Effect of degree,type, and position of unsaturation on the pKa of long-chain fatty acids

Titration of a series of C(18) fatty acids yields pK(a) values that decrease with an increasing degree of unsaturation in the fatty acid chain. The pK(a) values of stearic, elaidic, oleic, linoleic, and linolenic acids were studied and compared to values of area per molecule in a spread monolayer of these acids. The decrease in pK(a) was found to relate to melting point temperature and area per molecule in the spread fatty acid monolayer. The pK(a) value was determined by first dissolving the fatty acid in a high pH solution (pH>10) and subsequently titrating the solution with HCl to obtain the characteristic S-shaped curves used to calculate the pK(a) values. The pK(a) values of stearic, elaidic, oleic, linoleic, and linolenic acids were found to be 10.15, 9.95, 9.85, 9.24, and 8.28, respectively. These pK(a) values were in the same order as area per molecule values of fatty acids in spread monolayers. This suggests that as area per molecule increases the intermolecular distance increases and pK(a) decreases due to reduced cooperation between adjacent carboxyl groups.     

An accurate density functional theory based estimation of pK(a) values of polar residues combined with experimental data: from amino acids to minimal proteins

We report a scheme for estimating the acid dissociation constant (pK(a)) based on quantum-chemical calculations combined with a polarizable continuum model, where a parameter is determined for small reference molecules. We calculated the pK(a) values of variously sized molecules ranging from an amino acid to a protein consisting of 300 atoms. This scheme enabled us to derive a semiquantitative pK(a) value of specific chemical groups and discuss the influence of the surroundings on the pK(a) values. As applications, we have derived the pK(a) value of the side chain of an amino acid and almost reproduced the experimental value. By using our computing schemes, we showed the influence of hydrogen bonds on the pK(a) values in the case of tripeptides, which decreases the pK(a) value by 3.0 units for serine in comparison with those of the corresponding monopeptides. Finally, with some assumptions, we derived the pK(a) values of tyrosines and serines in chignolin and a tryptophan cage. We obtained quite different pK(a) values of adjacent serines in the tryptophan cage; the pK(a) value of the OH group of Ser13 exposed to bulk water is 14.69, whereas that of Ser14 not exposed to bulk water is 20.80 because of the internal hydrogen bonds.     

Influence of water content on the acidities in acetonitrile. Quantifying charge delocalization in anions

The effect of traces of water on the relative strengths of acids (ΔpK(a) values) in acetonitrile was quantitatively evaluated experimentally and computationally (COSMO-RS). Water affects first of all the anions by selective solvation. Expectedly, the more localized is the charge in acid anions the higher is the effect of water. The energetic effect of increasing water content from 0 to ca. 10,000 ppm on solvation enthalpies of anions ranged from 0.2-0.4 kcal mol?1 (anions with delocalized charges) to 15 kcal mol?1 in the case of the highly charge-localized acetate ion. In the case of ΔpK(a) values the change ranges from 0.01 to ca. 1.7 pK(a) units (acid pair involving acetic acid). The COSMO-RS method was found to satisfactorily describe the trends in ΔpK(a) values. To quantify the extent of charge localization/delocalization in anions a parameter, weighted average positive σ (WAPS), was introduced, which can be conveniently computed using the COSMO approach. WAPS characterizes the distribution of charge density across the molecular surface and was found to correlate well with the extent of water influence on the dissociation of the respective acid.     

Mechanisms of acid decomposition of dithiocarbamates. 3. Aryldithiocarbamates and the torsional effect

