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.

Elucidating the Activation Mechanism of the Proton-sensing GPR68 Receptor.

GPR68 is a proton-sensing G-protein Coupled Receptor (GPCR) involved in a variety of physiological processes and disorders including neoplastic pathologies. While GPR68 and few other GPCRs have been shown to be activated by a decrease in the extracellular pH, the molecular mechanism of their activation remains largely unknown. In this work, we used a combined computational and in vitro approach to provide new insight into the activation mechanism of the receptor. Molecular Dynamics simulations of GPR68 were used to model the changes in residue interactions and motions triggered by pH. Global and local rearrangements consistent with partial activation were observed upon protonation of the inactive state. Selected extracellular histidine and transmembrane acidic residues were found to have significantly upshifted pKa values during the simulations, consistently with their previously hypothesised role in activation through changes in protonation state. Moreover, a novel pairing between histidine and acidic residues in the extracellular region was highlighted by both sequence analyses and simulation data and tested through site-directed mutagenesis. At last, we identified a previously unknown hydrophobic lock in the extracellular region that might stabilise the inactive conformation and regulate the transition to the active state.

Acidity of persulfides and its modulation by the protein environments in sulfide quinone oxidoreductase and thiosulfate sulfurtransferase.

Persulfides (RSSH/RSS-) participate in sulfur metabolism and are proposed to transduce hydrogen sulfide (H2S) signaling. Their biochemical properties are poorly understood. Herein, we studied the acidity and nucleophilicity of several low molecular weight persulfides using the alkylating agent, monobromobimane. The different persulfides presented similar pKa values (4.6-6.3) and pH-independent rate constants (3.2-9.0 × 103 M-1 s-1), indicating that the substituents in persulfides affect properties to a lesser extent than in thiols because of the larger distance to the outer sulfur. The persulfides had higher reactivity with monobromobimane than analogous thiols and putative thiols with the same pKa, providing evidence for the alpha effect (enhanced nucleophilicity by the presence of a contiguous atom with high electron density). Additionally, we investigated two enzymes from the human mitochondrial H2S oxidation pathway that form catalytic persulfide intermediates, sulfide quinone oxidoreductase and thiosulfate sulfurtransferase (TST, rhodanese). The pH dependence of the activities of both enzymes was measured using sulfite and/or cyanide as sulfur acceptors. The TST half-reactions were also studied by stopped-flow fluorescence spectroscopy. Both persulfidated enzymes relied on protonated groups for reaction with the acceptors. Persulfidated sulfide quinone oxidoreductase appeared to have a pKa of 7.8 ± 0.2. Persulfidated TST presented a pKa of 9.38 ± 0.04, probably due to a critical active site residue rather than the persulfide itself. The TST thiol reacted in the anionic state with thiosulfate, with an apparent pKa of 6.5 ± 0.1. Overall, our study contributes to a fundamental understanding of persulfide properties and their modulation by protein environments.

Revisiting the pKa-Flux method for determining intrinsic membrane permeability.

Intrinsic membrane permeability is one of several factors that critically determine the intestinal absorption of a chemical. The intrinsic membrane permeability of a chemical is usually extracted from transwell experiments with Caco-2 or MDCK cells, preferably by the pKa-Flux method, which is considered the method of choice when aqueous boundary layer effects need to be excluded. The pKa-Flux method has two variants, the iso-pH method, where apical and basolateral pH are equal, and the gradient-pH method, where apical and basolateral pH are different. The most commonly used method is the gradient-pH method, as it is intended to reflect the pH-conditions in the gastrointestinal tract. However, concentration-shift effects caused by the applied pH-difference between apical and basolateral compartment in the gradient-pH method have not been considered in the evaluation of the experimental data in the past. Consequently, incorrect intrinsic membrane permeabilities have been determined. In this work, we present a revised method for extracting the intrinsic membrane permeability from gradient-pH data that considers concentration-shift effects in the basolateral aqueous boundary layer and filter as well as in the cytosol. Furthermore, we propose the use of the iso-pH method, where only concentration-shift effects in the cytosol need to be considered, as an alternative to the gradient-pH method. We use the five lipophilic bases amantadine, chloroquine, propranolol, venlafaxine and verapamil as examples to compare gradient-pH method and iso-pH method with regard to the extractability of the intrinsic membrane permeability. For lipophilic bases, the iso-pH method proves to be advantageous. All intrinsic membrane permeabilities determined in this work were substantially higher than the intrinsic membrane permeabilities reported in literature.

