Krisztina Németh

Enantiomeric separation of antimalarial drugs by capillary electrophoresis using neutral and negatively charged cyclodextrins

Capillary electrophoresis (CE) methods for chiral resolution of five antimalarial drugs (primaquine, tafenoquine, mefloquine, chloroquine and quinacrine) were developed by using a wide selection of neutral and anionic cyclodextrin (CD) derivatives. The use of sulfobutyl-β-CD and carboxymethyl-β-CD (CMBCD) resulted in good resolution of quinacrine and tafenoquine, respectively. New results are presented for resolutions of chloroquine and mefloquine. Application of carboxyalkyl- and sulfobutyl-CD derivatives provided improved resolution for primaquine. The impurity in primaquine sample detected by CE was identified as quinocide by MS and NMR. CMBCD provided not only the best separation of primaquine from quinocide but also the simultaneous complete resolution of both compounds.

Chiral separation by a monofunctionalized cyclodextrin derivative: From selector to permethyl-β-cyclodextrin bonded stationary phase

Preparation of (6-monoureido-6-monodeoxy) permethylated beta-cyclodextrin bonded chiral stationary phase from permethylated 6-monoamino-6-monodeoxy-beta-cyclodextrin is described. The optimized chiral stationary phase was evaluated by using HPLC separation of racemates of coumarin derivatives. Column characterization was performed by solid-state (13)C, (15)N, (29)Si NMR using cross-polarization at the magic angle spinning. The development process was supported by CE experiments where the complex formation between cyclodextrins and warfarin was investigated. The results demonstrate good enantio-discrimination for coumarin derivatives.

Separation of cis-β-lactam enantiomers by capillary electrophoresis using cyclodextrin derivatives

Chiral separation of 19 pairs of cis-beta-lactam (BL) stereoisomers of pharmacological importance was examined by capillary electrophoresis using cyclodextrin (CD) derivatives. In order to select the most effective conditions separating the highest number of stereoisomers of BLs, single carboxymethyl alpha-, beta- and gamma-, as well as sulfobutyl beta-CD derivatives were applied. Additionally, carboxymethyl and sulfobutyl beta-CD derivatives complemented with neutral beta-CD derivatives as dual CD systems were tested. Both the composition and concentration of applied selectors and the pH of background electrolyte were selected. In single systems the structural characteristics of BLs and the complex forming affinity were correlated. Most BLs provided optimal complexation with beta-CD derivatives. In conclusion, the efficiency of combining sulfobutyl-beta-CD and permethylated beta-CD was superior to other single and dual CD systems applied. This method successfully separated each pair of stereoisomers investigated.

Assessing toxicity of polyamidoamine dendrimers by neuronal signaling functions

Abstract We report for the first time on neuronal signaling for the evaluation of interactions between native plasmamembrane and polyamidoamine (PAMAM) dendrimers. Generation 5 polycationic (G5-NH2), novel β-D-glucopyranose-conjugated G5-NH2 and generation 4.5 polyanionic (G4.5-COONa) polyamidoamine (PAMAM) dendrimers (1–0.0001 mg/ml) were applied in acute brain slices. Functional toxicity assessments–validated by fluorescence imaging of dead cells–were performed by employing electrophysiological indicators of plasma membrane breakdown and synaptic transmission relapse. Irreversible membrane depolarization and decrease of membrane resistance predicted substantial functional neurotoxicity of unmodified G5-NH2, but not of the G4.5-COONa PAMAM dendrimers. Model calculations suggested that freely moving protonated NH2 groups of terminal monomeric units of PAMAM dendrimers may be able directly destroy the membrane or inhibit important K+ channel function via contacting the positively charged NH2. In accordance, conjugation of surface amino groups by β-D-glucopyranose units reduced functional neurotoxicity that may hold great potential for biomedical applications.

A Double-Clicking Bis-Azide Fluorogenic Dye for Bioorthogonal Self-Labeling Peptide Tags.

Herein, we give the very first example for the development of a fluorogenic molecular probe that combines the two-point binding specificity of biarsenical-based dyes with the robustness of bioorthogonal click-chemistry. This proof-of-principle study reports on the synthesis and fluorogenic characterization of a new, double-quenched, bis-azide fluorogenic probe suitable for bioorthogonal two-point tagging of small peptide tags by double strain-promoted azide-alkyne cycloaddition. The presented probe exhibits remarkable increase in fluorescence intensity when reacted with bis-cyclooctynylated peptide sequences, which could also serve as possible self-labeling small peptide tag motifs.

Structure and stability of warfarin-sodium inclusion complexes formed with permethylated monoamino-β-cyclodextrin.