The acid decomposition of some p-substituted aryldithiocarbamates (arylDTCs) was observed in 20% aqueous ethanol at 25 degrees C, mu = 1.0 (KCl, for pH > 0). The pH-rate profiles showed a dumbell shape with a plateau where the observed first-order rate constant k(obs) was equal to k(o), the rate constant of the decomposition of the dithiocarbamic acid species. The acid dissociation constants of the dithiocarbamic acids (pK(a)) and their conjugate acids (pK(+)) were calculated from the pH-rate profiles. Comparatively, k(o) was more than 10(4)-fold faster than alkyldithiocarbamates (alkDTCs) with similar pK(N) (the acid dissociation constant of the parent amine). It was observed that the values of pK(a) and pK(+)were 5 and 8 units of pK, respectively, higher than the expected values from the pK(N) of alkylDTCs. The higher values were attributed to the inhibition of the delocalization of the nitrogen electron pair into the benzene ring because of the strong electron withdrawal effect of the thiocarbonyl group. Comparison of the activation parameters showed that the rate acceleration was due to a decrease in the enthalpy of activation. Proton inventory indicated the existence of a multiproton transition state, and it was consistent with an S to N proton transfer through a water molecule. There are two hydrogens contributing to a secondary SIE, and there are also two protons that are being transferred at the transition state to form a zwitterion followed by fast C-N bond cleavage. The mechanism could also be a concerted asynchronic process where the N-protonation is more advanced than the C-N bond breakdown. The kinetic barrier is similar to the torsional barrier of thioamides, suggesting that the driving force to reach the transition state is the needed torsion of the C-N bond that inhibits the resonance with the thiocarbonyl group and the aromatic moiety, increasing the basicity of the nitrogen and making the proton transfer thermodynamically favorable.     

Theoretical evaluation of pK(a) in phosphoranes: implications for phosphate ester hydrolysis

Knowledge of the pK(a) of phosphoranes is important for the interpretation of phosphate ester hydrolysis. Calculated pK(a)'s of the model phosphorane, ethylene phosphorane, are reported. The method of calculation is based on the use of dimethyl phosphate as a reference state for evaluating relative pK(a) values, and on the optimization of the oxygen and acidic hydrogen van der Waals radii to give reasonable pK(1)(a), pK(2)(a), and pK(3)(a) for phosphoric acid in solution. Density functional theory is employed to calculate the gas-phase protonation energies, and continuum dielectric methods are used to determine the solvation corrections. The calculated pK(1)(a) and p(2)(a) for the model phosphorane are 7.9 and 14.3, respectively. These values are within the range of proposed experimental values, 6.5-11.0 for pK(1)(a), and 11.3-15.0 for pK(2)(a). The mechanistic implications of the calculated pK(a)'s are discussed.     

Application of Multiple Linear Regression and Extended Principal-Component Analysis to Determination of the Acid Dissociation Constant of 7-Hydroxycoumarin in Water/AOT/Isooctane Reverse Micelles

The apparent pK(a) of dyes in water-in-oil microemulsions depends on the charge of the acid and base forms of the buffers present in the water pool. Extended principal-component analysis allows the precise determination of the apparent pK(a) and of the spectra of the acid and base forms of the dye. Combination with multiple linear regression increases the precision. The pK(a) of 7-hydroxycoumarin (umbelliferone) was spectrophotometrically measured in a water/AOT/isooctane microemulsion in the presence of a series of buffers carrying different charges at various different water/surfactant ratios. The spectra of the acid and base forms of the dye in the microemulsion are very similar to those in bulk water in the presence of Tris and ammonia. The presence of carbonate changes somewhat the spectrum of the acid form. Results are discussed taking into account the profile of the electrostatic potential drop in the water pool and the possible partition of umbelliferone between the aqueous core and the surfactant. The pK(a) values corrected for these effects are independent of w(0) and are close to the value of the pK(a) in bulk water. Copyright 2000 Academic Press.     

Protonation studies of modified adenine and adenine nucleotides by theoretical calculations and (15)N NMR

