Protonation State Of Ligands Research Articles

Front Cover: Structural Changes to the Gd‐DTPA Complex at Varying Ligand Protonation State (Eur. J. Inorg. Chem. 6/2024)

Front Cover: Structural Changes to the Gd‐DTPA Complex at Varying Ligand Protonation State (Eur. J. Inorg. Chem. 6/2024)

Structural Changes to the Gd‐DTPA Complex at Varying Ligand Protonation State

AbstractDiethylenetriaminepentaacetic acid (DTPA) is a chelating agent whose complex with the Gd3+ ion is used in medical imaging. DTPA is also used in lanthanide‐actinide separation processes. As protonation of the DTPA ligand can facilitate dissociation of the Gd3+ ion from the Gd‐DTPA complex, this work investigates the coordination structures of the aqueous Gd3+ ion and its environment when chelated by DTPA in eight different DTPA protonation states. Both classical and ab initio molecular dynamics (MD) simulations are conducted to model the solvated complexes. Extended X‐ray absorption fine structure (EXAFS) measurements of the Gd3+aqua ion, and the Gd‐DTPA complex at pH 1 and 11, are compared to EXAFS spectra predicted from the MD simulations to verify the accuracy of the MD structures. The findings of this work provide atomic‐level details into the fluctuating Gd‐DTPA complex environment as the DTPA ligand gradually detaches from the Gd3+ ion with increased protonation.

Characterizing Protein Protonation Microstates Using Monte Carlo Sampling.

Proteins are polyelectrolytes with acidic and basic amino acids Asp, Glu, Arg, Lys, and His, making up ≈25% of the residues. The protonation state of residues, cofactors, and ligands defines a "protonation microstate". In an ensemble of proteins some residues will be ionized and others neutral, leading to a mixture of protonation microstates rather than in a single one as is often assumed. The microstate distribution changes with pH. The protein environment also modifies residue proton affinity so microstate distributions change in different reaction intermediates or as ligands are bound. Particular protonation microstates may be required for function, while others exist simply because there are many states with similar energy. Here, the protonation microstates generated in Monte Carlo sampling in MCCE are characterized in HEW lysozyme as a function of pH and bacterial photosynthetic reaction centers (RCs) in different reaction intermediates. The lowest energy and highest probability microstates are compared. The ΔG, ΔH, and ΔS between the four protonation states of Glu35 and Asp52 in lysozyme are shown to be calculated with reasonable precision. At pH 7 the lysozyme charge ranges from 6 to 10, with 24 accepted protonation microstates, while RCs have ≈50,000. A weighted Pearson correlation analysis shows coupling between residue protonation states in RCs and how they change when the quinone in the QB site is reduced. Protonation microstates can be used to define input MD parameters and provide insight into the motion of protons coupled to reactions.

Crystallographic and Computational Characterisation of the Potential PET Tracer 1,4,7‐Triazacyclononane‐1,4,7‐tri(methylenephosphonato)gallium(III)

AbstractThe macrocyclic chelator 1,4,7‐triazacyclononane‐1,4,7‐tri(methylenephosphonic acid) (H6NOTP) has shown significant promise for use in positron emission tomography with the positron‐emitting radionuclide 68Ga3+. We report the X‐ray crystal structure of the neutral complex 1,4,7‐triazacyclononane‐1,4,7‐tri(methylenephosphonato)gallium(III) ([Ga(H3NOTP)]), which crystallised in the centrosymmetric space group P21/n. The coordination geometry of the Ga3+ ion is distorted octahedral with three macrocyclic amine N donor atoms and three singly deprotonated phosphonato O donor atoms. Two discrete enantiomers, Δ(λλλ)‐SSS and Λ(δδδ)‐RRR, are present as a racemate within the unit cell. Intermolecular hydrogen bonds between phosphonic acid groups of adjacent complexes form a two‐dimensional sheet network. Density functional theory (DFT) calculations supported the observation of complexes with low electric field gradient values with slight but significant deviations from ideal octahedral geometry. The DFT calculations were also used to investigate the influence of intermolecular interactions and ligand protonation states on the coordination environment and surface charge distribution.

