Determination of pKa values of quinolones from mobility and spectroscopic data obtained by capillary electrophoresis and a diode array detector

This work compares the values of dissociation constants obtained from electrophoretic mobilities of a series of quinolones at different pH values and those obtained using absorbance spectra at the maximum of the eletrophoretic peaks. The results obtained show that the two methods are complementary and constitute a valuable means of obtaining better precision. The two methods proposed can be used simultaneously without an increase in the experimental time and allow confirmation of the results obtained.     

Acid-base equilibrium of β-adrenoceptor blocking agents in acetic acid medium and methods for their titration

The overall dissociation constants and the formation constants of the perchlorate salts, in anhydrous acetic acid, of a series of -adrenoceptor blocking agents have been determined. On the basis of these values simple potentiometric and visual titration methods for the assay of -adrenoceptor blocking drugs in acetic acid are described.     

Assignment of reference pH-values to primary standard buffer solutions for standardization of potentiometric sensors in acetonitrile-water mixtures

Standard pH-values pH(PS) for seven primary standard buffer solutions in 0, 10, 30, 40, 50, 70 and 100% (w/w) acetonitrile-water mixed solvents at 298.15 K were determined according to the criteria recently endorsed by IUPAC. The preferential solvation of the ions in acetonitrile-water mixtures was studied in order to clarify the acid-base behaviour of the solutes in such mixtures. The influence of variation in the solvent composition on pH(PS) values was considered with a view to obtaining correlations that can be used to determine the pH(PS) values in any acetonitrile-water mixture up to 70% (w/w) acetonitrile. The pH(PS) values were then correlated with the weight and volume percentages and molar fraction of acetonitrile and with the Kamlet-Taft, pi(*), alpha and beta solvatochromic parameters of the acetonitrile-water mixtures. The equations obtained permit the standardization of potentiometric sensors in these mixtures.     

Chemical and photochemical electron transfer of new helianthrone derivatives: aspects of their photodynamic activity

Helianthrones 2-4 are a new class of synthetic photosensitizers, which have a molecular skeleton related to that of hypericin. We established that irradiation of heliantrones with visible light leads to the formation of semiquinone radicals and reactive oxygen species. The structures of the paramagnetic anion species produced by electron transfer were calculated on the density functional level and investigated by cyclovoltammetry, UV/vis, and EPR/ENDOR spectroscopy. As with hypericin, the pi system of the helianthrones was found to be considerably deviated from planarity, and, upon electron transfer, deprotonation in the bay region occurs. The structure of the semiquinone radicals was found to be identical in THF, DMF, and aqueous buffered solutions regardless of the means by which reduction was achieved. Semiquinone radicals can be formed via self-electron transfer between the excited state and the ground state or via electron transfer from an electron donor to the excited state of helianthrone. Therefore, the presence of an electron donor significantly enhanced the photogeneration of semiquinone and superoxide radical. The kinetic studies showed that no significant photochemical destruction of helianthrones occurred upon irradiation. Generation of superoxide and singlet oxygen upon irradiation of helianthrones was established by spin trapping techniques. This shows that both type I and type II mechanisms are of importance for the photodynamic action of these compounds.     

Synergic effect studies of the bi-enzymatic system laccase-peroxidase in a voltammetric biosensor for catecholamines

Several bi-enzymatic carbon paste biosensors modified with enzymes laccase from Pleurotus ostreatus fungi and peroxidase from zucchini (Cucurbita pepo) were constructed for evaluating the synergic effect of the two enzymes on the voltammetric biosensor response for various catecholamines. Initially was investigated the effect of pH from 5.0 to 7.5, temperature from 25 to 50 °C, initial stirring time from 30 to 150 s, scan rate from 10 to 60 mV s⁻¹ and potential pulse amplitude from 10 to 60 mV on the biosensor response for several catecholamines such as dopamine, adrenaline, isoprenaline and l-dopa. It was observed a biosensor signal increase employing both enzymes, indicating thus there is a synergic effect between laccase and peroxidase, verified also in spectrophotometric studies, in the determination of these catecholamines.     

