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.     

Fast high-throughput method for the determination of acidity constants by capillary electrophoresis. II. Acidic internal standards

A fast method for the determination of acidity constants by CZE has been recently developed. This method is based on the use of an internal standard of pK(a) similar to that of the analyte. In this paper we establish the reference pK(a) values of a set of 24 monoprotic neutral acids of varied structure that we propose as internal standards. These compounds cover the most usual working pH range in CZE and facilitate the selection of adequate internal standards for a given determination. The reference pK(a) values of the acids have been established by the own internal standard method, i.e. from the mobility differences between different acids of similar pK(a) in the same pH buffers. The determined pK(a) values have been contrasted to the literature pK(a) values and confirmed by determination of the pK(a) values of some acids of the set by the classical CE method. Some systematic deviations of mobilities have been observed in NaOH buffer in reference to the other used buffers, overcoming the use of NaOH in the classical CE method. However, the deviations affect in a similar degree to the test compounds and internal standards allowing thus, the use of NaOH buffer in the internal standard method. This fact demonstrates the better performance of the internal standard method over the classical method to correct mobility deviations, which together with its fastness makes it an interesting method for the routine determination of accurate pK(a) values of new pharmaceutical drugs and drug precursors.     

Direct pK(a) measurement of the active-site cytosine in a genomic hepatitis delta virus ribozyme

Hepatitis delta virus ribozymes have been proposed to perform self-cleavage via a general acid/base mechanism involving an active-site cytosine, based on evidence from both a crystal structure of the cleavage product and kinetic measurements. To determine whether this cytosine (C75) in the genomic ribozyme has an altered pK(a) consistent with its role as a general acid or base, we used (13)C NMR to determine its microscopic pK(a) in the product form of the ribozyme. The measured pK(a) is moderately shifted from that of a free nucleoside or a base-paired cytosine and has the same divalent metal ion dependence as the apparent reaction pK(a)'s measured kinetically. However, under all conditions tested, the microscopic pK(a) is lower than the apparent reaction pK(a), supporting a model in which C75 is deprotonated in the product form of the ribozyme at physiological pH. While additional results suggest that the pK(a) is not shifted in the reactant state of the ribozyme, these data cannot rule out elevation of the C75 pK(a) in an intermediate state of the transesterification reaction.     

Concerted electron-proton transfer (EPT) in the oxidation of tryptophan with hydroxide as a base

Tryptophan is unique among the redox-active amino acids owing to its weakly acidic indolic proton (pK(a) ≈ 16) compared to the -O-H proton of tyrosine (pK(a) = 10.1) or the -S-H proton of cysteine (pK(a) = 8.2). Stopped-flow and electrochemical measurements have been used to explore the roles of proton-coupled electron transfer and concerted electron-proton transfer (EPT) in tryptophan oxidation. The results of these studies have revealed a role for OH(-) as a proton acceptor base in EPT oxidation of N-acetyl-tryptophan but not for other common bases. The reorganizational barrier for (N-acetyl-tryptophan)(+/?) self-exchange is also estimated.     

Dissociation constants, neutralization enthalpies and reactions of 3-styryl-2-mercaptopropenoic and 3-(1-naphthyl)-2-mercaptopropenoic acids

Dissociation constants (pK(a1) and pK(a2) in water-ethanol medium for 3-styryl-2-mercaptopropenoic and 3-(1-naphthyl)-2-mercaptopropenoic acid have been determined potentiometrically, and pK(a2) for both in aqueous medium, spectrophotometrically. Neutralization enthalpies in water-ethanol medium have been determined by thermometric titration. The reactions with metal ions have been studied, and the main reactions are described. The most sensitive reactions are with titanium(IV) (pD = 7.00) and nickel(II) (pD = 6.50).     

Acid-base equilibria in nonpolar media. 2.(1) Self-consistent basicity scale in THF solution ranging from 2-methoxypyridine to EtP(1)(pyrr) phosphazene

Relative ion-pair basicities Delta(pK)(ip) of 25 substituted aryl and alkyl iminophosphoranes (phosphazenes) and 20 other N-bases (various pyridines, amines, amidines) have been measured in THF medium using the UV-Vis and/or (13)C NMR methods. The Delta(pK)(ip) values were corrected for ion pairing using the Fuoss equation to obtain relative ionic basicities Delta(pK)(alpha). Based on the measurements, a basicity scale ranging from 2-methoxypyridine to EtP(1)(pyrr) and having a total span over 18 pK units has been created. The scale has been anchored to the pK(alpha) value of triethylamine (pK(alpha) = 12.5). The results are compared to pK(a) values in various other solvents and in the gas phase. The pK(alpha) values give better correlations than the pK(ip) values, thus indirectly validating the procedure of correction for ion pairing. The predictability of the basicity together with suitable spectral properties in the UV range make the phenylphosphazenes convenient neutral indicators in the high basicity range where the choice of neutral indicators is very limited.     

