ABSTRACT
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.
Subject(s)
Acids/chemistry , Epinephrine/chemistry , Norepinephrine/chemistry , Acid-Base Equilibrium , Catecholamines/chemistry , Dopamine/chemistry , Hydrogen-Ion Concentration , Magnetic Resonance Spectroscopy/methods , Protons , Species SpecificityABSTRACT
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.
ABSTRACT
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.
Subject(s)
Cell Membrane Permeability , Dopamine/pharmacokinetics , Models, Chemical , Protons , Cell Membrane/metabolism , Dopamine/chemistry , Hydrogen-Ion Concentration , Magnetic Resonance SpectroscopyABSTRACT
In the present study, several Cu chelators [2,2'-biquinoline, 8-hydroxiquinoline (oxine), ammonium pyrrolidinedithiocarbamate (APDTC), Dp44mT, dithizone, neocuproine] were used to study Cu uptake, depletion and localization in different cancer cell lines. To better understand the concentration dependent fluctuations in the Cu intracellular metal content and Cu-dependent in vitro antiproliferative data, the conditional stability constants of the Cu complex species of the investigated ligands were calculated. Each investigated chelator increased the intracellular Cu content on HT-29 cells causing Cu accumulation depending on the amount of the free Cu(II). Copper accumulation was 159 times higher for Dp44mT compared to the control. Investigating a number of other transition metals, intracellular accumulation of Cd was observed only for two chelators. Intracellular Zn content slightly decreased (cca. 10%) for MCF-7 cells, while a dramatic decrease was observed on MDA-MB-231 ones (cca. 50%). A similar decrease was observed for HCT-116, while Zn depletion for HT-29 corresponded to cca. 20%. The IC50 values were registered for the investigated four cell lines at increasing external Cu(II) concentration, namely, MDA-MB-231 cells had the lowest IC50 values for Dp44mT ranging between 7 and 35â¯nM. Thus, Zn depletion could be associated with lower IC50 values. Copper depletion was observed for all ligands being less pronounced for Dp44mT and neocuproine. Copper localization and its colocalization with Zn were determined by µ-XRF imaging. Loose correlation (0.57) was observed for the MCF-7 cells independently of the applied chelator. Similarly, a weak correlation (0.47) was observed for HT-29 cells treated with Cu(II) and oxine. Colocalization of Cu and Zn in the nucleus of HT-29 cells was observed for Dp44mT (correlation coefficient of 0.85).
Subject(s)
Chelating Agents/pharmacology , Copper/metabolism , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Copper/pharmacokinetics , Copper/toxicity , Dithizone/pharmacology , Humans , Metals/pharmacokinetics , Oxyquinoline/pharmacology , Phenanthrolines/pharmacology , Thiosemicarbazones/pharmacology , Zinc/metabolism , Zinc/pharmacokineticsABSTRACT
Biorelevant, isomer-specific physicochemical parameters of resveratrol, a multifunctional component in red wines, with cardioprotective, anti-Alzheimer and several other pharmacologic activities were determined. The parameters include site-specific basicities, lipophilicities, solubilities and diffusion constants for the two geometric isomers. The protonation equilibria of (E)- and (Z)-resveratrol were monitored by 1H NMR-pH titrations. Five closely related auxiliary compounds ((E)-pinostilbene, (Z)-pinostilbene, (E)-pterostilbene, (Z)-pterostilbene and resorcinol) were also studied. Combining the datasets, the group-specific protonation constants of resveratrol isomers were determined. The results show that (Z)-resveratrol is more basic at every protonation site than the (E)-isomer. Lipophilicities are quantified in terms of logP values and were determined by octanol/water partition experiments and quantitative NMR spectroscopy: (E)-resveratrol was found to be more lipophilic. Since the molecular geometries of the isomers differ, diffusion ordered NMR spectroscopy (DOSY) experiments were also carried out to quantify the diffusion capabilities of the isomers: (Z)-resveratrol of bent shape has a slightly higher diffusion coefficient than its extended (E) counterpart. A striking 10-fold difference of water solubility was found in favor of the (Z) isomer, due obviously to the reduced water-repellent character in the more compact molecule. This is so far the greatest recorded solubility difference between geometric isomers of any compounds.
Subject(s)
Stilbenes/chemistry , Hydrogen-Ion Concentration , Isomerism , Magnetic Resonance Spectroscopy/methods , Octanols/chemistry , Resveratrol , Solubility , Water/chemistryABSTRACT
The hydroxide-catalyzed non-enzymatic, simultaneous and consecutive hydrolyses of diacetylmorphine (DAM, heroin) are quantified in terms of 10 site- and species-specific rate constants in connection with also 10 site- and species-specific acid-base equilibrium constants, comprising all the 12 coexisting species in solution. This characterization involves the major and minor decomposition pathways via 6-acetylmorphine and 3-acetylmorphine, respectively, and morphine, the final product. Hydrolysis has been found to be 18-120 times faster at site 3 than at site 6, depending on the status of the amino group and the rest of the molecule. Nitrogen protonation accelerates the hydrolysis 5-6 times at site 3 and slightly less at site 6. Hydrolysis rate constants are interpreted in terms of intramolecular inductive effects and the concomitant local electron densities. Hydrolysis fraction, a new physico-chemical parameter is introduced and determined to quantify the contribution of the individual microspecies to the overall hydrolysis. Hydrolysis fractions are depicted as a function of pH.