Lipophilicity of fentalogs: Comparison of experimental and computationally derived data.

Although fentanyl and a small number of derivatives used for medical or veterinary procedures are well characterized, physiochemical properties have not been determined for many of the newer fentanyl analogs. Partition coefficients (Log P) were determined for 19 fentalogs using the shake-flask method and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Experimentally determined partition coefficients were compared with computationally derived data using six independent software sources (ACD/LogP, LogKOWWIN v 1.69, miLogP 2.2, OsirisP, XLOGP 3.0, ALogPS 2.1). Fentalogs with a wide variety of structural modifications were intentionally selected, yielding Log P values ranging from 1.21 to 4.90. Comparison of experimental and computationally derived Log P values were highly correlated (R2 0.854-0.967). Overall, substructure-based modeling using fragmental methods or property-based topological approaches aligned more closely with experimentally determined Log P values. LC-MS/MS was also used to estimate pKa values for fentalogs with no previously reported data. Lipophilicity and pKa are important considerations for analytical detection and toxicological interpretation. In silico methods allow the determination of physicochemical information prior to certified reference materials being readily available for in vitro or in vivo studies. Computationally derived data can provide insight regarding physiochemical characteristics of future fentalogs and other classes of synthetic analogs that have yet to emerge.

Avoid missing pKas: High-throughput workflow using solution pH-metric in tandem with UV-metric measurements.

We describe a new high-throughput automated pKa workflow using potentiometry starting with 10 mM DMSO stock (solution pH-metric). Two approaches using either neat DMSO stock solution or removal of DMSO were evaluated with different sample amounts and cosolvent schemes. These were validated against traditional potentiometric measurements for optimal conditions. Further, we detail how high throughput solution pH-metric experiments are performed in tandem with established UV-metric measurements to capitalize on the advantages of both approaches. This new workflow maintains the sample and time savings required for measuring a large number of samples in a drug discovery setting, while avoiding "missing pKas" due to lack of sufficient UV chromophores. The combination of the two assays is key to tackle the challenges of low solubility, overlapping pKas, and preliminary assignment of pKas for Structure-Activity Relationship (SAR) understanding.

A systematic review of various pKa determination techniques.

The pKa values of functional groups is crucial in determining the pharmacokinetic properties of a drug, affecting its absorption and thus bioavailability. This physicochemical property is also vital for the designing of drug excipients and vehicles. There are currently 13 known methods of determining a pKa value, namely: potentiometric titration, spectrometry, fluorometry, NMR, HPLC, conductometry, electrophoresis, voltammetry, solubility, partition coefficient, calorimetry, computational, and surface tension. Some of these techniques are more widely utilized and well-established compared to others, with each having their inherent advantages and disadvantages. This review discusses each of the aforementioned techniques with emphasis on their pros and cons.

Torque generation mechanism in Fo motor of ATP synthase elucidated by free-energy and Coulomb-energy landscapes along the c-ring rotation.

Fo portion of ATP synthase is a proton-motive rotary motor. The Coulombic attraction between the conserved acidic residues in the c-ring and the arginine in the a-subunit (aR) was early proposed to drive the c-ring rotation relative to the a-subunit, and has been actually observed in our previous molecular dynamics simulation with full atomistic description of Fo embedded in the membrane. In this study, to quantify the driving force, we conducted the umbrella sampling (US) and obtained the free-energy landscape for the c-ring rotation. We first show that the free-energy gradient toward the ATP-synthesis direction appears in the deprotonated state of cE. Using the sampled snapshots that cover a wide range of the rotational angle, we further analyzed the rotational-angle dependence of the hydration and the protonation states and obtained the Coulomb-energy landscapes with a focus on the cE-aR interaction. The results indicate that both the Coulombic solvation energy of cE and the interaction energy between cE and aR contribute to the torque generation for the c-ring rotation.

Spectroscopic and Density Functional Studies on the Interaction of a Naphthalene Derivative with Anions.