Inclusion complexes of warfarin enantiomers with permethylated monoamino-β-cyclodextrin (PMMABCD) were characterized using CE and (1)H NMR spectroscopy in aqueous solution. These techniques gave complementary information on the stability and the structure of the diastereomeric host-guest inclusion complexes. The stability constants were determined from CE experiments in a wide pH range. Change in the migration order on the variation of the pH was observed. (1)H NMR assignments have been established for the seven non-equivalent carbohydrate units of the host in the complex at pH 7-9. Specific H-H distance restraints were obtained from NOESY experiments and were introduced into molecular modeling to establish the geometry of the inclusion complexes. It was found that the open side chain warfarin enters the cavity from the primary side of the CD. The orientation of the coumarin ring within the cavity has the same preference for the two warfarin enantiomers owing to an ionic interaction with the amino group of the CD. Accordingly, enantioselectivity at pH 8.5 arises from the difference in the CH/π interactions between warfarin aromatics and the manifold of CH groups of the CD.

Cationic permethylated 6-monoamino-6-monodeoxy-β-cyclodextrin as chiral selector of dansylated amino acids in capillary electrophoresis.

The resolution power of permethylated 6-monoamino-6-monodeoxy-βCD (PMMABCD) - a single isomer, cationic CD derivative - developed previously for chiral analyses in capillary electrophoresis was further studied here. Dansylated amino acids (Dns-AA) were chosen as amphoteric chiral model compounds. Changes in the resolutions of Dns-AAs by varying pH and selector concentrations were investigated and correlated with their structures and chemical properties (isoelectric point and lipophilicity). Maximal resolutions could be achieved at pH 6 or pH 4. The separations improved with increasing concentration of the selector. Baseline or substantially better resolution for 8 pairs of these Dns-AAs could be achieved. Low CD concentration was enough for the separation of the most apolar Dns-AAs. Chiral discrimination ability of PMMABCD was demonstrated by the separation of an artificial mixture of 8 Dns-AA pairs.

Bisazide Cyanine Dyes as Fluorogenic Probes for Bis-Cyclooctynylated Peptide Tags and as Fluorogenic Cross-Linkers of Cyclooctynylated Proteins

Herein we present the synthesis and fluorogenic characterization of a series of double-quenched bisazide cyanine probes with emission maxima between 565 and 580 nm that can participate in covalent, two-point binding bioorthogonal tagging schemes in combination with bis-cyclooctynylated peptides. Compared to other fluorogenic cyanines, these double-quenched systems showed remarkable fluorescence intensity increase upon formation of cyclic dye-peptide conjugates. Furthermore, we also demonstrated that these bisazides are useful fluorogenic cross-linking platforms that are able to form a covalent linkage between monocyclooctynylated proteins.

Stereoselective analysis of endomorphin diastereomers: Resolution of biologically active analogues by capillary electrophoresis applying cyclodextrins as mobile phase additives

Seven diastereomer pairs of tetrapeptide analogues (TP) of endomorphin-1 and -2 were synthesized. A stereoselective capillary electrophoretic method was developed for controlling stereoisomeric ratio or purity. The isoelectric points of the tetrapeptides were between 8.3 and 8.9 as predicted and measured. A few of the analytes could be resolved without selectors due to the difference in their mobility. Neutral and anionic cyclodextrins (CDs) were applied in order to improve resolution. Stability constants as well as the mobilities of complexes were determined. Contributions of differences in the mobilities of free analytes and in the mobilities and stabilities of their complexes formed by CDs were equally important in the efficient resolution and migration order of diastereomers. As a result of the optimization of the pH of buffers and the concentration of the CD derivatives each diastereomer pair was resolved at baseline at least or better.

Development of novel chiral capillary electrophoresis methods for the serotonin receptor (5-HT2A) antagonist MDL 100,907 (volinanserin) and for its key intermediate compound

Enantioselective capillary electrophoretic methods were elaborated for the determination of the enantiomeric purity of (R)-MDL 100,907 and its preparatively resolved key intermediate compound during the synthesis route. The pKa values of the intermediate compound and the end product determined by CE were 10.5±0.1 and 9.0±0.1, respectively. The enantiopurity of the intermediate compound can be monitored in fully protonated state by applying 15mM sulfobutylether-β-cyclodextrin at pH 5 when the peak belonging to the impurity migrates before the main component. The fact that the consecutive steps of the synthesis do not affect the enantiomeric purity was verified by the other, newly developed CE method. The enantiomers of rac-MDL 100,907 were resolved by 15mM carboxymethyl-γ-cyclodextrin at pH 3. The applicability (selectivity, LOD, LOQ, repeatability, precision and accuracy) of the methods was studied as well.