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.     

Protonation of kanamycin A: Detailing of thermodynamics and protonation sites assignment

Protonation of an aminoglycoside antibiotic kanamycin A sulfate was studied by potentiometric titrations at variable ionic strength, sulfate concentration and temperature. From these results the association constants of differently protonated forms of kanamycin A with sulfate and enthalpy changes for protonation of each amino group were determined. The protonation of all amino groups of kanamycin A is exothermic, but the protonation enthalpy does not correlate with basicity as in a case of simple polyamines. The sites of stepwise protonation of kanamycin A have been assigned by analysis of ¹H-¹³C-HSQC spectra at variable pH in D₂O. Plots of chemical shifts for each H and C atom of kanamycin A vs. pH were fitted to the theoretical equation relating them to pK_a values of ionogenic groups and it was observed that changes in chemical shifts of all atoms in ring C were controlled by ionization of a single amino group with pK_a 7.98, in ring B by ionization of two amino groups with pK_a 6.61 and 8.54, but in ring A all atoms felt ionization of one group with pK_a 9.19 and some atoms felt ionization of a second group with pK_a 6.51, which therefore should belong to amino group at C3 in ring B positioned closer to the ring A while higher pK_a 8.54 can be assigned to the group at C1. This resolves the previously existed uncertainty in assignment of protonation sites in rings B and C.     

Ionization constants and thermodynamic parameters of histidine and derivatives

The pK_a values for the proton dissociation of carboxyl, imidazolium, and ammonium groups for histidine and ten of its derivatives were determined electrometrically at seven temperatures in the range 10–40°C. The ΔH and ΔS values were estimated from the temperature dependence of the dissociation constants of histidine and its derivatives. These results and the pK_a values compared in terms of inductive effect suggest an ion-dipole interaction between the protonated amino group and the unprotonated imidazole ring. The charge and the solvation effects of the neighboring groups are the main factors that determine the imidazole group pK_a in histidine and its studied derivatives. The N^τ-H tautomer is favored over the N^π-H by 1.6 kcal/mol, indicating that the inductive substituent effect at position 4 of the imidazole ring is the major component in determining this tautomeric preference.     

The pKa Values of Acidic and Basic Residues Buried at the Same Internal Location in a Protein Are Governed by Different Factors

The pK_a values of internal ionizable groups are usually very different from the normal pK_a values of ionizable groups in water. To examine the molecular determinants of pK_a values of internal groups, we compared the properties of Lys, Asp, and Glu at internal position 38 in staphylococcal nuclease. Lys38 titrates with a normal or elevated pK_a, whereas Asp38 and Glu38 titrate with elevated pK_a values of 7.0 and 7.2, respectively. In the structure of the L38K variant, the buried amino group of the Lys38 side chain makes an ion pair with Glu122, whereas in the structure of the L38E variant, the buried carboxyl group of Glu38 interacts with two backbone amides and has several nearby carboxyl oxygen atoms. Previously, we showed that the pK_a of Lys38 is normal owing to structural reorganization and water penetration concomitant with ionization of the Lys side chain. In contrast, the pK_a values of Asp38 and Glu38 are perturbed significantly owing to an imbalance between favorable polar interactions and unfavorable contributions from dehydration and from Coulomb interactions with surface carboxylic groups. Their ionization is also coupled to subtle structural reorganization. These results illustrate the complex interplay between local polarity, Coulomb interactions, and structural reorganization as determinants of pK_a values of internal groups in proteins. This study suggests that improvements to computational methods for pK_a calculations will require explicit treatment of the conformational reorganization that can occur when internal groups ionize.     

Triaryl (Z)-olefins suitable for radiolabeling with iodine-124 or fluorine-18 radionuclides for positron emission tomography imaging of estrogen positive breast tumors

A group of (Z)-1,2-diphenyl-1-[4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]but-1-enes were synthesized using methodologies that will allow incorporation of a [¹²⁴I]iodine substituent at the para-position of either the C-1 phenyl ring or the C-2 phenyl ring, or a [¹⁸F]OCH₂CH₂F substituent at the para-position of the C-2 phenyl ring. These [¹²⁴I] and [¹⁸F] radiotracers are designed as potential radiopharmaceuticals to image estrogen positive breast tumors using positron emission tomography (PET).     