The acid/base character of nucleobases affects phenomena such as self-association, interaction with metal ions, molecular recognition by proteins, and nucleic acid base-pairing. Therefore, the investigation of proton-transfer equilibria of natural and synthetic nucleos(t)ides is of great importance to obtain a deeper understanding of these phenomena. For this purpose, a set of ATP prototypes was investigated using (15)N NMR spectroscopy, and the corresponding adenine bases were investigated by theoretical calculations. (15)N NMR measurements provided not only acidity constants but also information on the protonation site(s) on the adenine ring and regarding the ratio of the singly protonated species in equilibrium. Substituents of different nature and position on the adenine ring did not change the preferred protonation site, which remained N1. However, for 2-thioether-ATP derivatives a mixed population of N1 and N7 singly protonated species was observed. Reduction of basicity of 0.4-1 pK(a) units relative to ATP was also observed for all evaluated ATP derivatives, except for 2-Cl-ATP, for which K(a) was ca. 10,000-fold lower. To explain the substitution-dependent variations in the experimental pK(a) values of the ATP analogues, gas-phase proton affinities (PA), Delta Delta G(hyd), and pK(a) values of the corresponding adenine bases were calculated using quantum mechanical methods. The computed PA and Delta Delta G(hyd) values successfully explained the experimental pK(a) values. A computational procedure for the prediction of accurate pK(a) values was developed using density functional theory and polarizable continuum model calculations. In this procedure, we developed a set of parameters for the polarizable continuum model that was fitted to reproduce experimental pK(a) values of nitrogen heterocycles. This method is proposed for the prediction of pK(a) values and protonation site(s) of purine analogues that have not been synthesized or analyzed.     

Determination of the intrinsic site pK(a) value and cooperativity of the symmetrical hexadentate chelator N,N',N'-tris[2-(3-hydroxy-2-oxo-1,2-dihydropyridin-1-yl)acetamido]ethylamine

The three overlapping pK(a) values of N,N',N'-tris[2-(3-hydroxy-2-oxo-1,2-dihydropyridin-1-yl)acetamido]ethylamine, a tripodal hexadentate chelator formed from three 3-hydroxy-2(1H)-pyridinone moieties amide linked to tris-(2-aminoethyl)amine, were determined by simultaneous spectrophotometric and potentiometric titration. The data was analysed by non-linear regression with constraints to deal with (a) the highly correlated absorptivities and (b) the highly correlated pK(a) values. The three pK(a) values were optimized first from the spectrophotometric data (absorbance vs. pH) by non-linear regression to a model in which the molar absorptivity of the ith species ((i)) was constrained by the correlation equation (i) = epsilon (0) + (epsilon (3) - epsilon (0))i 3 with i = 0, 1, 2, 3, where (3) and (0) represent the molar absorptivities of the most protonated and least protonated species, respectively. The molar absorbitivity of the four species defined by three pK(a) values is, therefore, linearly related to proton stoichiometry. The pK(a) values were then optimized from the potentiometric data (pH vs. titrant volume) by non-linear regression to a model in which the three pK(a) values were constrained by the correlation equation pK(a(i)) = pK(a(int)) + b(i - 1) + (i - 2)log(3) where i = 1, 2 or 3. This expresses the three pK(a) values in terms of only two optimizable parameters, the intrinsic site pK(a) (pK(a(int))) and the interaction energy between sites (b). The fixed term (i - 2)log(3) accounts for the statistical effect on the pK(a) values of three equivalent ionizable sites. The modified analytical derivatives required for optimization of these parameters by the Gauss-Newton-Marquardt algorithm and the merits of optimizing pK(a) values with these two correlation equations are discussed. The optimized pK(a) values were 9.31 +/- 0.01, 8.75 +/- 0.01 and 8.19 +/- 0.01. The separation between pK(a) values is 0.58 comprising 0.477 for the statistical effect and 0.081 for the interaction energy while the intrinsic site pK(a) is 8.672 +/- 0.005. The tertiary amine at the centre of the tripodal backbone has a pK(a) of 5.88 +/- 0.03.     

