Theoretical prediction of relative and absolute pKa values of aminopyridines

This work presents a study aimed at the theoretical prediction of pK(a) values of aminopyridines, as a factor responsible for the activity of these compounds as blockers of the voltage-dependent K(+) channels. To cover a large range of pK(a) values, a total of seven substituted pyridines is considered as a calibration set: pyridine, 2-aminopyridine, 3-aminopyridine, 4-aminopyridine, 2-chloropyridine, 3-chloropyridine, and 4-methylpirydine. Using ab initio G1, G2 and G3 extrapolation methods, and the CPCM variant of the Polarizable Continuum Model for solvation, we calculate gas phase and solvation free energies. pK(a) values are obtained from these data using a thermodynamic cycle for describing protonation in aqueous and gas phases. The results show that the relatively inexpensive G1 level of theory is the most accurate at predicting pK(a) values in aminopyridines. The highest standard deviation with respect to the experimental data is 0.69 pK(a) units for absolute values calculations. The difference increases slightly to 0.74 pK(a) units when the pK(a) is computed relative to the pyridine molecule. Considering only compounds at least as basic as pyridine (the values of interest for bioactive aminopyridines) the error falls to 0.10 and 0.12 pK(a) units for the absolute and relative computations, respectively. The technique can be used to predict the effect of electronegative substituents in the pK(a) of 4-AP, the most active aminopyridine considered in this work. Thus, 2-chloro and 3-chloro-4-aminopyridine are taken into account. The results show a decrease of the pK(a), suggesting that these compounds are less active than 4-AP at blocking the K(+) channel.     

The pKa Cooperative: a collaborative effort to advance structure-based calculations of pKa values and electrostatic effects in proteins

The pK(a) Cooperative (http://www.pkacoop.org) was organized to advance development of accurate and useful computational methods for structure-based calculation of pK(a) values and electrostatic energies in proteins. The Cooperative brings together laboratories with expertise and interest in theoretical, computational, and experimental studies of protein electrostatics. To improve structure-based energy calculations, it is necessary to better understand the physical character and molecular determinants of electrostatic effects. Thus, the Cooperative intends to foment experimental research into fundamental aspects of proteins that depend on electrostatic interactions. It will maintain a depository for experimental data useful for critical assessment of methods for structure-based electrostatics calculations. To help guide the development of computational methods, the Cooperative will organize blind prediction exercises. As a first step, computational laboratories were invited to reproduce an unpublished set of experimental pK(a) values of acidic and basic residues introduced in the interior of staphylococcal nuclease by site-directed mutagenesis. The pK(a) values of these groups are unique and challenging to simulate owing to the large magnitude of their shifts relative to normal pK(a) values in water. Many computational methods were tested in this first Blind Prediction Challenge and critical assessment exercise. A workshop was organized in the Telluride Science Research Center to objectively assess the performance of many computational methods tested on this one extensive data set. This volume of Proteins: Structure, Function, and Bioinformatics introduces the pK(a) Cooperative, presents reports submitted by participants in the Blind Prediction Challenge, and highlights some of the problems in structure-based calculations identified during this exercise.     

On the NMR analysis of pKa values in the unfolded state of proteins by extrapolation to zero denaturant

Detailed knowledge of the pH-dependence in both folded and unfolded states of proteins is essential to understand the role of electrostatics in protein stability. The increasing number of natively disordered proteins constitutes an excellent source for the NMR analysis of pKa values in the unfolded state of proteins. However, the tendency of many natively disordered proteins to aggregate via intermolecular hydrophobic clusters limits their NMR analysis over a wide pH range. To assess whether the pKa values in natively disordered polypeptides can be extrapolated from NMR measurements in the presence of denaturants, the natively disordered backbone of the C-terminal fragment 75 to 105 of Human Thioredoxin was studied. First, assignments using triple resonance experiments were performed to confirm lack of secondary structure. Then the pH-dependence of the amides and carboxylate side chains of Glu residues (Glu88, Glu95, Glu98, and Glu103) in the pH range from 2.0 to 7.0 was monitored using 2D 1H15N HSQC and 3D C(CO)NH experiments, and the behavior of their amides and corresponding carboxyl groups was compared to confirm the absence of nonlocal interactions. Lastly, the effect of increasing dimethyl urea concentration on the pKa values of these Glu residues was monitored. The results indicate that: (i) the dispersion in the pKa of carboxyl groups and the pH midpoints of amides in Glu residues is about 0.5 pH units and 0.6 pH units, respectively; (ii) the backbone amides of the Glu residues exhibit pH midpoints which are within 0.2 pH units from those of their carboxylates; (iii) the addition of denaturant produces upshifts in the pKa values of Glu residues that are nearly independent of their position in the sequence; and (iv) these upshifts show a nonlinear behavior in denaturant concentration, complicating the extrapolation to zero denaturant. Nevertheless, the relative ordering of the pKa values of Glu residues is preserved over the whole range of denaturant concentrations indicating that measurements at high denaturant concentration (e.g. 4 M dimethyl urea) can yield a qualitatively correct ranking of the pKa of these residues in natively disordered proteins whose pH-dependence cannot be monitored directly by NMR.     

