PKA17—A Coarse-Grain Grid-Based Methodology and Web-Based Software for Predicting Protein pK a Shifts

We have developed and tested PKA17, a coarse-grain grid-based model for predicting protein pK _a shifts. Our pK _a predictor is currently deployed via a website interface. We have carried out parameter fitting using 442 Asp, Glu, His, Lys, and Arg residues for which experimental results are available in the literature. PROPKA software has been used for benchmarking. The average unsigned error and root-mean-square deviation (RMSD) have been found to be 0.628 and 0.831 pH units, respectively, for PKA17. The corresponding results with PROPKA are 0.761 and 1.063 units. We have assessed the robustness of the developed PKA17 methodology with a number of tests and have also explored the possibility of using a combination of PROPKA and PKA17 calculations in order to improve the accuracy of predicted pK _a values for protein residues. We have also once again confirmed that protein acidity constants are influenced almost entirely by residues in the immediate spatial proximity of the ionizable amino acids. The resulting PKA17 software has been deployed online with a web-based interface at http://users.wpi.edu/~jpcvitkovic/pka_calc.html . © 2019 Wiley Periodicals, Inc.     

SAMPL6: calculation of macroscopic p<Emphasis Type="Italic">K</Emphasis><Subscript>a</Subscript> values from ab initio quantum mechanical free energies

Macroscopic pK_a values were calculated for all compounds in the SAMPL6 blind prediction challenge, based on quantum chemical calculations with a continuum solvation model and a linear correction derived from a small training set. Microscopic pK_a values were derived from the gas-phase free energy difference between protonated and deprotonated forms together with the Conductor-like Polarizable Continuum Solvation Model and the experimental solvation free energy of the proton. pH-dependent microstate free energies were obtained from the microscopic pK_as with a maximum likelihood estimator and appropriately summed to yield macroscopic pK_a values or microstate populations as function of pH. We assessed the accuracy of three approaches to calculate the microscopic pK_as: direct use of the quantum mechanical free energy differences and correction of the direct values for short-comings in the QM solvation model with two different linear models that we independently derived from a small training set of 38 compounds with known pK_a. The predictions that were corrected with the linear models had much better accuracy [root-mean-square error (RMSE) 2.04 and 1.95 pK_a units] than the direct calculation (RMSE 3.74). Statistical measures indicate that some systematic errors remain, likely due to differences in the SAMPL6 data set and the small training set with respect to their interactions with water. Overall, the current approach provides a viable physics-based route to estimate macroscopic pK_a values for novel compounds with reasonable accuracy.     

p<Emphasis Type="Italic">K</Emphasis><Subscript>a</Subscript> measurements for the SAMPL6 prediction challenge for a set of kinase inhibitor-like fragments

Determining the net charge and protonation states populated by a small molecule in an environment of interest or the cost of altering those protonation states upon transfer to another environment is a prerequisite for predicting its physicochemical and pharmaceutical properties. The environment of interest can be aqueous, an organic solvent, a protein binding site, or a lipid bilayer. Predicting the protonation state of a small molecule is essential to predicting its interactions with biological macromolecules using computational models. Incorrectly modeling the dominant protonation state, shifts in dominant protonation state, or the population of significant mixtures of protonation states can lead to large modeling errors that degrade the accuracy of physical modeling. Low accuracy hinders the use of physical modeling approaches for molecular design. For small molecules, the acid dissociation constant (pK_a) is the primary quantity needed to determine the ionic states populated by a molecule in an aqueous solution at a given pH. As a part of SAMPL6 community challenge, we organized a blind pK_a prediction component to assess the accuracy with which contemporary pK_a prediction methods can predict this quantity, with the ultimate aim of assessing the expected impact on modeling errors this would induce. While a multitude of approaches for predicting pK_a values currently exist, predicting the pK_as of drug-like molecules can be difficult due to challenging properties such as multiple titratable sites, heterocycles, and tautomerization. For this challenge, we focused on set of 24 small molecules selected to resemble selective kinase inhibitors—an important class of therapeutics replete with titratable moieties. Using a Sirius T3 instrument that performs automated acid–base titrations, we used UV absorbance-based pK_a measurements to construct a high-quality experimental reference dataset of macroscopic pK_as for the evaluation of computational pK_a prediction methodologies that was utilized in the SAMPL6 pK_a challenge. For several compounds in which the microscopic protonation states associated with macroscopic pK_as were ambiguous, we performed follow-up NMR experiments to disambiguate the microstates involved in the transition. This dataset provides a useful standard benchmark dataset for the evaluation of pK_a prediction methodologies on kinase inhibitor-like compounds.     