Subject(s)
Heroin/chemistry , Catalysis , Hydrogen-Ion Concentration , Hydrolysis , Hydroxides/chemistry , Morphine/chemistry , Morphine Derivatives/chemistry , Nitrogen/chemistry , Solutions/chemistryABSTRACT
The site-specific acid-base properties of ovothiol A disulfide (OvSSOv), the smallest hexabasic multifunctional biomolecule with complex interdependent moieties, were studied with (1)H NMR-pH and potentiometric titrations. The unprecedented complexity of the protonation microequilibria could be overcome by taking into account the mirror-image molecular symmetry, synthesizing and studying auxiliary model compounds and developing a custom-tailored evaluation method. The amino, imidazole, and carboxylate moieties are quantified in terms of 192 microscopic protonation constants and 64 microspecies, 96 and 36 of which are chemically different ones, respectively. Nine pairwise interactivity parameters also characterize the OvSSOv-proton system at the level of molecular subunits. These data allow understanding and influencing the co-dependent acid-base and redox properties of the highly complex OvSH-OvSSOv and related thiol-disulfide systems, which provide protection against oxidative stress. This work is the first complete microspeciation of a hexabasic molecule.
Subject(s)
Disulfides/chemistry , Methylhistidines/chemistry , Acids/chemistry , Alkalies/chemistry , Hydrogen-Ion Concentration , Magnetic Resonance Spectroscopy/methods , ProtonsABSTRACT
Resveratrol is a polyphenol that can be found in various plants, including grapes. Wines therefore also contain this compound. The famous phenomenon, named "French paradox" is considered to be an effect of resveratrol: the regular, modest consumption of red wine causes low incidence of cardiovascular diseases. Resveratrol is also reported to have anti-carcinogenic, anti-inflammatory, neuroprotective, and several further beneficial effects. As yet, resveratrol is not a registered drug in Hungary; nevertheless it is a common food supplement. Paradoxically, this is the very danger of its use: it can have harmful side-effects with several drugs. The bioavailability of resveratrol is, however, not favorable, due to its poor water-solubility and extensive metabolism. Our current knowledge is still far from being sufficient to understand its mode of action at a molecular level. Only a few of the most important physicochemical properties has been determined so far. The two major directions of pharmaceutical research of resveratrol are the elaboration of formulation systems that can improve the water solubility; the other is the development of analogous agents of enhanced effects.
Subject(s)
Stilbenes/pharmacology , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Anticarcinogenic Agents/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Antioxidants/pharmacology , Blood Glucose/drug effects , Cardiovascular System/drug effects , Chemistry, Pharmaceutical , Dietary Supplements , Humans , Hungary , Obesity/drug therapy , Obesity/prevention & control , Resveratrol , Solubility , Stilbenes/chemistry , Stilbenes/pharmacokineticsABSTRACT
Ovothiol A, a small biomolecule with highly potent antioxidant capacity, and three newly synthesized derivatives were studied by (1)H NMR, (15)N NMR, UV-pH titrations, and a customized evaluation method. The omni-interactive imidazole, amino, carboxylate, and thiolate moieties of ovothiol A are quantified in terms of 32 microscopic protonation constants, the relative concentrations of 16 microspecies, 6 pairwise interactivity parameters, and 8 protonation shifts. The highest and lowest imidazole basicities differ by a record-breaking five orders of magnitude, and the predominant thiolate protonation constant is by far the smallest known thiolate logK value. The latter provides an indication as to why ovothiol A occurs naturally under deep-water circumstances only. Since thiolate basicities are in correlation with thiol-disulfide redox potentials, the eight different, fine-tunable thiolate basicities offer versatile and highly specific antioxidant capacities within one single molecular skeleton. This work is the first complete microspeciation of a tetrabasic, nonsymmetrical natural compound.