Macroscopic and microscopic ionization constants of the thiol and amine groups in 1-methyl-4-mercaptopiperidine

The acid dissociation constants of 1-methyl-4-mercaptopiperidine (pK(1) = 9.51, pK(2) = 11.33), the 1,1-dimethyl-4-mercaptopiperidinium ion (pK(A) = 9.59) and 1-methyl-4-(methylthio)piperidine (pK(B) = 10.18) have been determined potentiometrically in 3M sodium perchlorate (10% methanol) medium. The ultraviolet absorption of the mercaptide ion has been used to determine the relative proton affinity of the sulphur and nitrogen functions in 1-methyl-4-mercaptopiperidine under the same conditions, and its four microscopic constants (pK(a) = 9.49, pK(b) = 10.23, pK(c) = 11.34, pK(d) = 10.60) have been calculated; pK(A) has also been determined spectrophotometrically. From the results obtained, it can be concluded that the thiol group is more acidic than the amine group and that the Adams relation, K(a) + K(b) = K(1), holds very well when it is assumed that the spectrophotometric values for K(a), and K(b), can be replaced by K(A) and K(B) respectively.     

Accurate determination of low pK values by (1)H NMR titration

The NMR titration methodology to determine acid dissociation constants in aqueous solutions is extended for pK(a) values between 0 and 2, where potentiometric titrations are no longer applicable. (1)H NMR spectra are acquired for single samples of constant acid concentration (e.g. 0.02M), controlled ionic strength (I=1M with HNO(3)/NaNO(3)) and varying pH. To avoid biased pH readings due to the acid error of the glass electrode, true, concentration-based pH values are deduced by combination of the charge balance equation with information from (1)H NMR chemical shifts of the investigated acid. The method has been tested on histidine (pK(1)=1.83+/-0.02) and yielded the dissociation constant of dichloroacetic acid (pK=1.06+/-0.01) for the first time with good accuracy and precision. Dichloroacetic acid is recommended as an NMR spectroscopical "indicator molecule" for in situ monitoring the pH in strong acidic solutions of other equilibrium systems.     

DFT and AIM study of the protonation of nitrous acid and the pKa of nitrous acidium ion

The gas phase and aqueous thermochemistry, NMR chemical shifts, and the topology of chemical bonding of nitrous acid (HONO) and nitrous acidium ion (H(2)ONO(+)) have been investigated by ab initio methods using density functional theory. By the same methods, the dissociation of H(2)ONO(+) to give the nitrosonium ion (NO(+)) and water has also been investigated. We have used Becke's hybrid functional (B3LYP), and geometry optimizations were performed with the 6-311++G(d,p) basis set. In addition, highly accurate ab initio composite methods (G3 and CBS-Q) were used. Solvation energies were calculated using the conductor-like polarizable continuum model, CPCM, at the B3LYP/6-311++G(d,p) level of theory, with the UAKS cavity model. The pK(a) value of H(2)ONO(+) was calculated using two different schemes: the direct method and the proton exchange method. The calculated pK(a) values at different levels of theory range from -9.4 to -15.6, showing that H(2)ONO(+) is a strong acid (i.e., HONO is only a weak base). The equilibrium constant, K(R), for protonation of nitrous acid followed by dissociation to give NO(+) and H(2)O has also been calculated using the same methodologies. The pK(R) value calculated by the G3 and CBS-QB3 methods is in best (and satisfactory) agreement with experimental results, which allows us to narrow down the likely value of the pK(a) of H(2)ONO(+) to about -10, a value appreciably more acidic than literature values.     

Multiwavelength spectrophotometric determination of acid dissociation constants Part IV. Water-insoluble pyridine derivatives

A multiwavelength spectrophotometric (WApH) titration method for determination of acid dissociation constants (pK(a) values) of ionizable compounds developed previously was applied in the case of pyridine derivatives of pharmaceutical interest. Specifically, UV absorption spectra of the drug solution are acquired in the course of a pH-metric titration using an optical device based on a fibre optics dip probe, a light source and a diode array detector. Target factor analysis was applied to deduce the pK(a) values from the spectral data recorded at different pH. Using this technique, the pK(a) values of six pyridine derivatives were determined successfully. It was demonstrated that the WApH technique in this case outperforms conventional pH-metric methods with respect to the measurement of pK(a) values of the sparingly water soluble samples reported in this study.     