Absolute and relative p<Emphasis Type="Italic">K</Emphasis><Subscript>a</Subscript> predictions via a DFT approach applied to the SAMPL6 blind challenge

In this work, quantum mechanical methods were used to predict the microscopic and macroscopic pK_a values for a set of 24 molecules as a part of the SAMPL6 blind challenge. The SMD solvation model was employed with M06-2X and different basis sets to evaluate three pK_a calculation schemes (direct, vertical, and adiabatic). The adiabatic scheme is the most accurate approach (RMSE?=?1.40 pK_a units) and has high correlation (R²?=?0.93), with respect to experiment. This approach can be improved by applying a linear correction to yield an RMSE of 0.73 pK_a units. Additionally, we consider including explicit solvent representation and multiple lower-energy conformations to improve the predictions for outliers. Adding three water molecules explicitly can reduce the error by 2–4 pK_a units, with respect to experiment, whereas including multiple local minima conformations does not necessarily improve the pK_a prediction.     

Acid-catalysed liquid-to-solid transitioning of arylazoisoxazole photoswitches

Molecular photoswitches play a vital role in the development of responsive materials. These molecular building blocks are particularly attractive when multiple stimuli can be combined to bring about physical changes, sometimes leading to unexpected properties and functions. The arylazoisoxazole molecular switch was recently shown to be capable of efficient photoreversible solid-to-liquid phase transitions with application in photoswitchable surface adhesion. Here, we show that the arylazoisoxazole forms thermally stable and photoisomerisable protonated Z- and E-isomers in an apolar aprotic solvent when the pK_a of the applied acid is sufficiently low. The tuning of isomerisation kinetics from days to seconds by the pK_a of the acid not only opens up new reactivity in solution, but also the solid-state photoswitching of azoisoxazoles can be efficiently reversed with selected acid vapours, enabling acid-gated photoswitchable surface adhesion.

Molecular photoswitches are versatile components for materials with bistable and photoreversible properties. Here, we enrich the functionality of the arylazoisoxazole molecular switch by analyzing its photoresponsive protonated Z- and E-isomers.     

Benchmarking and validating algorithms that estimate p<Emphasis Type="Italic">K</Emphasis><Subscript>a</Subscript> values of drugs based on their molecular structures

The REGDIA regression diagnostics algorithm in S-Plus is introduced in order to examine the accuracy of pK _a predictions made with four updated programs: PALLAS, MARVIN, ACD/pKa and SPARC. This report reviews the current status of computational tools for predicting the pK _a values of organic drug-like compounds. Outlier predicted pK _a values correspond to molecules that are poorly characterized by the pK _a prediction program concerned. The statistical detection of outliers can fail because of masking and swamping effects. The Williams graph was selected to give the most reliable detection of outliers. Six statistical characteristics (F _exp, R ², , MEP, AIC, and s(e) in pK _a units) of the results obtained when four selected pK _a prediction algorithms were applied to three datasets were examined. The highest values of F _exp, R ², , the lowest values of MEP and s(e), and the most negative AIC were found using the ACD/pK _a algorithm for pK _a prediction, so this algorithm achieves the best predictive power and the most accurate results. The proposed accuracy test performed by the REGDIA program can also be applied to test the accuracy of other predicted values, such as log P, log D, aqueous solubility or certain physicochemical properties of drug molecules.     

