Determination Of pKa Values Research Articles

Direct observation of the complex S(IV) equilibria at the liquid-vapor interface

The multi-phase oxidation of S(IV) plays a crucial role in the atmosphere, leading to the formation of haze and severe pollution episodes. We here contribute to its understanding on a molecular level by reporting experimentally determined pKa values of the various S(IV) tautomers and reaction barriers for SO2 formation pathways. Complementary state-of-the-art molecular-dynamics simulations reveal a depletion of bisulfite at low pH at the liquid-vapor interface, resulting in a different tautomer ratio at the interface compared to the bulk. On a molecular-scale level, we explain this with the formation of a stable contact ion pair between sulfonate and hydronium ions, and with the higher energetic barrier for the dehydration of sulfonic acid at the liquid-vapor interface. Our findings highlight the contrasting physicochemical behavior of interfacial versus bulk environments, where the pH dependence of the tautomer ratio reported here has a significant impact on both SO2 uptake kinetics and reactions involving NOx and H2O2 at aqueous aerosol interfaces.

Determination of the pKa Value of a Brønsted Acid by 19F NMR Spectroscopy.

Brønsted acids, such as phosphoric acids derived from chiral 1,1'-bi-2-naphthol (BINOL), are important catalysts in the formation of carbon-carbon and carbon-heteroatom bonds, for example. The catalytic activity of these Brønsted acids is strongly linked to their acidity, and as such, the evaluation of compounds to determine pKa values provides insight into their catalytic activity. Herein, a 19F{1H} NMR methodology is detailed to determine the pKa of a fluorinated binaphthyl-derived phosphinic acid, rac-1, in acetonitrile and in the presence of a fluorinated sulfonamide reference compound (2-4). The approach was tested initially using 2 and 3, with the ΔpKa (0.08) in strong agreement with previously reported values (6.6 for 2 and 6.68/6.73 for 3). Sigmoidal curves of normalised chemical shift change (Δδ) against equivalents of the base phosphazene P1-tBu added overlapped for 2 and 3, but in the case of rac-1 and either 2, 3 or 4, there was significant separation. A variety of different approaches for determining the ΔpKa were compared. Values of pKa determined when the normalised Δδ was 90% were optimal for 2 and 3, whereas a normalised Δδ of 75% was optimal for 4, resulting in the pKa of rac-1 being determined to be 8.47-8.71.

Highly Acidic Electron-Rich Brønsted Acids Accelerate Asymmetric Pictet-Spengler Reactions by Virtue of Stabilizing Cation-π Interactions.

Electron-rich heteroaromatic imidodiphosphorimidates (IDPis) catalyze the asymmetric Pictet-Spengler reaction of N-carbamoyl-β-arylethylamines with high stereochemical precision. This particular class of catalysts furthermore provides a vital rate enhancement compared to related Brønsted acids. Here we present experimental studies on the underlying reaction kinetics that shed light on the specific origins of rate acceleration. Analysis of Hammett plots, kinetic isotope effects, reaction orders, Eyring plots, and isotopic scrambling experiments, allowed us to gather insights into the molecular interactions between the chiral Brønsted acid and catalytically formed intermediates. Based on rigorously determined pKa values as well as the experimental evidence, we propose that attractive intermolecular forces offered by electron-rich π-surfaces of the chiral counteranion enthalpically stabilize cationic intermediates and transition states by way of cation-π interactions. This view is furthermore supported by in-depth density functional theory calculations. Our deepened understanding of the reaction mechanism allowed us to develop a method for accessing 1-aryltetrahydroisoquinolines from aromatic dimethyl acetals, a substrate class that was thus far inaccessible via catalytic asymmetric Pictet-Spengler reactions.

Amino-Acid-Derived Anionic Polyacrylamides with Tailored Hydrophobicity-Physicochemical Properties and Cellular Interactions.