Chemistry of covalent inhibition of the gastric (H+, K+)-ATPase by proton pump inhibitors

Proton pump inhibitors (PPIs), drugs that are widely used for treatment of acid related diseases, are either substituted pyridylmethylsulfinyl benzimidazole or imidazopyridine derivatives. They are all prodrugs that inhibit the acid-secreting gastric (H(+), K(+))-ATPase by acid activation to reactive thiophiles that form disulfide bonds with one or more cysteines accessible from the exoplasmic surface of the enzyme. This unique acid-catalysis mechanism had been ascribed to the nucleophilicity of the pyridine ring. However, the data obtained here show that their conversion to the reactive cationic thiophilic sulfenic acid or sulfenamide depends mainly not on pyridine protonation but on a second protonation of the imidazole component that increases the electrophilicity of the C-2 position on the imidazole. This protonation results in reaction of the C-2 with the unprotonated fraction of the pyridine ring to form the reactive derivatives. The relevant PPI pK(a)'s were determined by UV spectroscopy of the benzimidazole or imidazopyridine sulfinylmethyl moieties at different medium pH. Synthesis of a relatively acid stable analogue, N(1)-methyl lansoprazole, (6b), allowed direct determination of both pK(a) values of this intact PPI allowing calculation of the two pK(a) values for all the PPIs. These values predict their relative acid stability and thus the rate of reaction with cysteines of the active proton pump at the pH of the secreting parietal cell. The PPI accumulates in the secretory canaliculus of the parietal cell due to pyridine protonation then binds to the pump and is activated by the second protonation on the surface of the protein to allow disulfide formation.     

Tunable pK of amino acid residues at the air-water interface gives an L-zyme (langmuir enzyme)

Various amino acid-carrying amphiphiles were synthesized, and the pK values of the attached amino acid residues were investigated at the air-water interface and in aqueous vesicles using pi-A isotherm measurements, (1)H NMR titration, and IR spectroscopy in reflection-adsorption mode. The epsilon-amino group of the Lys residue embedded at the air-water interface displays a significant pK shift (4 or 5 unit) compared with that observed in bulk water, while the pK shift in aqueous vesicles was not prominent (ca. 1 unit). Moreover, pK values of the amino acids at the air-water interface can be tuned simply by control of the subphase ionic strength as well as by molecular design of the amphiphiles. A simple equation based on the dominant contribution by the electrostatic energy to the pK shift reproduces well the surface pressure difference between protonated and unprotonated species, suggesting a reduction in the apparent dielectric constant at the air-water interface. Hydrolysis of a p-nitrophenyl ester derivative was used as a model reaction to demonstrate the use of the Lys-functionalized monolayer. Efficient hydrolysis was observed, even at neutral pH, after tuning of pK for the Lys residue in the monolayer, which is a similar case to that occurring in biological catalysis.     

Determination of pK(a) values of diastereomers of phosphinic pseudopeptides by CZE

A CE method was used for the determination of acidity constants (pK(a)) of a series of ten phosphinic pseudopeptides, which varied in number and type of ionogenic groups. Effective electrophoretic mobilities were measured in the 1.8-12.0 pH range in the BGEs of constant ionic strength of 25 mM. Effective electrophoretic mobilities, corrected to standard temperature of 25 degrees C, were subjected to non-linear regression analysis and the obtained apparent pK(a) values were recalculated to thermodynamic pK(a)'s by extrapolation to zero ionic strength according to the extended Debye-Hückel model. The pK(a) values of the phosphinic acid group fell typically in the 1.5-2.25 interval, C-terminal carboxylic groups in the 2.94-3.50 interval, carboxylic groups of the lateral chain of glutamate and aspartate in the 4.68-4.97 interval, imidazolyl moiety of histidine in the 6.55-8.32 interval, N-terminal amino groups in the 7.65-8.28 interval and epsilon-amino group of the lateral chain of lysine in the 10.46-10.61 interval. Further, separation of diastereomers of the phosphinic pseudopeptides was investigated in achiral BGEs. Evaluation of the resolution of the diastereomers as a function of pH of the BGE revealed that most suitable pH region for separation of the diastereomers is around the pK(a) values of the central phosphinic acid group of the pseudopeptides. Successful separation of some diastereomers was, however, achieved in the neutral and alkaline BGEs as well.     

