Determination of the pKa values of titratable groups of an antigen-antibody complex, HyHEL-5-hen egg lysozyme

The titration behavior of the ionizable residues of the HyHEL-5–henegg lysozyme complex and its individual components has beenstudied using continuum electrostatic calculations. Severalresidues of HyHEL-5 had pK_a values shifted away from model valuesfor isolated residues by more than three pH units. Shifts awayfrom the model values were smaller for the residues of hen egglysozyme. A moderate variation in the pK_a values of the titratablegroups was observed upon increase of the ionic strength from0 to 100 mM, amounting to 1–2 pH units in most cases.Under physiological conditions, the net charge of HyHEL-5 wasopposite that for hen egg lysozyme. Several residues, includingthose involved in the Arg–Glu salt bridges that have beenproposed to be important in antibody-antigen binding, had pK_avalues that were changed significantly upon binding. The maintitration event upon antibody-antigen binding appears to beloss of a proton from residue GluH50 of the Fv molecule. Thelimitations of our calculation methods and the role they mightplay in the design of antibodies for use in assays, sensorsand separations are discussed     

Preclinical Characterization of Acyl Sulfonimidamides: Potential Carboxylic Acid Bioisosteres with Tunable Properties

Herein we present the preclinical characterization of novel compounds containing the linear acyl sulfonimidamide functionality. Specifically, we studied the pK_a, lipophilicity, in vitro metabolic stability, plasma protein binding, Caco‐2 permeability, and aqueous solubility for nine aryl acyl sulfonimidamides. In comparison with widely used carboxylic acid bioisosteres, the acyl sulfonimidamides were found to be less acidic and more lipophilic depending on the substitution pattern in the studied compounds. Importantly, the pK_a values (5.9–7.6) were significantly influenced by substituents on the nitrogen atom and the aryl substituents. Moreover, the acyl sulfonimidamides displayed membrane permeabilities ranging from moderate to very high, which correlated with decreased pK_a and low to negligible efflux ratios. We foresee that the chiral sulfur center and the two handles for structural diversity of linear acyl sulfonimidamides will offer new opportunities for drug design and for improving the oral bioavailability of acidic drug candidates.     

Effects of the Protonation State of Titratable Residues and the Presence of Water Molecules on Nocodazole Binding to β‐Tubulin

Regulation of microtubule assembly by antimitotic agents is a potential therapeutic strategy for the treatment of cancer, parasite infections, and neurodegenerative diseases. One of these agents is nocodazole (NZ), which inhibits microtubule polymerization by binding to β‐tubulin. NZ was recently co‐crystallized in Gallus gallus tubulin, providing new information about the features of interaction for ligand recognition and stability. In this work, we used state‐of‐the‐art computational approaches to evaluate the protonation effects of titratable residues and the presence of water molecules in the binding of NZ. Analysis of protonation states showed that residue E198 has the largest modification in its pK_a value. The resulting E198 pK_a value, calculated with pH‐REMD methodology (pK_a=6.21), was higher than the isolated E amino acid (pK_a=4.25), thus being more likely to be found in its protonated state at the binding site. Moreover, we identified an interaction between a water molecule and C239 and G235 as essential for NZ binding. Our results suggest that the protonation state of E198 and the structural water molecules play key roles in the binding of NZ to β‐tubulin.     

Dissociation constants of long chain fatty acids in methanol-water and ethanol-water mixtures

The pK_a values of lauric acid, myristic acid, palmitic acid and stearic acid in methanol-water and ethanol-water mixtures of different compositions at 303 K and ionic strength, I=0.1M(KNO₃) have been determined. The pK_a values of the fatty acids have been found to increase with the alcohol content of the solvent. It has also been found that, in general, the pK_a values in the ethanolic solvent are greater than those in the methanolic solvent. In a given solvent mixture the observed pK_a value decreases in the order of stearic acid > palmitic acid > myristic acid > lauric acid. Departure from the predicted linearity of the plots of pK_a against the inverse of the dielectric constant, 1/D indicates that the electrostatic effects taken into account by the Born theory are not adequate enough to represent the total medium effects on pK_a.     

