Selenate-An internal chemical shift standard for aqueous 77 Se NMR spectroscopy.

The 77 Se NMR spectra of selenate were studied under various circumstances, such as concentration, pH, temperature, ionic strength, and D2 O:H2 O ratio, in order to examine its potential as a water-soluble internal chemical shift standard. The performance of selenate as a chemical shift reference and that of other attempted ones from the literature (dimethyl selenide, tetramethylsilane/TMS, and 3-(trimethylsilyl)propane-1-sulfonate/DSS) was also explored. The uncertainty in the resulting chemical shift relative to the effective spectral width is comparable to that of DSS. Compared to the currently prevalent water-soluble external chemical shift reference, selenic acid solution, the properties of internal selenate are much more favorable in terms of ease of use. We have also demonstrated that selenate can be used in reducing media, which is inevitable for the analysis of selenol compounds. Thus, it can be stated that sodium selenate is a robust internal chemical shift reference in aqueous media for 77 Se NMR measurements; the chemical shift of this reference in a solution containing 5 V/V% D2 O at 25°C and 0.15 mol·dm-3 ionic strength is 1048.65 ppm relative to 60 V/V% dimethyl selenide in CDCl3 and 1046.40 ppm relative to the 1 H signal of 0.03 V/V% TMS in CDCl3 . In summary, a water-soluble, selenium-containing internal chemical shift reference compound was introduced for 77 Se NMR measurements for the first time in the literature, and with the aforementioned results all previous 77 Se measurements can be converted to a unified scale defined by the International Union of Pure and Applied Chemistry.

Solution Structure and Acid-Base Properties of Reduced α-Conotoxin MI.

The reduced derivative of α-conotoxin MI, a 14 amino acid peptide is characterized by NMR-pH titrations and molecular dynamics simulations to determine the protonation constants of the nine basic moieties, including four cysteine thiolates, and the charge-dependent structural properties. The peptide conformation at various protonation states was determined. The results show that the disulfide motifs in the native globular α-conotoxin MI occur between those cysteine moieties that exhibit the most similar thiolate basicities. Since the basicity of thiolates correlates to its redox potential, this phenomenon can be explained by the higher reactivity of the two thiolates with higher basicities. The folding of the oxidized peptide is further facilitated by the loop-like structure of the reduced form, which brings the thiolate groups into sufficient proximity. The 9 group-specific protonation constants and the related, charge-dependent, species-specific peptide structures are presented.

Characterization of the Site-Specific Acid-Base Equilibria of 3-Nitrotyrosine.

The complete macro- and microequilibrium analyses of 3-nitrotyrosine, a biomarker of oxidative stress damage, are presented for the first time. The protonation macroconstants were determined by 1 H-NMR-pH titration, while microconstants were elucidated by a combination of deductive and NMR methods, in which properties of the methyl ester derivative as an auxiliary compound were also studied. Combination of the NMR-pH characterization of the title and auxiliary compounds and the pair-interactivity parameters of 3-iodotyrosine provided the sufficient system to evaluate all the microconstants. NMR-pH profiles, macroscopic and microscopic protonation schemes, and species-specific distribution diagrams are included. The phenolate basicity of 3-nitrotyrosine is 500 times below that of tyrosine, and it is even lower than that of 3-iodotyrosine. This phenomenon can be explained by the stronger electron withdrawing and the negative mesomeric effect of the nitro group. Based on our results, 89 % of the phenolic OH groups are deprotonated in 3-NT molecules at the pH of the blood plasma.

Prediction of Antioxidant Capacity of Thiolate-Disulfide Systems Using Species-Specific Basicity Values.

The principal reactions that maintain redox homeostasis in living systems are the deprotonation of thiols, followed by the oxidative conversion of the produced thiolates into disulfides, which thus reduce the harmful oxidizing agents. The various biological thiols have different molecule-specific propensities to carry on the co-dependent deprotonation and redox processes. This study utilizes the known correlation between thiolate basicities and oxidizabilities, to quantify antioxidant or reducing capacities and pH-dependences of thiol-disulfide antioxidant systems, as a tool to find adequate molecules against oxidative stress.

