Bio- és gyógyszermolekulák mikrospeciációja = Microspeciation of bio- and drug molecules

A kutatás csoportosított eredményei az alábbiak: 1) Új elvek és módszerek bio- és gyógyszermolekulák mikrospeciációjában 1)a) A mikroszkópikus lipofilitás meghatározása, különös tekintettel az ikerionos és töltésmentes protonáltsági izomerekre A hatóanyagok felszívódásában kulcsfontosságú mikroszkópikus lipofilitás jellemzéséhez levezettük a szükséges összefüggéseket és meghatároztuk konkrét értéküket több jelentős molekulára (morfin, tiroxin, nifluminsav, vinpocetin) 1)b) Torzításmentes, erősen lúgos oldatokban is használható, NMR alapú pH-meghatározási módszerkidolgozása és alkalmazása 2) Bio-, gyógyszer- és gyógyszerjelölt molekulák részecskéinek receptor-kötési vizsgálata in silico módszerekkel 3) Biomolekulák metabonomikai analízise 4) Biológiai alapmolekulák (szerotonin, tiroxin, arginin) mikrospeciációja 5) Néhány hatóanyag (cetirizin, famotidin, tolperizon, fluorokinolonok) mikrospeciációja 6) Bio- és gyógyszermolekulák ciklodextrin komplexeinek részecske-specifikus egyensúlyi és szerkezeti jellemzése 7) Új gyógyszerjelölt vegyületek szintézise és jellemzése 8) Összefoglaló közlemények megjelentetése a témakörben Ezen eredmények 31 közlemény és egy könyvrészlet formájában jelentek meg jelentős nemzetközi folyóiratokban ( J. Med. Chem, J. Proteom. Res, Anal. Bioanal. Chem, J. Pharm. Biomed. Anal., Eur, J. Pharm. Sci., Electrophoresis, Tetrahedron Letters, stb.) | Results of the funded research can be sorted as follows: 1) New principles and methods in the microspeciation of bio and drug molecules 1)a) Determination of microscopic lipophilicity, with special regard to the noncharged and zwitterionic protonation isomers. Physicochemical relationships of microscopic lipophilicity, the key parameter in pharmacokinetics have been deduced, and real microscopic lipophilicities have been determined for such important molecules as morphine, thyroxin, niflumic acid, vinpocetine. 1)b) Elaboration and application of an undistorted, NMR-based pH determination method that can be used in highly basic solutions too. 2) Receptor-binding studies on bio-, drug and drug candidate molecules, by in silico techniques. 3) Metabonomic analysis of biomolecules 4) Microspeciation of some fundamental biomolecules (serotonin, thyroxin, arginin) 5) Microspeciation of some therapeutic agents (cetirizin, famotidin, tolperizon, fluorokinolons9 6) Species-specific equilibrium and structural characterization of cyclodextrin complexation of bio- and drug molecules 7) Synthesis and characterization of new drug candidates 8) Publishing related review papers These results have been published as 31 papers and one book chapter in prestigious international journals, including J. Med. Chem., J. Proteome. Res, Anal. Bioanal. Chem, J. Pharm. Biomed. Anal., Eur, J. Pharm. Sci., Electrophoresis, Tetrahedron Letters

