Protonated benzofuran,anthracene, naphthalene,benzene, ethene,and ethyne: measurements and estimates of pK(a) and pK(R)

Aqueous solvolyses of acyl derivatives of hydrates (water adducts) of anthracene and benzofuran yield carbocations which undergo competitive deprotonation to form the aromatic molecules and nucleophilic reaction with water to give the aromatic hydrates. Trapping experiments with azide ions yield rate constants k(p) for the deprotonation and k(H2O) for the nucleophilic reaction based on the "azide clock". Combining these with rate constants for (a) the H(+)-catalyzed reaction of the hydrate to form the carbocation and (b) hydrogen isotope exchange of the aromatic molecule (from the literature) yields pK(R) = -6.0 and -9.4 and pK(a) = -13.5 and -16.3 for the protonated anthracene and protonated benzofuran, respectively. These pK values may be compared with pK(R) = -6.7 for naphthalene hydrate (1-hydroxy-1,2-dihydronaphthalene), extrapolated to water from measurements by Pirinccioglu and Thibblin for acetonitrile-water mixtures, and pK(a) = -20.4 for the 2-protonated naphthalene from combining k(p) with an exchange rate constant. The differences between pK(R) and pK(a) correspond to pK(H2O), the equilibrium constant for hydration of the aromatic molecule (pK(H2O) = pK(R) - pK(a)). For naphthalene and anthracene values of pK(H2O) = +13.7 and +7.5 compare with independent estimates of +14.2 and +7.4. For benzene, pK(a) = -24.3 is derived from an exchange rate constant and an assigned value for the reverse rate constant close to the limit for solvent relaxation. Combining this pK(a) with calculated values of pK(H2O) gives pK(R) = -2.4 and -2.1 for protonated benzenes forming 1,2- and 1,4-hydrates, respectively. Coincidentally, the rate constant for protonation of benzene is similar to those for protonation of ethylene and acetylene (Lucchini, V.; Modena, G. J. Am. Chem Soc. 1990, 112, 6291). Values of pK(a) for the ethyl and vinyl cations (-24.8) may thus be derived in the same way as that for the benzenonium ion. Combining these with appropriate values of pK(H2O) then yields pK(R) = -39.8 and -29.6 for the vinyl and ethyl cations, respectively.     

Amplified potentiometric determination of pK(00), pK(0), pK(l), and pK(2) of hydrogen sulfides with Ag(2)S ISE

The chemical capacitor theory has been applied to accurately determine dissociation constants of H(2)S with the Ag(2)S ion-selective electrode (ISE). The theory's principle is based on the measurement of the change in electrode charge density as a result of protonated or unprotonated sulfide adsorbed on the electrode surface. This charge density is related to the potential. Connection of each individual capacitor in series amplifies the potential according to the equation, E(total)=E(1)+E(2)+E(3)+cdots, three dots, centeredE(n). As the charges of individual capacitors are concentrated to one capacitor area, the charge density rises, and the potential increases. The pK(00), pK(0), pK(1), and pK(2) are reported as 1.8, 2.12, 7.05, and 12.0, respectively. The pK(00) and pK(0) are reported here for the first time. The pK(1) agrees well with the literature values; however, the pK(2) differs from those reported recently under extreme conditions. Reasons for disproving the unreasonably high pK(2)>17-19 values are given based on calculations. Mainly, when pK(2)>17-19, the experimental results do not fit the equilibrium equations, pH=(pK(1)+pK(2))/2, pK(1)=(pK(0)+pK(2))/2, and pH=pK(2)+log(HS(-))/(S(2-)).     

Improved pK(a) prediction: combining empirical and semimicroscopic methods

Using three different methods we tried to compute 171 experimentally known pK(a) values of ionizable residues from 15 different proteins and compared the accuracies of computed pK(a) values in terms of the root mean square deviation (RMSD) from experiment. One method is based on a continuum electrostatic model of the protein including conformational flexibility (KBPLUS). The others are empirical approaches with PROPKA deploying physically motivated energy terms with adjustable parameters and PKAcal using an empirical function with no physical basis. PROPKA reproduced the pK(a) values with highest overall accuracy. Differentiating the data set into weakly and strongly shifted experimental pK(a) values, however, we found that PROPKA's accuracy is better if the pK(a) values are weakly shifted but on equal footing with that of KBPLUS for more strongly shifted values. On the other hand, PKAcal reproduces strongly shifted pK(a) values badly but weakly shifted values with the same accuracy as PROPKA. We tested different consensus approaches combining data from all three methods to find a general procedure for most accurate pK(a) predictions. In most of the cases we found that the consensus approach reproduced experimental data with better accuracy than any of the individual methods alone.     