Negatively charged residues and hydrogen bonds tune the ligand histidine pKa values of Rieske iron-sulfur proteins

Rieske proteins carry a redox-active iron-sulfur cluster, which is bound by two histidine and two cysteine side chains. The reduction potential of Rieske proteins depends on pH. This pH dependence can be described by two pK(a) values, which have been assigned to the two iron-coordinating histidines. Rieske proteins are commonly grouped into two major classes: Rieske proteins from quinol-oxidizing cytochrome bc complexes, in which the ligand histidines titrate in the physiological pH range, and bacterial ferredoxin Rieske proteins, in which the ligand histidines are protonated at physiological pH. In the study presented here, we have calculated pK(a) values of the cluster ligand histidines using a combined density functional theory/continuum electrostatics approach. Experimental pK(a) values for a bc-type and a ferredoxin Rieske protein could be reproduced. We could identify functionally important differences between the two proteins: hydrogen bonds toward the cluster, which are present in bc-type Rieske proteins, and negatively charged residues, which are present in ferredoxin Rieske proteins. We removed these differences by mutating the proteins in our calculations. The Rieske centers in the mutated proteins have very similar pK(a) values. We thus conclude that the studied structural differences are the main reason for the different pH-titration behavior of the proteins. Interestingly, the shift caused by neutralizing the negative charges in ferredoxin Rieske proteins is larger than the shift caused by removing the hydrogen bonds toward the cluster in bc-type Rieske proteins.     

Electrostatic effects in highly charged proteins: salt sensitivity of pKa values of histidines in staphylococcal nuclease

The pK(a) values of most histidines in small peptides and in myoglobin increase on average by 0.30 unit between 0.02 and 1.5 M NaCl [Kao et al. (2000) Biophys. J. 79, 1637]. The DeltapK(a) values reflect primarily the ionic strength dependence of the solvation energy; screening of Coulombic interactions contributes only in a minor way. This implies that Coulombic interactions are weak, or that attractive and repulsive contributions to the pK(a) values are balanced. To distinguish experimentally between these two possibilities, and to further characterize the magnitude and salt sensitivity of surface electrostatic interactions in proteins, the salt dependence of pK(a) values of histidines in staphylococcal nuclease was measured by (1)H NMR spectroscopy. Three of the four histidines titrated with significantly depressed pK(a) values, and the salt sensitivity of all histidine pK(a) values was substantial. In three cases, the pK(a) values increased by a full unit between 0.01 and 1.5 M KCl. Anion-specific effects were found; the pK(a) values measured under equivalent ionic strengths in SCN(-) and SO(4)(2-) were higher than in Cl(-); the order of the sensitivity of pK(a) values to anions was SCN(-) > Cl(-) > SO(4)(2-). Structure-based pK(a) calculations with continuum methods were performed to interpret the measured effects structurally and to test their ability to capture the experimental behavior. Calculations in which the protein interior was treated empirically with a dielectric constant of 20 reproduced the pK(a) values and their dependence on the concentration of Cl(-). According to the calculations, the pK(a) values are depressed because of unfavorable self-energies and repulsive Coulombic interactions. Their striking salt sensitivity reflects screening of weak, repulsive, Coulombic interactions among charges separated by more than 10 A. Long-range Coulombic interactions on the surfaces of proteins are weak, but they can add up to produce substantial electrostatic effects when positive and negative charges are not balanced.     