Simultaneous determination of the acid and basic ionization constants of imidazole

The acid and base ionization constants of imidazole (LH) have been determined simultaneously by potentiometry. Concentration constants were obtained at 10.0, 15.0, 25.0 and 40.0 degrees with I = 0.1M (KNO(3)), the values (and standard deviations) at 25.0 degrees being pK(a1)(LH(2)(+)) = 7.002 +/- 0.006 and pK(a2)(LH(2)(+)) = 12.588 +/- 0.004. The following values were obtained for the corresponding thermodynamic parameters: DeltaH(1) =38 and DeltaH(2) = 38 kJ mole ; DeltaS(1) = -8 and DeltaS(2)= -112 J.mole(-1).K(-1).     

Effects of pH on protein association: modification of the proton-linkage model and experimental verification of the modified model in the case of cytochrome c and plastocyanin.

Effects of pH on protein association are not well understood. To understand them better, we combine kinetic experiments, calculations of electrostatic properties, and a new theoretical treatment of pH effects. The familiar proton-linkage model, when used to analyze the dependence of the association constant K on pH, reveals little about the individual proteins. We modified this model to allow determination not only of the numbers of the H+ ions involved in the association but also of the pK(a) values, in both the separate and the associated proteins, of the side chains that are responsible for the dependence of K on pH. Some of these side chains have very similar pK(a) values, and we treat them as a group having a composite pK(a) value. Use of these composite pK(a) values greatly reduces the number of parameters and allows meaningful interpretation of the experimental results. We experimentally determined the variation of K in the interval 5.4 < or = pH < or = 9.0 for four diprotein complexes, those that the wild-type cytochrome c forms with the wild-type plastocyanin and its mutants Asp42Asn, Glu59Gln, and Glu60Gln. The excellent fittings of the experimental results to the modified model verified this model and revealed some unexpected and important properties of these prototypical redox metalloproteins. Protein association causes a decrease in the pK(a) values of the acidic side chains and an increase in the pK(a) values of the basic side chains. Upon association, three carboxylic side chains in wild-type plastocyanin each release a H+ ion. These side chains in free plastocyanin have an anomalously high composite pK(a) value, approximately 6.3. Upon association, five or six side chains in cytochrome c, likely those of lysine, each take up a H+ ion. Some of these side chains have anomalously low pK(a) values, less than 7.0. The unusual pK(a) values of the residues in the recognition patches of plastocyanin and cytochrome c may be significant for the biological functions of these proteins. Although each mutation in plastocyanin markedly, and differently, changed the dependence of K on pH, the model consistently gave excellent fittings. They showed decreased numbers of H+ ions released or taken up upon protein association and altered composite pK(a) values of the relevant side chains. Comparisons of the fitted composite pK(a) values with the theoretically calculated pK(a) values for plastocyanin indicated that Glu59 and Asp61 in the wild-type plastocyanin each release a H+ ion upon association with cytochrome c. Information of this kind cannot readily be obtained by spectroscopic methods. Our modification of the proton-linkage model is a general one, applicable also to ligands other than H+ ion and to processes other than association.     

Fluorimetric determination of dissociation constants and ph-controlled fluorescence analysis of purines and pyrimidines

Fluorimetrically determined pH-titration curves have been obtained for twelve purines and pyrimidines at room temperature, in aqueous solution. The pK(a) values for these compounds have been determined fluorimetrically and potentiometrically. Except for 6-mercaptopurine and uric acid, there is close agreement between the two sets of pK(a) values, but a large difference for the corresponding excited singlet-state values, pK(S1)(a), which were calculated previously. This indicates that the excited singlet-state proton-transfer rate is much slower than the fluorescence-decay rate of purines and pyrimidines in our experimental conditions. A direct, simple, pH-controlled fluorimetric method is proposed for the determination of purines and pyrimidines. The limits of detection vary between 60 ng ml and 5.4 mug ml .     

