Neutral Tautomers Research Articles

Constant-pH Simulations with the Polarizable Atomic Multipole AMOEBA Force Field.

Accurately predicting protein behavior across diverse pH environments remains a significant challenge in biomolecular simulations. Existing constant-pH molecular dynamics (CpHMD) algorithms are limited to fixed-charge force fields, hindering their application to biomolecular systems described by permanent atomic multipoles or induced dipoles. This work overcomes these limitations by introducing the first polarizable CpHMD algorithm in the context of the Atomic Multipole Optimized Energetics for Biomolecular Applications (AMOEBA) force field. Additionally, our implementation in the open-source Force Field X (FFX) software has the unique ability to handle titration state changes for crystalline systems including flexible support for all 230 space groups. The evaluation of constant-pH molecular dynamics (CpHMD) with the AMOEBA force field was performed on 11 crystalline peptide systems that span the titrating amino acids (Asp, Glu, His, Lys, and Cys). Titration states were correctly predicted for 15 out of the 16 amino acids present in the 11 systems, including for the coordination of Zn2+ by cysteines. The lone exception was for a HIS-ALA peptide where CpHMD predicted both neutral histidine tautomers to be equally populated, whereas the experimental model did not consider multiple conformers and diffraction data are unavailable for rerefinement. This work demonstrates the promise polarizable CpHMD simulations for pKa predictions, the study of biochemical mechanisms such as the catalytic triad of proteases, and for improved protein-ligand binding affinity accuracy in the context of pharmaceutical lead optimization.

Semiempirical Approach to the Fukui Function Analysis of Uric Acid under Different pH Conditions.

Analytic Fukui functions calculated at a first-principles level are combined with experimental pKa values and the calculation of tautomerization energies to obtain the effective regioselectivity of uric acid toward electron-transfer reactions under different pH conditions. Second-order electron binding energies are also computed to determine which of the tautomers is more likely to participate in the electron transfer. A comparison of vertical and adiabatic proton detachment energies allows us to conclude that tautomerization is not mediating deprotonation and that two monoanionic species are of comparable relevance. The main difference between these monoanionic species is the ring that has been deprotonated. Both monoanionic species are produced from a single neutral tautomer and mainly produce a single dianionic tautomer. As a method for the analysis of systems affected by pH such as uric acid, we propose to plot condensed Fukui functions versus pH, allowing us to draw the effect of pH on the regioselectivity of electron transfer in a single image.

Effect of solutes structure and pH on the n-octanol/water partition coefficient of ionizable organic compounds.

Liquid-liquid partition coefficient is a useful tool to predict biological and environmental fate of organic compounds, for example bioaccumulation or toxicity of lipophilic contaminants. Conversely, the partitioning of ionizable compounds is poorly studied in contrast to that of neutral compounds. Yet, such topic deserves attention, since numerous organic contaminants are ionizable as well as their degradation products. Hence, the contribution of charged species has to be considered in order to model accurately the mass balance or partition of ionizable compounds. In this context, we investigated the liquid-liquid partition of 13 ionizable compounds (oxalic acid, histidine, benzimidazole, etc.), covering various classes of compounds (carboxylic acids, amino-acids, etc.). The n-octanol/water partition coefficient was measured from pH 1 up to 13, in order to fully gather the distribution of both neutral and charged species. Empirical models describing these results are reviewed and partition parameters adjusted for charged species. The study of benzoic acid derivatives (benzoic, salicylic, ortho- and iso-phthalic acids) provides insights on the influence of chemical groups on the partitioning. In the case of tryptophan, the use of acid/base microconstants allowed to estimate the partition of both the zwitterion and its neutral tautomer. Despite a major zwitterionic form (log PZ(tryptophan)=-1.58±0.30), the minor but neutral tautomer (log PN(tryptophan)=+0.03±0.30) drives the partition equilibrium. Overall, the provided data may be useful to assess the retention of contaminants, its dependency on pH and salinity variations, and thus understanding their environmental fate. Such data may also be useful as well for molecular simulation involving solvation of organic ions in aqueous and non-aqueous solvents.