The SAMPL6 challenge on predicting aqueous p<Emphasis Type="Italic">K</Emphasis><Subscript>a</Subscript> values from EC-RISM theory

The “embedded cluster reference interaction site model” (EC-RISM) integral equation theory is applied to the problem of predicting aqueous pK_a values for drug-like molecules based on an ensemble of tautomers. EC-RISM is based on self-consistent calculations of a solute’s electronic structure and the distribution function of surrounding water. Following-up on the workflow developed after the SAMPL5 challenge on cyclohexane-water distribution coefficients we extended and improved the methodology by taking into account exact electrostatic solute–solvent interactions taken from the wave function in solution. As before, the model is calibrated against Gibbs energies of hydration from the “Minnesota Solvation Database” and a public dataset of acidity constants of organic acids and bases by adjusting in total 4 parameters, among which only 3 are relevant for predicting pK_a values. While the best-performing training model yields a root-mean-square error (RMSE) of 1 pK unit, the corresponding test set prediction on the full SAMPL6 dataset of macroscopic pK_a values using the same level of theory exhibits slightly larger error (1.7 pK units) than the best test set model submitted (1.7 pK units for corresponding training set vs. test set performance of 1.6). Post-submission analysis revealed a number of physical optimization options regarding the numerical treatment of electrostatic interactions and conformational sampling. While the experimental test set data revealed after submission was not used for reparametrizing the methodology, the best physically optimized models consequentially result in RMSEs of 1.5 if only improved electrostatic interactions are considered and of 1.1 if, in addition, conformational sampling accounts for quantum-chemically derived rankings. We conclude that these numbers are probably near the ultimate accuracy achievable with the simple 3-parameter model using a single or the two best-ranking conformations per tautomer or microstate. Finally, relations of the present macrostate approach to microstate pK_a results are discussed and some illustrative results for microstate populations are presented.     

Efficient construction of the hexacyclic ring core of palau'amine: the pKa concept for proceeding with unfavorable equilibrium reactions

Palau''amine has received a great deal of attention as an attractive synthetic target due to its intriguing molecular architecture and significant immunosuppressive activity, and we achieved its total synthesis in 2015. However, the synthesized palau''amine has not been readily applicable to the mechanistic study of immunosuppressive activity, because it requires 45 longest linear steps from a commercially available compound. Here, we report the short-step construction of the ABCDEF hexacyclic ring core of palau''amine. The construction of the CDE tricyclic ring core in a single step is achieved by our pK_a concept for proceeding with unfavorable equilibrium reactions, and a palau''amine analog without the aminomethyl and chloride groups is synthesized in 20 longest linear steps from the same starting material. The palau''amine analog is confirmed to retain the immunosuppressive activity. The present synthetic approach for a palau''amine analog has the potential for use in the development of palau''amine probes for mechanistic elucidation.

A palau''amine analog (2) was synthesized from 2-cyclopentenone in 20 steps. The construction of the CDE tricyclic ring core in a single step is achieved by our pK_a concept for proceeding with the unfavorable equilibrium reactions.     

The SAMPL5 challenge for embedded-cluster integral equation theory: solvation free energies,aqueous p<Emphasis Type="Italic">K</Emphasis><Subscript>a</Subscript>, and cyclohexane–water log <Emphasis Type="Italic">D</Emphasis>

We predict cyclohexane–water distribution coefficients (log D _7.4) for drug-like molecules taken from the SAMPL5 blind prediction challenge by the “embedded cluster reference interaction site model” (EC-RISM) integral equation theory. This task involves the coupled problem of predicting both partition coefficients (log P) of neutral species between the solvents and aqueous acidity constants (pK _a) in order to account for a change of protonation states. The first issue is addressed by calibrating an EC-RISM-based model for solvation free energies derived from the “Minnesota Solvation Database” (MNSOL) for both water and cyclohexane utilizing a correction based on the partial molar volume, yielding a root mean square error (RMSE) of 2.4 kcal mol^?1 for water and 0.8–0.9 kcal mol^?1 for cyclohexane depending on the parametrization. The second one is treated by employing on one hand an empirical pK _a model (MoKa) and, on the other hand, an EC-RISM-derived regression of published acidity constants (RMSE of 1.5 for a single model covering acids and bases). In total, at most 8 adjustable parameters are necessary (2–3 for each solvent and two for the pK _a) for training solvation and acidity models. Applying the final models to the log D _7.4 dataset corresponds to evaluating an independent test set comprising other, composite observables, yielding, for different cyclohexane parametrizations, 2.0–2.1 for the RMSE with the first and 2.2–2.8 with the combined first and second SAMPL5 data set batches. Notably, a pure log P model (assuming neutral species only) performs statistically similarly for these particular compounds. The nature of the approximations and possible perspectives for future developments are discussed.     