Estimating the Bias of Model Compounds for the Determination of Species-Specific Protonation Constants.

The previously unknown extent of the goodness of using model compounds for the microspeciation of polyprotic systems was studied. Mirror-symmetric dibasic compounds and their monosubstituted derivatives were investigated to quantify how the derivatives are appropriate models of the minor microspecies to be mimicked in various microspeciation systems. The results were analyzed using statistical methods. It was found that the respective O-methyl and S-methyl derivatives of phenols and thiols as well as the methyl esters of carboxylic acids are sufficiently good derivatives for microspeciation. It was also found that the methyl esters are superior to the carboxylic amides for modeling the -COOH moiety.

Properties of Selenolate-Diselenide Redox Equilibria in View of Their Thiolate-Disulfide Counterparts.

Selenium, the multifaceted redox agent, is characterized in terms of oxidation states, with emphasis on selenol and diselenide in proteinogenic compounds. Selenocysteine, selenocystine, selenocysteamine, and selenocystamine are depicted in view of their co-dependent, interfering acid-base, and redox properties. The pH-dependent, apparent (conditional), and pH-independent, highly specific, microscopic forms of the redox equilibrium constants are described. Experimental techniques and evaluation methods for the determination of the equilibrium and redox parameters are discussed, with a focus on nuclear magnetic resonance spectroscopy, which is the prime technique to observe selenium properties in organic compounds. The correlation between redox, acid-base, and NMR parameters is shown in diagrams and tables. The fairly accessible NMR and acid-base parameters are discussed to assess the predictive power of these methods to estimate the site-specific redox properties of selenium-containing moieties in large molecules.

Understanding the pH Dependence of Supersaturation State-A Case Study of Telmisartan.

Creating supersaturating drug delivery systems to overcome the poor aqueous solubility of active ingredients became a frequent choice for formulation scientists. Supersaturation as a solution phenomenon is, however, still challenging to understand, and therefore many recent publications focus on this topic. This work aimed to investigate and better understand the pH dependence of supersaturation of telmisartan (TEL) at a molecular level and find a connection between the physicochemical properties of the active pharmaceutical ingredient (API) and the ability to form supersaturated solutions of the API. Therefore, the main focus of the work was the pH-dependent thermodynamic and kinetic solubility of the model API, TEL. Based on kinetic solubility results, TEL was observed to form a supersaturated solution only in the pH range 3-8. The experimental thermodynamic solubility-pH profile shows a slight deviation from the theoretical Henderson-Hasselbalch curve, which indicates the presence of zwitterionic aggregates in the solution. Based on pKa values and the refined solubility constants and distribution of macrospecies, the pH range where high supersaturation-capacity is observed is the same where the zwitterionic form of TEL is present. The existence of zwitterionic aggregation was confirmed experimentally in the pH range of 3 to 8 by mass spectrometry.

Inclusion complexation of the anticancer drug pomalidomide with cyclodextrins: fast dissolution and improved solubility.