Excited-state behavior and photoinduced electron transfer of pH-sensitive Ir(III) complexes with cyclometallation (C/N–) ratios between 0/6 and 3/3

The first coordination sphere of Ir(III) 2,2′-bipyridine / 2-phenylpyridine complexes can be tuned to achieve either C– or N–chelation in ratios that range between 0/6 and 3/3. Of particular interest is the synthesis of Ir(III) complexes bearing a 2,2′-bipyridine ligand coordinated in a N,C3 pattern, leaving an exposed pyridine moiety, accessible for acid-base chemistry or coordination to a second transition metal center. The protonated forms of these “rolled-over” Ir(III) complexes were isolated in a straight-forward procedure using trifluoroacetic acid. The photophysical, photochemical and electrochemical properties of both the protonated and unprotonated Ir(III) complexes were investigated by steady-state and time-resolved spectroscopies, as well as by density functional theory calculations. The nature of the excited states was shown to depend on both the ligand coordination pattern and protonation state of the complex. In addition, the unprotonated and protonated analogues were efficiently quenched by hydroquinone and benzoquinone in acetonitrile with quenching rate constants close to the solvent diffusion limit. The results presented herein have direct implications for proton sensitive photoredox chemistry and the development of photo-acids and photo-bases.

A Ligand Protonation Series in Aluminum(III) Complexes of Tridentate Bis(enol)amine Ligand

A series of aluminum(III) complexes supported by the tridentate bis(enol)amine ligand (ONO, 1,1′-azanediylbis(3,3-dimethylbutan-2-one)) in two protonation states have been synthesized and characterized structurally. Reaction of AlCl3 with singly deprotonated H2ONO– afforded pseudo-octahedral [(H2ONO–)2Al][AlCl4] (1). AlCl3 was also reacted with doubly deprotonated HONO2– to afford the five-coordinate, pseudotrigonal bipyramidal complex (HONO2–)AlCl(THF) (2). The reaction of complex 2 with HCl yielded complex 1, which demonstrates reversible protonation of the ligand backbone. Synthesis of an Al(III) complex of ONO3– was not achieved using a variety of Lewis bases. An attempt to deprotonate complex 2 with tBuOK yielded [(HONO2–)Al](μ-tBuO) (3), with no change to the ligand protonation state.

How Ligand Protonation State Controls Water in Protein-Ligand Binding.

The role of water in protein-ligand binding has been an intensely studied topic in recent years; however, how ligand protonation state change perturbs water has not been considered. Here we show that water dynamics and interactions can be controlled by the protonation state of ligand using continuous constant pH molecular dynamics simulations of two closely related model systems, β-secretase 1 and 2 (BACE1 and BACE2), in complex with a small-molecule inhibitor. Simulations revealed that, upon binding, the inhibitor pyrimidine ring remains deprotonated in BACE1 but becomes protonated in BACE2. Pyrimidine protonation results in water displacement, rigidification of the binding pocket, and shift in the ligand binding mode from water-mediated to direct hydrogen bonding. These findings not only support but also rationalize the most recent structure-selectivity data in BACE1 drug design. Binding-induced protonation state changes are likely common; our work offers a glimpse at how modeling protein-ligand binding while allowing ligand titration can further advance the understanding of water and structure-based drug design.

Investigation of the binding mode of a novel cruzain inhibitor by docking, molecular dynamics, ab initio and MM/PBSA calculations.

Chagas disease remains a major health problem in South America, and throughout the world. The two drugs clinically available for its treatment have limited efficacy and cause serious adverse effects. Cruzain is an established therapeutic target of Trypanosoma cruzi, the protozoan that causes Chagas disease. Our group recently identified a competitive cruzain inhibitor (compound 1) with an IC50 = 15 µM that is also more synthetically accessible than the previously reported lead, compound 2. Prior studies, however, did not propose a binding mode for compound 1, hindering understanding of the structure-activity relationship and optimization. Here, the cruzain binding mode of compound 1 was investigated using docking, molecular dynamics (MD) simulations with ab initio derived parameters, ab initio calculations, and MM/PBSA. Two ligand protonation states and four binding poses were evaluated. A careful ligand parameterization method was employed to derive more physically meaningful parameters than those obtained by automated tools. The poses of unprotonated 1 were unstable in MD, showing large conformational changes and diffusing away from the binding site, whereas the protonated form showed higher stability and interaction with negatively charged residues Asp161 and Cys25. MM/PBSA also suggested that these two residues contribute favorably to binding of compound 1. By combining results from MD, ab initio calculations, and MM/PBSA, a binding mode of 1 is proposed. The results also provide insights for further optimization of 1, an interesting lead compound for the development of new cruzain inhibitors.

PH-Dependent spin state population and 19F NMR chemical shift via remote ligand protonation in an iron(ii) complex.