Acidity and alkali metal adsorption on the SiO2-aqueous solution interface

The intrinsic deprotonation constant (pK(a(2))(int)) and the intrinsic ion exchange constants (pK(Me(+))(int)) of Li(+), Na(+), and K(+) on SiO(2) were uniquely determined at 30 degrees C by using the potentiometric titration data, the Gouy-Chapman-Stern-Grahame (GSCG) model for the structure of the electrical double-layer (edl) and the double-extrapolation method. The values of these constants were pK(a(2))(int) = 6.57, pK(Li(+))(int) = pK(Na(+))(int) = pK(K(+))(int) = 5.61. The chemical meaning of intrinsic equilibrium constants and the equality in the values of pK(Li(+))(int), pK(Na(+))(int) and pK(K(+))(int) were discussed.     

Kinetics and mechanism of the pyridinolysis of S-2,4-dinitrophenyl 4-substituted thiobenzoates

[reaction: see text] The reactions of S-2,4-dinitrophenyl 4-methyl (1), S-2,4-dinitrophenyl 4-H (2), S-2,4-dinitrophenyl 4-chloro (3), and S-2,4-dinitrophenyl 4-nitro (4) thiobenzoates with a structurally homogeneous series of pyridines are subjected to a kinetic investigation in 44 wt % ethanol-water, at 25.0 degrees C and an ionic strength of 0.2 M (KCl). The reactions are studied spectrophotometrically (420 nm) by monitoring the appearance of 2,4-dinitrobenzenethiolate anion. Pseudo-first-order rate coefficients (k(obsd)) are obtained for all the reactions, employing excess of amine. The plots of k(obsd) vs [free pyridine] at constant pH are linear with the slopes (k(N)) independent of pH. The Br?nsted-type plots (log k(N) vs pK(a) of the conjugate acid of the pyridines) are curved for all the reactions. The Br?nsted curves are in accordance with stepwise mechanisms, through a zwitterionic tetrahedral intermediate (T(+/-)), and a change in the rate-limiting step. An equation based on this hypothesis accounts well for the experimental points. The Br?nsted lines were calculated with the following parameters: Reactions of thiolbenzoate 1: beta(1) 0.33 (slope at high pK(a)), beta(2) 0.95 (slope at low pK(a)), and pK(a)(0) = 8.5 (pK(a) at the curvature center); thiolbenzoate 2: beta(1) 0.30, beta(2) 0.88, and pK(a)(0) = 8.9; thiolbenzoate 3: beta(1) 0.33, beta(2) 0.89, and pK(a)(0) = 9.5; thiolbenzoate 4: beta(1) 0.21, beta(2) 0.97, and pK(a)(0) = 9.9. The increase of the pK(a)(0) value with the increase of the electron-withdrawing effect of the acyl substituent is explained by the argument that the rate of pyridine expulsion from T(+/-) (k(-)(1)) is favored over that of 2,4-dinitrobenzenethiolate leaving (k(2)), i.e., k(-)(1)/k(2) increases, as the acyl group becomes more electron withdrawing. The pK(a)(0) values for the title reactions are smaller than those for the reactions of the corresponding 4-nitrophenyl 4-substituted thiolbenzoates with the same pyridine series. This is explained by the larger k(2) value for 2,4-dinitrobenzenethiolate leaving from T(+/-) compared with 4-nitrobenzenethiolate, which results in lower k(-)(1)/k(2) ratios for the dinitro derivatives. The pK(a)(0) value obtained for the pyridinolysis of thiolbenzoate 2 (pK(a)(0) = 8.9) is smaller than that found for the same aminolysis of 2,4-dinitrophenyl benzoate (pK(a)(0) = 9.5). This is attributed to the greater nucleofugality from T(+/-) of 2,4-dinitrobenzenethiolate (pK(a) of conjugate acid 3.4) relative to 2,4-dinitrophenoxide (pK(a) of conjugate acid 4.1). The title reactions are also compared with the aminolysis of similar esters to assess the effect of the amine nature and leaving and acyl groups on the kinetics and mechanism.