Biomolekulák mikrospeciációja = Microspeciation and Biomolecules

Biomolekulák mikrospeciációja Az élő világ sokféleségének egyik oka, hogy ugyanazon molekula többféle töltéssel és szerkezettel (azaz mikrorészecske, mikrospecies formájában) fordulhat elő és vehet részt az egyes kémiai és biokémiai folyamatokban, változatos reakciótermékeket eredményezve. E nanosecundum egyedi élettartamú, egymásba folytonosan átalakuló, ezért elválaszthatatlan, csak közvetett módszerekkel vizsgálható részecskék koncentrációjának és tulajdonságainak meghatározásában (mikrospeciációjában) - többek közt - megvalósítottuk: Új típusú, az irodalomban le nem írt mikroszkópikus fizikai-kémiai mennyiségek bevezetését (rotamer-specifikus megoszlási hányados, észter hidrolízis sebességi mikroállandó) Élettani vagy gyógyszertani jelentőségű molekulák jellemzését részecske-specifikus paraméterekkel (stressz elleni glutation, magzatvédő fólsav, tumorgátló metotrexát és imatinib, köhögéscsillapító folkodin, penicillin és kefalosporin antibiotikumok, gyulladásgátló tenoxikám, stb.) A mikro- és szubmikro speciáció körébe tartozó elvi kérdések tisztázását (mikroállandók és rotamer populációk NMR-alapú meghatározásának kritikai értékelése, az első irodalmi négycsoportos mikrospeciáció, a hisztamin biológiai kémiája). Eredményeink világszínvonalú nemzetközi folyóiratokban kaptak nyilvánosságot (J. Med. Chem., J. Phys. Chem. B, Anal. Bioanal. Chem., Eur. J. Pharm, Electrophoresis, stb.) | MICROSPECIATION OF BIOMOLECULES The chemical and biological diversity of the world stems partly from the fact that the molecules - especially the bio- and drug molecules - occur and react in various forms of protonation and conformation (i.e. microspecies), resulting in a cornucopia of products. Microspecies are of nanoseconds individual lifetime, which makes them inseparable, coexisting ones. In the research of their analytical determination and physicochemical characterization (i.e. microspeciation) our results include: Introduction and determination of previously undefined microscopic physicochemical quantities (rotamer-specific partition coefficient, microscopic rate constants of ester hydrolysis) Characterization of bio- and drug molecules in terms of species-specific parameters (anti-stress glutathione, fetus-protective folic acid, anti-cancer methotrexate and imatinib, anti inflammatory tenoxicame, penicilline and cephalosporine antibiotics, glutamate receptor ligand NMDA, etc.) Elucidation of some principal questions concerning microspeciation (critical reviews on NMR methods to evaluate microconstants and rotamer populations, microscopic characterization of a tetradentate ligand for the first time, the biological chemistry of histamine) Our results appeared in world-class journals of the field (J. Med. Chem., J. Phys. Chem. B, Anal. Bioanal. Chem., Eur. J. Pharm, Electrophoresis, etc.

NMR assignments and the acid-base characterization of the pomegranate ellagitannin punicalagin in the acidic pH-range.

In exploring the capability of nuclear magnetic resonance (NMR) spectroscopy for pomegranate juice analysis, the eight aromatic singlet resonances of alpha- and beta-punicalagin were clearly identified in the 1H NMR spectra of juice samples. The four downfield resonances were found to be sensitive to small pH changes around pH 3.50 where the NMR spectra of the juice samples were recorded. To understand this unusual behavior, the 1H and 13C resonance assignments of the punicalagin anomers were determined in aqueous solution and pH titrations with UV and 1H NMR detection carried out to characterize the acid-base properties of punicalagin over the pH range 2-8. Simultaneous fitting of all of the pH-sensitive 1H NMR signals produced similar but significantly different pK a values for the first two deprotonation equilibria of the gallagic acid moiety of the punicalagin alpha- (pK a1 = 4.57 +/- 0.02, pK a2 = 5.63 +/- 0.03) and beta- (pK a1 = 4.36 +/- 0.01, pK a2 = 5.47 +/- 0.02) anomers. Equivalent pK a values, (alpha : 6.64 +/- 0.01, beta : 6.63+/- 0.01) were measured for the third deprotonation step involving the ellagic acid group, in good agreement with a prior literature report. The punicalagin anomer equilibrium readjusts in parallel with the proton dissociation steps as the pH is raised such that beta-punicalagin becomes the most abundant anomer at neutral pH. The unusual upfield shifts observed for the glucose H3 and H5 resonances with increasing pH along with the shift in the alpha/beta anomer equilibrium are likely the consequence of a conformational rearrangement

The interaction of enoxaparin and fondaparinux with calcium.