Assessment of antidotal efficacy of cholinesterase reactivators against paraoxon: In vitro reactivation kinetics and physicochemical properties

The search of proficient oximes as reactivators of irreversibly inhibited-AChE by organophosphate poisoning necessitates an appropriate assessment of their physicochemical properties and reactivation kinetics. Therefore, herein acid dissociation constant; pK_a, lipophilicity; log P, polar surface area, hydrogen bond donor and acceptor counts of structurally different oximes (two tertiary oximes and thirteen pyridinium aldoxime derivatives) have been evaluated. The experimentally obtained data for pK_a has been comparatively analyzed by using non-linear regression. Further the tested oximes were screened through in vitro reactivation kinetics against paraoxon-inhibited AChE. The pK_a values of all the examined oximes were within the range of 7.50–9.53. pK_a values of uncharged and mono-pyridinium oximes were in good correlation with their reactivation potency. The high negative log P values of pyridinium oxime reactivators indicate their high hydrophilic character; hence oximes with improved lipophilicity should be designed for the development of novel and more potent antidotes. Propane and butane linked oximes were superior reactivators than xylene linked bis-oxime reactivators. It is concluded from the present study that pK_a value is not only ruled by the position of oximino functionality in the pyridinium ring, but also by the position of linker. Although, pyridinium oximes are proved to be better reactivators but their lipophilicity has to be improved.     

Novel antagonists of serotonin-4 receptors: Synthesis and biological evaluation of pyrrolothienopyrazines

Based on the definition of a 5-HT₄ receptor antagonist pharmacophore, a series of pyrrolo[1,2-a]thieno[3,2-e] and pyrrolo[1,2-a]thieno[2,3-e] pyrazine derivatives were designed, prepared, and evaluated to determine the properties necessary for high-affinity binding to 5-HT₄ receptors. The compounds were synthesized by substituting the chlorine atom of the pyrazine ring with various N-alkyl-4-piperidinylmethanolates. They were evaluated in binding assays with [³H]GR113808 (1) as the 5-HT₄ receptor radioligand. The affinity values (K_i or inhibition percentages) were affected by both the substituent on the aromatic ring and the substituent on the lateral piperidine chain. A methyl group on the tricyclic ring produced a marked increase in affinity while an N-propyl or N-butyl group gave compounds with nanomolar affinities. Among the most potent ligands, 34d was selected for further pharmacological studies and evaluated in vivo. This compound acts as an antagonist/weak partial agonist in COS-7 cells stably expressing the 5-HT_4(a) receptor and is of great interest as a peripheral antinociceptive agent.     

Axial perturbations on the electronic and magnetic properties of ferric porphyrins I. Effect of axial ligand basicity in bis-substituted pyridine complexes of synthetic porphyrins

The proton NMR spectra of the bis-4-substituted pyridinates of ferric tetrapheylporphyrin and octaethylporphyrin complexes have been recorded and analyzed fort he purpose of ascertaining the influence of variable axial lignad basicity on the bonding and magnetic properties of the iron. Under the conditions of slow ligand exhange where the bis stoichiometry can be established, all complexes exist exlusively in the low-spin, $\text{S}\text{=}\text{1}\text{2}$, state. The hyperfine shifts at ?60° C for both the porphyrins and axial ligands are shown to be very sensitive to the basicity of the substituted pyridine, as measured by its pK_a. For the tetraphenylporphyrin complexes, we illustrate that the pattern of the meso-phenyl hyperfine shifts permits a quantitative separation of the contact and dipolar contributions to these shifts. This separation reveals that the shift variations with pyridine pK_a are dominated by changes in the magnetic susceptibility anisotropy (dipolar shift), which decreases markedly upon lowering the pyridine basicity; ESR data support this conclusion in the few samples investigated. However, this trend in magnetic anisotropy with ligand basicity is not valid when comparing pyridines with other ligands such as imidazoles. The important change in the contact shift reflects a decrease in porphyrin → iron π change transfer as the ligand basicity is lowered. A correlation between increase in proton NMR linewidth and magnetic anisotrophy of the iron suggests that electron spin relaxation occurs via a process which couples the same levels that control the magnetic anisotropy.     