Free radical chemistry of flavan-3-ols: determination of thermodynamic parameters and of kinetic reactivity from short (ns) to long (ms) time scale

The physical chemistry and the free radical chemistry of the most abundant polyphenolic flavan-3-ols in food, catechin, its methylated metabolites, and several methylated analogues, have been investigated by laser flash photolysis and cyclic voltammetry studies. Two independent phenoxyl radicals formed upon oxidation of flavan-3-ols have been characterized and identified unambiguously: a short-lived resorcinol-like radical characterized by an absorption band at lambda = 495 nm and a long-lived catechol-like transient absorbing at lambda = 380 nm. The determination of all the thermodynamic constants of each phenolic function of flavan-3-ols, namely, redox potential (E degrees (3') = 0.13(5) V/SCE, E degrees (4') = 0.11(0) V/SCE, E degrees (5) = 0.28(5) V/SCE) and microscopic dissociation constants (pK(a3') = 9.02, pK(a4') = 9.12, pK(a5) = 9.43, pK(a7) = 9.58) were performed. These values are discussed and compared to the prediction of the density functional theory calculations made on the different species catechin, catechin phenoxyl, and catechin phenolate for each phenolic site.     

Direct NMR observation and pKa determination of the Asp102 side chain in a serine protease

The pK(a) value of aspartic acid in the catalytic triad of serine proteases has been a pivotal element in essentially every mechanism proposed for these enzymes over the past 40 years, but has, until now, eluded direct determination. Here, we have used the multinuclear 3D-NMR pulse programs HCACO and HCCH-TOCSY to directly identify and study the side-chain resonances of the aspartate and glutamate residues in uniformly (13)C-labeled α-lytic protease. Resonances from four of the six residues were detected and assigned, including that of Asp(102), which is notably the weakest of the four. pH titrations have shown all of the carboxylate (13)C signals to have unusually low pK(a) values: 2.0, 3.2, and 1.7 for Glu(129), Glu(174), and Glu(229), respectively, and an upper limit of 1.5 for Asp(102). The multiple H-bonds to Asp(102), long known from X-ray crystal studies, probably account for its unusually low pK(a) value through preferential stabilization of its anionic form. These H-bonds probably also contribute to the weakness of the NMR resonances of Asp(102) by restricting its mobility. The Asp(102)(13)C(γ) atom responds to the ionization of His(57) in the resting enzyme and to the inhibitor-derived oxyanion in a chloromethyl ketone complex, observations that strongly support the assignment. The low pK(a) value of Asp(102) would appear to be incompatible with mechanisms involving strong Asp(102)-His(57) H-bonds or high pK(a) values, but is compatible with mechanisms involving normal Asp(102)-His(57) H-bonds and moving His(57) imidazole rings, such as the reaction-driven ring flip.     

An active-site guanine participates in glmS ribozyme catalysis in its protonated state

Active-site guanines that occupy similar positions have been proposed to serve as general base catalysts in hammerhead, hairpin, and glmS ribozymes, but no specific roles for these guanines have been demonstrated conclusively. Structural studies place G33(N1) of the glmS ribozyme of Bacillus anthracis within hydrogen-bonding distance of the 2'-OH nucleophile. Apparent pK(a) values determined from the pH dependence of cleavage kinetics for wild-type and mutant glmS ribozymes do not support a role for G33, or any other active-site guanine, in general base catalysis. Furthermore, discrepancies between apparent pK(a) values obtained from functional assays and microscopic pK(a) values obtained from pH-fluorescence profiles with ribozymes containing a fluorescent guanosine analogue, 8-azaguanosine, at position 33 suggest that the pH-dependent step in catalysis does not involve G33 deprotonation. These results point to an alternative model in which G33(N1) in its neutral, protonated form donates a hydrogen bond to stabilize the transition state.     

Acid dissociation and metal chelate formation equilibria of some halogenated diphenylthiocarbazones

The acid dissociation constants (K(a)) of di-p-fluoro-, di-p-chloro-, di-p-bromo-, di-p-iodo-and di-m-trifluoromethylphenylthiocarbazones and the equilibrium formation constants (K(f(1))) of their 1:1 complexes with Co(II), Ni and Zn have been determined at 25 degrees in 50% v v aqueous dioxan at 0.10 M ionic strength. Each of the electron-withdrawing substituents gives a reduction in pK(a) roughly proportional to its Hammett sigma value, and log K(f(1)) increases linearly with pK(a).     