Polyanions can internalize into cells via endocytosis without any cell disruption and are therefore interesting materials for biomedical applications. In this study, amino-acid-derived polyanions with different alkyl side-chains are synthesized via postpolymerization modification of poly(pentafluorophenyl acrylate), which is synthesized via reversible addition-fragmentation chain-transfer (RAFT) polymerization, to obtain polyanions with tailored hydrophobicity and alkyl branching. The success of the reaction is verified by size-exclusion chromatography, NMR spectroscopy, and infrared spectroscopy. The hydrophobicity, surface charge, and pH dependence are investigated in detail by titrations, high-performance liquid chromatography, and partition coefficient measurements. Remarkably, the determined pK a-values for all synthesized polyanions are very similar to those of poly(acrylic acid) (pK a = 4.5), despite detectable differences in hydrophobicity. Interactions between amino-acid-derived polyanions with L929 fibroblasts reveal very slow cell association as well as accumulation of polymers in the cell membrane. Notably, the more hydrophobic amino-acid-derived polyanions show higher cell association. Our results emphasize the importance of macromolecular engineering toward ideal charge and hydrophobicity for polymer association with cell membranes and internalization. This study further highlights the potential of amino-acid-derived polymers and the diversity they provide for tailoring properties toward drug delivery applications.

Determination of acidity constants of pyridines, imidazoles, and oximes by capillary electrophoresis.

The acidity constant in the form of pKa is one of the most important physicochemical quantities. There are prediction tools available for calculating the pKa , but they only deliver precise calculated values for a relatively small set of chemicals. For complex structures with multiple functional groups in particular, the error in the predicted pKa is high due to the application domain of the corresponding models. Thus, we aim to enlarge the dataset of experimentally determined pKa values using capillary electrophoresis. We, therefore, selected various pyridines, imidazoles, and oximes to determine the pKa values using the internal standard approach and the classical method. Especially oximes were not investigated in the past, and predictions for them include larger errors. Thus, our experimentally determined values could contribute to an improved understanding of various functional groups impacting the pKa values and serve as additional datasets to develop improved pKa prediction tools.

Evaluation of In Vitro Distribution and Plasma Protein Binding of Selected Antiviral Drugs (Favipiravir, Molnupiravir and Imatinib) against SARS-CoV-2.

There are a number of uncertainties regarding plasma protein binding and blood distribution of the active drugs favipiravir (FAVI), molnupiravir (MOLNU) and imatinib (IMA), which were recently proposed as therapeutics for the treatment of COVID-19 disease. Therefore, proton dissociation processes, solubility, lipophilicity, and serum protein binding of these three substances were investigated in detail. The drugs display various degrees of lipophilicity at gastric (pH 2.0) and blood pH (pH 7.4). The determined pKa values explain well the changes in lipophilic character of the respective compounds. The serum protein binding was studied by membrane ultrafiltration, frontal analysis capillary electrophoresis, steady-state fluorometry, and fluorescence anisotropy techniques. The studies revealed that the ester bond in MOLNU is hydrolyzed by protein constituents of blood serum. Molnupiravir and its hydrolyzed form do not bind considerably to blood proteins. Likewise, FAVI does not bind to human serum albumin (HSA) and α1-acid glycoprotein (AGP) and shows relatively weak binding to the protein fraction of whole blood serum. Imatinib binds to AGP with high affinity (logK' = 5.8-6.0), while its binding to HSA is much weaker (logK' ≤ 4.0). The computed constants were used to model the distribution of IMA in blood plasma under physiological and 'acute-phase' conditions as well.

Calculation-assisted regioselective functionalization of the imidazo[1,2-a]pyrazine scaffold via zinc and magnesium organometallic intermediates.