Differences in lysine pKa values may be used to improve NMR signal dispersion in reductively methylated proteins

Reductive methylation of lysine residues in proteins offers a way to introduce ¹³C methyl groups into otherwise unlabeled molecules. The ¹³C methyl groups on lysines possess favorable relaxation properties that allow highly sensitive NMR signal detection. One of the major limitations in the use of reductive methylation in NMR is the signal overlap of ¹³C methyl groups in NMR spectra. Here we show that the uniform influence of the solvent on chemical shifts of exposed lysine methyl groups could be overcome by adjusting the pH of the buffering solution closer to the pKa of lysine side chains. Under these conditions, due to variable pKa values of individual lysine side chains in the protein of interest different levels of lysine protonation are observed. These differences are reflected in the chemical shift differences of methyl groups in reductively methylated lysines. We show that this approach is successful in four different proteins including Ca²⁺-bound Calmodulin, Lysozyme, Ca²⁺-bound Troponin C, and Glutathione S-Transferase. In all cases significant improvement in NMR spectral resolution of methyl signals in reductively methylated proteins was obtained. The increased spectral resolution helps with more precise characterization of protein structural rearrangements caused by ligand binding as shown by studying binding of Calmodulin antagonist trifluoperazine to Calmodulin. Thus, this approach may be used to increase resolution in NMR spectra of ¹³C methyl groups on lysine residues in reductively methylated proteins that enhances the accuracy of protein structural assessment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Toward accurate prediction of pKa values for internal protein residues: the importance of conformational relaxation and desolvation energy

Proton uptake or release controls many important biological processes, such as energy transduction, virus replication, and catalysis. Accurate pK(a) prediction informs about proton pathways, thereby revealing detailed acid-base mechanisms. Physics-based methods in the framework of molecular dynamics simulations not only offer pK(a) predictions but also inform about the physical origins of pK(a) shifts and provide details of ionization-induced conformational relaxation and large-scale transitions. One such method is the recently developed continuous constant pH molecular dynamics (CPHMD) method, which has been shown to be an accurate and robust pK(a) prediction tool for naturally occurring titratable residues. To further examine the accuracy and limitations of CPHMD, we blindly predicted the pK(a) values for 87 titratable residues introduced in various hydrophobic regions of staphylococcal nuclease and variants. The predictions gave a root-mean-square deviation of 1.69 pK units from experiment, and there were only two pK(a)'s with errors greater than 3.5 pK units. Analysis of the conformational fluctuation of titrating side-chains in the context of the errors of calculated pK(a) values indicate that explicit treatment of conformational flexibility and the associated dielectric relaxation gives CPHMD a distinct advantage. Analysis of the sources of errors suggests that more accurate pK(a) predictions can be obtained for the most deeply buried residues by improving the accuracy in calculating desolvation energies. Furthermore, it is found that the generalized Born implicit-solvent model underlying the current CPHMD implementation slightly distorts the local conformational environment such that the inclusion of an explicit-solvent representation may offer improvement of accuracy.     

Electrostatic calculations of the pKa values of ionizable groups in bacteriorhodopsin.

The effects of solvation and charge-charge interactions on the pKa of ionizable groups in bacteriorhodopsin have been studied using a macroscopic dielectric model with atom-level detail. The calculations are based on the atomic model for bacteriorhodopsin recently proposed by Henderson et al. Even if the structural data are not resolved at the atomic level, such calculations can indicate the quality of the model, outline some general aspects of electrostatic interactions in membrane proteins, and predict some features. The effects of structural uncertainties on the calculations have been investigated by conformational sampling. The results are in reasonable agreement with experimental measurements of several unusually large pKa shifts (e.g. the experimental findings that Asp96 and Asp115 are protonated in the ground state over a wide pH range). In general, we find that the large unfavorable desolvation energies of forming charges in the protein interior must be compensated by strong favorable charge-charge interactions, with the result that the titrations of many ionizable groups are strongly coupled to each other. We find several instances of complex titration behavior due to strong electrostatic interactions between titrating sites, and suggest that such behavior may be common in proton transfer systems. We also propose that they can help to resolve structural ambiguities in the currently available density map. In particular, we find better agreement between theory and experiment when a structural ambiguity in the position of the Arg82 side-chain is resolved in favor of a position near the Schiff base.     

pKa values of the 8 alpha-imidazole substituents in selected flavoenzymes containing 8 alpha-histidylflavins

