Biophysical Features of Bacillithiol,the Glutathione Surrogate of Bacillus subtilis and other Firmicutes

Bacillithiol (BSH) is the major low‐molecular‐weight (LMW) thiol in many low‐G+C Gram‐positive bacteria (Firmicutes). Evidence now emerging suggests that BSH functions as an important LMW thiol in redox regulation and xenobiotic detoxification, analogous to what is already known for glutathione and mycothiol in other microorganisms. The biophysical properties and cellular concentrations of such LMW thiols are important determinants of their biochemical efficiency both as biochemical nucleophiles and as redox buffers. Here, BSH has been characterised and compared with other LMW thiols in terms of its thiol pK_a, redox potential and thiol–disulfide exchange reactivity. Both the thiol pK_a and the standard thiol redox potential of BSH are shown to be significantly lower than those of glutathione whereas the reactivities of the two compounds in thiol–disulfide reactions are comparable. The cellular concentration of BSH in Bacillus subtilis varied over different growth phases and reached up to 5 mM , which is significantly greater than previously observed from single measurements taken during mid‐exponential growth. These results demonstrate that the biophysical characteristics of BSH are distinctively different from those of GSH and that its cellular concentrations can reach levels much higher than previously reported.     

Thiolated chitosans: In vitro comparison of mucoadhesive properties

The aim of this study was to compare the mucoadhesive properties of thiolated chitosans with regard to their molecular mass and type of immobilized thiol ligand. Mediated by a carbodiimide, aromatic‐ and aliphatic‐thiol‐bearing compounds were covalently attached to low‐ and medium‐molecular‐mass chitosan. All synthesized conjugates displayed on average 320 ± 50 μmol of immobilized free thiol groups per gram of polymer. The rheological synergy was observed by the mixture of equal volumes of polymer with mucin solution. Because of the increase in viscosity of the conjugate/mucin mixture, the self‐crosslinking properties and the interaction of thiomers with the mucus layer could be confirmed. Further mucoadhesion of the chitosan conjugates was evaluated in vitro with the rotating cylinder method and tensile studies on excised porcine intestinal mucosa. The results show a significantly enhanced residence time (p < 0.05) on the mucosa of all thiolated chitosans compared to the unmodified polymer. Among all of the conjugates tested, the following rank order of mucoadhesion could be determined: Chitosan–thiobutylamidine > Chitosan–4‐mercaptobenzoic acid > Chitosan–glutathione > Chitosan–6‐mercaptonicotinic acid > Chitosan–N‐acetyl cysteine > Chitosan–thioglycolic acid > Unmodified chitosan. The charge, pK_a, and reactivity of the attached compounds were found to be important factors influencing the mucoadhesive potential of the polymer. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

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.     

Thiol Redox and pKa Properties of Mycothiol,the Predominant Low‐Molecular‐Weight Thiol Cofactor in the Actinomycetes

The thiol pK_a and standard redox potential of mycothiol, the major low‐molecular‐weight thiol cofactor in the actinomycetes, are reported. The measured standard redox potential reveals substantial discrepancies in one or more of the other previously measured intracellular parameters that are relevant to mycothiol redox biochemistry.     

Experimental pKa Value Determination of All Ionizable Groups of a Hyperstable Protein

Electrostatic interactions significantly contribute to the stability and function of proteins. The stabilizing or destabilizing effect of local charge is reflected in the perturbation of the pK_a value of an ionizable group from the intrinsic pK_a value. Herein, the charge network of a hyperstable dimeric protein (ribbon–helix–helix (rhh) protein from plasmid pRN1 from Sulfolobus islandicus) is studied through experimental determination of the pK_a values of all ionizable groups. Transitions were monitored by multiple NMR signals per ionizable group between pH 0 and 12.5, prior to a global analysis, which accounted for the effects of neighboring residues. It is found that for several residues involved in salt bridges (four Asp and one Lys) the pK_a values are shifted in favor of the charged state. Furthermore, the pK_a values of residues C40 and Y47, both located in the hydrophobic dimer interface, are shifted beyond 13.7. The necessary energy for such a shift is about two-thirds of the total stability of the protein, which confirms the importance of the hydrophobic core to the overall stability of the rhh protein.     

Ionic end‐capping of (semi)telechelic polymers by mesogens

Two methods have been used to end‐cap linear polymer chains at one end or at both ends by a mesogen through ionic bonding. These polymers are designated as liquid‐crystalline halato(semi)telechelic polymers (LC H(S)TPs). The first method relies on the ion exchange reaction between the metal counterion of halato(semi)telechelic polymers and an ionic mesogen. The second method is based on the proton transfer from a sulfonic or carboxylic acid end‐group to a tertiary aliphatic amine, this approach being controlled by the relative pK_a values of the acidic and basic groups. If the pK_a difference is not large enough, strong hydrogen‐bonding is observed by Fourier‐transform infrared (FTIR) spectroscopy rather than proton transfer. The resulting materials have been characterized by differential scanning calorimetry (DSC), polarized optical microscopy (POM) and small‐angle X‐ray scattering (SAXS). © 2000 Society of Chemical Industry     

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.     