Electrostatic contributions to the stability of the GCN4 leucine zipper structure

Ion pairs are ubiquitous in X-ray structures of coiled coils, and mutagenesis of charged residues can result in large stability losses. By contrast, pK_a values determined by NMR in solution often predict only small contributions to stability from charge interactions. To help reconcile these results we used triple-resonance NMR to determine pK_a values for all groups that ionize between pH 1 and 13 in the 33 residue leucine zipper fragment, GCN4p. In addition to the native state we also determined comprehensive pK_a values for two models of the GCN4p denatured state: the protein in 6 M urea, and unfolded peptide fragments of the protein in water. Only residues that form ion pairs in multiple X-ray structures of GCN4p gave large pK_a differences between the native and denatured states. Moreover, electrostatic contributions to stability were not equivalent for oppositely charged partners in ion pairs, suggesting that the interactions between a charge and its environment are as important as those within the ion pair. The pH dependence of protein stability calculated from NMR-derived pK_a values agreed with the stability profile measured from equilibrium urea-unfolding experiments as a function of pH. The stability profile was also reproduced with structure-based continuum electrostatic calculations, although contributions to stability were overestimated at the extremes of pH. We consider potential sources of errors in the calculations, and how pK_a predictions could be improved. Our results show that although hydrophobic packing and hydrogen bonding have dominant roles, electrostatic interactions also make significant contributions to the stability of the coiled coil.     

Kinetic trans effect of phosphine ligands in anation reactions of aquocobaloximes

Both electronic and steric effects concur to determine the trans effect of the P ligands in the anation reactions of phosphino aquocobaloximes, electronic effects being prevailing.The anation reactions and the pK_a values of these complexes vary in a rather restricted range with variation of the phosphine ligands.A fairly linear relationship is observed between pK_a and log k₁, suggesting that the labilizing effect of the phosphine mainly acts through a weakening of the trans bond in the ground state.     

Triaryl (Z)-olefins suitable for radiolabeling with carbon-11 or fluorine-18 radionuclides for positron emission tomography imaging of cyclooxygenase-2 expression in pathological disease

A group of (Z)-1,1-diphenyl-2-(4-methylsulfonylphenyl)alk-1-enes were synthesized using methodologies that will allow incorporation of a [¹¹C]OCH₃ substituent at the para-position of the C-1 phenyl ring, a [¹¹C]SO₂CH₃ substituent at the para-position of the C-2 phenyl ring, a [¹⁸F]OCH₂CH₂F substituent at the para-position of the C-1 phenyl ring, and a [¹⁸F]CH₂CH₂F substituent at the C-2 position of the olefinic bond. The [¹¹C] and [¹⁸F] radiotracers are designed as potential radiopharmaceuticals to image cyclooxygenase-2 (COX-2) expression in any organ where COX-2 is upregulated. The COX-1/COX-2 inhibition data acquired suggest that compounds having a [¹¹C]OMe or [¹⁸F]OCH₂CH₂F substituent at the para-position of the C-1 phenyl ring may be more suitable for imaging COX-2 expression in view of their ability to exclusively inhibit the COX-2 isozyme.     

Heme-linked protonation of HCN, CO, NO and O2 complexes of reduced horseradish peroxidases.

Small reversible changes in the absorption spectra of HCN, CO, NO and O₂ complexes of ferrous diacetyldeuteroperoxidase A, not hitherto observed, were attributed to proton dissociation of a distal amino acid residue. From spectrophotometric titration data the pK_a was measured as 5.5 (HCN), 5.6 (ligand free), 6.0 (CO), 6.55 (NO) and 8.0 (O₂). The value of 8.0 for the pK_a of the O₂ complex was also obtained from a curve of pH dependence of proton uptake in the reaction of the ferrous enzyme with O₂. Absorption bands in the visible region were shifted to longer wavelengths in the order of CO to NO to O₂ which is the decreasing order of the energy of π¹ level of these diatomic ligands.The pK_a values for CO complexes of ferroperoxidases, isoenzymes A and (B+C) were varied with substituents at the 2 and 4 positions of deuterohemin IX, and the $\text{Δp}\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{Δp}\text{K}{}_3$ ratio was about 0.3 in both series of isoenzyme preparations, where pK₃ is a measure of basicity of pyrrole nitrogen.The present data support the previous conclusion (Yamada and Yamazaki (1974) Arch. Biochem. Biophys., 165, 728) that the pK_a for ferroperoxidases, measured from small reversible changes in the absorption spectra, represents a proton dissociation constant of a distal amino acid residue and that there is hydrogen bonding between the residue and a ligand atom directly bound to the iron atom.     