How \u03bc-opioid receptor recognizes fentanyl

Roughly half of the drug overdose-related deaths in the United States are related to synthetic opioids represented by fentanyl which is a potent agonist of mu-opioid receptor (mOR). In recent years, X-ray crystal structures of mOR in complex with morphine derivatives have been determined; however, structural basis of mOR activation by fentanyl-like opioids remains lacking. Exploiting the X-ray structure of BU72-bound mOR and several molecular simulation techniques, we elucidated the detailed binding mechanism of fentanyl. Surprisingly, in addition to the salt-bridge binding mode common to morphinan opiates, fentanyl can move deeper and form a stable hydrogen bond with the conserved His2976.52, which has been suggested to modulate mOR’s ligand affinity and pH dependence by previous mutagenesis experiments. Intriguingly, this secondary binding mode is only accessible when His2976.52 adopts a neutral HID tautomer. Alternative binding modes may represent a general mechanism in G protein-coupled receptor-ligand recognition.

Infrared Multiple Photon Dissociation Spectroscopy of Protonated Cyameluric Acid.

The present study reports the first structural characterization of protonated cyameluric acid ([CA + H]+) in the gas phase, which paves the way for prospective bottom-up research on the condensed-phase chemistry of CA in the protonated form. A number of [CA + H]+ keto-enol isomers can a priori be produced as a result of protonation at available N and O positions of precursor neutral CA tautomers, yet ab initio computations predict different reduced [CA + H]+ isomer populations dominating the solution and gas phases that are involved in the ion generation process (i.e., electrospray ionization). Infrared multiple photon dissociation spectra were recorded in the 990-1900 and 3300-3650 cm-1 regions and compared with theoretical [B3LYP/6-311++G(d,p)] IR absorption spectra of several [CA + H]+ isomers, providing a satisfactory agreement for the most stable monohydroxy form in the gas phase, [1358a]+, yet the contribution of its nearly isoenergetic OH rotamer, [1358b]+, cannot be neglected. This is indicative of the occurrence of [CA + H]+ isomer interconversion reactions, assisted by protic solvent molecules, during their transfer into the gas phase. The results suggest that available O positions on neutral CA are energetically favored protonation sites in the gas phase.

How does α1Histidine102 affect the binding of modulators to α1β2γ2GABAAreceptors? molecular insights fromin silicoexperiments

The activation of GABAA receptors by the neurotransmitter gamma-aminobutyric acid mediates the rapid inhibition response in the central nervous system of mammals. Many neurological and mental health disorders arise from alterations in the structure or function of these pentameric ion channels. GABAA receptors are targets for numerous drugs, including benzodiazepines, which bind to α1β2γ2 GABAA receptors with high affinity to a site in the extracellular domain, between subunits α1 and γ2. It has been established experimentally that the binding of these drugs depends on the presence of one particular amino acid in the α1 subunit: histidine 102. However, the specific role it plays in the intermolecular interaction has not been elucidated. In this work, we applied in silico methods to understand whether certain protonation and rotamer states of α1His102 facilitate the binding of modulators. We analysed diazepam binding, a benzodiazepine, and the antagonist flumazenil to the GABAA receptor using molecular dynamics simulations and adaptive biasing force simulations. The binding free energy follows changes in the protonation state for both ligands, and rotameric states of α1His102 were specific for the different compounds, suggesting distinct preferences for positive allosteric modulators and antagonists. Moreover, in the presence of diazepam and favoured by a neutral tautomer, we identified a water molecule that links loops A, B, and C and may be relevant to the modulation mechanism.

Keto‐enol tautomerism of the 4,5‐dimethyl‐2‐(2′‐hydroxyphenyl)imidazole in water solution: Modeling equilibrium between neutral forms and accurate assignment of the absorption bands

AbstractTautomerizarion between keto, cis‐ and trans‐enol forms of the 4,5‐dimethyl‐2‐(2′‐hydroxyphenyl)imidazole (DMHI) is studied using molecular dynamics simulations with the quantum mechanics/molecular mechanics (QM/MM) potentials. We applied the live solvent selection approach that presumes division of water molecules to either QM or MM subsystems on the fly depending on their distances from the chromophore. This allowed us to treat a water shell around the chromophore at the QM level and to describe properly all chromophore‐water interactions. According to the calculated Gibbs energy profiles the most stable is a keto form and a cis‐enol form is ~1 kcal/mol higher. The trans‐enol form is much higher in energy and should not be observed in experiment. Vertical S0,min − S1 excitation energies of all neutral tautomers were calculated at the XMCQDPT2 level: both enol forms have similar values ~4 eV and keto form is red‐shifted, with the excitation energy of 3.3 eV. Thus, we conclude that two absorption bands in experimental absorption spectrum of DMHI in water solution are due to the presence of cis‐enol and keto forms of the chromophore.