Acid-Base Equilibria and the Proton Pump in Bacteriorhodopsin

The light-induced proton pump in bacteriorhodopsin is reviewed with emphasis on acid-base equilibria of protein residues and of the retinal Schiff base moiety. Pump mechanisms in bR and in some of its mutants are classified in terms of light-induced pK_a changes (class I) or light-induced exposure changes, in which the proton accessibility of the protein changes from the outside to the inside of the membrane (class II). A discussion of the theoretical basis of the factors which determine the pK_a of ionizable protein groups is followed by a review of the experimental phenomena associated with the titration of residues in both unphotolyzed bR and during its photocycle. The time-resolved titrations of the Schiff base and of the Asp-85 residue are discussed in terms of the accessibility of the two groups to external protons. Finally, the molecular aspects of the pH-dependent proton pump in native bR and in various mutants are analyzed, focusing on the mechanism of the initial proton release reaction and on the subsequent molecular switch which allows reprotonation from the inside of the cell. Special attention is devoted to the question of coupling between the photocycle intermediates (primary M formation and decay) and the transmembrane proton translocation. Recent work with bR mutants raise the question as to whether proton transfer from the Schiff base to Asp-85 at the M stage is directly responsible for proton translocation, as well as for the reprotonation switch.     

Stabilizing p-Dithiobenzyl Urethane Linkers without Rate-Limiting Self-Immolation for Traceless Drug Release

Exploiting the redox sensitivity of disulfide bonds is a prevalent strategy in targeted prodrug designs. In contrast to aliphatic disulfides, p-thiobenzyl-based disulfides have rarely been used for prodrug designs, given their intrinsic instability caused by the low pK_a of aromatic thiols. Here, we examined the interplay between steric hindrance and the low-pK_a effect on thiol–disulfide exchange reactions and uncovered a new thiol–disulfide exchange process for the self-immolation of p-thiobenzyl-based disulfides. We observed a central leaving group shifting effect in the α,α-dimethyl-substituted p-dithiobenzyl urethane linkers (DMTB linkers), which leads to increased disulfide stability by more than two orders of magnitude, an extent that is significantly greater than that observed with typical aliphatic disulfides. In particular, the DMTB linkers display not only high stability, but also rapid self-immolation kinetics due to the low pK_a of the aromatic thiol, which can be used as a general and robust linkage between targeting reagents and cytotoxic drugs for targeted prodrug designs. The unique and promising stability characteristics of the present DMTB linker will likely inspire the development of novel targeted prodrugs to achieve traceless release of drugs into cells.     

Context-Dependence of the Reactivity of Cysteine and Lysine Residues

The S-alkylation of Cys residues with a maleimide and the N^ϵ-acylation of Lys residues with an N-hydroxysuccinimide (NHS) ester are common methods for bioconjugation. Using Cys and Lys derivatives as proxies, we assessed differences in reactivity depending on the position of Cys or Lys in a protein sequence. We find that Cys position is exploitable to improve site-selectivity in maleimide-based modifications. Reactivity decreases substantially in the order N-terminal>in-chain>C-terminal Cys due to modulation of sulfhydryl pK_a by the α-ammonium and carboxylate groups at the termini. A lower pK_a value yields a larger fraction thiolate, which promotes selectivity while somewhat decreasing thiolate nucleophilicity in accord with =0.41. Lowering pH and salt concentration enhances selectivity still further. In contrast, differences in the reactivity of Lys towards an NHS ester were modest due to an appreciable decrease in amino group nucleophilicity with a lower pK_a of its conjugate acid. Hence, site-selective Lys modification protocols will require electrophiles other than NHS esters.     