Studies on aroyl- and aryl-hydrazide derivatives from d-glycero-d-gulo-heptono-1,4-lactone

A series of aroyl- and aryl-hydrazide derivatives was prepared from d-glycero-d-gulo-heptono-1,4-lactone (1). The reactivity of the NH proton in these hydrazides, in terms of their dissociation constants (pK_a), was determined from their electronic spectra, and correlated to the Hammett σ values of the substituents. Comparable reactivities of the NH protons for the compounds, and the effect of the substituent, were studied by n.m.r. spectroscopy. Decomposition of the aroylhydrazides with copper(II) sulfate or nitrous acid resulted in the regeneration of 1.     

Electrostatic effects in a network of polar and ionizable groups in staphylococcal nuclease

His121 and His124 are embedded in a network of polar and ionizable groups on the surface of staphylococcal nuclease. To examine how membership in a network affects the electrostatic properties of ionizable groups, the tautomeric state and the pK_a values of these histidines were measured with NMR spectroscopy in the wild-type nuclease and in 13 variants designed to disrupt the network. In the background protein, His121 and His124 titrate with pK_a values of 5.2 and 5.6, respectively. In the variants, where the network was disrupted, the pK_a values range from 4.03 to 6.46 for His121, and 5.04 to 5.99 for His124. The largest decrease in a pK_a was observed when the favorable Coulomb interaction between His121 and Glu75 was eliminated; the largest increase was observed when Tyr91 or Tyr93 was substituted with Ala or Phe. In all variants, the dominant tautomeric state at neutral pH was the N^ε2 state. At one level the network behaves as a rigid unit that does not readily reorganize when disrupted: crystal structures of the E75A or E75Q variants show that even when the pivotal Glu75 is removed, the overall configuration of the network was unaffected. On the other hand, a few key hydrogen bonds appear to govern the conformation of the network, and when these bonds are disrupted the network reorganizes. Coulomb interactions within the network report an effective dielectric constant of 20, whereas a dielectric constant of 80 is more consistent with the magnitude of medium to long-range Coulomb interactions in this protein. The data demonstrate that when structures are treated as static, rigid bodies, structure-based pK_a calculations with continuum electrostatics method are not useful to treat ionizable groups in cases where pK_a values are governed by short-range polar and Coulomb interactions.     

Steric Enhancement of Imidazole Basicity incis-Urocanic Acid Derivatives: Models for the Action of Chymotrypsin

To test the hypothesis that substrate-induced steric compression between His 57 and Asp 102 at the active site of chymotrypsin can increase the basicity of His 57, we have synthesized thecis- andtrans-isomers of 2-bromo-3-(N-tritylimidazole)-2-propenoic acid and 2-chloro-3-(N-tritylimidazole)-2-propenoic acid and compared selected properties with those ofcis-andtrans-urocanic acids. Thecis-isomers display low field¹H NMR signals at 17 ppm in dimethylsulfoxide, similar tocis-urocanic acid; whereas thetrans-isomers do not show strong hydrogen bonds. Increasing the size of the C2 substituent (H < Cl < Br) in thecis-isomers increases the pK_aof the imidazolium group from 6.78 for H to 7.81 and 9.10 for Cl and Br, respectively; whereas the pK_as of thetransisomers are all 6.0 ± 0.1. The results indicate that thecis-urocanic acid derivatives with large substituents at C2 act as proton sponges in water, and they support the concept that steric compression in the catalytic triad of chymotrypsin can increase the basicity of His 57.     