Fmoc-POAC: [(9-fluorenylmethyloxycarbonyl)-2,2,5,5-tetramethylpyrrolidine-N-oxyl-3-amino-4-carboxylic acid]: a novel protected spin labeled beta-amino acid for peptide and protein chemistry

The stable free radical 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC) is the only spin labeled amino acid that has been used to date to successfully label peptide sequences for structural studies. However, severe difficulty in coupling the subsequent amino acid has been the most serious shortcoming of this paramagnetic marker. This problem stems from the low nucleophilicity of TOAC's amine group towards the acylation reaction during peptide chain elongation. The present report introduces the alternative beta-amino acid 2,2,5,5-tetramethylpyrrolidine-N-oxyl-3-amino-4-carboxylic acid (POAC), potentially useful in peptide and protein chemistry. Investigations aimed at addressing the stereochemistry of this cyclic molecule through X-ray diffraction measurements of crystalline and bulk samples revealed that it consists only of the trans conformer. The 9-fluorenylmethyloxycarbonyl group (Fmoc) was chosen for temporary protection of the POAC amine function, allowing insertion of the probe at any position in a peptide sequence. The vasoactive octapeptide angiotensin II (All, DRVYIHPF) was synthesized by replacing Pro7 with POAC. The reaction of Fmoc-POAC with the peptidyl-resin occurred smoothly, and the coupling of the subsequent amino acid showed a much faster reaction when compared with TOAC. POAC7-AII was obtained in good yield, demonstrating that, in addition to TOAC, POAC is a convenient amino acid for the synthesis of spin labeled peptide analogues. The present findings open the possibility of a wide range of chemical and biological applications for this novel beta-amino acid derivative, including structural investigations involving its differentiated bend-inducing characteristics.     

Liquid chromatography-mass spectrometry approach for the characterization and purification of crude synthetic peptide hormones

In this study, conditions for the optimal separation by LC-UV and characterisation by LC-ES-MS of crude mixtures generated during SPPS of several peptide hormones are compiled. The linear solvation energy relationship (LSER) methodology has been used to predict the retention of the target peptides and their side products and then to develop a separation LC methodology with applicability on both the analytical and preparative scale. Identification of these side products by LC-ES-MS analysis has been made on the basis of their calculated molecular masses. This method may be regarded as a key tool for the optimisation of the synthetic procedures and for complying with regulatory agencies' requirements before commercialisation of a safe and effective peptide-based pharmaceutical drug.Awarded a prize as outstanding poster on the occasion of Jornadas de Análisis Instrumental (JAI), November 26 to 29, 2002, Barcelona, Spain     

Prediction of LC retention of steroids using solvatochromic parameters

Summary The proportion of organic modifier and pH of the hydroorganic mobile phase were optimized to separate a series of nine important steroids by LC. It has been shown that linear plots of logk′ against Reichardt’sE _T ^N parameter for the mobile phase is observed over the whole range of compositions of practical importance. Data from these plots summarize an approach for optimization of resolution from only two retention measurements for each compound. pH is shown to be irrelevant. This behaviour could be extrapolated to other steroids and drastically reduce the number of retention values needed for mobile phase optimization for screening analysis of steroids in doping investigations.     

Hypericin derivatives: substituent effects on radical-anion formation

The electron-transfer properties of the hypericin derivatives, dibromo-, hexaacetyl-, hexamethyl- and desmethylhypericin, were studied. Cyclovoltammetric measurements revealed that dibromo- and desmethylhypericin have almost the same redox potentials as the parent hypericin. Substitution of the hydroxyl groups by acetoxy leads to less negative E1/2 values, whereas methoxy substitution induces more negative values. Electron paramagnetic resonance (EPR)/electron nuclear double resonance/general TRIPLE spectroscopy and quantum mechanical calculations were used to establish the structure of the one-electron reduced stages of hypericin derivatives. Proton loss in the bay region, already demonstrated for hypericin, was also found for dibromo- and desmethylhypericin. The spin and charge of the radical ions are predominately confined to the central biphenoquinone moiety of the hypericin skeleton. Generation of the radical ions by in situ electrolysis indicates that the redox potentials of hypericin, dibromo- and desmethylhypericin, containing hydroxyls at the 1, 3, 4, 6, 8 and 13 positions, largely depend on the solvent. With phosphate-buffered saline (pH 7.4)/dimethylsulfoxide (DMSO) as the solvent the EPR spectra of the corresponding radical ions appear at markedly lower potentials than in pure DMSO and N,N'-dimethylformamide. However, this effect is not observable for hexaacetyl- and hexamethyl-hypericin-lacking hydroxyl groups. In all cases the EPR data and calculations revealed the presence of 7,14 tautomers.