Advanced Pyrrolidine-Carbamate Self-Immolative Spacer with Tertiary Amine Handle Induces Superfast Cyclative Drug Release

Amine-carbamate self-immolative (SI) spacers represent practical and versatile tools in targeted prodrugs, but their slow degradation mechanism limits drug activation at the site of disease. We engineered a pyrrolidine-carbamate SI spacer with a tertiary amine handle which strongly accelerates the spacer cyclization to give a bicyclic urea and the free hydroxy groups of either cytotoxic (Camptothecin) or immunostimulatory (Resiquimod) drugs. In silico conformational analysis and pK_a calculations suggest a plausible mechanism for the superior efficacy of the advanced SI spacer compared to state-of-art analogues.     

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.     

Phospholipid Cyclosporine Prodrugs Targeted at Inflammatory Bowel Disease (IBD) Treatment: Design,Synthesis, and in Vitro Validation

Novel phospholipid (PL)-cyclosporine conjugates were prepared and studied as potential prodrugs for inflammatory bowel disease (IBD). Our approach relies on phospholipase A₂ (PLA₂), which is overexpressed in the inflamed intestinal tissues, as the prodrug activator to potentially release cyclosporine at the site of inflammation. PL-cyclosporine prodrug conjugates with methylene linkers of various lengths between the sn-2 position of the PL and cyclosporine were synthesized and evaluated for in vitro activation. Surprisingly, despite previous work indicating that conjugates with six methylene linkers between the lipid and drug would suffer rapid enzymatic hydrolysis, with cyclosporine this was not observed. However, compounds with longer linkers (n=10, 12 methylene units) display complete release of the drug by PLA₂-catalyzed hydrolysis, thus demonstrating the importance and profound impact of structural fine-tuning. This study represents a proof-of-concept for our hypothesis and a first step towards a truly targeted IBD treatment with cyclosporine that could be administered throughout the GI tract.     

Chiral Molecular Clefts For Dicarboxylic Acid Complexation

Three efficient cleft-type receptors, 3,5 , and 6 have been prepared by attachment of two amidopyridine units as hydrogen bonding centers to either the 2,2′-positions of 9, 9′-spirobifluorene or the 6,6′-positions of 1,1′-binaphthyl spacers. The easy availability of these compounds in short synthetic routes make them attractive complexing agents for aliphatic and aromatic dicarboxylic acids which undergo bidentate binding in chloroform solution. ¹H NMR binding studies show that substrates of different size can be accommodated into the clefts and form 1:1 complexes that are predominantly stabilized by multiple host-guesthydrogen bonds. The flexible aliphatic substrates diethylmalonic, 2,2-diphenylsuccinic, glutaric, and pimelic acid form complexes with association constants K_a ranging from 10³ to 10⁴ L mol⁻¹. Significantly more stable complexes (K_a > 10⁵ L mol⁻¹) are obtained with the more rigid, preorganized substrate 5-dodecyloxyisophthalic acid.     

Reverse osmosis separation of amino acids in aqueous solutions using porous cellulose acetate membranes

Polar and steric effects together govern the reverse osmosis separation of amino acids in single-solute aqueous solution systems. The solute transport parameter for the completely ionized aliphatic amino acids (with no additional polar groups other than one ? NH₂ and one ? COOH) in the pK₁ range of 4.03 to 1.71 can be represented as a function of pK₁ and the steric parameter ΣE_s. The latter parameter has a relatively greater influence with respect to the separation of zwitter ions. The effect of the polar parameter pK₁ on solute separation increases with increase in the concentration of the ionic species R⁺ (or decrease in the concentration of the ionic species R^?) in the feed solution. The effect of the presence of additional polar groups in the amino acid molecule is to increase its basicity. Experiments with p-aminobenzoic acid solutions indicate that the undissociated acid is preferentially sorbed at the membrane–solution interface. With respect to both aliphatic and aromatic amino acid ions, solute separation is in the order R^? > R^± > R⁺ for the cellulose acetate membrane material studied.     

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.     

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.     

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.     

Appended Aromatic Moieties in Flexible Bis-3-chloropiperidines Confer Tropism against Pancreatic Cancer Cells

Nitrogen mustards (NMs) are an old but still largely diffused class of anticancer drugs. However, spreading mechanisms of resistance undermine their efficacy and therapeutic applicability. To expand their antitumour value, we developed bis-3-chloropiperidines (B-CePs), a new class of mustard-based alkylating agent, and we recently reported the striking selectivity for BxPC-3 pancreatic tumour cells of B-CePs bearing aromatic moieties embedded in the linker. In this study, we demonstrate that such tropism is shared by bis-3-chloropiperidines bearing appended aromatic groups in flexible linkers, whereas esters substituted by aliphatic groups or by efficient DNA-interacting groups are potent but nonselective cytotoxic agents. Besides, we describe how the critical balance between water stability and DNA reactivity can affect the properties of bis-3-chloropiperidines. Together, these findings support the exploitation of B-CePs as potential antitumour clinical candidates.     