Difference absorption spectroscopy as a function of pH is described as a probe to determine the pKa values of the 8 alpha-imidazole substituent in flavoenzymes containing 8 alpha-histidylflavin coenzymes. Reversible absorption difference spectra are observed in the pH range 5.5 to 8.5 when synthetic 8 alpha-imidazolyl-FMN is bound to the apoflavodoxins from Azotobacter vinelandii and from Clostridium pasterianum. The observed spectral perturbations of these two flavodoxin complexes follow a single proton ionization dependence with respective pKa values of 6.7 and 6.8. No pH-induced spectral perturbations were observed when 8 alpha-(N-CH3)-imidazolium FMN was bound to either flavodoxin. Similar approaches are described to determine the 8 alpha-imidazolyl pKa values of the 8 alpha-histidyl-FAD coenzyme of the cholesterol oxidases from Schizophyllum commune and from Gleocystidium chrysocreas. Previous work has shown the former enzyme contains an 8 alpha-N1-histidyl-FAD (W. C. Kenney et al. (1979) J. Biol. Chem. 254, 4689-4690) while experiments reported here show the latter enzyme also contains one 8 alpha-N1-histidyl-FAD per mole of enzyme. The pKa value for the 8 alpha-imidazole substituent on the flavin of S. commune cholesterol oxidase is 5.4 while that determined for the G. chrysocreas enzyme is 6.2. These results demonstrate that the pKa of the 8 alpha-imidazole substituent can be determined in enzymes containing an 8 alpha-histidylflavin, provided that the enzyme is stable in the pH range required to observe ionization. Furthermore it is shown this the pKa value can differ even on comparison of enzymes from different sources that catalyze the same reaction.     

Highly perturbed pKa values in the unfolded state of hen egg white lysozyme

The majority of pK_a values in protein unfolded states are close to the amino acid model pK_a values, thus reflecting the weak intramolecular interactions present in the unfolded ensemble of most proteins. We have carried out thermal denaturation measurements on the WT and eight mutants of HEWL from pH 1.5 to pH 11.0 to examine the unfolded state pK_a values and the pH dependence of protein stability for this enzyme. The availability of accurate pK_a values for the folded state of HEWL and separate measurements of mutant-induced effects on the folded state pK_a values, allows us to estimate the pK_a values of seven acidic residues in the unfolded state of HEWL. Asp-48 and Asp-66 display pK_a values of 2.9 and 3.1 in our analysis, thus representing the most depressed unfolded state pK_a values observed to date. We observe a strong correlation between the folded state pK_a values and the unfolded state pK_a values of HEWL, thus suggesting that the unfolded state of HEWL possesses a large degree of native state characteristics.     

Developing hybrid approaches to predict pKa values of ionizable groups

Accurate predictions of pKa values of titratable groups require taking into account all relevant processes associated with the ionization/deionization. Frequently, however, the ionization does not involve significant structural changes and the dominating effects are purely electrostatic in origin allowing accurate predictions to be made based on the electrostatic energy difference between ionized and neutral forms alone using a static structure and the subtle structural changes be accounted by using dielectric constant larger than two. On another hand, if the change of the charge state is accompanied by a large structural reorganization of the target protein, then the relevant conformational changes have to be explicitly taken into account in the pKa calculations. Here we report a hybrid approach that first predicts the titratable groups whose ionization is expected to cause large conformational changes, termed "problematic" residues, and then applies a special protocol on them, while the rest of the pK(a)s are predicted with rigid backbone approach as implemented in multi-conformation continuum electrostatics (MCCE) method. The backbone representative conformations for "problematic" groups are generated with either molecular dynamics simulations with charged and uncharged amino acid or with ab-initio local segment modeling. The corresponding ensembles are then used to calculate the titration curves of the "problematic" residues and then the results are averaged to obtain the corresponding pKa.     

pKa values of estrone, 17 beta-estradiol and 2-methoxyestrone

The acid ionization constants of estrone (10.77), 17 beta-estradiol (10.71) and 2-methoxyestrone (10.81) have been determined spectrophotometrically and shown to be consistent with the additivity of substituent effects of the phenol ring. Previously published values for estrone (10.914) and 17 beta-estradiol (10.078) are shown to be incorrect, at variance with the established trend for phenols, and inconcsistent with the similarity of the compounds.     