Salts, Cocrystals, and Ionic Cocrystals of a “Simple” Tautomeric Compound

We have investigated the cocrystallization behavior of AHMP (2-amino-6-methyl-1,4-dihydropyrimidin-4-one) with a series of acids and imide coformers. Being a base able to tautomerize, at the molecular level, AHMP may exist as three different neutral tautomers or, if protonated, in the form of several cationic species. Crystal engineering concepts were used in order to attempt selective isolation of several tautomers. For this, a series of acids and imides were used as coformers. Cocrystallization with acids resulted in 11 new solid forms including salts, ionic cocrystals, and cocrystals. Cocrystallizations with imides did not afford any new forms, while cocrystallization with saccharin afforded two polymorphic salts and an ionic cocrystal. Two of the possible tautomers were obtained as well as one of the cationic species. DFT calculations were carried out in order to rationalize the molecular species obtained as well as the supramolecular motifs. Despite being a small “simple” molecule, AHMP has a very ri...

Distinct Differences in Structural States of Conserved Histidines in Two Related Proteins: NMR Studies of the Chemokines CXCL1 and CXCL8 in the Free Form and Macromolecular Complexes.

Hydrogen-bonding and ionic interactions play fundamental roles in macromolecular recognition and function. In contrast to lysines and arginines, how histidines mediate these interactions is less well-understood due to the unique properties of its side chain imidazole that include an aromatic ring with two titratable nitrogens, a p Ka that can vary significantly, and the ability to exist in three distinct forms: protonated imidazolium and two tautomeric neutral (Nδ1 and Nε2) states. Here, we characterized the structural features of histidines in the chemokines CXCL8 and CXCL1 in the free, GAG heparin-bound, and CXCR2 receptor N-terminal domain-bound states using solution NMR spectroscopy. CXCL8 and CXCL1 share two conserved histidines, one in the N-loop and the other in the 30s loop. In CXCL8, both histidines exist in the Nε2 tautomeric state in the free, GAG-bound, and receptor-bound forms. On the other hand, in unliganded CXCL1, each of the two histidines exists in two states, as the neutral Nε2 tautomer and charged imidazolium. Further, both histidines exclusively exist as the imidazolium in the GAG-bound and as the Nε2 tautomer in the receptor-bound forms. The N-loop histidine alone in both chemokines is involved in direct GAG and receptor interactions, indicating the role of the 30s loop varies between the chemokines. Our observation that the structural features of conserved histidines and their functional role in two related proteins can be quite different is novel. We further propose that directly probing the imidazole structural features is essential to fully appreciate the molecular basis of histidine function.

Identifying Cytosine-Specific Isomers via High-Accuracy Single Photon Ionization.

Biological entities, such as DNA bases or proteins, possess numerous tautomers and isomers that lie close in energy, making the experimental characterization of a unique tautomer challenging. We apply VUV synchrotron-based experiments combined with state-of-the-art ab initio methodology to determine the adiabatic ionization energies (AIEs) of specific gas-phase cytosine tautomers produced in a molecular beam. The structures and energetics of neutral and cationic cytosine tautomers were determined using explicitly correlated methods. The experimental spectra correspond to well-resolved bands that are attributable to the specific contributions of five neutral tautomers of cytosine prior to ionization. Their AIEs are experimentally determined for the first time with an accuracy of 0.003 eV. This study also serves as an important showcase for other biological entities presenting a dense pattern of isomeric and tautomeric forms in their spectra that can be investigated to understand the charge redistribution in these species upon ionization.