Subject(s)
Antioxidants/chemistry , Methylhistidines/chemistry , Hydrogen-Ion Concentration , Molecular StructureABSTRACT
Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) is a potential candidate in chelation therapy as an iron chelator. This study showed that a combined treatment with 2µM easily available Fe(II), Cu(II) and Zn(II) each and 5µM Dp44mT on eight different cancer cell lines resulted in a 10-40-fold increase in the intracellular Cu content compared to control samples. The uptake of Cu and Cu-dependent cytotoxicity strictly depend on the Cu concentration of the culture medium. Even as low concentration of Dp44mT as 0.1µM can transport high amounts of copper inside the cells. The Cu accumulation and toxicity through Dp44mT can hardly be influenced by Fe. Copper uptake and toxicity triggered by 2µM extracellular Cu(II) and 5µM Dp44mT could not be influenced by Fe(II) extracellular concentrations even 50-times higher than that of Cu(II). A 50-times higher Co(II) extracellular concentration hindered the Cu(II) uptake almost completely and a 10-times higher Co(II) concentration already decreased the Dp44mT-mediated Cu toxicity. Conditional complex stability constant determinations for Dp44mT with Cu(II), Co(II), Fe(II), Ni(II) and Zn(II) revealed that the metal-to-ligand ratio is 1:1 in [Cu(II)Dp44mT] complex, while for Co(II), Fe(II) and Ni(II) is 1:2. The highest stability constant was obtained for Cu(II) (lg ß=7.08±0.05) and Co(II) (lg ß2=12.47±0.07). According to our results, Dp44mT in combination with Cu is highly toxic in vitro. Therefore, the use of Dp44mT as an iron chelator is limited if biologically available Cu is also present even at low concentrations.
Subject(s)
Chelating Agents/chemistry , Chelating Agents/toxicity , Thiosemicarbazones/chemistry , Thiosemicarbazones/toxicity , Cell Line, Tumor , Cobalt/chemistry , Cobalt/metabolism , Copper/metabolism , Copper/pharmacokinetics , Dose-Response Relationship, Drug , HT29 Cells/drug effects , HT29 Cells/metabolism , Humans , Iron/metabolism , Iron Chelating Agents/chemistry , Iron Chelating Agents/toxicity , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Protons , Thiosemicarbazones/metabolism , Thiosemicarbazones/pharmacokinetics , Zinc/chemistry , Zinc/metabolismABSTRACT
Streptomycin, the classical aminoglycoside antibiotic, generally considered the most basic drug compound was characterized in terms of protonation macro- and microconstants. ¹H NMR-pH and ¹H-¹³C HSQC-pH titrations were carried out on streptomycin and streptidine, a symmetrical constituent compound of reduced complexity to monitor the proton-binding processes of the basic sites. Accurate, undistorted, electrodeless pH measurement was ensured by a new set of in tube indicators. The microscopic protonation constants of the two guanidino groups of streptomycin were calculated by evaluating the various NMR-pH data and transferring the pair-interactivity parameter from streptidine to streptomycin. Inherent guanidino basicities fall in the range of 13.03-13.39 log k units, which drop to 12.48-12.85 upon protonation of the other site. pH-dependent distribution of the major microspecies and charge-related biological consequences are provided.
Subject(s)
Anti-Bacterial Agents/analysis , Guanidines/analysis , Hexosamines/analysis , Models, Chemical , Protons , Streptomycin/analysis , Anti-Bacterial Agents/chemistry , Guanidines/chemistry , Hexosamines/chemistry , Hydrogen-Ion Concentration , Magnetic Resonance Spectroscopy , Molecular Structure , Streptomycin/chemistryABSTRACT
(1)H NMR-pH titrations of arginine, the most basic natural amino acid and citrulline, its neutral counterpart were carried out. Two other closely related auxiliary compounds were also studied. The 8 macroscopic protonation constants were determined. Combining the four datasets in a deductive method, all the 12 microconstants of arginine, the 4 microconstants of citrulline and arginine amide were calculated. An error-propagation analysis and the pH-dependent distribution of the 8 arginine microspecies are provided.
Subject(s)
Arginine/analysis , Citrulline/analysis , Arginine/chemistry , Chemistry, Pharmaceutical , Citrulline/chemistry , Hydrogen-Ion Concentration , Isoelectric Point , Magnetic Resonance Spectroscopy , Models, Chemical , Molecular Structure , Potentiometry , ProtonsABSTRACT
A set of indicator molecules was selected and applied to elaborate an NMR-based pH determination method, free of glass electrode errors in highly basic media. Accurate measurement of pH values and protonation constants was achieved by a successive build-up of overlapping, increasingly high pH solutions, using a collection of 8 compounds of appropriately incremented basicities. In order to verify the method, acid-base properties were quantified for two compounds with very high basicities in conflicting reports: two pharmaceutically important biguanidine drugs, metformin and phenformin.
Subject(s)
Magnetic Resonance Spectroscopy , Pharmaceutical Preparations/analysis , Technology, Pharmaceutical/methods , Hydrogen-Ion Concentration , Metformin/analysis , Metformin/chemistry , Molecular Structure , Osmolar Concentration , Pharmaceutical Preparations/chemistry , Phenformin/analysis , Phenformin/chemistry , Potentiometry , Protons , Reproducibility of Results , SolutionsABSTRACT
The rate constants of spontaneous and hydroxide-catalyzed decomposition and the tautomer-specific protonation constants of tolperisone, a classical muscle relaxant were determined. A solution NMR method without any separation techniques was elaborated to quantitate the progress of decomposition. All the rate and equilibrium constants were determined at four different temperatures and the activation parameters were calculated. The molecular mechanism of decomposition is proposed.