The Dynamic Range of Acidity: Tracking Rules for the Unidirectional Penetration of Cellular Compartments

Labeled ammonium cations with pK_a∼7.4 accumulate in acidic organelles because they can be neutralized transiently to cross the membrane at cytosolic pH 7.2 but not at their internal pH<5.5. Retention in early endosomes with less acidic internal pH was achieved recently using weaker acids of up to pK_a 9.8. We report here that primary ammonium cations with higher pK_a 10.6, label early endosomes more efficiently. This maximized early endosome tracking coincides with increasing labeling of Golgi networks with similarly weak internal acidity. Guanidinium cations with pK_a 13.5 cannot cross the plasma membrane in monomeric form and label the plasma membrane with selectivity for vesicles embarking into endocytosis. Self-assembled into micelles, guanidinium cations enter cells like arginine-rich cell-penetrating peptides and, driven by their membrane potential, penetrate mitochondria unidirectionally despite their high inner pH. The resulting tracking rules with an approximated dynamic range of pK_a change ∼3.5 are expected to be generally valid, thus enabling the design of chemistry tools for biology research in the broadest sense. From a practical point of view, most relevant are two complementary fluorescent flipper probes that can be used to image the mechanics at the very beginning of endocytosis.     

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.     

Synthesis and characterization of sulfonated polyimides containing aliphatic linkages in the main chain

A series of six‐membered sulfonated polyimides with aliphatic linkages (SPIAs) was successfully synthesized using 1,4,5,8‐naphthalenetetracarboxylic dianhydride (NTDA), 4,4′‐diaminobiphenyl 2,2′‐disulfonic acid (BDSA) as the sulfonated diamine, and aliphatic diamines H₂N(CH₂)_nNH₂ where n = 6, 8, 10, 12. These SPIAs were evaluated for thermal stability, ion exchange capacity (IEC), water uptake, proton conductivity, and hydrolytic stability. Proton conductivity and hydrolytic stability of the SPIAs were compared with the fully aromatic polyimide (MDA‐SPI) prepared from 4,4′‐methylenedianiline (MDA), BDSA, and NTDA. All the SPIAs exhibited high thermal stability. As the chain length of the aliphatic diamine decreased, the IEC and water uptake of the SPIAs increased. The SPIAs showed higher proton conductivity than commercially available membranes such as Nafion 117 at high temperatures and higher proton conductivity than MDA‐SPI at all temperatures. All SPIAs exhibited a hydrolytic stability more than twice as high as that of MDA‐SPI. Copyright © 2006 Society of Chemical Industry     

Examinations of Dimethyl Sulfoxide-Phase Equilibrium Acidities of Selected Polycyclic Aromatic Compounds

Comparisons of dimethyl sulibxide (DMSO)-phase pK _a's (in parentheses) for 10-cyano-9-methoxymethy-lanthracene (17.35), 10-cyano-9-phenoxymethylanthracene (14.9), 9-cyanomethoxymethylanthracene (20.4), and (α-methoxy) phenylacetonitrile (23.0) with literature DMSO-phase pK _a's for 9-methylanth-racene, 9-methoxymethylanthracene, 9-phenoxymethylanthracene, 9-cyanomethylanthracene, 10-cyano-9-methylanthracene, and phenylacetonitrile (31.1, 30.6, 30.2, 19.7, 20.0, and 21.9, respectively), indicate that: (a) α-methoxy and α-phenoxy substituents acidify 10-cyano-9-methylanthracene; (b) an α-methoxy moiety deacidifies 9-cyanomethylanthracene and phenylacetonitrile; and (c) 10-cyano substituents acidify 9-methylanthracene, 9-methoxymethylanthracene, and 9-phenoxymethylanthracene by 11.1, 13.25, and 15.3 pK _a units, respectively. These data are indicative of steric inhibition of resonance, resonance saturation, and effects due to cross conjugation, in carbanions derived from variously substituted 9-methylanthracenes. DMSO-phase pK _a for phenalene (18.2) and benzanthrene (20.2) have also been determined. In DMSO, phenalene is therefore 4.4 pK units more acidic than its isomeric analogue fluorene (22.6), while benzanthrene is 0.5 pK units less acidic than its isomeric analogue 1,2-benzofluorene (19.7). Specific solvation of the allylic-like phenalenide anion is a likely reason for its enhanced acidity, relative to benzanthrene.