Replacement of Oxygen by Sulfur in Small Organic Molecules. 3. Theoretical Studies on the Tautomeric Equilibria of the 2OH and 4OH-Substituted Oxazole and Thiazole and the 3OH and 4OH-Substituted Isoxazole and Isothiazole in the Isolated State and in Solution.

This follow-up paper completes the author’s investigations to explore the in-solution structural preferences and relative free energies of all OH-substituted oxazole, thiazole, isoxazole, and isothiazole systems. The polarizable continuum dielectric solvent method calculations in the integral-equation formalism (IEF-PCM) were performed at the DFT/B97D/aug-cc-pv(q+(d))z level for the stable neutral tautomers with geometries optimized in dichloromethane and aqueous solution. With the exception of the predictions for the predominant tautomers of the 3OH isoxazole and isothiazole, the results of the IEF-PCM calculations for identifying the most stable tautomer of the given species in the two selected solvents agreed with those from experimental investigations. The calculations predict that the hydroxy proton, with the exception for the 4OH isoxazole and 4OH isothiazole, moves preferentially to the ring nitrogen or to a ring carbon atom in parallel with the development of a C=O group. The remaining, low-fraction OH tautomers will not be observable in the equilibrium compositions. Relative solvation free energies obtained by the free energy perturbation method implemented in Monte Carlo simulations are in moderate accord with the IEF-PCM results, but consideration of the ΔGsolv/MC values in calculating ΔGstot maintains the tautomeric preferences. It was revealed from the Monte Carlo solution structure analyses that the S atom is not a hydrogen-bond acceptor in any OH-substituted thiazole or isothiazole, and the OH-substituted isoxazole and oxazole ring oxygens may act as a weak hydrogen-bond acceptor at most. The molecules form 1.0−3.4 solute−water hydrogen bonds in generally unexplored numbers at some specific solute sites. Nonetheless, hydrogen-bond formation is favorable with the NH, C=O and OH groups.

A computational study of azaphospholes: anions and neutral tautomers

One hundred and eleven azaphospholes (31 anions and 80 neutral compounds) have been studied theoretically at the B3LYP/6-311++G(d,p) level. Among the analyzed properties, there are the geometries, mainly the non-planarity of the PH tautomers, the isomerism of the anions, the NH/PH tautomerism of neutral compounds, the chemical shifts and spin–spin coupling constants. The aromaticity has been assessed through Schleyer’s NICS values, preferably NICS(1). Finally, we have compared our calculations with previous ones as well as with the available experimental data for the calculated compounds and for related ones.

Structure of protonated thymidine characterized by infrared multiple photon dissociation and quantum calculations.

Many fundamental studies are motivated by the probable relationship between the presence of rare enol tautomers of nucleobases and point mutation developing during nucleic acid replication. The evaluation of the tautomeric behaviour of nucleobases is therefore of fundamental importance. This can be probed in the gas phase by combining action spectroscopy and mass spectrometry. Experimental Infrared Multiple Photon Dissociation spectra in the fingerprint region of electrospray-generated and subsequently selected ions were recorded at the CLIO free electron laser (FEL) facility, by coupling the FEL to a quadrupole ion trap, and compared to calculated harmonic vibrational infrared spectra of the different low-lying isomers computed at the B3LYP/6-31++G(d,p) level. Relative energies were refined using the extended basis set 6-311++G(3df,2p). The Density Functional Theory (DFT) study shows that, as for protonated thymine, the global energy minimum of protonated thymidine corresponds to an enol tautomer, whose infrared absorption spectrum is found to be in very good agreement with the experimental IRMPD spectrum. A very weak IRMPD signal observed at ~1780 cm(-1) is very likely the signature of an oxo tautomer. Consequently, as for thymine, protonated thymidine generated by electrospray corresponds to a mixture of at least two tautomeric forms. Tautomerization can be characterized by IRMPD spectroscopy. Interestingly, the dominant enolic tautomeric form(s) presently observed cannot be directly generated from the most stable neutral tautomer of the thymine residue.