Determination of pK a Values of Oxocholanoic Acids by Potentiometric Titration

The pK _a and the maximum solubility values of cholic, deoxycholic salts and their oxo-derivatives have been measured by the method of potentiometric titration. In the monomer phase (under the critical micellar concentration, CMC), the bile salts have different pK _a values, as a result of their structural differences (the number of hydroxyl and oxo groups in the steroid skeleton) and different hydration properties of the acid anions. In the micellar phase (above the CMC), the bile salts have higher pK _a values than in the monomer phase (under the CMC). This increase in the pK _a values is greater in more hydrophobic bile salts (cholate and deoxycholate), than in less hydrophobic oxo derivatives, which can be explained by the different aggregation numbers of these bile salts. The oxo-derivatives are more likely to form dimeric micelles, where the carboxylic groups are situated on the two sides of the micelle, not causing any electrostatic repulsion. In the more hydrophobic bile salts, aggregation numbers are higher, which causes electrostatic repulsion of the nearby situated carboxylic anions and consequential protonation of these anions (which leads to the stabilization of the micelle). The maximum solubility values are higher for the oxo-derivatives. If the steroid skeleton of the bile salt is more hydrophobic, the capacity to solubilize the unionized bile acid is higher, i.e. a smaller amount of the bile acid anion is needed for the solubilization of the bile acid monomer. The oxo-derivatives are less hydrophobic, but alongside their hydrophobicity, the structure of the micelle determines the solubilization capacities.     

Cucurbit[n]uril Host-Guest Complexes of Acids,Photoacids, and Super Photoacids

The supramolecular chemistry of host-guest complexes of cucurbit[n]urils (CB[n]) with acidic guests in the ground (HG⁺) and excited states (HG⁺*) are reviewed. The effects of CB[n] complexation on the guests’ pK_a and/or pK_a* values are related to relative binding constants and host-guest structures of the acid form of the guest and its conjugate base. Included are carbon acids, guests of biological and medicinal interest, dyes and related polyaromatic guests, and other organic and organometallic guests. The applications of the pK_a shifts to the solubility, stability, and bioavailabilty of drug molecules, the stability and enhanced spectral properties of dyes, and in pH-induced self-sorting, micelle formation, host-guest shuttling, and controlled guest release, are discussed.     

A Chemogenomic Analysis of Ionization Constants—Implications for Drug Discovery

Chemogenomics methods seek to characterize the interaction between drugs and biological systems and are an important guide for the selection of screening compounds. The acid/base character of drugs has a profound influence on their affinity for the receptor, on their absorption, distribution, metabolism, excretion and toxicity (ADMET) profile and the way the drug can be formulated. In particular, the charge state of a molecule greatly influences its lipophilicity and biopharmaceutical characteristics. This study investigates the acid/base profile of human small‐molecule drugs, chemogenomics datasets and screening compounds including a natural products set. We estimate the acid‐ionization constant (pK_a) values of these compounds and determine the identity of the ionizable functional groups in each set. We find substantial differences in acid/base profiles of the chemogenomic classes. In many cases, these differences can be linked to the nature of the target binding site and the corresponding functional groups needed for recognition of the ligand. Clear differences are also observed between the acid/base characteristics of drugs and screening compounds. For example, the proportion of drugs containing a carboxylic acid was 20 %, in stark contrast to a value of 2.4 % for the screening set sample. The proportion of aliphatic amines was 27 % for drugs and only 3.4 % for screening compounds. This suggests that there is a mismatch between commercially available screening compounds and the compounds that are likely to interact with a given chemogenomic target family. Our analysis provides a guide for the selection of screening compounds to better target specific chemogenomic families with regard to the overall balance of acids, bases and pK_a distributions.     

Stability constants of polymer‐bound iminodiacetate‐type chelating agents with some transition‐metal ions