Straightforward calculations such as determinations of pKa values and N-basicities have allowed the development of a set of organometallic reactions for the regioselective functionalization of the underexplored fused N-heterocycle imidazo[1,2-a]pyrazine. Thus, regioselective metalations of 6-chloroimidazo[1,2-a]pyrazine using TMP-bases (TMP = 2,2,6,6-tetramethylpiperidyl) such as TMPMgCl·LiCl and TMP2Zn·2MgCl2·2LiCl provided Zn- and Mg-intermediates, that after quenching with various electrophiles gave access to polyfunctionalized imidazopyrazine heterocycles. Additionally, the use of TMP2Zn·2MgCl2·2LiCl as base for the first metalation allowed an alternative regioselective metalation. Nucleophilic additions at position 8 as well as selective Negishi cross-couplings complete the set of methods for selectively decorating this heterocycle of the future.

Reliable and Accurate Prediction of Single-Residue pKa Values through Free Energy Perturbation Calculations.

Accurate prediction of the pKa's of protein residues is crucial to many applications in biological simulation and drug discovery. Here, we present the use of free energy perturbation (FEP) calculations for the prediction of single-protein residue pKa values. We begin with an initial set of 191 residues with experimentally determined pKa values. To isolate sampling limitations from force field inaccuracies, we develop an algorithm to classify residues whose environments are significantly affected by crystal packing effects. We then report an approach to identify buried histidines that require significant sampling beyond what is achieved in typical FEP calculations. We therefore define a clean data set not requiring algorithms capable of predicting major conformational changes on which other pKa prediction methods can be tested. On this data set, we report an RMSE of 0.76 pKa units for 35 ASP residues, 0.51 pKa units for 44 GLU residues, and 0.67 pKa units for 76 HIS residues.

AMOEBA Force Field Trajectories Improve Predictions of Accurate pKa Values of the GFP Fluorophore: The Importance of Polarizability and Water Interactions.

Precisely quantifying the magnitude, direction, and biological functions of electric fields in proteins has long been an outstanding challenge in the field. The most widely implemented experimental method to measure such electric fields at a particular residue in a protein has been through changes in pKa of titratable residues. While many computational strategies exist to predict these values, it has been difficult to do this accurately or connect predicted results to key structural or mechanistic features of the molecule. Here, we used experimentally determined pKa values of the fluorophore in superfolder green fluorescent protein (GFP) with amino acid mutations made at position Thr 203 to evaluate the pKa prediction ability of molecular dynamics (MD) simulations using a polarizable force field, AMOEBA. Structure ensembles from AMOEBA were used to calculate pKa values of the GFP fluorophore. The calculated pKa values were then compared to trajectories using a conventional fixed charge force field (Amber03 ff). We found that the position of water molecules included in the pKa calculation had opposite effects on the pKa values between the trajectories from AMOEBA and Amber03 force fields. In AMOEBA trajectories, the inclusion of water molecules within 35 Å of the fluorophore decreased the difference between the predicted and experimental values, resulting in calculated pKa values that were within an average of 0.8 pKa unit from the experimental results. On the other hand, in Amber03 trajectories, including water molecules that were more than 5 Å from the fluorophore increased the differences between the calculated and experimental pKa values. The inaccuracy of pKa predictions determined from Amber03 trajectories was caused by a significant stabilization of the deprotonated chromophore's free energy compared to the result in AMOEBA. We rationalize the cutoffs for explicit water molecules when calculating pKa to better predict the electrostatic environment surrounding the fluorophore buried in GFP. We discuss how the results from this work will assist the prospective prediction of pKa values or other electrostatic effects in a wide variety of folded proteins.

Hydrogen Atom Transfer Thermodynamics of Homologous Co(III)- and Mn(III)-Superoxo Complexes: The Effect of the Metal Spin State.