Two-dimensional IR spectroscopy of the anti-HIV agent KP1212 reveals protonated and neutral tautomers that influence pH-dependent mutagenicity

Antiviral drugs designed to accelerate viral mutation rates can drive a viral population to extinction in a process called lethal mutagenesis. One such molecule is 5,6-dihydro-5-aza-2'-deoxycytidine (KP1212), a selective mutagen that induces A-to-G and G-to-A mutations in the genome of replicating HIV. The mutagenic property of KP1212 was hypothesized to originate from its amino-imino tautomerism, which would explain its ability to base pair with either G or A. To test the multiple tautomer hypothesis, we used 2D IR spectroscopy, which offers subpicosecond time resolution and structural sensitivity to distinguish among rapidly interconverting tautomers. We identified several KP1212 tautomers and found that >60% of neutral KP1212 is present in the enol-imino form. The abundant proportion of this traditionally rare tautomer offers a compelling structure-based mechanism for pairing with adenine. Additionally, the pKa of KP1212 was measured to be 7.0, meaning a substantial population of KP1212 is protonated at physiological pH. Furthermore, the mutagenicity of KP1212 was found to increase dramatically at pH <7, suggesting a significant biological role for the protonated KP1212 molecules. Overall, our data reveal that the bimodal mutagenic properties of KP1212 result from its unique shape shifting ability that utilizes both tautomerization and protonation.

Geometric and energetic consequences of prototropy for adenine and its structural models – a review

Prototropy for adenine and its convenient models causes parallel changes of geometric (HOMED) and energetic (ΔE) parameters for neutral tautomers.

Pseudo-cyclic structures of mono- and di-azaderivatives of malondialdehydes. Synthesis and conformational disentanglement by computational analyses.

Mono- and diaza-derivatives of malondialdehydes, namely 3-alkyl(aryl)amino-2-arylacroleins and 1,5-dialkyl(aryl)-3-arylvinamidines are open-chain systems in which extended electron delocalization and pseudoaromaticity can be envisaged. A set of diversely functionalized compounds has been synthesized and characterized by spectroscopic data and X-ray diffractometry. Quantum-chemical calculations were performed for all possible neutral tautomers and conformers in the gas phase and compared to those in polar solvents (CHCl3, DMSO, and EtOH) at the M06-2X/6-311++G(d,p) level. Tautomeric equilibria and conformational preferences can be rationalized in terms of structural factors, which can be roughly estimated as summation or subtractions of intramolecular interactions. As expected, a key role is played by intramolecular hydrogen bonds whose strength varies from the gas phase to polar ethanol. This issue also delves into the concept of resonance-assisted H-bond, where the donor and acceptor atoms are connected by a π-conjugated system. The most stable conformers (structures a and c) possess a high degree of pseudoaromaticity as inferred from HOMA indexes and other delocalization parameters.

Electron delocalization and relative stabilities for the favored and rare tautomers of hydroxyazines in the gas phase – A comparison with aminoazines

Quantum-chemical calculations {G2 and/or B3LYP/6-311+G(d,p)} were performed in the gas phase for neutral and ionized hydroxyazines (2- and 4-hydroxy derivatives of pyridine and pyrimidine). For each derivative, all possible tautomeric conversions were considered and all possible prototropic tautomers (OH, NH, and CH) analyzed. Rotational isomerism of the OH group was also taken into account. An interesting change of the relative stabilities takes place when proceeding from the neutral to ionized forms. Positive ionization favors the NH tautomers, and negative ionization the CH ones, except 2-hydroxypyrimidine for which the NH tautomer predominates for both ionized forms. A good relation exists between prototropy and electron delocalization solely for the neutral NH and CH tautomers. The OH forms are separated from the subfamily of the NH and CH tautomers due to significant differences between the stabilities of the tautomeric oxo and hydroxy groups. Intramolecular interactions affect both the geometric and energetic parameters, but the relative stabilities in higher degree. When compared to series of aminoazines, the total geometric effects of the exo OH/O and NH2/NH groups are similar for neutral azines.