Electrostatic pKa computations in proteins: role of internal cavities

The solvent accessible surface area (SASA) algorithm is conventionally used to characterize protein surfaces in electrostatic energy computations of proteins. Unfortunately, it often fails to find narrow cavities inside a protein. As a consequence pK(a) computations based on this algorithm perform badly. In this study a new cavity-algorithm is introduced, which solves this problem and provides improved pK(a) values. The procedure is applied to 20 pK(a) values of titratable groups introduced as point mutations in SNase variants, where crystal structures are available. The computations of these pK(a)s are particular challenging, since they are placed in a rather hydrophobic environment. For nine mutants, where the titratable residue is in contact with a large cavity, the RMSD(pKa) between computed and measured pK(a) values is 2.04, which is a considerable improvement as compared to the original results obtained with Karlsberg(+) (http://agknapp.chemie.fu-berlin.de/karlsberg/) that yielded an RMSD(pKa) of 8.8. However, for 11 titratable residues the agreement with experiments remains poor (RMSD(pKa) = 6.01). Considering 15 pK(a)s of SNase, which are in a more conventional less hydrophobic protein environment, the RMSD(pKa) is 2.1 using the SASA-algorithm and 1.7 using the new cavity-algorithm. The agreement is reasonable but less good than what one would expect from the general performance of Karlsberg(+) indicating that SNase belongs to the more difficult proteins with respect to pK(a) computations. We discuss the possible reasons for the remaining discrepancies between computed and measured pK(a)s.     

NMR determination of lysine pKa values in the Pol lambda lyase domain: mechanistic implications

The base excision repair (BER) process requires removal of an abasic deoxyribose-5-phosphate group, a catalytic activity that has been demonstrated for the N-terminal 8 kDa domain of DNA polymerase beta (Pol beta), and for the homologous domain of DNA polymerase lambda (Pol lambda). Previous studies have demonstrated that this activity results from formation of a Schiff base adduct of the abasic deoxyribose C-1' with a lysine residue (K312 in the case of Pol lambda), followed by a beta-elimination reaction. To better understand the underlying chemistry, we have determined pKa values for the lysine residues in the Pol lambda lyase domain labeled with [epsilon-13C]lysine. At neutral pH, the H(epsilon) protons on 3 of the 10 lysine residues in this domain, K287, K291, and K312, exhibit chemical shift inequivalence that results from immobilization of the lysyl side chains. For K287 and K291, this results from the K287-E261 and K291-E298 salt bridge interactions, while for K312, immobilization apparently results from steric and hydrogen-bonding interactions that constrain the position of the lysine side chain. The pKa value of K312 is depressed to 9.58, a value indicating that at physiological pH K312 will exist predominantly in the protonated form. Titration of the domain with hairpin DNA containing a 5'-tetrahydrofuran terminus to model the abasic site produced shifts of the labeled lysine resonances that were in fast exchange but appeared to be complete at a stoichiometry of approximately 1:1.3, consistent with a dissociation constant of approximately 1 microM. The epsilon-proton shifts of K273 were the most sensitive to the addition of the DNA, apparently due to changes in the relative orientation between K273 and W274 in the DNA complex. The average pKa values increased by 0.55, consistent with the formation of some DNA-lysine salt bridges and with the general pH increase expected to result from a reduction in the net positive charge of the complex. A general increase in the Hill coefficients observed in the complex is consistent with the screening of the interacting lysine residues by the DNA. The pKa of K312 residue increased to 10.58 in the complex, probably due to salt bridge formation with the 5'-phosphate group of the DNA. The pKa values obtained for the lyase domain of Pol lambda in the present study are consistent with recent crystallographic studies of Pol beta complexed with 5-phosphorylated abasic sugar analogues in nicked DNA which reveal an open site with no obvious interactions that would significantly depress the pK value for the active site lysine residue. It is suggested that due to the heterogeneity of the damaged DNA substrates with which Pol lambda as well as other related polymerases may be required to bind, the unexpectedly poor optimization of the lyase catalytic site may reflect a compromise of flexibility with catalytic efficiency.     

The intrinsic pKa values for phosphatidylserine and phosphatidylethanolamine in phosphatidylcholine host bilayers.

Potentiometric titrations and surface potential measurements have been used to determine the intrinsic pKa values of both the carboxyl and amino groups of phosphatidylserine (PS) in mixed vesicles of PS and phosphatidylcholine (PC), and also of the amino group of phosphatidylethanolamine (PE) in mixed PE-PC vesicles. The pKa of the carboxyl group of PS in liposomes with different PS/PC lipid ratios measured by the two different methods is 3.6 +/- 0.1, and the pKa of its amino group is 9.8 +/- 0.1. The pKa of the amino group of PE in PE-PC vesicles, determined solely by surface potential measurements, is 9.6 +/- 0.1. These pKa values are independent of the aqueous phase ionic strength and of the effect of the liposome's surface potential due to the presence of these partially charged lipids.     