This article highlights the investigation of anion interactions and recognition abilities of naphthalene derivative, [(E)-1-(((4-nitrophenyl)imino)methyl)naphthalen-2-ol], (NIMO) by UV-visible spectroscopically and colorimetrically. NIMO shows selective recognition of F- ions colorimetrically, and a visual color change from yellow to pink is observed by the naked eye. The F- ions recognition is fully reversible in the presence of HSO4- ions. The limit of F- ions detection by NIMO could be possible down to 0.033 ppm-level. A paper strips-based test kit has been demonstrated to detect F- ions selectively by the naked eye, and a smartphone-based method for real sample analysis in the non-aqueous medium has also been demostrated. Spectroscopic behavior is well supported by pKa value calculation and DFT analysis, to find a correlation with receptor analyte interaction. The optical response of NIMO towards the accumulation of F- ions and, subsequently, HSO4- ions as chemical inputs provides an opportunity to construct INH and IMP molecular logic gates.

Machine learning methods for pKa prediction of small molecules: Advances and challenges.

The acid-base dissociation constant (pKa) is a fundamental property influencing many ADMET properties of small molecules. However, rapid and accurate pKa prediction remains a great challenge. In this review, we outline the current advances in machine-learning-based QSAR models for pKa prediction, including descriptor-based and graph-based approaches, and summarize their pros and cons. Moreover, we highlight the current challenges and future directions regarding experimental data, crucial factors influencing pKa and in silico prediction tools. We hope that this review can provide a practical guidance for the follow-up studies.

The role of lipid components in lipid nanoparticles for vaccines and gene therapy.

Lipid nanoparticles (LNPs) play an important role in mRNA vaccines against COVID-19. In addition, many preclinical and clinical studies, including the siRNA-LNP product, Onpattro®, highlight that LNPs unlock the potential of nucleic acid-based therapies and vaccines. To understand what is key to the success of LNPs, we need to understand the role of the building blocks that constitute them. In this Review, we discuss what each lipid component adds to the LNP delivery platform in terms of size, structure, stability, apparent pKa, nucleic acid encapsulation efficiency, cellular uptake, and endosomal escape. To explore this, we present findings from the liposome field as well as from landmark and recent articles in the LNP literature. We also discuss challenges and strategies related to in vitro/in vivo studies of LNPs based on fluorescence readouts, immunogenicity/reactogenicity, and LNP delivery beyond the liver. How these fundamental challenges are pursued, including what lipid components are added and combined, will likely determine the scope of LNP-based gene therapies and vaccines for treating various diseases.

Determination of proton dissociation constants (pKa) of hydroxyl groups of 2,5-dihydroxy-1,4-benzoquinone (DHBQ) by UV-Vis, fluorescence and ATR-FTIR spectroscopy.

The dissociation constant is an important physicochemical parameter of amolecule. The protonation state of a molecule reflects its reactivity, solubility or ability to chemically interact with other molecules. In the present study, dissociation constants (pKa) values of 2,5-dihydroxy-1,4-benzoquinone (DHBQ) were determined by UV-Vis, fluorescence and ATR-FTIR spectroscopy at 25 °C. The resulting pKa values for DHBQ were 2.95 and 5.25. We have also experimentally found out that the monoanionic form (HBQ-) provides weak fluorescence in the pH range of about 3-6. This allowed us to determine not only the pKa in the ground but also the excited state of the molecule (pKa1* = 4.38 andpKa2* = 5.27).

Determination of the aqueous pKa of very insoluble drugs by capillary electrophoresis: Internal standards for methanol-water extrapolation.

A fast determination of acidity constants (pKa) of very insoluble drugs has become a necessity in drug discovery process because it often produces molecules that are highly lipophilic and sparingly soluble in water. In this work the high throughput internal standard capillary electrophoresis (IS-CE) method has been adapted to the determination of pKa of water insoluble compounds by measurement in methanol/aqueous buffer mixtures. For this purpose, the reference pKa values for a set of 46 acid-base compounds of varied structure (internal standards) have been established in methanol-water mixtures at several solvent composition levels (with a maximum of 40% methanol). The IS-CE method has been successfully applied to seven test drugs of different chemical nature with intrinsic solubilities lower than 10-6 M. pKa values have been determined at different methanol/aqueous buffer compositions and afterwards Yasuda-Shedlovsky extrapolation method has been applied to obtain the aqueous pKa. The obtained results have successfully been compared to literature ones obtained by other methods. It is concluded that the IS-CE method allows the determination of aqueous pKa values using low proportions of methanol, becoming then more accurate in the extrapolation procedure than other reference methods.