Functional tuning of the catalytic residue pKa in a de novo designed esterase

AlleyCatE is a de novo designed esterase that can be allosterically regulated by calcium ions. This artificial enzyme has been shown to hydrolyze p‐nitrophenyl acetate (pNPA) and 4‐nitrophenyl‐(2‐phenyl)‐propanoate (pNPP) with high catalytic efficiency. AlleyCatE was created by introducing a single‐histidine residue (His¹⁴⁴) into a hydrophobic pocket of calmodulin. In this work, we explore the determinants of catalytic properties of AlleyCatE. We obtained the pK_a value of the catalytic histidine using experimental measurements by NMR and pH rate profile and compared these values to those predicted from electrostatics pK_a calculations (from both empirical and continuum electrostatics calculations). Surprisingly, the pK_a value of the catalytic histidine inside the hydrophobic pocket of calmodulin is elevated as compared to the model compound pK_a value of this residue in water. We determined that a short‐range favorable interaction with Glu¹²⁷ contributes to the elevated pK_a of His¹⁴⁴. We have rationally modulated local electrostatic potential in AlleyCatE to decrease the pK_a of its active nucleophile, His¹⁴⁴, by 0.7 units. As a direct result of the decrease in the His¹⁴⁴ pK_a value, catalytic efficiency of the enzyme increased by 45% at pH 6. This work shows that a series of simple NMR experiments that can be performed using low field spectrometers, combined with straightforward computational analysis, provide rapid and accurate guidance to rationally improve catalytic efficiency of histidine‐promoted catalysis. Proteins 2017; 85:1656–1665. © 2017 Wiley Periodicals, Inc.     

1H nuclear magnetic resonance studies of histidine-containing di- and tripeptides. Estimation of the effects of charged groups on the pKa value of the imidazole ring.

The nmr titration curves of chemical shifts versus pH were observed for the protons of various histidine-containing di- and tripeptides. With these results, the macroscopic pK_a values and the chemical shifts intrinsic to each ionic species were determined by a computer curve-fitting based on a simple acid dissociation sequence. The pK_a value of the imidazole ring in N-acetyl-L -histidine methylamide was assumed to represent the intrinsic (or unperturbed) pK_a of the imidazole rings of histidine having peptide linkages at both the CO and NH sides. The pK_a values of the imidazole rings observed for most di- and tripeptides were reasonably reproduced by simple calculations using the intrinsic value and the perturbations due to the CO₂^? and NH₃⁺ groups located at various positions. Some other factors affecting the pK_a value of the imidazole ring are also discussed.     

Thermodynamics and dissociation constants of carboxylic acids at high ionic strength and temperature

Dissociation constants (pK_a) of oxalic, iminodiacetic, citric, nitrilotriacetic, ethylenediaminetetraacetic, trans-1,2 diaminocyclohexanetetraacetic acid and diethylenetriaminepentaacetic acid have been determined potentiometrically using a glass electrode at an ionic strength of 6.60 m (NaClO₄) and temperatures of 0-60 °C. The constants of iminodiacetic, nitrilotriacetic and diethylenetriaminepentaacetic acid were measured at 25 °C at ionic strengths from 0.30 to 6.60 m (NaClO₄). The thermodynamic parameters for the dissociation of these carboxylic acids were derived from the temperature dependence of the dissociation constants. The Specific Ion Interaction Theory (SIT) and the Parabolic model successfully described the ionic strength dependencies of the pK_a values. The variation of the pK_a values at high ionic strengths as a function of the type and concentration of supporting electrolyte is discussed and compared with literature data.     

Design,synthesis and evaluation of 2,4-disubstituted pyrimidines as cholinesterase inhibitors

A group of 2,4-disubstituted pyrimidine derivatives (7a–e, 8a–e and 9a–d) that possess a variety of C-2 aliphatic five- and six-membered heterocycloalkyl ring in conjunction with a C-4 arylalkylamino substituent were designed, synthesized and evaluated as cholinesterase (ChE) inhibitors. The steric and electronic properties at C-2 and C-4 positions of the pyrimidine ring were varied to investigate their effect on ChE inhibitory potency and selectivity. The structure–activity relationship (SAR) studies identified N-benzyl-2-thiomorpholinopyrimidin-4-amine (7c) as the most potent cholinesterase inhibitor (ChEI) with an IC₅₀ = 0.33 μM (acetylcholinesterase, AChE) and 2.30 μM (butyrylcholinesterase, BuChE). The molecular modeling studies indicate that within the AChE active site, the C-2 thiomorpholine substituent was oriented toward the cationic active site region (Trp84 and Phe330) whereas within the BuChE active site, it was oriented toward a hydrophobic region closer to the active site gorge entrance (Ala277). Accordingly, steric and electronic properties at the C-2 position of the pyrimidine ring play a critical role in ChE inhibition.