Simple method for determining nucleobase pK(a) values by indirect labeling and demonstration of a pK(a) of neutrality in dsDNA

Phosphorothioates were substituted into double-stranded DNA to study protonated Class I A+.C basepairs by 31P NMR. The method was effective in reporting the A+.C pKa. pKa values near 7.0 were found with optimal nearest-neighbor partners. Such pKa values could expand the catalytic repertoire of nucleic acids.     

The pK(a) values of N,N,N',N'-tetrakis-(2-hydroxypropyl)ethylenediamine

Aqueous solutions of the industrially important chelating agent N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine exhibit basic properties. The proton dissociation constants were determined to be 8.99 +/- 0.04 (pK(1)) and 4.30 +/- 0.04 (pK(2)) by potentiometric titration at 25 degrees in 0.15M sodium chloride.     

Cationic sigma-phenylplatinum(II) complexes with carboxylic acid functionality: pK(a) determinations and x-ray structures

The preparation and characterization of a novel series of cationic sigma-phenylplatinum(II) complexes of the type trans-[Pt(sigma-C(6)H(5))(L)(2)A]OTf (A = picolinic acid, L = PPh(3) (4) and PMePh(2) (7); A = nicotinic acid, L = PPh(3) (5) and PMePh(2) (8); A = isonicotinic acid, L = PPh(3) (6), PMePh(2) (9), and PEt(3) (10)) are described. The pK(a) value for the carboxylic acid functionality in selected complexes was found to follow the order 7 (pK(a) = 5.23 +/- 0.09) > 8 (4.85 +/- 0.10) > 9 (3.51 +/- 0.08) > 6 (3.26 +/- 0.07) approximately 10 (3.21 +/- 0.08) by means of potentiometric titration experiments in 50% (v/v) EtOH/H(2)O solution at 295 K. The X-ray crystal structures of 9 and 10 were also determined. The asymmetric unit of each of 9 and 10 comprises a univalent complex cation, a triflate anion, and a solvent CH(2)Cl(2) molecule of crystallization. Centrosymmetrically related pairs of complex cations in 9 associate via the familiar carboxylic acid dimer motif, whereas with 10, the carboxylic acid dimer motif is absent. Instead, the carboxylic acid residue forms both donor and acceptor interactions to the triflate anion and CH(2)Cl(2) solvent of crystallization, respectively, to afford a 10-membered ring structure. Possible reasons for the observed differences in the solid-state structures of 9 and 10 are presented.     

Determination of pK(a) of acetic and benzoic acids in pyridine

The pK(a)'s of acetic acid and benzoic acid in pyridine as solvent are found to be 10.1 and 9.8, respectively, at 25 degrees . These results are based on measurements of hydrogen ion activities in mixtures of the acids and their tetrabutylammonium salts. Supplementary studies of differential vapour pressure characteristics of solutions of the acids and the acid-salt mixtures, and conductance of tetrabutylammonium benzoate solutions are also incorporated.     

First-principles prediction of the pK(a)s of anti-inflammatory oxicams

The gas- and aqueous-phase acidities of a series of oxicams have been computed by combining M05-2X/6-311+G(3df,2p) gas-phase free energies with solvation free energies from the CPCM-UAKS, COSMO-RS, and SMD solvent models. To facilitate accurate gas-phase calculations, a benchmarking study was further carried out to assess the performance of various density functional theory methods against the high-level composite method G3MP2(+). Oxicams are typically diprotic acids, and several tautomers are possible in each protonation state. The direct thermodynamic cycle and the proton exchange scheme have been employed to compute the microscopic pK(a)s on both solution- and gas-phase equilibrium conformers, and these were combined to yield the macroscopic pK(a) values. Using the direct cycle of pK(a) calculation, the CPCM-UAKS model delivered reasonably accurate results with MAD ~ 1, whereas the SMD and COSMO-RS models' performance was less satisfactory with MAD ~ 3. Comparison with experiment also indicates that direct cycle calculations based on solution conformers generally deliver better accuracy. The proton exchange cycle affords further improvement for all solvent models through systematic error cancellation and therefore provides better reliability for the pK(a) prediction of compounds of these types. The latter approach has been applied to predict the pK(a)s of several recently synthesized oxicam derivatives.     