Fluorimetric and absorptiometric determination of dissociation constants and chromogenic reactions of benzyl 2-pyridyl ketone 2-quinolylhydrazone

The dissociation constants in the ground state of benzyl 2-pyridyl ketone 2-quinolylhydrazone (BPKQH) have been determined fluorimetrically and spectrophotometrically in aqueous ethanol medium. The pK(a) values of this compound in the excited state have also been established. The analytical properties of the reagent and its chromogenic reactions with some metal ions were studied.     

Lithium and Cesium Ion-Pair Acidities of Some Terminal Acetylenes and Aggregation in Tetrahydrofuran(1)

The equilibrium lithium acidities in THF have been determined for 4-ethynylbiphenyl, EB (pK 21.5-22.3), 3,3,3-triphenylpropyne, TPP (pK 22.3-22.7), and 1-ethynyladamantane, EA (pK 23.7). Ion pairs of (4-ethynylbiphenylyl)lithium are aggregated in the concentration range from 10(-)(4) to 10(-)(3) M, with an average aggregation number of 2.5. (3,3,3-Triphenylpropynyl)lithium ion pairs are partially aggregated at concentrations from 10(-)(5) to 10(-)(3) M; the average aggregation number is 1.2. Cesium acidities in THF have been determined for 3,3,3-triphenylpropyne (pK 29.1-29.9) and 1-ethynyladamantane (pK 31.56). The average aggregation number of (3,3,3-triphenylpropynyl)cesium ion pairs is 6.2 at concentrations of 10(-)(4)-10(-)(3) M.     

Acid-base equilibria in nonpolar media. Absolute pK(a) scale of bases in tetrahydrofuran

The acidity constants (pKa) of 11 bases (amines, anilines, pyridines, pyrrolidines, and iminophosphoranes) have been determined in tetrahydrofuran by potentiometry, complemented by conductometric measurements. The pK(a) values of the studied bases cover a wide absolute pH range of acidity in tetrahydrofuran, from 7.4 to 21.7. From the pK(a) values obtained, a scale of absolute acidity in tetrahydrofuran has been established, which has allowed calculation of the absolute pKa values of 77 bases from literature relative pK(a) data.     

Synthesis of thiomorpholino buffers for isoelectric focusing in immobilized pH gradients

The two commercially available Immobilines having a pK of 6.2 (2-morpholino ethyl acrylamide) and 7.0 (3-morpholinopropylacrylamide) have been modified and two new buffers have been synthesized: 2-thiomorpholinoethylacrylamide, pK 6.6, and 3-thiomorpholinopropyl acrylamide, pK 7.4. The replacement of an oxygen with a sulfur atom in the morpholino ring is thus seen to shift the pK values of these two bases by +0.4 pH units. In formulations in which the two new bases replaced the standard morpholino derivatives, identical pH profiles and protein patterns were obtained. The reason for this work was to try to close the gap between the pK 7.0 and 8.5 species and to provide the users of immobilized pH gradients with more buffers in the neutral pH region. The two new thiomorpholino derivatives are an important step in this direction.     

60]Fullerene as a substituent

The substituent effect of the dihydro[60]fullerenyl group and its hydrophobic parameters have been evaluated quantitatively. The substituent constant has been determined from the pK value of a fullerene-based, para-substituted benzoic acid 1 in 80% dioxane/water (v/v) by NMR spectroscopy. The resulting Hammett sigma value of 0.06, consistent with a small electron-withdrawing effect of C(60), is a consequence of the fact that only inductive effects can be transmitted through the two tetracoordinate carbon atoms between the fullerene pi system and the para-position of the benzoic acid moiety in 1. The parameter pi, which describes the hydrophobic character of the substituent C(60), has been evaluated as the difference between that of 1 and model compound 2. The pi value, which is larger than 3, indicates that the fullerene cage imparts high hydrophobicity to the molecule to which it is attached. Finally, we have evaluated how the fullerene spheroid influences the acid-base properties and nucleophilicity of the pyrrolidine nitrogen in a suitably functionalized fulleropyrrolidine. The fulleropyrrolidine 4 (pK(BD)(+)=5.6) is six orders of magnitude less basic and 1000 times less reactive than its model 3 (pK(BD)(+)=11.6). This may be related to through-space interactions of the nitrogen lone pair and the fullerene pi system.     