A chelating vinyl monomer, glycidyl methacrylate (GMA)–iminodiacetic acid (IDA), was formed by the reaction between GMA and IDA. Three polymeric chelating agents, PGMA–IDA, PGMA–IDA‐co‐methyl acrylate (MA), and PGMA–IDA‐co‐acrylamide (AAm), were also synthesized. Acid dissociation constants and stability constants of these chelating agents with Ni(II), Zn(II), and Co(II) were determined by means of potentiometric titration and ultraviolet–visible spectrophotometry, respectively. The values of K_a1 and K_a2 of all the polymeric chelating agents were smaller than those of GMA–IDA. The stability constants of all the polymeric chelating agents were larger than those of GMA–IDA. Increasing the MA content within PGMA–IDA‐co‐MA affected the stability constant only slightly. A proper molar ratio of AAm in PGMA–IDA‐co‐AAm, stability constants was 30–60 times greater than that of GMA–IDA. However, as the molar content of AAm increased, the stability constant of PGMA–IDA‐co‐AAm decreased. The results obtained in the polymer system are explained in terms of the polymer's stereo and entanglement structure, the neighboring effect, and the hydrophobic/hydrophilic nature of MA or AAm. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 1986–1994, 2002     

Interfacial chemistry on carboxylate-functionalized monolayer assemblies

Deposition of trichlorosilanes with ester groups at their remote termini provides a convenient entry to carboxylic acid-bearing siloxane-anchored self-assembled monolayers. The de-esterification of these esters has been optimized to minimize monolayer damage, and their quantitative re-esterification provides clear evidence for the stability of these systems. Both the structure of the ester-terminated monolayer and its de-esterification/esterification chemistry can be easily monitored by FTIR-ATR measurements. This spectroscopic tool, together with a liquid cell that enables IR spectra to be measured in an aqueous environment, enables a detailed structural analysis of the carboxylic acid-bearing siloxane-anchored self-assembled monolayers and an assessment of their acid/base behavior (by in situ titration). The use of D₂O instead of H₂O for the in situ titration experiments also improves the available IR window. Both monomeric and dimeric/oligomeric acid groups are seen, and the relative ease of deprotonation of these various species can be directly monitored. Monomers of alkyl carboxylic acids that are hydrogen bonded only to surrounding water molecules have a pK_a = 4.9, while the pK_a for the aggregated molecules is 9.3. Similar behavior is seen for surface-bound benzoic acids, where the two pK_a values are 4.7 and 9.0. The influence of temperature on these structures and their chemistry has been explored to a limited extent as well. When the alkylcarboxylic acid system is cooled to 10 °C, the pK_a value for the acid monomers is reduced from 4.9 to 4.5 and increases from 9.3 to 10.3 for the aggregates.     

Efficiency of different clay minerals modified with a cationic surfactant in the adsorption of pesticides: Influence of clay type and pesticide hydrophobicity

In the present work we studied the efficiency of a series of clay minerals (montmorillonite, illite, muscovite, sepiolite and palygorskite) modified with the cationic surfactant octadecyltrimetylammonium bromide (ODTMA) in the adsorption of the pesticides penconazole, linuron, alachlor, atrazine and metalaxyl. A study was also carried out on the effect of the structure (expansible and non-expansible layered, and non-layered), the surface area and charge density of the clay minerals, and the hydrophobicity of the pesticides (K_ow) on the adsorption process. Adsorption–desorption isotherms of the pesticides by clay minerals were obtained and the constants of the Freundlich model (K_f and K_fd) (natural clays) and of the linear model (K and K_des) (ODTMA-clays) were determined. Correlation coefficient values (r²) found between K and K_des, and organic matter (OM) content of ODTMA-clays indicate a more effective partitioning of the pesticides in the organic phase of ODTMA after desorption. Furthermore the positive significant correlation found between the K_desom values and the charge density of different ODTMA-clays indicates that a higher density of ODTMA in the clay gives rise to the formation of an organic phase more effective for the partition of the pesticides. This correlation explained that the highest K_desom value obtained was for kaolinite and the lowest K_desom value was for montmorillonite in the adsorption of all pesticides by the different organo clays. Simple correlations between adsorption constants and K_ow values, and multiple correlations between these constants and clay OM contents and K_ow values were also calculated. The results obtained indicate the interest of ODTMA-clays in the adsorption of hydrophobic pesticides. Non-expandible layered and fibrous clay minerals may also be of interest as adsorbents derived from their density charge, and these minerals, when modified with ODTMA, and used in appropriate amounts, higher than that of montmorillonite, or when present in soils with high clay contents, even in the absence of montmorillonite, may be good adsorbents for hydrophobic pesticides. According to the results of the study, ODTMA-clays and ODTMA-soils could be used as barriers to prevent the mobility of certain hydrophobic pesticides from a point source of pollution.     