Modulating physicochemical properties of tetrahydropyridine-2-amine BACE1 inhibitors with electron-withdrawing groups: A systematic study.

A common challenge for medicinal chemists is to reduce the pKa of strongly basic groups' conjugate acids into a range that preserves the desired effects, usually potency and/or solubility, but avoids undesired effects like high volume of distribution (Vd), limited membrane permeation, and off-target binding to, notably, the hERG channel and monoamine receptors. We faced this challenge with a 3,4,5,6-tetrahydropyridine-2-amine scaffold harboring an amidine, a key structural component of potential inhibitors of BACE1, the rate-limiting enzyme in the production of Aß species that make up amyloid plaques in Alzheimer's disease. In our endeavor to balance potency with desirable properties to achieve brain penetration, we introduced a diverse set of groups in beta position of the amidine that modulate logD, PSA and pKa. Given the synthetic challenge to prepare these highly functionalized warheads, we first developed a design flow including predicted physicochemical parameters which allowed us to select only the most promising candidates for synthesis. For this we evaluated a set of commercial packages to predict physicochemical properties, which can guide medicinal chemists in their endeavors to modulate pKa values of amidine and amine bases.

Molecular interactions in remdesivir-cyclodextrin systems.

Remdesivir (REM) is the first antiviral drug (Veklury™) approved by the Food and Drug Administration for the therapy of COVID-19. Due to its poor water solubility, the preparation of Veklury™ requires a suitable solubilizing excipient at pH 2 conditions. For this purpose, the final formulation contains the randomly substituted sulfobutylether-ß-cyclodextrin (SBEßCD) as a complexing agent. Herein, extensive NMR spectroscopic study with various cyclodextrin (CD) derivatives were conducted to understand the interactions in SBEßCD - REM systems at the molecular level. The pKa value of REM has been determined experimentally for the first time, as the protonation state of the aminopyrrolo-triazine moiety can play a key role in CD-REM inclusion complex formation as SBEßCD has permanent negative charges. The UV-pH titration experiments yielded a pKa of 3.56, thus the majority of REM bears a positive charge at pH 2.0. NMR experiments were performed on ß- and γCD derivatives to determine complex stabilities, stoichiometries and structures. The stability constants were determined by nonlinear curve fitting based on 1H NMR titrations at pH 2.0, while Job's method was used to determine the stoichiometries. ßCD complexes were one order of magnitude more stable than their γCD counterparts. Sulfobutylation resulted in a significant increase in stability and the single isomer derivatives showed unexpectedly high stability values (logK = 4.35 for REM - per-6-SBEßCD). In the case of ßCDs, the ethylbutyl-moiety plays a key role in complexation immersing into the ßCD cavity, while the phenoxy-moiety overtakes and drives the inclusion of REM in the case of γCDs. This is the first comprehensive study of REM-CD complexation, allowing the design of new CD derivatives with tailored stabilities, thereby aiding the formulation or production and even the analytical characterization of REM.

Transient kinetic analysis for studying ionizations in RNA modification enzyme mechanisms.

The application of in vitro kinetic tools has the potential to provide important insight into the molecular mechanisms of RNA modification enzymes. Utilizing quantitative biochemical approaches can reveal information about enzyme preferences for specific substrates that are relevant for understanding modification reactions in their biological contexts. Moreover, kinetic tools have been powerfully applied to identify and characterize roles for specific amino acid residues in catalysis, which can be essential information for understanding the molecular basis for human disease, as well as for targeting these enzymes for potential therapeutic interventions. RNA methyltransferases are a particularly interesting group of RNA modification enzymes because of the diversity in structure and mechanism that has been revealed among members of this group, even including some examples of enzymes that use entirely distinct reaction mechanisms to form identical methylated nucleotides in RNA. Yet, many questions remain unanswered about how these distinct catalytic strategies are facilitated by the relevant enzyme families. We have applied in vitro kinetic analysis to specifically focus on catalytically relevant ionizations in the context of tRNA methyltransferase reactions, by measuring rates under conditions of varied pH. This analysis can be applied broadly to RNA methyltransferases to expand our understanding of these important enzymes.

