Rather, this analysis focuses attention on the GDP leaving group, including the beta-gamma bridge oxygen of GTP, the atom that undergoes the largest change in charge in going from the ground state to the transition state. This leads to a new catalytic proposal in which a hydrogen bond from the backbone amide of Gly-13 to this bridge oxygen is strengthened in the transition state relative to the ground state, within an active site that provides a template complementary to the transition state. Strengthened transition state interactions of the active site lysine, Lys-16, with the beta-nonbridging phosphoryl oxygens and a network of interactions that positions the nucleophilic water molecule and gamma-phosphoryl group with respect to one another may also contribute to catalysis. It is speculated that a significant fraction of the GAP-activated GTPase activity of Ras arises from an additional interaction of the beta-gamma bridge oxygen with an Arg side chain that is provided in trans by GAP. The conclusions for Ras and related G proteins are expected to apply more widely to other enzymes that catalyze phosphoryl (-PO(3)2-) transfer, including kinases and phosphatases.

Calorimetric study of the effect of water quantity on tetramethoxysilane hydrolysis under ultrasound stimulation

The ultrasound stimulated and oxalic acid-catalyzed hydrolysis of tetramethoxysilane (TMOS) was studied by means of a heat flux calorimetric method as a function of the initial water/TMOS molar ratio (r) ranging from 2 to 10. The method is based on the time recording of the hydrolysis exothermic heat peak. which takes place in acidulated heterogeneous water-TMOS mixtures under ultrasonic stimulation, accounting for the instantaneous hydrolysis rate. The hydrolysis rate increases from zero up to a maximum value during the heterogeneous step of the process and then diminishes naturally according to the reactant consumption. The total hydrolyzed quantity was found to be slightly increasing with r. The immiscibility gap of the TMOS- water system in the presence of the hydrolysis products has been inferred from the evaluation of the reacted quantity during the heterogeneous step of the reaction and it has been represented in a ternary diagram in the studied r-range.

Study of the hydrolysis of TEOS-TMOS mixtures under ultrasound stimulation

Pure and mixed tetramethoxysilane (TMOS) and tetraethoxysilane (TEOS) were hydrolyzed at 35 degrees C, using oxalic acid as a catalyst and ultrasound stimulation. The hydrolysis reaction was carried out in a specially designed device, in which a heat flow steady state, between the ultrasound source and an external thermostatic bath, was maintained, in the absence of reactions. The exothermic hydrolysis causes a time dependent thermal peak. An induction time is apparent in pure TEOS before the hydrolysis peaks starts, which has been explained by the initial immiscibility gap of the TEOS-water system. The induction time was found to be approximately of the same magnitude as in the HCl catalyzed hydrolysis, in spite of the uncertainty accompanying the peak definition. No induction period is apparent in pure TMOS, so that the hydrolysis starts with its maximum rate. Two independent thermal peaks in the mixed TMOS-TEOS samples were found, both associated to the respective hydrolyses of the pure component. The induction time for the TEOS hydrolysis is decreased as more alcohol (and silanol) is produced in the earlier TMOS hydrolysis. This effect is explained by improvement of homogenization by alcohol.

A CALORIMETRIC STUDY OF THE ULTRASOUND-STIMULATED HYDROLYSIS OF SOLVENTLESS TEOS-WATER MIXTURES

The kinetics of ultrasound-stimulated and HCl-catalyzed hydrolysis of solventless TEOS-water mixtures was studied as a function of temperature ranging from 10 degrees C up to 65 degrees C by means of flux calorimetry measurements. A specially designed device was utilized for this purpose. The exothermic peak arising few minutes after sonication began has been attributed mainly to the hydrolysis reaction. The overall hydrolysis process, which was measured through the irradiation time up to the hydrolysis peak, was found to be thermally activated, with an apparent activation energy Delta E = 36.4 kJ/mol. The alcohol produced at the early hydrolysis due to sonication seems to further enhance the reaction, via a parallel autocatalytic path, which is controlled by a faster pseudo second order rate constant (k'). Our modeling yielded k' = 6.3 x 10(-2) M(-1) min(-1) at 20 degrees C, which is in a reasonable agreement with the literature, and an activation energy Delta E = 40.4 kJ/mol for the specific process of hydrolysis in presence of alcohol.