Intramolecular hydrogen bond strength and pK(a) determination of N,N'-disubstituted imidazole-4,5-dicarboxamides

N,N'-Disubstituted imidazole-4,5-dicarboxamides (I45DCs) form an intramolecular hydrogen bond worth an estimated 14 +/- 1 kcal/mol, as measured with a model structure in DMSO-d6 at 3 mM, thereby predisposing the molecular conformation to a folded rather than extended form. The I45DCs also show evidence of aggregation in both CDCl3 (>1 mM) and DMSO-d6 (>10 mM) solutions. These compounds are uncharacteristically weak bases in comparison with imidazoles bearing similar electron-withdrawing groups.     

Sequence diversity, metal specificity, and catalytic proficiency of metal-dependent phosphorylating DNA enzymes

Although DNA has not been found responsible for biological catalysis, many artificial DNA enzymes have been created by "in vitro selection." Here we describe a new selection approach to assess the influence of four common divalent metal ions (Ca(2+), Cu(2+), Mg(2+), and Mn(2+)) on sequence diversity, metal specificity, and catalytic proficiency of self-phosphorylating deoxyribozymes. Numerous autocatalytic DNA sequences were isolated, a majority of which were selected using Cu(2+) or Mn(2+) as the divalent metal cofactor. We found that Cu(2+)- and Mn(2+)-derived deoxyribozymes were strictly metal specific, while those selected by Ca(2+) and Mg(2+) were less specific. Further optimization by in vitro evolution resulted in a Mn(2+)-dependent deoxyribozyme with a k(cat) of 2.8 min(-1). Our findings suggest that DNA has sufficient structural diversity to facilitate efficient catalysis using a broad scope of metal cofactor utilizing mechanisms.     

Proton binding to proteins: pK(a) calculations with explicit and implicit solvent models

Ionizable residues play important roles in protein structure and activity, and proton binding is a valuable reporter of electrostatic interactions in these systems. We use molecular dynamics free energy simulations (MDFE) to compute proton pKa shifts, relative to a model compound in solution, for three aspartate side chains in two proteins. Simulations with explicit solvent and with an implicit, dielectric continuum solvent are reported. The implicit solvent simulations use the generalized Born (GB) model, which provides an approximate, analytical solution to Poisson's equation. With explicit solvent, the direction of the pKa shifts is correct in all three cases with one force field (AMBER) and in two out of three cases with another (CHARMM). For two aspartates, the dielectric response to ionization is found to be linear, even though the separate protein and solvent responses can be nonlinear. For thioredoxin Asp26, nonlinearity arises from the presence of two substates that correspond to the two possible orientations of the protonated carboxylate. For this side chain, which is partly buried and has a large pKa upshift, very long simulations are needed to correctly sample several slow degrees of freedom that reorganize in response to the ionization. Thus, nearby Lys57 rotates to form a salt bridge and becomes buried, while three waters intercalate along the opposite edge of Asp26. Such strong and anisotropic reorganization is very difficult to predict with Poisson-Boltzmann methods that only consider electrostatic interactions and employ a single protein structure. In contrast, MDFE with a GB dielectric continuum solvent, used for the first time for pKa calculations, can describe protein reorganization accurately and gives encouraging agreement with experiment and with the explicit solvent simulations.     

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.     