An ab initio analysis of the structure of l-tryptophan tautomers in microhydrated environments, in water and in hydrophobic solvents

The free energy of tryptophan (Trp) tautomers and conformers in different aqueous and non-aqueous environments has been calculated at the MP2/aug-cc-pVDZ//B3LYP/6-31+G(d,p) level of theory. Comparison with glycine (Gly) has been done in order to discuss the influence of the hydrophobic side chain. A detailed structural analysis has been first carried out to identify the relevant neutral (non-ionized) and zwitterionic (ionized) conformations. In water solution, explicit consideration of the first solvation shell (eight water molecules) has been found to be mandatory in order to correctly describe the system and therefore we have used a combined discrete–continuum solvation model to obtain free energies in this medium. Calculations in non-aqueous solvents have been done with the continuum model. The study shows that the hydrophobicity of the indole group of the Trp side chain does not influence much the tautomerization energy of the amino acid in water. Thus, the computed free energy of Trp tautomerization amounts −8.93kcal/mol, which is not far from the theoretical and experimental values reported for the simplest natural amino acid glycine. Conversely, the free energy of transfer from water to cyclohexane has been estimated to 9.79kcal/mol, which is much smaller than the energy reported before for glycine at a similar theoretical level (18.2kcal/mol). The analysis of the results shows that the indole group yields a straightforward transfer of the neutral tautomer from the polar to the apolar phase, which requires only 0.86kcal/mol instead of the 10kcal/mol required for the neutral tautomer of glycine. The estimated energy of transfer is consistent with available experimental data for analogous systems.

Theoretical study on the static and dynamic first-order hyperpolarisabilities of adenine tautomers

Static and dynamic electronic and vibrational first-order hyperpolarisabilities (β) of the lowest energy neutral adenine tautomers (amine forms A7 and A9) were obtained in gaseous and aqueous phases by using Hartree–Fock, Møller–Plesset second-order and fourth-order perturbation theory (MP2 and MP4-SDQ) and conventional and long-range corrected density functional theory methods with the Dunning's correlation-consistent cc-pVDZ, aug-cc-pVDZ, aug-cc-pVTZ and d-aug-cc-pVDZ basis sets. Frequency-dependent properties were calculated at the characteristic wavelength of the Nd:YAG laser (1064 nm) for the second harmonic generation and electro-optical Pockels effect nonlinear optical processes. Solvent effects were introduced under the polarised continuum model approximation. The electronic βe values of the investigated isomers are noticeably affected by the theoretical level, basis set and solvation. In vacuum, the static and dynamic βe values of A9 are greater than the corresponding data of A7, whereas the contribution of the solvent significantly enhances the hyperpolarisabilities of the A7 tautomer, resulting in βe(A9)/βe(A7) ratios between 0.5 and 0.6. The vibrational hyperpolarisabilities of the adenine tautomers are quite close to each other.

Divergent Mechanisms for Enzymatic Excision of 5-Formylcytosine and 5-Carboxylcytosine from DNA

5-Methylcytosine (mC) is an epigenetic mark that impacts transcription, development, and genome stability, and aberrant DNA methylation contributes to aging and cancer. Active DNA demethylation involves stepwise oxidation of mC to 5-hydroxymethylcytosine, 5-formylcytosine (fC), and potentially 5-carboxylcytosine (caC), excision of fC or caC by thymine DNA glycosylase (TDG), and restoration of cytosine via follow-on base excision repair. Here, we investigate the mechanism for TDG excision of fC and caC. We find that 5-carboxyl-2'-deoxycytidine ionizes with pK(a) values of 4.28 (N3) and 2.45 (carboxyl), confirming that caC exists as a monoanion at physiological pH. Calculations do not support the proposal that G·fC and G·caC base pairs adopt a wobble structure that is recognized by TDG. Previous studies show that N-glycosidic bond hydrolysis follows a stepwise (S(N)1) mechanism, and that TDG activity increases with pyrimidine N1 acidity, that is, leaving group quality of the target base. Calculations here show that fC and the neutral tautomers of caC are acidic relative to other TDG substrates, but the caC monoanion exhibits poor acidity and likely resists TDG excision. While fC activity is independent of pH, caC excision is acid-catalyzed, and the pH profile indicates that caC ionizes in the enzyme-substrate complex with an apparent pKa of 5.8, likely at N3. Mutational analysis reveals that Asn191 is essential for excision of caC but dispensable for fC activity, indicating that N191 may stabilize N3-protonated forms of caC to facilitate acid catalysis and suggesting that N191A-TDG could potentially be useful for studying DNA demethylation in cells.