The main sites of calcium binding were determined for the low molecular weight heparin drug enoxaparin and the synthetic pentasaccharide Arixtra (fondaparinux). [1H,13C] HSQC pH titrations were carried out to characterize the acid-base properties of these samples both in the presence and absence of calcium. The differences in the titration curves were used to determine the structural components of enoxaparin and fondaparinux responsible for Ca2+ binding. In enoxaparin both unsubstituted and 2-O-sulfated iduronic acid residues are important in calcium binding and the presence of the 2-O-sulfo group does not seem to influence the Ca2+ binding capability of the iduronate ring. In fondaparinux changes in chemical shifts upon Ca2+ binding were smaller than observed for enoxaparin, and were observed for both the glucuronic acid and 2-O-sulfated iduronic acid residues. In enoxaparin significant perturbations of the chemical shift of the N-sulfoglucosamine anomeric carbon in residues connected to 2-O-sulfated iduronic acid were detected on Ca2+ binding, however it was not possible to determine whether these changes reflect direct involvement in calcium complexation or result from through space interactions or conformational changes

Effect of Substitution Degree and Homogeneity on Cyclodextrin-Ligand Complex Stability: Comparison of Fenbufen and Fenoprofen Using CD and NMR Spectroscopy

The stability of host–guest complexes of two NSAID drugs with similar physicochemical properties, fenbufen and fenoprofen, was investigated by comparing induced circular dichroism and 1H nuclear magnetic resonance methods using eight cyclodextrins of different degrees of substitution and isomeric purity as guest compounds. These cyclodextrins include native β-cyclodextrin (BCyD), 2,6-dimethyl-β-cyclodextrin 50 (DIMEB50), 80 (DIMEB80) and 95% (DIMEB95) isomerically pure versions, low-methylated CRYSMEB, randomly methylated β-cyclodextrin (RAMEB) and 4.5 and 6.3 average substitution grade hydroxypropyl-β-cyclodextrin (HPBCyD). The stability constants obtained by the two methods show good agreement in most cases. For fenbufen complexes, there is a clear trend that the stability constant increases with the degree of substitution while isomer purity has a smaller effect on the magnitude of stability constants. A significant difference was found in the case of DIMEB50 when compared to DIMEB80/DIMEB95, while the latter two are similar. In the fenbufen–fenoprofen comparison, fenbufen, with its linear axis, gives a more stable complex, while fenoprofen shows lower constants and poorly defined trends

Identification and quantification of lignans and sesquilignans in the fruits of Cnicus benedictus L.: Quantitative chromatographic and spectroscopic approaches

Identification, quantification and isolation of lignans (Lig-s: arctiin, arctigenin and matairesinol) and sesquilignans (SLig-s: lappaol A, isolappaol A, lappaol C and isolappaol C), in Cnicus benedictus L. fruit (CBfr) were performed for the first time. Identity of Lig-s and SLig-s was confirmed by gas chromatography-mass spectrometry (GC-MS) and by high performance liquid chromatography-time-of-flight (HPLC-TOF) MS, while their detailed structures were defined by nuclear magnetic resonance (NMR) spectroscopy. As a novelty to the field, fruit part-specific accumulation of Lig-s and SLig-s under germination and their transformation during acidic and enzymatic hydrolyses were followed in a quantitative manner by HPLC-UV. It was shown that during germination, the spontaneous separation of Lig-s and SLig-s occurs: the fruit wall part accumulates SLig-s, while the embryo accumulates the Lig arctiin. It was confirmed that under optimized mild acidic conditions (50. °C, 2. M trifluoroacetic acid, TFA), lappaol C and isolappaol C can be transformed into lappaol A and isolappaol A, quantitatively. Analytical performance characteristics (reliability and reproducibility), in the HPLC-UV quantifications of Lig-s and SLig-s were defined by the relative standard deviation percentages (RSD%-s) of analyses (averages of RSD%-s. ≤. 3.7). Due to our new harmonized analysis system, CBfr proved to be a new and rich source of SLig-s: levels as high as 5.8. mmol/100. g were determined compared to the highest level (0.72. mmol/100. g) reported so far. © 2014 Elsevier B.V