An FeII complex that features a pH-dependent spin state population, by virtue of a variable ligand protonation state, is described. This behavior leads to a highly pH-dependent 19F NMR chemical shift with a sensitivity of 13.9(5) ppm per pH unit at 37 °C, thereby demonstrating the potential utility of the complex as a 19F chemical shift-based pH sensor.

Toward Quantitatively Accurate Calculation of the Redox-Associated Acid–Base and Ligand Binding Equilibria of Aquacobalamin

Redox processes in complex transition metal-containing species are often intimately associated with changes in ligand protonation states and metal coordination number. A major challenge is therefore to develop consistent computational approaches for computing pH-dependent redox and ligand dissociation properties of organometallic species. Reduction of the Co center in the vitamin B12 derivative aquacobalamin can be accompanied by ligand dissociation, protonation, or both, making these properties difficult to compute accurately. We examine this challenge here by using density functional theory and continuum solvation to compute Co-ligand binding equilibrium constants (Kon/off), pKas, and reduction potentials for models of aquacobalamin in aqueous solution. We consider two models for cobalamin ligand coordination: the first follows the hexa, penta, tetra coordination scheme for Co(III), Co(II), and Co(I) species, respectively, and the second model features saturation of each vacant axial coordination site on Co(II) and Co(I) species with a single, explicit water molecule to maintain six directly interacting ligands or water molecules in each oxidation state. Comparing these two coordination schemes in combination with five dispersion-corrected density functionals, we find that the accuracy of the computed properties is largely independent of the scheme used, but including only a continuum representation of the solvent yields marginally better results than saturating the first solvation shell around Co throughout. PBE performs best, displaying balanced accuracy and superior performance overall, with RMS errors of 80 mV for seven reduction potentials, 2.0 log units for five pKas and 2.3 log units for two log Kon/off values for the aquacobalamin system. Furthermore, we find that the BP86 functional commonly used in corrinoid studies suffers from erratic behavior and inaccurate descriptions of Co-axial ligand binding, leading to substantial errors in predicted pKas and Kon/off values. These findings demonstrate the effectiveness of the present approach for computing electrochemical and thermodynamic properties of a complex transition metal-containing cofactor.

The importance of protonation and tautomerization in relative binding affinity prediction: a comparison of AMBER TI and Schrödinger FEP.

In drug discovery, protonation states and tautomerization are easily overlooked. Through a Merck-Rutgers collaboration, this paper re-examined the initial settings and preparations for the Thermodynamic Integration (TI) calculation in AMBER Free-Energy Workflows, demonstrating the value of careful consideration of ligand protonation and tautomer state. Finally, promising results comparing AMBER TI and Schrödinger FEP+ are shown that should encourage others to explore the value of TI in routine Structure-based Drug Design.

A Proton-Switchable Bifunctional Ruthenium Complex That Catalyzes Nitrile Hydroboration.

A new bifunctional pincer ligand framework bearing pendent proton-responsive hydroxyl groups was prepared and metalated with Ru(II) and subsequently isolated in four discrete protonation states. Stoichiometric reactions with H2 and HBPin showed facile E-H (E = H or BPin) activation across a Ru(II)-O bond, providing access to unusual Ru-H species with strong interactions with neighboring proton and boron atoms. These complexes were found to promote the catalytic hydroboration of ketones and nitriles under mild conditions, and the activity was highly dependent on the ligand's protonation state. Mechanistic experiments revealed a crucial role of the pendent hydroxyl groups for catalytic activity.

On the Basicity of 8-Phenylsulfanyl Quipazine Derivatives: New Potential Serotonergic Agents

A protonation state of serotonergic ligands plays a crucial role in their pharmacological activity. In this research, the basicity of 8-phenylsulfanyl quipazine derivatives as new potential serotonergic agents was studied. The most favorable protonation sites were determined in the gas and aqueous phases. In water, a solvation effect promoting the protonation of the N3 atom overcomes a positive charge delocalization phenomenon favoring a N1 atom protonation. The most stable conformations of neutral and protonated molecules in gas and water were found. It was demonstrated that a diprotonation reaction may occur. The most favorable among the diprotonated structures is the molecule with the N1 and N3 atoms protonated. A calculation of the pKa and pKa2 in water of a set of monosubstituted 8-phenylsulfanyl quipazine derivatives was performed using B3LYP/6-31G(d) and the SMD continuum solvation model. Enthalpic and entropic contributions to the pKa and pKa2 in gas and water were separated for a rationalization of a substituent effect on values of the pKa and pKa2. The relationship of the proton affinity and the solvation enthalpy in water with some reactivity descriptors, such as the Fukui function, the molecular electrostatic potential (MEP), and the global softness, was investigated. The order of the pKa values is the most controlled by the entropy. The diprotonation reaction, despite having an unfavorable enthalpy in water, is driven entropically. Final state effects in the diprotonated species were analyzed with the triadic formula. Results of a calculation of the theoretical basicity of the 8-phenylsulfanyl quipazines indicate that they should be monoprotonated on the N3 atom in the CNS environment. Diprotonation of the studied compounds may occur in very acidic body fluids such as the gastric juice.