Prediction of n-octanol/water partition coefficients and acidity constants (pKa) in the SAMPL7 blind challenge with the IEFPCM-MST model.

Within the scope of SAMPL7 challenge for predicting physical properties, the Integral Equation Formalism of the Miertus-Scrocco-Tomasi (IEFPCM/MST) continuum solvation model has been used for the blind prediction of n-octanol/water partition coefficients and acidity constants of a set of 22 and 20 sulfonamide-containing compounds, respectively. The log P and pKa were computed using the B3LPYP/6-31G(d) parametrized version of the IEFPCM/MST model. The performance of our method for partition coefficients yielded a root-mean square error of 1.03 (log P units), placing this method among the most accurate theoretical approaches in the comparison with both globally (rank 8th) and physical (rank 2nd) methods. On the other hand, the deviation between predicted and experimental pKa values was 1.32 log units, obtaining the second best-ranked submission. Though this highlights the reliability of the IEFPCM/MST model for predicting the partitioning and the acid dissociation constant of drug-like compounds compound, the results are discussed to identify potential weaknesses and improve the performance of the method.

Evaluation of log P, pKa, and log D predictions from the SAMPL7 blind challenge.

The Statistical Assessment of Modeling of Proteins and Ligands (SAMPL) challenges focuses the computational modeling community on areas in need of improvement for rational drug design. The SAMPL7 physical property challenge dealt with prediction of octanol-water partition coefficients and pKa for 22 compounds. The dataset was composed of a series of N-acylsulfonamides and related bioisosteres. 17 research groups participated in the log P challenge, submitting 33 blind submissions total. For the pKa challenge, 7 different groups participated, submitting 9 blind submissions in total. Overall, the accuracy of octanol-water log P predictions in the SAMPL7 challenge was lower than octanol-water log P predictions in SAMPL6, likely due to a more diverse dataset. Compared to the SAMPL6 pKa challenge, accuracy remains unchanged in SAMPL7. Interestingly, here, though macroscopic pKa values were often predicted with reasonable accuracy, there was dramatically more disagreement among participants as to which microscopic transitions produced these values (with methods often disagreeing even as to the sign of the free energy change associated with certain transitions), indicating far more work needs to be done on pKa prediction methods.

SAMPL7 blind challenge: quantum-mechanical prediction of partition coefficients and acid dissociation constants for small drug-like molecules.

The physicochemical properties of a drug molecule determine the therapeutic effectiveness of the drug. Thus, the development of fast and accurate theoretical approaches for the prediction of such properties is inevitable. The participation to the SAMPL7 challenge is based on the estimation of logP coefficients and pKa values of small drug-like sulfonamide derivatives. Thereby, quantum mechanical calculations were carried out in order to calculate the free energy of solvation and the transfer energy of 22 drug-like compounds in different environments (water and n-octanol) by employing the SMD solvation model. For logP calculations, we studied eleven different methodologies to calculate the transfer free energies, the lowest RMSE value was obtained for the M06L/def2-TZVP//M06L/def2-SVP level of theory. On the other hand, we employed an isodesmic reaction scheme within the macro pKa framework; this was based on selecting reference molecules similar to the SAMPL7 challenge molecules. Consequently, highly well correlated pKa values were obtained with the M062X/6-311+G(2df,2p)//M052X/6-31+G(d,p) level of theory.

Using QSAR Models to Predict Mitochondrial Targeting by Small-Molecule Xenobiotics Within Living Cells.

Prediction of mitochondrial targeting, or prediction of exclusion from mitochondria, of small-molecule xenobiotics (biocides, drugs, probes, toxins) can be achieved using an algorithm derived from QSAR modeling. Application of the algorithm requires knowing the chemical structures of all ionic species of the xenobiotic compound in question, and for certain numerical structure parameters (AI, CBN, log P, pK a, and Z) to be obtained for all such species. Procedures for specification of the chemical structures; estimation of the structure parameters; and application of the algorithm are described in an explicit protocol.