Systematic investigations on H atom transfer (HAT) thermodynamics of metal O2 adducts is of fundamental importance for the design of transition metal catalysts for substrate oxidation and/or oxygenation directly using O2. Such work should help elucidate underlying electronic-structure features that govern the OO–H bond dissociation free energies (BDFEs) of metal-hydroperoxo species, which can be used to quantitatively appraise the HAT activity of the corresponding metal-superoxo complexes. Herein, the BDFEs of two homologous CoIII- and MnIII-hydroperoxo complexes, 3-Co and 3-Mn, were calculated to be 79.3 and 81.5 kcal/mol, respectively, employing the Bordwell relationship based on experimentally determined pKa values and redox potentials of the one-electron-oxidized forms, 4-Co and 4-Mn. To further verify these values, we tested the HAT capability of their superoxo congeners, 2-Co and 2-Mn, toward three different substrates possessing varying O–H BDFEs. Specifically, both metal-superoxo species are capable of activating the O–H bond of 4-oxo-TEMPOH with an O–H BDFE of 68.9 kcal/mol, only 2-Mn is able to abstract a H atom from 2,4-di-tert-butylphenol with an O–H BDFE of 80.9 kcal/mol, and neither of them can react with 3,5-dimethylphenol with an O–H BDFE of 85.6 kcal/mol. Further computational investigations suggested that it is the high spin state of the MnIII center in 3-Mn that renders its OO–H BDFE higher than that of 3-Co, which features a low-spin CoIII center. The present work underscores the role of the metal spin state being as crucial as the oxidation state in modulating BDFEs.

Structure-Activity Relationships of 8-Hydroxyquinoline-Derived Mannich Bases with Tertiary Amines Targeting Multidrug-Resistant Cancer.

A recently proposed strategy to overcome multidrug resistance (MDR) in cancer is to target the collateral sensitivity of otherwise resistant cells. We designed a library of 120 compounds to explore the chemical space around previously identified 8-hydroxyquinoline-derived Mannich bases with robust MDR-selective toxicity. We included compounds to study the effect of halogen and alkoxymethyl substitutions in R5 in combination with different Mannich bases in R7, a shift of the Mannich base from R7 to R5, as well as the introduction of an aromatic moiety. Cytotoxicity tests performed on a panel of parental and MDR cells highlight a strong influence of experimentally determined pKa values of the donor atom moieties, indicating that protonation and metal chelation are important factors modulating the MDR-selective anticancer activity of the studied compounds. Our results identify structural requirements increasing MDR-selective anticancer activity, providing guidelines for the development of more effective anticancer chelators targeting MDR cancer.

Imidazo[4,5-b]pyridine derived iminocoumarins as potential pH probes: Synthesis, spectroscopic and computational studies of their protonation equilibria

We describe the synthesis, spectroscopic characterization and computational analysis of the protonation equilibria of several newly designed imidazo[4,5-b]pyridine derived iminocoumarins to confirm their potential as pH sensing molecules in different solvents. The photophysical characterization and acid/base properties of these chromophores have been carried out in several polar and non-polar organic solvents as well as buffered aqueous solution with different pH values using UV–Vis and fluorescence spectroscopies. Obtained results revealed that both the solvent polarity and the electronic nature of substituents placed at the iminocoumarin and imidazo[4,5-b]pyridine cores significantly influence spectral responses of studied systems. An excellent agreement in experimentally measured and computationally determined pKa values and electronic excitations suggests that all systems are monoprotonated cations at neutral pH, while their transition to diprotonated forms occurs at pH values between 3.4 and 4.4, whereas they get reverted to neutral unionized molecules between 10.4 and 13.7. These small differences in narrow ranges of borderline pH values are predominantly brought about by variations in a single substituent on the iminocoumarin core, thereby allowing a simple strategy to further modify the observed sensing features and easily tune them for a specific application.

Applicability of hybrid micelle liquid chromatography for practical determination of acid dissociation constant

Hybrid micellar liquid chromatography was used for practical determination of acid dissociation constant (pKa) of three analytes, namely, ibuprofen (IBU), naproxen (NPX) and benzoic acid (BZA). The proposed method was established based on the principle adopting the dependence of the retention time of ionizable analytes on the mobile phase pH. Simple data manipulation was carried out without the need of specialized software, allowing estimation of practically determined pKa values, which were found to be in accordance with calculated and experimentally determined values, emphasizing on the versatility and applicability of the proposed method. The present methodology carries unique features which support its utility over traditional techniques adopted for pKa determination, like its solubilizing ability, overcoming long retention times experienced in reversed phase chromatography, non-limitation to a specified chemical class and possible justification of the determined pKa values using linear regression equations.