Insights into the Mechanism of Fe(II) Adsorption and Oxidation at Fe–Clay Mineral Surfaces from First-Principles Calculations

Interfacial reactivity of redox-active iron-bearing mineral surfaces plays a crucial role in many environmental processes including biogeochemical cycling of various elements and contaminants. Herein, we apply density functional theory (DFT) calculations to provide atomistic insights into the heterogeneous reaction between aqueous Fe(II) and the Fe-bearing clay mineral nontronite Fe2Si4O10(OH)2 by studying its adsorption mechanism and interfacial Fe(II)–Fe(III) electron transfer (ET) at edge and basal surfaces. We find that edge-bound Fe(II) adsorption complexes at different surface sites (ferrinol, silanol, and mixed) may coexist on both (010) and (110) edge facets, with complexes at ferrinol FeO(H) sites being the most energetically favorable and coupled to proton transfer. Calculation of the ET activation energy suggests that interfacial ET into dioctahedral Fe(III) sheets is probable at the clay edges and occurs predominantly but not exclusively through the complexes adsorbed at ferrinol sites and might also involve mixed sites. No clear evidence is found for complexes on basal surface that are compatible with ET through the basal sheet despite this experimentally hypothesized ET interface. This study suggests a strong pH-dependence of Fe(II) surface complexation at basal versus edge facets and highlights the importance of the protonation state of bridging ligands and proton coupled electron transfer to facilitate ET into Fe-rich clay minerals.

Sensitivity of X-ray Core Spectroscopy to Changes in Metal Ligation: A Systematic Study of Low-Coordinate, High-Spin Ferrous Complexes

In order to assess the sensitivity and complementarity of X-ray absorption and emission spectroscopies for determining changes in the metal ligation sphere, a systematic experimental and theoretical study of iron model complexes has been carried out. A series of high-spin ferrous complexes, in which the ligation sphere has been varied from a three-coordinate complex, [L(tBu)Fe(SPh)] (1) (where L(tBu) = bulky β-diketiminate ligand; SPh = phenyl thiolate) to four-coordinate complexes [L(tBu)Fe(SPh)(X)] (where X = CN(t)Bu (2); 1-methylimidazole (3); or N,N-dimethylformamide (DMF) (4)), has been investigated using a combination of Fe K-edge X-ray absorption (XAS) and Kβ X-ray emission (XES) spectroscopies. The Fe K XAS pre-edge and edge of all four complexes are consistent with a high-spin ferrous assignment, with the largest differences in the pre-edge intensities attributed to changes in covalency of the fourth coordination site. The X-ray emission spectra show pronounced changes in the valence to core region (V2C) as the identity of the coordinated ligand is varied. The experimental results have been correlated to density functional theory (DFT) calculations, to understand key molecular orbital contributions to the observed absorption and emission features. The calculations also have been extended to a series of hypothetical high-spin iron complexes to understand the sensitivity of XAS and XES techniques to different ligand protonation states ([L(tBu)Fe(II)(SPh)(NHn)](3-n) (n = 3, 2, 1, 0)), metal oxidation states [L(tBu)Fe(SPh)(N)](n-) (n = 3, 2, 1), and changes in the ligand identity [L(tBu)Fe(IV)(SPh)(X)](n-) (X = C(4-), N(3-), O(2-); n = 2, 1, 0). This study demonstrates that XAS pre-edge data have greater sensitivity to changes in oxidation state, while valence to core (V2C) XES data provide a more sensitive probe of ligand identity and protonation state. The combination of multiple X-ray spectroscopic methods with DFT results thus has the potential to provide for detailed characterization of complex inorganic systems in both chemical and biological catalysis.