Genome-based prediction of testcross values in maize

This is the first large-scale experimental study on genome-based prediction of testcross values in an advanced cycle breeding population of maize. The study comprised testcross progenies of 1,380 doubled haploid lines of maize derived from 36 crosses and phenotyped for grain yield and grain dry matter content in seven locations. The lines were genotyped with 1,152 single nucleotide polymorphism markers. Pedigree data were available for three generations. We used best linear unbiased prediction and stratified cross-validation to evaluate the performance of prediction models differing in the modeling of relatedness between inbred lines and in the calculation of genome-based coefficients of similarity. The choice of similarity coefficient did not affect prediction accuracies. Models including genomic information yielded significantly higher prediction accuracies than the model based on pedigree information alone. Average prediction accuracies based on genomic data were high even for a complex trait like grain yield (0.72–0.74) when the cross-validation scheme allowed for a high degree of relatedness between the estimation and the test set. When predictions were performed across distantly related families, prediction accuracies decreased significantly (0.47–0.48). Prediction accuracies decreased with decreasing sample size but were still high when the population size was halved (0.67–0.69). The results from this study are encouraging with respect to genome-based prediction of the genetic value of untested lines in advanced cycle breeding populations and the implementation of genomic selection in the breeding process.     

The determination of the pKa of histidine residues in proteins by Raman difference spectroscopy

Sensitive Raman difference spectroscopy was used to monitor the protonation and deprotonation of histidine residues in apo-transferrin. We have shown previously that the behavior of small molecules and/or small molecular groups bound to proteins or other large macromolecules can be studied by Raman difference spectroscopy (Yue, K.T. et al. (1989) J. Raman Spectrosc. 20, 541-545). Using this method, we have measured the Raman difference spectra of human transferrin at different pH values with respect to pH 8.9, titrating its various histidine residues. About 12 +/- 2 of the 19 residues were titrated. The pH difference spectrum of transferrin obtained is very similar to that of histidine in solution, but with clear differences in the 1200-1400 cm-1 region. A titration curve with pKa of 6.08 +/- 0.01 fit the data of histidine in solution and a value of 6.56 +/- 0.02 was found for the average value of the 12 histidine residues inside transferrin. The technique has enough sensitivity at present to monitor a single histidine residue in a 130 kDa molecule and to determine the titration curve of one residue in a 40 kDa protein.     

Electrostatic forces in two lysozymes: calculations and measurements of histidine pKa values.

In order to examine the electrostatic forces in globular proteins, pKa values and their ionic strength dependence of His residues of hen egg white lysozyme (HEWL) and human lysozyme (HUML) were measured, and they were compared with those calculated numerically. pKa values of His residues in HEWL, HUML, and short oligopeptides were determined from chemical shift changes of His side chains by 1H-nmr measurements. The associated changes in pKa values in HEWL and HUML were calculated by solving the Poisson-Boltzmann equations numerically for macroscopic dielectric models. The calculated pKa changes and their ionic strength dependence agreed fairly well with the observed ones. The contribution from each residue of each alpha-helix dipole to the pKa values and their ionic strength dependence was analyzed using Green's reciprocity theorem. The results indicate that (1) the pKa of His residues are largely affected by surrounding ionized and polar groups; (2) the ionic strength dependence of the pKa values is determined by the overall charge distributions and their accessibilities to solvent; and (3) alpha-helix dipoles make a significant contribution to the pKa, when the His residue is close to the helix terminus and not fully exposed to the solvent.     

Ionic interactions for substituted MCH1R inhibitors studied by pKa values

MCH1R inhibitors with the quinoline moiety having the aromatic amine and aliphatic amine chain were selected, and then the effect of substituents of the quinoline ring on the ionic interaction were studied by calculating pK_a values for these amines at the B3LYP/6-311++G(d,p)//B3LYP/6-31+G(d) level in the gas phase and in water. For substituent with C, N, O, and S atoms next to the quinoline ring, respectively, the pK_a values of aromatic amines are estimated to be 8.98, 12.19, 4.64, and 4.33 and those of the aliphatic amines are 12.65, 10.82, 9.94, and 11.55, respectively.