Lipase-catalyzed kinetic resolution of beta-borylated carboxylic esters

The kinetic resolution of chiral beta-borylated carboxylic esters via lipase-catalyzed hydrolysis and transesterification reactions was studied. The enantioselective hydrolysis catalyzed by CAL-B furnished the beta-borylated carboxylic acid with reasonable enantiomeric excess (62% ee), while both methyl and ethyl beta-borylated carboxylic esters were recovered with excellent ee (>99%). Meanwhile, the transesterification reaction of beta-borylated carboxylic esters and several alcohols, catalyzed by CAL-B, only indicated a high selectivity when ethanol and methyl-(beta-pinacolylboronate)-butanoate were used as substrates, which gave ethyl-(beta-pinacolylboronate)-butanoate with >99% ee. (C) 2012 Elsevier Ltd. All rights reserved.

Kinetic characterization for pretreatment of timber varieties and switchgrass using diluted acid hydrolysis

In recent years, growing attention has been devoted to the use of lignocellulosic biomass as a feedstock to produce renewable carbohydrates as a source of energy products, including liquid alternatives to fossil fuels. The benefits of developing woody biomass to ethanol technology are to increase the long-term national energy security, reduce fossil energy consumption, lower greenhouse gas emissions, use renewable rather than depletable resources, and create local jobs. Currently, research is driven by the need to reduce the cost of biomass-ethanol production. One of the preferred methods is to thermochemically pretreat the biomass material and subsequently, enzymatically hydrolyze the pretreated material to fermentable sugars that can then be converted to ethanol using specialized microorganisms. The goals of pretreatment are to remove the hemicellulose fraction from other biomass components, reduce bioconversion time, enhance enzymatic conversion of the cellulose fraction, and, hopefully, obtain a higher ethanol yield. The primary goal of this research is to obtain kinetic detailed data for dilute acid hydrolysis for several timber species from the Upper Peninsula of Michigan and switchgrass. These results will be used to identify optimum reaction conditions to maximize production of fermentable sugars and minimize production of non-fermentable byproducts. The structural carbohydrate analysis of the biomass species used in this project was performed using the procedure proposed by National Renewable Energy Laboratory (NREL). Subsequently, dilute acid-catalyzed hydrolysis of biomass, including aspen, basswood, balsam, red maple, and switchgrass, was studied at various temperatures, acid concentrations, and particle sizes in a 1-L well-mixed batch reactor (Parr Instruments, ii Model 4571). 25 g of biomass and 500 mL of diluted acid solution were added into a 1-L glass liner, and then put into the reactor. During the experiment, 5 mL samples were taken starting at 100°C at 3 min intervals until reaching the targeted temperature (160, 175, or 190°C), followed by 4 samples after achieving the desired temperature. The collected samples were then cooled in an ice bath immediately to stop the reaction. The cooled samples were filtered using 0.2 μm MILLIPORE membrane filter to remove suspended solids. The filtered samples were then analyzed using High Performance Liquid Chromatography (HPLC) with a Bio-Rad Aminex HPX-87P column, and refractive index detection to measure monomeric and polymeric sugars plus degradation byproducts. A first order reaction model was assumed and the kinetic parameters such as activation energy and pre-exponential factor from Arrhenius equation were obtained from a match between the model and experimental data. The reaction temperature increases linearly after 40 minutes during experiments. Xylose and other sugars were formed from hemicellulose hydrolysis over this heat up period until a maximum concentration was reached at the time near when the targeted temperature was reached. However, negligible amount of xylose byproducts and small concentrations of other soluble sugars, such as mannose, arabinose, and galactose were detected during this initial heat up period. Very little cellulose hydrolysis yielding glucose was observed during the initial heat up period. On the other hand, later in the reaction during the constant temperature period xylose was degraded to furfural. Glucose production from cellulose was increased during this constant temperature period at later time points in the reaction. The kinetic coefficient governing the generation of xylose from hemicellulose and the generation of furfural from xylose presented a coherent dependence on both temperature and acid concentration. However, no effect was observed in the particle size. There were three types of biomass used in this project; hardwood (aspen, basswood, and red maple), softwood (balsam), and a herbaceous crop (switchgrass). The activation energies and the pre-exponential factors of the timber species and switchgrass were in a range of 49 - 180 kJ/mol and from 7.5x104 - 2.6x1020 min-1, respectively, for the xylose formation model. In addition, for xylose degradation, the activation energies and the preexponential factors ranged from 130 - 170 kJ/mol and from 6.8x1013 - 3.7x1017 min-1, respectively. The results compare favorably with the literature values given by Ranganathan et al, 1985. Overall, up to 92 % of the xylose was able to generate from the dilute acid hydrolysis in this project.