A theoretical study of the limits of the acidity of carbon acids in phase transfer catalysis in water and in liquid ammonia

The acidities of a large number of carbon acids have been theoretically calculated for the gas-phase and for DMSO solution. The gas-phase values, both ΔH and ΔG, are very well correlated with the available experimental data. From the calculated ΔG values in DMSO and the pKas in the same solvent, a homogeneous set of pK _a (DMSO) values was devised that was used to generate pK _a (water). These last pK _as were used to establish the limits of the acidity of carbon acids for reactions under PTC conditions both alkylations and H/D exchange. A step further led to the pK _as in liquid ammonia and from them to the virtual use of PTC using liquid ammonia instead of water.      

What are the pKa values of organophosphorus compounds?

A first-principle theoretical protocol was developed, which could successfully predict the pK_a values of a number of amines and thiols in DMSO with a precision of about 1.1 pK_a unit. Using this protocol we calculated the pK_a values of diverse types of organophosphorus compounds in DMSO. The accuracy of these predicted values was estimated to be about 1.1 pK_a because phosphorus is in the same group as nitrogen and in the same period as sulfur. The theoretical predictions were also consistent with all the available experimental data. Thus, a scale of reliable pK_a values was constructed for the first time for organophosphorus. These pK_a values would be helpful to synthetic chemists who need to design the experimental conditions for handling deprotonated organophosphorus. On the basis of these pK_a values we also studied, for the first time, some interesting topics such as the substituent effects on the pK_a values of various types of organophosphorus, and the differences between the pK_a values of organophosphorus and organic amines.     

Prediction of partition and distribution coefficients in various solvent pairs with COSMO-RS

Performance of COSMO-RS method as a tool for partition and distribution modeling in 20 solvent pairs—composed of neutral or acidic aqueous solution and organic solvents of different polarity, ranging from alcohols to toluene and hexane—was evaluated. Experimental partition/distribution data of lignin-related and drug-like compounds (neutral, acidic, moderately basic) were used as reference. Several aspects of partition modeling were addressed: accounting for mutual saturation of aqueous and organic phases, variability of systematic prediction errors across solvent pairs, taking solute ionization into account. COSMO-RS was found to predict extraction outcome for both ligneous and drug-like compounds in various solvent pairs fairly well without any additional empirical input. The solvent-specific systematic errors were found to be moderate, despite being statistically significant, and related to the solvent hydrophobicity. Accounting for mutual solubilities of the two liquids was proven crucial in cases where water was considerably soluble in the organic solvent. The root mean square error of a priori logP prediction varied, depending mainly on the solvent pair, from 0.2 to 0.7, overall value being 0.6 log units. The accuracy was higher in case of hydrophilic than hydrophobic solvents. The logD predictions were less accurate, due to pK_a prediction being an additional source of error, and also because of the complexity of modeling the behaviour of ionic species in the two-phase system. A simple correction for partitioning of free ions was found to notably improve logD prediction accuracy in case of the most hydrophilic organic phase (butanol/water).     

Palladium-catalyzed asymmetric allylic alkylation (AAA) with alkyl sulfones as nucleophiles

An efficient palladium-catalyzed AAA reaction with a simple α-sulfonyl carbon anion as nucleophiles is presented for the first time. Allyl fluorides are used as superior precursors for the generation of π-allyl complexes that upon ionization liberate fluoride anions for activation of silylated nucleophiles. With the unique bidentate diamidophosphite ligand ligated palladium as catalyst, the in situ generated α-sulfonyl carbon anion was quickly captured by the allylic intermediates, affording a series of chiral homo-allylic sulfones with high efficiency and selectivity. This work provides a mild in situ desilylation strategy to reveal nucleophilic carbon centers that could be used to overcome the pK_a limitation of “hard” nucleophiles in enantioselective transformations.