Assessment of a semi‐quantitative method for estimation of the rejection of organic compounds in aqueous solution in nanofiltration

A large number of different mechanisms describing the retention of dissolved organic compounds in nanofiltration have been proposed. A recent review identified the parameters possibly involved in the separation performance and suggested a qualitative classification of dissolved compounds. Continuing this approach, a semi‐quantitative assessment of the observed rejections in nanofiltration is given in this paper, based on threshold values of key parameters such as molecular weight and molecular weight cut‐off (MWCO), molecular size, pH and pK_a, hydrophobicity (logK_ow) and membrane charge. Experimental values and literature data were used to provide a broad basis for comparison. It was concluded that (a) all categories that contain hydrophobic components are badly defined, in particular for small components, with rejections varying from low to very high, (b) all components that contain hydrophilic components have relatively high rejections and (c) all categories that contain charged components have well‐defined, high rejections (intermediate for membranes with low surface charge). In all cases, the average rejection is higher when the component's molecular weight is larger than the MWCO of the membrane and when the molecular size is larger than the pore size of the membrane. Copyright © 2006 Society of Chemical Industry     

Directed Evolution of a Formate Dehydrogenase for Increased Tolerance to Ionic Liquids Reveals a New Site for Increasing the Stability

The formate dehydrogenase (FDH) from Candida boidinii is a well‐known enzyme in biocatalysis for NADH regeneration. Nevertheless, it has low activity in a water‐miscible ionic liquid (1,3‐dimethylimidazolium dimethyl phosphate, [MMIm][Me₂PO₄]). In this work, this enzyme was subjected to directed evolution by using error‐prone PCR, and a mutant (N187S/T321S) displaying higher activity was obtained following selection based on the formazan‐based colorimetric assay. The mutation N187S is responsible for improved activity both in aqueous solution and in [MMIm][Me₂PO₄], through an enhancement of the k_cat value by a factor of 5.8. Fluorescence experiments performed in the presence of a quenching agent revealed that the mutant does not unfold in the presence of 50 % (v/v) [MMIm][Me₂PO₄] whereas the wild‐type enzyme does. Molecular modelling revealed that the mutation is located at the monomer–monomer interface and causes an increase in the pK_a of residue E163 from 4.8 to 5.5. Calculation of the pK_a of this residue in other microbial FDHs showed that thermostable FDHs have a highly basic glutamate at this position (pK_a up to 6.2). We have identified a new site for improving FDH thermostability and tolerance to ionic liquids, and it is linked to the local charge of the enzymes in this class.     

Steady-state fluorescence and NMR study on self-association behavior of poly(methacrylamides) bearing hydrophobic amino acid residues

Self-association properties of poly(N-methacryloylphenylalanine) and poly(N-methacryloyltryptophan) (pMPhe and pMTrp, respectively) were investigated by several characterization techniques, including steady-state fluorescence and NMR. These characterization data revealed similarities and distinctions of their self-association properties.The pH dependencies of association properties of pMPhe and pMTrp are practically the same. Apparent pK_a values for pMPhe and pMTrp were determined to be 5.7 and 5.8, respectively, by potentiometric titration. Steady-state fluorescence measurements at varying pH using pyrene as fluorescence probe indicated that, at pH≈5 (< apparent pH), hydrophobic microdomains were formed, while, at pH≈7 and 9 (> apparent pK_a), hydrophobic microdomains were not formed significantly. ¹H NMR spectra for both the polymers measured in D₂O exhibited that a significant fraction of aromatic rings in amino acid residues were located close to the polymer main chain, and that, at pH≈5, the mobility of the polymer main chain and the aromatic ring was extremely restricted.The polymer concentration (C_p) dependencies of association properties of pMPhe and pMTrp at pH≈5 are distinct. Steady-state fluorescence data at varying C_p indicated that pMPhe was more hydrophobic microscopically than pMTrp. Dynamic light scattering data indicated that pMTrp had a stronger tendency for interpolymer association than pMPhe did at pH≈5. It is concluded that the distinction in C_p dependency of the self-association properties of pMPhe and pMTrp is due to the differences in the bulkiness and the hydrophobicity of the substituents of amino acid residues.     