p-cresol as a reversible acylium ion scavenger in solid-phase peptide synthesis

In this work we have defined the nature of the p-cresol and p-thiocresol adducts generated from acylium ions during HF cleavage, following contemporary Boc/benzyl solid-phase peptide synthesis. Contrary to the results in previous reports, we found that both p-cresol and p-thiocresol predominantly form. aryl esters under typical cleavage conditions. Initially we investigated a number of small peptides containing either a single glutamate residue or a C-terminal long-chain amino acid which allowed us to unambiguously characterize the scavenged side products. Whereas, the p-cresol esters are stable at 0 degrees C they rearrange irreversibly at higher temperatures (5-20 degrees C) to form aryl ketones. By contrast, p-thiocresol esters do not undergo a Fries rearrangement but readily undergo further additions of p-thiocresol to form ketenebisthioacetals and trithio ortho esters, even at low temperatures. Importantly, we found by LC/MS and FT-ICR MS analysis that peptides containing p-cresol esters at glutamyl side chains are susceptible to amidation and fragmentation reactions at these sites during standard mild base workup procedures. The significance of these side reactions was further demonstrated in the synthesis of neutrophil immobilization factor, a 26-residue peptide, containing four glutamic acid residues. The side reactions were largely avoided by mild hydrogen peroxide-catalyzed hydrolysis which converted the p-cresol adducts to the free carboxylic acids in near quantitative yield. The choice of p-cresol as a reversible acylium ion scavenger when coupled with the simple workup conditions described is broadly applicable to Boc/benzyl peptide synthesis and will significantly enhance the quality of peptides produced.

Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and metalloproteases

Two intramolecularly quenched fluorogenic peptides containing o-aminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acid residues, Abz-DArg-Arg-Leu-EDDnp (Abz-DRRL-EDDnp) and Abz-DArg-Arg-Phe-EDDnp (Abz-DRRF-EDDnp), were selectively hydrolyzed by neutral endopeptidase (NEP, enkephalinase, neprilysin, EC 3.4.24.11) at the Arg-Leu and Arg-Phe bonds, respectively. The kinetic parameters for the NEP-catalyzed hydrolysis of Abz-DRRL-EDDnp and Abz-DRRF-EDDnp were Km = 2.8 µM, kcat = 5.3 min-1, kcat/Km = 2 min-1 µM-1 and Km = 5.0 µM, kcat = 7.0 min-1, kcat/Km = 1.4 min-1 µM-1, respectively. The high specificity of these substrates was demonstrated by their resistance to hydrolysis by metalloproteases [thermolysin (EC 3.4.24.2), angiotensin-converting enzyme (ACE; EC 3.4.24.15)], serineproteases [trypsin (EC 3.4.21.4), a-chymotrypsin (EC 3.4.21.1)] and proteases present in tissue homogenates from kidney, lung, brain and testis. The blocked amino- and carboxyl-terminal amino acids protected these substrates against the action of aminopeptidases, carboxypeptidases and ACE. Furthermore, DR amino acids ensured total protection of Abz-DRRL-EDDnp and Abz-DRRF-EDDnp against the action of thermolysin and trypsin. Leu-EDDnp and Phe-EDDnp were resistant to hydrolysis by a-chymotrypsin. The high specifity of these substrates suggests their use for specific NEP assays in crude enzyme preparations