A variety of “hard” α-sulfonyl carbanions of aryl, heteroaryl and alkyl sulfones were successfully employed as nucleophiles in palladium-catalyzed asymmetric allylic alkylation with excellent enantioselectivities.     

Inducing a pH-dependent conformational response by competitive binding to Zn2+ of a series of chiral ligands of disparate basicity

Molecules that change shape in response to environmental conditions are central to biological molecular communication devices and their synthetic chemical analogues. Here we report a molecular system in which a series of chiral anionic ligands of differing basicity are selectively protonated according to the pH of the medium. A cationic circular dichroism (CD) reporter complex responds to anion binding by selecting one of two alternative enantiomeric conformations. Exploiting the principle that less basic anions have, in general, weaker electrostatic interactions than more basic anions, a set of three chiral acids with large (>5 unit) pK_a differences and differing configurations were sequentially deprotonated in acetonitrile by addition of base, allowing the most basic anion in the mixture at any time to bind to the reporter complex. A characteristic CD output resulted, which changed in sign as the next-most basic anion was revealed by the next deprotonation in the series. Four cycles of switching between three ligand-bound states were achieved with minimal changes in signal magnitude, by alternating addition of base and acid. The pH-dependent conformational response was used to transduce a signal by appending to the binding site a 2-aminoisobutyric acid (Aib) oligomer, whose M or P helical conformation depended on the chirality of the bound ligand, and was reported by a remote ¹³C-labelled NMR reporter group. The multicomponent system thus converts a pH signal into a programmable conformational response which induces a remote spectroscopic effect.

A chemical system comprising a series of chiral acids of differing pK_a, a metal, and a conformationally labile chromophore responds to pH with a CD spectrum that reports the absolute stereochemistry of the most basic anion in the mixture.     

Thermodynamics and kinetics of protonated merocyanine photoacids in water

Metastable-state photoacids (mPAHs) are chemical species whose photo-activated state is long-lived enough to allow for proton diffusion. Liao''s photoacid (1) represents the archetype of mPAHs, and is being widely used on account of its unique capability to change the acidity of aqueous solutions reversibly. The behavior of 1 in water, however, still remains poorly understood. Herein, we provide in-depth insights on the thermodynamics and kinetics of 1 in water through a series of comparative ¹H NMR and UV-Vis studies and relative modelling. Under dark conditions, we quantified a three-component equilibrium system where the dissociation (K_a) of the open protonated form (MCH) is followed by isomerization (K_c) of the open deprotonated form (MC) to the closed spiropyran form (SP) – i.e., in the absence of light, the ground state acidity can be expressed as K^GS_a = K_a(1 + K_c). On the other hand, under powerful and continuous light irradiation we were able to assess, for the first time experimentally, the dissociation constant (K^MS_a) of the protonated metastable state (cis-MCH). In addition, we found that thermal ring-opening of SP is always rate-determining regardless of pH, whereas hydrolysis is reminiscent of what is found for Schiff bases. The proposed methodology is general, and it was applied to two other compounds bearing a shorter (ethyl, 2) and a longer (butyl, 3) alkyl-1-sulfonate bridge. We found that the pK_a remains constant, whereas both pK_c and pK^MS_a linearly increase with the length of the alkyl bridge. Importantly, all results are consistent with a four-component model cycle, which describes perfectly the full dynamics of proton release/uptake of 1–3 in water. The superior hydrolytic stability and water solubility of compound 3, together with its relatively high pK^GS_a (low K_c), allowed us to achieve fully reversible jumps of 2.5 pH units over 18 consecutive cycles (6 hours).

We rationalize the behaviour of protonated merocyanines in water through cross-validation of ¹H NMR, UV-Vis and pH measurements, and show their capability to act as reversible photoacids along light/dark cycles can be described by a four-state model.     