Acid Dissociation Constants of the Benzimidazole Unit in the Polybenzimidazole Chain: Configuration Effects.

The acid dissociation constant of three benzimidazoles, namely 2,2′-bibenzo[d]imidazole, 2,5′-bibenzo[d]imidazole, and 5,5′-bibenzo[d]imidazole, have been investigated by means of density functional theory calculations in gas phase and in aqueous solution. The theoretical approach was validated by the comparing of predicted and experimentally determined pKa values in imidazole, benzimidazole, and 2-phenylbenzimidazole. From the studied compounds, 2,2′-bibenzo[d]imidazole was found to be the most acidic, which made it a valuable candidate as a material for polymer electrolyte membrane fuel cells.

Spectroscopic and Computational Study of the Protonation Equilibria of Amino-Substituted benzo[b]thieno[2,3-b]pyrido[1,2-a]benzimidazoles as Novel pH-Sensing Materials

We present the synthesis and analytical, spectroscopic and computational characterization of three amino-substituted benzo[b]thieno[2,3-b]pyrido[1,2-a]benzimidazoles as novel pH probes with a potential application in pH-sensing materials. The designed systems differ in the number and position of the introduced isobutylamine groups on the pentacyclic aromatic core, which affects their photophysical and acid-base properties. The latter were investigated by UV-Vis absorption and fluorescence spectroscopies and interpreted by DFT calculations. An excellent agreement in experimentally measured and computationally determined pKa values and electronic excitations suggests that all systems are unionized at neutral pH, while their transition to monocationic forms occurs at pH values between 3 and 5, accompanied by substantial changes in spectroscopic responses that make them suitable for detecting acidic conditions in solutions. Computations identified imidazole imino nitrogen as the most favorable protonation site, further confirmed by analysis of perturbations in the chemical shifts of 1H and 13C NMR, and showed that the resulting basicity emerges as a compromise between the basicity-reducing effect of a nearby nitrile and a favorable contribution from the attached secondary amines. With this in mind, we designed a system with three amino substituents for which calculations predict pKa = 7.0 that we suggest as an excellent starting point for a potential pH sensor able to capture solution changes during the transition from neutral towards acidic media.

HPLC determination of pKa value of benfotiamine

Physicochemical characteristics such as the dissociation constant (pKa) and lipophilicity parameter are very important for the properties of compounds and their determination is of great interest to the research field. They affect the solubility and permeability of the active pharmaceutical ingredients and respectively the penetration through the cellular membrane. The higher value of the lipophilicity parameter contributes to the better interaction of the investigated substances with the cellular membrane and boosts their biological action. Utilizing HPLC methods for the determination of such parameters helps to overlap challenges such as poor solubility and purity of the investigated substances. An analytical HPLC method for the determination of the pKa values and the lipophilicity parameter of benfotiamine is used in the current work.

Differentiation of isomeric metabolites of carbamazepine based on acid-base properties; Experimental vs theoretical approach

Metabolism of carbamazepine is complex and leads to the three isomeric derivatives whose occurrence is dependent on the type of sample material. Their unambiguous differentiation is overall important. In this work, the qualitative analysis of 2-hydroxycarbamazepine, 3-hydroxycrbamazepine and carbamazepine-10,11-epoxide was attempted for the first time using capillary zone electrophoresis, based on the models linking electrophoretic mobility with pKa value determining the acidity of the hydroxyl groups. For this purpose, pKa values ​​were determined using electrophoretic and theoretical methods, and then the compliance of the obtained mobility models with the measured values ​​was analyzed. Despite the slight difference in acidity ​​(0.3-0.4 pH unit), the obtained results prove that the correct identification of the metabolites under consideration, and reliable prediction of the selectivity of their separation, are possible on the basis of experimentally determined pKa values, even with highly simplified methods assuming the lack of certain data. However, it is important to choose the optimal pH value, which should be close to pKa. On the other hand, worse results were obtained for the theoretically determined mobilities, which differed significantly from the experimental values. An attempt was also made to explain the acidity of hydroxycarbamazepines and the associated thermodynamic parameters - deprotonation enthalpy and entropy, with respect to their structure. The lack of intramolecular hydrogen bonds and the significant contribution of entropic effects stabilizing the protonated form seems to be significant. The higher pKa value for CBZ-2-OH probably results from the stronger effect of the energetically unfavorable organization of solvent dipoles due to ionization.