Reversible multicomponent self-assembly mediated by bismuth ions

Bi(III) ions are capable of reversible, multicomponent self assembly with suitable tris-coordinate ligands. The nature of the self-assembled structures observed are dependent on the ligand coordination geometry, ligand protonation state and Bi concentration. These assemblies can exploit the maximum number of coordination sites at the Bi vertices (nine), and the self-assembly process has been studied by 1D NMR, Diffusion NMR, ESI-MS and X-ray crystallographic analysis. V-shaped coordinating ligands reversibly form discrete M2L4, M2L3, and M2L2 complexes dependent on ligand/bismuth concentration, whereas a linear coordinating ligand forms a single discrete M3L3 assembly.

Mapping histamine H4receptor–ligand binding modes

Computational prediction of ligand binding modes in G protein-coupled receptors (GPCRs) remains a challenging task. Systematic consideration of different protein modelling templates, ligand binding poses, and ligand protonation states in extensive molecular dynamics (MD) simulation studies enabled the prediction of ligand-specific mutation effects in the histamine H4receptor, a key player in inflammation.

Effect of atomic charge, solvation, entropy, and ligand protonation state on MM‐PB(GB)SA binding energies of HIV protease

The molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) and MM-generalized-Born surface area (MM-GBSA) approaches are commonly used in molecular modeling and drug design. Four critical aspects of these approaches have been investigated for their effect on calculated binding energies: (1) the atomic partial charge method used to parameterize the ligand force field, (2) the method used to calculate the solvation free energy, (3) inclusion of entropy estimates, and (4) the protonation state of the ligand. HIV protease has been used as a test case with six structurally different inhibitors covering a broad range of binding strength to assess the effect of these four parameters. Atomic charge methods are demonstrated to effect both the molecular dynamics (MD) simulation and MM-PB(GB)SA binding energy calculation, with a greater effect on the MD simulation. Coefficients of determination and Spearman rank coefficients were used to quantify the performance of the MM-PB(GB)SA methods relative to the experimental data. In general, better performance was achieved using (i) atomic charge models that produced smaller mean absolute atomic charges (Gasteiger, HF/STO-3G and B3LYP/cc-pVTZ), (ii) the MM-GBSA approach over MM-PBSA, while (iii) inclusion of entropy had a slightly positive effect on correlations with experiment. Accurate representation of the ligand protonation state was found to be important. It is demonstrated that these approaches can distinguish ligands according to binding strength, underlining the usefulness of these approaches in computer-aided drug design.

Comparative Assessment of the Composition and Charge State of Nitrogenase FeMo-Cofactor

A significant limitation in our understanding of the molecular mechanism of biological nitrogen fixation is the uncertain composition of the FeMo-cofactor (FeMo-co) of nitrogenase. In this study we present a systematic, density functional theory-based evaluation of spin-coupling schemes, iron oxidation states, ligand protonation states, and interstitial ligand composition using a wide range of experimental criteria. The employed functionals and basis sets were validated with molecular orbital information from X-ray absorption spectroscopic data of relevant iron-sulfur clusters. Independently from the employed level of theory, the electronic structure with the greatest number of antiferromagnetic interactions corresponds to the lowest energy state for a given charge and oxidation state distribution of the iron ions. The relative spin state energies of resting and oxidized FeMo-co already allowed exclusion of certain iron oxidation state distributions and interstitial ligand compositions. Geometry-optimized FeMo-co structures of several models further eliminated additional states and compositions, while reduction potentials indicated a strong preference for the most likely charge state of FeMo-co. Mössbauer and ENDOR parameter calculations were found to be remarkably dependent on the employed training set, density functional, and basis set. Overall, we found that a more oxidized [Mo(IV)-2Fe(II)-5Fe(III)-9S(2-)-C(4-)] composition with a hydroxyl-protonated homocitrate ligand satisfies all of the available experimental criteria and is thus favored over the currently preferred composition of [Mo(IV)-4Fe(II)-3Fe(III)-9S(2-)-N(3-)] from the literature.

LigpKa – a database of pKa values for small molecule ligands designed for the use in structure-based pKa calculations

Electrostatic forces play a large role in determining the strength of protein-ligand interactions, and the calculation of pKa value shifts upon ligand binding is therefore an important component of any accurate protein-ligand binding calculation. However, such pKa calculations are rarely performed in virtual screening experiments due to the unavailability of ligand solution pKa values and the difficulty in generating the required charge distributions for each ligand protonation state. We present a freely available web-based database of small-molecule pKa values that automates the task of retrieving protonation states and solution pKa values for small-molecule ligands so that these can be used with tools such as PRODRG and PDB2PQR for generating the necessary parameters for a structure-based pKa calculation of a protein-ligand complex. The database contains a total of 348 pKa values and represents a significant step towards performing accurate automated pKa value calculations in virtual screening experiments.