Pomalidomide (POM), a potent anticancer thalidomide analogue was characterized in terms of cyclodextrin complexation to improve its aqueous solubility and maintain its anti-angiogenic activity. The most promising cyclodextrin derivatives were selected by phase-solubility studies. From the investigated nine cyclodextrins - differing in cavity size, nature of substituents, degree of substitution and charge - the highest solubility increase was observed with sulfobutylether-ß-cyclodextrin (SBE-ß-CD). The inclusion complexation between POM and SBE-ß-CD was further characterized with a wide variety of state-of-the-art analytical techniques, such as nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (IR), circular dichroism spectroscopy, fluorescence spectroscopy as well as X-ray powder diffraction method (XRD). Job plot titration by NMR and the AL-type phase-solubility diagram indicated 1:1 stoichiometry in a liquid state. Complementary analytical methods were employed for the determination of the stability constant of the complex; the advantages and disadvantages of the different approaches are also discussed. Inclusion complex formation was also assessed by molecular modelling study. Solid state complexation in a 1:1 M ratio was carried out by lyophilization and investigated by IR and XRD. The complex exhibited fast-dissolution with immediate release of POM, when compared to the pure drug at acidic and neutral pH. Kinetic analysis of POM release from lyophilized complex shows that Korsmeyer-Peppas and Weibull model described the best the dissolution kinetics. The cytotoxicity of the complex was tested against the LP-1 human myeloma cell line which revealed that supramolecular interactions did not significantly affect the anti-cancer activity of the drug. Overall, our results suggest that the inclusion complexation of POM with SBE-ß-CD could be a promising approach for developing more effective POM formulations with increased solubility.

Species-Specific, pH-Independent, Standard Redox Potential of Selenocysteine and Selenocysteamine.

Microscopic redox equilibrium constants and standard redox potential values were determined to quantify selenolate-diselenide equilibria of biological significance. The highly composite, codependent acid-base and redox equilibria of selenolates could so far be converted into pH-dependent, apparent parameters (equilibrium constants, redox potentials) only. In this work, the selenolate-diselenide redox equilibria of selenocysteamine and selenocysteine against dithiothreitol were analyzed by quantitative nuclear magnetic resonance (NMR) methods to characterize the interfering acid-base and redox equilibria. The directly obtained, pH-dependent, conditional redox equilibrium constants were then decomposed by our method into pH-independent, microscopic constants, which characterize the two-electron redox transitions of selenocysteamine and selenocysteine. The 12 different, species-specific parameter values show close correlation with the respective selenolate basicities, providing a tool to estimate otherwise inaccessible site-specific selenolate-diselenide redox potentials of related moieties in large peptides and proteins.

Characterization of the species-specific acid-base equilibria of adrenaline and noradrenaline.

Adrenaline, noradrenaline, the biogenic catecholamines of vital importance, and four closely related compounds were studied by 1H NMR-pH titrations, and the resulting acid-base properties are quantified in terms of three macroscopic and twelve microscopic protonation constants for both molecules. The species-specific basicities are interpreted by means of inductive and shielding effects by comparing the protonation constants of the catecholamines, including dopamine. The site-specific basicities determined this way could be key parameters for the interpretation of biochemical behavior.

Dopamine: Acid-base properties and membrane penetration capacity.

Dopamine and 4 related compounds were studied by 1H NMR-pH titrations and a case-tailored evaluation method. The resulting acid-base properties of dopamine are quantified in terms of 3 macroscopic and 12 microscopic protonation constants and the concomitant 3 interactivity parameters. The species- and site-specific basicities are interpreted by means of inductive and shielding effects through various intra- and intermolecular comparisons. The site-specific basicities determined this way are key parameters for the prediction of pharmacokinetic behavior and receptor-binding at the molecular level.

Population, basicity and partition of short-lived conformers. Characterization of baclofen and pregabalin, the biaxial, doubly rotating drug molecules.

Populations, protonation constants and octanol-water partition coefficients were determined and assigned specifically to fast interconverting individual conformers, exemplified in baclofen and pregabalin, the GABA-related drug molecules of biaxial, double rotations. Rotamer statuses along both axes in water and octanol were elucidated from 1H NMR vicinal coupling constants. Conformer abundances were obtained by the appropriate combination of the rotamer populations in the two adjacent moieties in the molecule. The bulky aromatic group in baclofen versus the aliphatic side chain of pregabalin explains why baclofen exists mainly in trans-trans conformeric form, throughout the pH range, unlike pregabalin that has no any highly dominant form. Characteristically enough, for pregabalin, the lipophilicity of the conformers is primarily influenced by the conformation state. Conformers in gauche state are of higher lipophilicity. The conformers of the two compounds were ranked by their membrane-influx and -outflow propensities.