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.

Close correlation between thiolate basicity and certain NMR parameters in cysteine and cystine microspecies.

The imbalance between prooxidants and antioxidants in biological systems, known as oxidative stress, can lead to a disruption of redox signaling by the reactive oxygen/nitrogen species and is related to severe diseases. The most vulnerable moiety targeted by oxidant species in the redox signaling pathways is the thiol (SH) group in the cysteine residues, especially in its deprotonated (S-) form. Cysteine, along with its oxidized, disulfide-containing form, cystine, constitute one of the most abundant low molecular weight biological redox couples, providing a significant contribution to the redox homeostasis in living systems. In this work, NMR spectra from cysteine, cystine, and cysteine-containing small peptides were thoroughly studied at the submolecular level, and through the chemical shift data set of their certain atoms it is possible to estimate either thiolate basicity or the also related standard redox potential. Regression analysis demonstrated a strong linear relationship for chemical shift vs thiolate logK of the cysteine microspecies data. The αCH 13C chemical shift is the most promising estimator of the acid-base and redox character.

ß-cyclodextrin complex formation and protonation equilibria of morphine and other opioid compounds of therapeutic interest.

The inclusion complex formation of morphine and its 18 opioid derivatives with ß-cyclodextrin has been studied using nuclear magnetic resonance spectroscopy. Initially, the protonation equilibria and the acid-base properties of dibasic opioid compounds have been fully characterized. Apparent protonation constants and the relative concentration of the microspecies in cyclodextrin excess were also determined. The 1:1 complex stoichiometry was confirmed by the continuous variation method of Job using UV-VIS spectroscopy. The stability constants of the different protonation forms were determined by 1H NMR titrations. The highest stability was observed in highly alkaline solutions where the amino group is in its unprotonated, neutral state. The structures of the complexes were investigated by two-dimensional ROESY experiments. Based on the stability constants and ROESY experiments, morphine derivatives with longer side chain on the nitrogen atom such as nalbuphine and naltrexone show stronger complexation. The protonation state of the phenolate group, positioned outside the CD cavity, has only a slight influence on the complex stability.

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.

Tissue-Specific Accumulation and Isomerization of Valuable Phenylethanoid Glycosides from Plantago and Forsythia Plants.

A comparative phytochemical study on the phenylethanoid glycoside (PhEG) composition of the underground organs of three Plantago species (P. lanceolata, P. major, and P. media) and that of the fruit wall and seed parts of Forsythia suspensa and F. europaea fruits was performed. The leaves of these Forsythia species and six cultivars of the hybrid F. × intermedia were also analyzed, demonstrating the tissue-specific accumulation and decomposition of PhEGs. Our analyses confirmed the significance of selected tissues as new and abundant sources of these valuable natural compounds. The optimized heat treatment of tissues containing high amounts of the PhEG plantamajoside (PM) or forsythoside A (FA), which was performed in distilled water, resulted in their characteristic isomerizations. In addition to PM and FA, high amounts of the isomerization products could also be isolated after heat treatment. The isomerization mechanisms were elucidated by molecular modeling, and the structures of PhEGs were identified by nuclear magnetic resonance spectroscopy (NMR) and high-resolution mass spectrometry (HR-MS) techniques, also confirming the possibility of discriminating regioisomeric PhEGs by tandem MS. The PhEGs showed no cytostatic activity in non-human primate Vero E6 cells, supporting their safe use as natural medicines and allowing their antiviral potency to be tested.

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.

Physicochemical Properties of Zwitterionic Drugs in Therapy.

In solution, amphoteric compounds exist in anionic, uncharged, zwitterionic and cationic forms. The importance of zwitterionic drugs is currently under-represented in the literature. Herein, the acid-base parameters, lipophilicity and solubility of such compounds are discussed to deepen the molecular-level understanding of their pharmacokinetic and pharmacodynamic behaviour. Our recent studies show there are many drug molecules, including thyroid hormones and 5-hydroxytryptophan, the precursor of the neurotransmitter serotonin, for which the contribution of the zwitterionic microspecies to the overall lipophilicity exceeds that of the uncharged one, which is of higher individual lipophilicity, but occurs in much lower concentration. The second part of the minireview highlights the most important zwitterionic compounds in therapy, grouped into therapeutic classes. The importance of the charge of the molecules is emphasized in their binding to the target molecules.

Reversed-phase HPLC enantioseparation of pantoprazole using a teicoplanin aglycone stationary phase-Determination of the enantiomer elution order using HPLC-CD analyses.