Amines That Transport Protons across Bilayer Membranes: Synthesis, Lysosomal Neutralization, and Two-Phase pK(a) Values by NMR

It is desirable to be able to control the pH of lysosomes. A collection of lipophilic, nitrogenous bases, designed to act as membrane-active, catalytic proton transfer agents, were prepared and their effective pK(a)s measured in a vigorously stirred, two-phase system. One phase was a phosphate buffer whose pH was varied over the range ca. 1-11. The other was an immiscible, deuterated organic solvent in which the compounds preferentially resided even when protonated. When chemical shift changes versus the pH of the buffer were plotted, clear pK(a) curves were generated that are relevant to transmembrane proton transfer behavior. The two-phase pK(a)s increased with increasing counterion lipophilicity and with increasing organic solvent polarity. The compounds were also tested for their ability to neutralize the acidity of lysosomes, a model for other acidic vesicles involved in drug sorting. The most successful of these, imidazole 6a, has >100 times the neutralizing power of ammonia, a standard lysosomotropic amine, causing a 1.7 unit rise in lysosomal pH of RAW cells at 0.1 mM, compared to a 0.2 and 1.4 unit rise for ammonium chloride at 0.1 and 10 mM, respectively.     

Determination of pK(a) values of N-heterocyclic bases by fluorescence spectrophotometry

Fluorimetry is a relatively fast and accurate means of determining the dissociation constants of sparingly soluble heterocyclic bases. Complications can arise, however, from the dependence of fluorescence on excited-state as well as ground-state acid-base chemistry. Several approaches to circumventing or compensating for this difficulty are discussed. To demonstrate the utility of the methods, the pK(a), values of the conjugate acids of two bases are evaluated by the methods described.     

Zinc(II) complexes of ubiquitin: speciation, affinity and binding features

Intraneuronal inclusions consisting of hypermetallated, (poly-)ubiquitinated proteins are a hallmark of neurodegeneration. To highlight the possible role played by metal ions in the dysfunction of the ubiquitin-proteasome system, here we report on zinc(II)/ubiquitin binding in terms of affinity constants, speciation, preferential binding sites and effects on protein stability and self-assembly. Potentiometric titrations allowed us to establish that at neutral pH only two species, ZnUb and Zn(2)Ub, are present in solution, in line with ESI-MS data. A change in the diffusion coefficient of ubiquitin was observed by NMR DOSY experiments after addition of Zn(II) ions, and thus indicates metal-promoted formation of protein assemblies. Analysis of (1)H, (15)N, (13)Cα and (13)CO chemical-shift perturbation after equimolar addition of Zn(II) ions to ubiquitin outlined two different metal-binding modes. The first involves a dynamic equilibrium in which zinc(II) is shared between a region including Met1, Gln2, Ile3, Phe4, Thr12, Leu15, Glu16, Val17, Glu18, Ile61 and Gln62 residues, which represent a site already described for copper binding, and a domain comprising Ile23, Glu24, Lys27, Ala28, Gln49, Glu51, Asp52, Arg54 and Thr55 residues. A second looser binding mode is centred on His68. Differential scanning calorimetry evidenced that addition of increasing amounts of Zn(II) ions does not affect protein thermal stability; rather it influences the shape of thermograms because of the increased propensity of ubiquitin to self-associate. The results presented here indicate that Zn(II) ions may interact with specific regions of ubiquitin and promote protein-protein contacts.     

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.     

Novel methods for the prediction of logP,pK(a), and logD

Novel methods for predicting logP, pK(a), and logD values have been developed using data sets (592 molecules for logP and 1029 for pK(a)) containing a wide range of molecular structures. An equation with three molecular properties (polarizability and partial atomic charges on nitrogen and oxygen) correlates highly with logP (r2 = 0.89). The pK(a)s are estimated for both acids and bases using a novel tree structured fingerprint describing the ionizing centers. The new models have been compared with existing models and also experimental measurements on test sets of common organic compounds and pharmaceutical molecules.     

Potentiometric saccharide detection based on the pK(a) changes of poly(aniline boronic acid)

A novel approach for the potentiometric detection of saccharides using poly(aniline boronic acid) is presented. A model is described in which the electrochemical potential is sensitive to the change in the pK(a) of the conducting polymer as a result of boronic acid-diol complexation. In this system, boronic acid complexation is the mode of transduction and it is manifested as changes in the electrochemical potential of the polymer with remarkable selectivity. Characteristics of both transient and steady-state response associated with the complexation are discussed. The presence of Nafion and fluoride during the electrochemical polymerization of 3-aminophenylboronic acid are shown to impact the sensitivity and the stability of the electrode response. The sensor sensitivity is improved significantly by increasing the concentration of sodium fluoride during the polymerization. Finally, the nature of the selectivity of the boronic acid-diol reaction under these conditions is explored by using molecular orbital calculations.