pH-Dependent Protonation of Surface Carboxylate Groups in PsbO Enables Local Buffering and Triggers Structural Changes

Photosystem II (PSII) catalyzes the splitting of water, releasing protons and dioxygen. Its highly conserved subunit PsbO extends from the oxygen-evolving center (OEC) into the thylakoid lumen and stabilizes the catalytic Mn₄CaO₅ cluster. The high degree of conservation of accessible negatively charged surface residues in PsbO suggests additional functions, as local pH buffer or by affecting the flow of protons. For this discussion, we provide an experimental basis, through the determination of pK_a values of water-accessible aspartate and glutamate side-chain carboxylate groups by means of NMR. Their distribution is strikingly uneven, with high pK_a values around 4.9 clustered on the luminal PsbO side and values below 3.5 on the side facing PSII. pH-dependent changes in backbone chemical shifts in the area of the lumen-exposed loops are observed, indicating conformational changes. In conclusion, we present a site-specific analysis of carboxylate group proton affinities in PsbO, providing a basis for further understanding of proton transport in photosynthesis.     

Partitioning of water-soluble vitamins in biodegradable aqueous two-phase systems: Electrolyte perturbed-chain statistical associating fluid theory predictions and experimental validation

Partition coefficients (K) of vitamins (riboflavin, nicotinic acid, nicotinamide, folic acid, cyanocobalamin) in aqueous two-phase systems (ATPS) composed by polyethylene glycol (PEG 4000, PEG 6000) and organic salt (sodium citrate and sodium tartrate) at T = 298.15 K and p = 1 bar have been studied. Data on liquid–liquid equilibria of the ATPS considered in this study have been taken from the literature (PEG-Na₃Citrate) or measured in this work (PEG-Na₂Tartrate) for PEG 4000 and PEG 6000 at T = 298.15 K and p = 1 bar. The experimental K values were validated by electrolyte perturbed-chain-statistical associating fluid theory predictions. The neutral cyanocobalamin has the highest K values among all studied vitamins at any ATPS studied in this work. This finding contrasted with expectations based on literature data which let assume that charged species have typically the highest K values in the considered ATPS. Thus, besides the typically strong charge–charge interactions especially specific forces (e.g., hydrogen bonding) explains the strong PEG-cyanocobalamin interaction resulting in the high K values.     

The Effects of Cyclic Terminal Groups in 4-Aminoazo-benzene and Related Azo Dyes: Part 21—pKa Values of Some Monoazo Dyes Derived from

Basicity values have been determined for series of monoazo dyes incorporating a pyrrolidino or a piperidino moiety. In general, the pK_a value is found to decrease as the donor capacity of the terminal nitrogen atom decreases.The basicities of the 4'-substituted piperidinoazobenzenes decrease as the electron withdrawing strength of the 4'-substituent increases. However, the pK_a values of the related pyrrolidino compounds appear to be insensitive to the substituent.     

Encapsulation of Drug Molecules by Cucurbiturils: Effects on their Chemical Properties in Aqueous Solution

The effects of the encapsulation of drugs and other molecules of biomedical interest by cucurbit[n]uril (n=5–8, 10) host molecules on the chemical properties of the drugs in aqueous solution are reviewed. The cucurbituril complexation of drug molecules has been shown to generally increase the guests’ pK_a values through preferential inclusion of the protonated species, modulate other equilibria involving the guest, improve the solubility in aqueous solution, reduce the toxicity and other side effects, as well as enhance the stability and targeted delivery of the drug molecule. These benefits have led to an increasing interest in the applications of cucurbit[n]urils in novel drug formulations.