Dissociation constants of thymolphthalexone

For the dissociation constants of thymolphthalexone the following values have been found: pK(3) = 7.03 +/- 0.02, pK(4) = 8.05 +/- 0.09 (by potentiometric titration), pK(5) = 10.83 +/- 0.10, pK(6) = 12.99 +/- 0.11 (by spectrophotometry). They were determined at I = 0.4 and at 25 degrees.     

Effect of changing electrophilic center from C=O to C=S on rates and mechanism: pyridinolyses of O-2,4-dinitrophenyl thionobenzoate and its oxygen analogue

Second-order rate constants have been measured spectrophotometrically for the reactions of O-2,4-dinitrophenyl thionobenzoate (1) and 2,4-dinitrophenyl benzoate (2) with a series of substituted pyridines in 80 mol % H(2)O/20 mol % DMSO at 25.0 +/- 0.1 degrees C. The Br?nsted-type plots obtained are nonlinear with beta(1) = 0.26, beta(2) = 1.07, and pK(a) degrees = 7.5 for the reactions of 1 and beta(1) = 0.40, beta(2) = 0.90, and pK(a) degrees = 9.5 for the reactions of 2, suggesting that the pyridinolyses of 1 and 2 proceed through a zwiterionic tetrahedral intermediate T(+/-) with a change in the rate-determining step at pK(a) degrees = 7.5 and 9.5, respectively. The thiono ester 1 is more reactive than its oxygen analogue 2 except for the reaction with the strongest basic pyridine studied (pK(a) = 11.30). The k(1) value is larger for the reactions of 1 than for those of 2 in the low pK(a) region, but the difference in the k(1) value becomes negligible with increasing the basicity of pyridines. On the other hand, 1 exhibits slightly larger k(2)/k(-1) ratio than 2 in the low pK(a) region but the difference in the k(2)/k(-1) ratio becomes more significant with increasing the basicity of pyridines. Pyridines are more reactive than alicyclic secondary amines of similar basicity toward 2 in the pK(a) above ca. 7.2 but less reactive in the pK(a) below ca. 7.2. The k(1) value is slightly larger, but the k(2)/k(-1) ratio is much smaller for the reactions of 2 with pyridines than with isobasic secondary amines in the low pK(a) region, which is responsible for the fact that the weakly basic pyridines are less reactive than isobasic secondary amines.     

Effect of CTAB and SDS micelles on the excited state equilibria of some indole probes

The absorption and fluorescence spectral characteristics of some biologically active indoles have been studied as a function of acidity and basicity (H_/pH/H(o)) in cationic (cetyltrimethylammonium bromide, CTAB), anionic (sodium dodecylsulphate, SDS) and aqueous phases at a given surfactant concentration. The prototropic equilibrium reactions of these probes have been studied in aqueous and micellar phases and apparent excited state acidity constant (pK(a)(*)) values are calculated. The probes show formation of different species on changing pH. Various species present in water, CTAB and SDS have been identified and the equilibrium constants have been determined by Fluorimetric Titration method. The fluorescence spectral data suggest the formation of oxonium ion through the excited state proton transfer reaction in highly acidic media and formation of photoproducts due to the base catalyzed auto-oxidative reaction in basic aqueous solutions. Variations in the apparent pK(a)(*) value have been observed in different media. The change in the apparent pK(a) values depends upon the solubilising power of the micelles, as well as on the location of the protonating site in the molecule. The observation about increase in pK(a)(*) values in SDS and decrease in CTAB compared to pure water for various equilibria is consistent with the pseudophase ion-exchange (PIE) model.     

Protolytic reactions of 3,3'-dimethylnaphthidine and 3,3'-dimethoxybenzidine

The dissociation constants of diprotonated 3,3'-dimethylnaphthidine (DMN) and 3,3'-dimethoxybenzidine (DMB) have been determined spectrophotometrically. They are: pK(a1) = 2.62 +/- 0.03, pK(a2) = 3.33 +/- 0.09 for DMN: pK(a1) = 2.83 +/- 0.07, pK(a2) = 4.05 +/- 0.12 for DMB. The molar absorptivities (l.mole(-1).cm(-1)) of all forms of the indicators have been also determined: epsilon(B) = 1.68 x 10(4), epsilon(BH(+)) = 9.34 x 10(3), epsilon(BH(2+)(2)) = 1.80 x 10(3) at 300 nm for DMB; epsilon(B) = 7.33 x 10(3), epsilon(BH(+)) = 3.73 x 10(3), epsilon(BH(2+)(2)) = 0 at 330 nm for DMN.