Ratiometric Near-Infrared Fluorescent Probes Based on Hemicyanine Dyes Bearing Dithioacetal and Formal Residues for pH Detection in Mitochondria.

Ratiometric near-infrared fluorescent probes (AH+ and BH+) have been prepared for pH determination in mitochondria by attaching dithioacetal and formal residues onto a hemicyanine dye. The reactive formyl group on probe BH+ allows for retention inside mitochondria as it can react with a protein primary amine residue to form an imine under slightly basic pH 8.0. Probes AH+ and BH+ display ratiometric fluorescent responses to pH changes through the protonation and deprotonaton of a hydroxy group in hemicyanine dyes with experimentally determined pKa values of 6.85 and 6.49, respectively. Calculated pKa values from a variety of theoretical methods indicated that the SMDBONDI method of accounting for solvent and van der Waals radii plus including a water molecule located near the site of protonation produced the closest overall agreement with the experimental values at 7.33 and 6.14 for AH+ and BH+ respectively.

Neutral pH monosaccharide receptor based on boronic acid decorated poly(2-hydroxyethyl methacrylate): Spectral Methods for determination of glucose-binding and ionization constants

Herein, a glucose-responsive polymer featuring boronic acid has been synthesized that operates under physiological conditions. Phenylboronic acid-containing polymer has been successfully synthesized in two reaction steps using atom transfer radical polymerization (ATRP) of 2-hydroxyethyl methacrylate (HEMA) and post-polymerization modification of the obtained polyHEMA with 4-carboxyphenylboronic acid. The structure of the synthesized polyHEMA and polyHEMA-PBA has been investigated by size exclusion chromatography (SEC), proton nuclear magnetic resonance (1H NMR), and Fourier-transform infrared spectrometer (FT-IR). Interaction of polyHEMA-PBA with D(+)-glucose has been studied by Ultraviolet visible (UV–vis.) spectroscopy. Determined pKa values were approximately 7.8 and 7.9 by pH titration and Spectral Difference Methods, respectively. Also, the binding constant of polyHEMA-PBA with glucose has been determined 121 M−1 by Spectral Difference Methods. Dynamic light scattering (DLS) and field emission scanning electron microscopy (FE-SEM) have been utilized to investigate dual pH- and diol (glucose)-responsive behavior of polyHEMA-PBA. Aggregates and unimers (smaller objects) have been observed below and above the pKa of the polyHEMA-PBA, respectively. Upon addition of glucose to polyHEMA-PBA solution at pH = 7.4 aggregates dissociated into molecularly dissolved unimers of polyHEMA-PBA.

Theoretical Study on Ionization of Boric Acid in Aqueous Solution by Ab Initio and DFT Methods at T=298.15 K.

The aim of this research work was to theoretically calculate the pKa value of boric acid in aqueous solution by theoretical methods at T=298.15 K. Boric acid has antifungal and antiviral properties. It is used in various prescription pharmaceutical products. The ab initio and density functional theory (DFT) methods were used in this research work. To explain the determined acidic dissociation constant, the various molecular conformations and solute-solvent interactions of the species of boric acid were considered. The basis set at the B3LYP/6-31+G (d) level of theory was selected for DFT calculations. We analyzed the formation of intermolecular hydrogen bonds between several species of boric acid and water molecules through Tomasi's method. The result showed that there was comparable agreement between the experimentally and theoretically determined pKa values for boric acid.