Evaluation of Acid Dissociation Constants in DMSO and DMF by Quantum-Chemical Methods

We have calculated total electronic energies (E) and Gibbs energies (G) of a large number of acids and their anions in water, dimethylsulfoxide, and dimethylformamide using the hybrid B3LYP functional DFT method in the 6-31++G(d,p) basis set, taking into account the solvent effect by the conductor-like polarizable continuum model method. A linear correlation has been found between the experimental values of acid dissociation constants (pK_a) of different nature and the difference between anion and acid E values, and between pK_a and the difference between anion and acid G values. The obtained correlations allowed us to evaluate the pK_a values of both inorganic and organic acids. Such an evaluation is of special importance for nonaqueous solvents as it is quite problematic to determine these dissociation constants.     

Nonspherical anion sequestration by C–H hydrogen bonding

Macrocyclic arenes laid the foundations of supramolecular chemistry and their study established the fundamentals of noncovalent interactions. Advancing their frontier, here we designed rigidified resorcin[4]arenes that serve as hosts for large nonspherical anions. In one synthetic step, we vary the host''s anion affinity properties by more than seven orders of magnitude. This is possible by engineering electropositive aromatic C–H bond donors in an idealized square planar geometry embedded within the host''s inner cavity. The hydrogen atom''s electropositivity is tuned by introducing fluorine atoms as electron withdrawing groups. These novel macrocycles, termed fluorocages, are engineered to sequester large anions. Indeed, experimental data shows an increase in the anion association constant (K_a) as the number of F atoms increase. The observed trend is rationalized by DFT calculations of Hirshfeld Charges (HCs). Most importantly, fluorocages in solution showed weak-to-medium binding affinity for large anions like [PF₆]⁻ (10²< K_a <10⁴ M⁻¹), and high affinity for [MeSO₃]⁻ (K_a >10⁶).

Fluorocages: new class of rigidified host utilizing nontraditional C–H hydrogen bonds to capture the nonspherical anions.     

A direct link between the Passerini reaction and α-lactams

α-Lactams (aziridinones) can function to replace two of the three reactants, the oxo-compound and the isonitrile, in the Passerini reaction. Four α-lactams (5a-d) were reacted with mono- and dicarboxylic acids of positive pK_a values to give 2-acyloxycarboxamides (4) and bis-2-acyloxycarboxamide products 12 and 13, respectively. The same compounds were also prepared via the Passerini reaction. Acids with a negative pK_a decarbonylate α-lactams to give immonium salts. The main path of the reaction depends on the pK_a of the acid component, the reactivity of the α-lactam, and the reaction conditions.     

Discovery of phosphotyrosine-binding oligopeptides with supramolecular target selectivity

We demonstrate phage-display screening on self-assembled ligands that enables the identification of oligopeptides that selectively bind dynamic supramolecular targets over their unassembled counterparts. The concept is demonstrated through panning of a phage-display oligopeptide library against supramolecular tyrosine-phosphate ligands using 9-fluorenylmethoxycarbonyl-phenylalanine-tyrosine-phosphate (Fmoc-FpY) micellar aggregates as targets. The 14 selected peptides showed no sequence consensus but were enriched in cationic and proline residues. The lead peptide, KVYFSIPWRVPM-NH₂ (P7) was found to bind to the Fmoc-FpY ligand exclusively in its self-assembled state with K_D = 74 ± 3 μM. Circular dichroism, NMR and molecular dynamics simulations revealed that the peptide interacts with Fmoc-FpY through the KVYF terminus and this binding event disrupts the assembled structure. In absence of the target micellar aggregate, P7 was further found to dynamically alternate between multiple conformations, with a preferred hairpin-like conformation that was shown to contribute to supramolecular ligand binding. Three identified phages presented appreciable binding, and two showed to catalyze the hydrolysis of a model para-nitro phenol phosphate substrate, with P7 demonstrating conformation-dependent activity with a modest k_cat/K_M = 4 ± 0.3 × 10⁻⁴ M⁻¹ s⁻¹.

Phage-display screening on self-assembled tyrosine-phosphate ligands enables the identification of oligopeptides selective to dynamic supramolecular targets, with the lead peptide showing a preferred hairpin-like conformation and catalytic activity.