A direct HPLC method was developed for the enantioseparation of pantoprazole using macrocyclic glycopeptide-based chiral stationary phases, along with various methods to determine the elution order without isolation of the individual enantiomers. In the preliminary screening, four macrocyclic glycopeptide-based chiral stationary phases containing vancomycin (Chirobiotic V), ristocetin A (Chirobiotic R), teicoplanin (Chirobiotic T), and teicoplanin-aglycone (Chirobiotic TAG) were screened in polar organic and reversed-phase mode. Best results were achieved by using Chirobiotic TAG column and a methanol-water mixture as mobile phase. Further method optimization was performed using a face-centered central composite design to achieve the highest chiral resolution. Optimized parameters, offering baseline separation (resolution = 1.91 ± 0.03) were as follows: Chirobiotic TAG stationary phase, thermostated at 10°C, mobile phase consisting of methanol/20mM ammonium acetate 60:40 v/v, and 0.6 mL/min flow rate. Enantiomer elution order was determined using HPLC hyphenated with circular dichroism (CD) spectroscopy detection. The online CD signals of the separated pantoprazole enantiomers at selected wavelengths were compared with the structurally analogous esomeprazole enantiomer. For further verification, the inline rapid, multiscan CD signals were compared with the quantum chemically calculated CD spectra. Furthermore, docking calculations were used to investigate the enantiorecognition at molecular level. The molecular docking shows that the R-enantiomer binds stronger to the chiral selector than its antipode, which is in accordance with the determined elution order on the column-S- followed by the R-isomer. Thus, combined methods, HPLC-CD and theoretical calculations, are highly efficient in predicting the elution order of enantiomers.

Physicochemical Profiling of Baicalin Along with the Development and Characterization of Cyclodextrin Inclusion Complexes.

Baicalin is a flavone glycoside extracted from Scutellaria baicalensis, a traditional Chinese herbal medicine. Numerous pharmacological effects of baicalin were reported (e.g. antioxidant, anxiolytic); nevertheless, the most important physicochemical properties influencing the pharmacokinetic behaviour and the concomitant oral bioavailability have not yet been described in a comprehensive study. The aim of this project was to characterize the acid-base, lipophilicity, biorelevant solubility and permeability properties of the drug substance and providing scientific data to support the dosage form design. Another important objective was the comparative evaluation of six various baicalin-cyclodextrin (CD) inclusion complexes along with the creation of a suitable Drug Delivery System (DDS) for this BCS IV drug. Biorelevant profiling was carried out by NMR-pH titrations, saturation shake-flask and distribution coefficients (logP) measurements, while CD inclusion studies were fulfilled by experimental methods (phase solubility, 1H/13C NMR, 2D ROESY) and computational approaches. Due to low aqueous solubility (67.03 ± 1.60 µg/ml) and low permeability (Papp = 0.037 × 10-6 cm/s), baicalin is classified as BCS IV. The γ-CD complexation significantly increased the solubility of baicalin (~ 5 times). The most promoted chemical shift change occurred in baicalin-γ-CD complex. Computational studies showed disparate binding pattern for baicalin in case of ß- and γ-CD; furthermore, the calculated complexation energy was - 162.4 kJ mol-1 for ß-CD, while it was significantly stronger for γ-CD (- 181.5 kJ mol-1). The physicochemical and structural information of baicalin and its CD complexes introduced herein can create molecular basis for a promising DDS with enhanced bioavailability containing a bioactive phytopharmacon.

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.

Advances in the Physicochemical Profiling of Opioid Compounds of Therapeutic Interest.

This review focuses on recent developments in the physicochemical profiling of morphine and other opioids. The acid-base properties and lipophilicity of these compounds is discussed at the microscopic, species-specific level. Examples are provided where this type of information can reveal the mechanism of pharmacokinetic processes at the submolecular level. The role of lipophilicity in quantitative structure-activity relationship (QSAR) studies of opioids is reviewed. The physicochemical properties and pharmacology of the main metabolites of morphine are also discussed. Recent studies indicate that the active metabolite morphine-6-glucuronide (M6G) can contribute to the analgesic activity of systemically administered morphine. The unexpectedly high lipophilicity of M6G partly accounts for its analgesic activity. When administered parenterally, another suspected minor metabolite, morphine-6-sulfate (M6S) has superior antinociceptive effects to those of morphine. However, because sulfate esters of morphine derivatives cannot cross the blood-brain barrier these esters may be good candidates to develop peripheral analgesic drugs.

Chiral separation of lansoprazole and rabeprazole by capillary electrophoresis using dual cyclodextrin systems.