Gas-Phase Acylium Ion Transfer Reactions Mediated by a Proton Shuttle Mechanism

The mechanism and the energy profile of the gas-phase reaction that mimics esterification under acidic conditions have been investigated at different levels of theory. These reactions are known to proceed with rate constants close to the collision limit in the gas-phase and questions have been raised as to whether the typical addition-elimination mechanism via a tetrahedral intermediate can explain the ease of these processes. Because these reactions are common to many organic and biochemical processes it is important to understand the intrinsic reactivity of these systems. Our calculations at different levels of theory reveal that a stepwise mechanism via a tetrahedral species is characterized by energy barriers that are inconsistent with the experimental results. For the thermoneutral exchange between protonated acetic acid and water and the exothermic reaction of protonated acetic acid and methanol our calculations show that these reactions proceed initially by a proton shuttle between the carbonyl oxygen and the hydroxy oxygen of acetic acid mediated by water, or methanol, followed by displacement at the acylium ion center. These findings suggest that the reactions in the gas-phase should be viewed as an acylium ion transfer reaction. (C) 2010 Wiley Periodicals, Inc. Int J Quantum Chem 111: 1596-1606, 2011

Synthesis and optimization of colloidal silica nanoparticles and their functionalization with methacrylic acid

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Small-angle X-ray scattering and nitrogen adsorption study of the nanoporosity elimination in TEOS sonohydrolysis-derived xerogels

Small-angle X-ray scattering (SAXS) and nitrogen adsorption techniques were used to study the temperature and time structural evolution of the nanoporosity in silica xerogels prepared from acid- and ultrasound-catalyzed hydrolysis of tetraetboxysilane (TEOS). Silica xerogels present a structure of nanopores of fully random shape, size, and distribution, which can be described by an exponential correlation function gamma(r) = exp (-r/a), where a is the correlation distance, as predicted by the Debye, Anderson, and Brumberger (DAB) model. The mean pore size was evaluated as about 1.25 nm from SAXS and about 1.9 nm from nitrogen adsorption. The nanopore elimination in TEOS sonohydrolysis-derived silica xerogels is readily accelerated at temperatures around 900 degrees C probably by the action of a viscous flow mechanism. The nanopore elimination process takes place in such a way that the pore volume fraction and the specific surface are reduced while the mean pore size remains constant. (c) 2005 WILEY-VCH Verlag GmbH S Co. KGaA, Weinheim.

A nonisothermal study of the kinetics of the nanoporosity elimination in sonogels-derived silica xerogels

A nonisothermal study of the kinetics of the nanoporosity elimination in monolithic silica xerogels, prepared from acid and ultrasound catalyzed hydrolysis of tetraethylortosilicate (TEOS), has been carried out by means of in situ linear shrinkage measurements performed with different heating rates. The study could be applied up to almost alpha similar to 0.6 of the volume fraction alpha of eliminated pores. The activation energy was found increasing from about 3.2 x 10(2) kJ/mol for alpha similar to 0.06 up to about 4.4 x 10(2) kJ/mol for alpha. similar to 0.44. The sintering process accompanying the nanopore elimination in this set of xerogels is in agreement with a viscous flux sintering process with the hydroxyl content diminishing with the volume fraction of eliminated pores. All the volume fraction of eliminated pores versus temperature (T) curves can be matched onto a unique curve with an appropriate rescaling of the T axis, independent of the heating rate. This scaling property suggests that the path of sintering seems the same, regardless of the heating rate; the difference is that the rate is faster at higher temperature.