Novel capillary electrophoresis methods using CDs as chiral selectors were developed and validated for the chiral separation of lansoprazole and rabeprazole, two proton pump inhibitors. Fourteen different neutral and anionic CDs were screened at pH 4 and 7 in the preliminary analysis. Sulfobutyl-ether-ß-CD with a degree of substitution of 6.5 and 10 at neutral pH proved to be the most suitable chiral selector for both compounds. Various dual CD systems were also compared, and the possible mechanisms of enantiomer separation were investigated. A dual selector system containing sulfobutyl-ether-ß-CD degree of substitution 6.5 and native γ-CD proved to be the most adequate system for the separations. Method optimization was carried out using an experimental design approach, performing an initial fractional factorial screening design, followed by a central composite design to establish the optimal analytical conditions. The optimized methods (25 mM phosphate buffer, pH 7, 10 mM sulfobutyl-ether-ß-CD/20 mM γ-CD, +20 kV voltage; 17°C temperature; 50 mbar/3 s injection, detection at 210 nm for lansoprazole; 25 mM phosphate buffer, pH 7, 15 mM sulfobutyl-ether-ß-CD/30 mM γ-CD, +20 kV voltage; 18°C temperature; 50 mbar/3 s injection, detection at 210 nm for rabeprazole) provided baseline separation for lansoprazole (Rs = 2.91) and rabeprazole (Rs = 2.53) enantiomers with favorable migration order (in both cases the S-enantiomers migrates first). The optimized methods were validated according to current guidelines and proved to be reliable, linear, precise, and accurate for the determination of 0.15% distomer as chiral impurity in dexlansoprazole and dexrabeprazole samples.

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.

Chiral separation of rasagiline using sulfobutylether-ß-cyclodextrin: capillary electrophoresis, NMR and molecular modeling study.

Pressure-assisted stereospecific capillary electrophoresis method was developed for the determination of enantiomeric purity of the antiparkinsonian agent (R)-rasagiline. The optimized method, 50 mM glycine-HCl buffer pH 2, supplied with 30 mM sulfobutylether-ß-cyclodextrin, at 35°C, applying 12 kV in reversed polarity, and -8 mbar pressure (vacuum), short-end injection with -25 mbar × 2 s, was successful for baseline separation of rasagiline enantiomers (Rs = 3.5 ± 0.1) in a short analysis time. The method was validated according to current guidelines and proved to be reliable, linear, precise and accurate for determination of 0.15% S-enantiomer as chiral impurity in R-rasagiline sample, as well as quantification of the eutomer. Method application was tested on a commercial tablet formulation. Determination of spatial structure of diastereomeric associates was based on 1 H and 2D ROESY NMR, indicating that the aromatic moiety of the molecule can enter the cyclodextrin cavity. NMR titration and molecular modeling revealed that S-rasagiline formed a more stable inclusion complex with sulfobutylether-ß-cyclodextrin, than its antipode, which is in agreement with electrophoretic results.

Phenolic composition, antioxidant and antinociceptive activities of Syringa vulgaris L. bark and leaf extracts.

Metabolite profile, antioxidant and antinociceptive activities of Syringa vulgaris bark and leaf methanolic extracts were investigated. By means of HPLC-DAD-ESI-TOF and HPLC-DAD-ESI-MS/MS, a total of 33 phenolics were identified, including 15 secoiridoids, 6 phenylpropanoids, 3 flavonoids, 3 lignans and 6 low molecular weight phenols. Validated quantitative analysis show that syringin (2.52%) and rutin (1.13%) are the main phenolic compounds in bark and leaf, respectively. Notable radical scavenging and antinociceptive activities of the bark and leaf extracts were confirmed by in vitro DPPH● and ABTS●+ assays and by in vivo hot-plate method in mice, respectively. Our results could lay the scientific basic of future clinical perspectives of lilac bark and leaf.

NMR-Based Determination of pH, Free of Electrodes and Reference Compounds.

A novel method was elaborated, and a set of 1H NMR-pH indicator molecules was selected to develop an NMR-based pH determination method, which does not need any glass or other electrodes or any separate reference compound, being thus devoid of the usual acid and base errors of the glass and combined electrodes. The method utilizes organic compounds of accurately known basicity and two or more carbon-bound protons, whose chemical shifts respond differently to pH changes. Accurate determination of protonation constants in pH extrema was achieved by the method of relative basicities using compounds of sequentially incremented protonation constants. The entire pH scale can be covered by six indicator molecules. The method was developed for two ionic strengths, 1.00 and 0.15 M.

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.

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.