Leopold Kremser

pKa shift‐associated effects in enantioseparations by cyclodextrin‐mediated capillary zone electrophoresis

Enantioselective migration of dansylated (Dns) amino acids in the presence of hydroxypropylated‒β‒cyclodextrin under acidic conditions near the pI value of the analytes was investigated by means of capillary zone electrophoresis. Based on the migration data, the pH dependence of the complexation constants was evaluated, as well as the variation of the complex mobilities with pH. As a result of these data, the migration behavior in the pH region near the pI could be understood, which, in some instances, includes the reversal of migration order upon variation of selector concentration. The enantioselective pKa shifts upon complexation could be quantitated for the carboxylic and the amino group separately. pKa shifts were found in the order of 0.8 pI units, the differences between the enantiomers being up to 0.25 pH units. These data were in agreement with the pI shifts reported from isoelectric focusing experiments. The accurate determination of the pI values of the Dns amino acids makes it possible to calibrate the pI scale in isoelectric focusing in the presence of chiral selectors.

Twelve receptor molecules attach per viral particle of human rhinovirus serotype 2 via multiple modules

The crystallographic T=1 (pseudo T=3) icosahedral symmetry of the human rhinovirus capsid dictates the presence of 60 identical, symmetry related surface structures that are available for antibody and receptor binding. X‐ray crystallography has shown that 60 individual very‐low density lipoprotein receptor (VLDLR) modules bind to HRV2. Their arrangement around the fivefold axes of the virion suggested that tandem oligomers of such modules could attach simultaneously to symmetry‐related sites. By resolving virus particles carrying various numbers of artificial recombinant concatemers of VLDLR repeat 3 (V33333) by capillary electrophoresis and extrapolation of the measured mobilities to that at saturation of all binding sites, we present evidence for up to 12 molecules of the concatemer to bind one single virion.

Enantiomeric resolution of galanthamine and related drugs used in anti-Alzheimer therapy by means of capillary zone electrophoresis employing derivatized cyclodextrin selectors

An analytical assay is presented for the determination of the enantiomeric composition of galanthamine and related synthetic and natural compounds. (-)-Galanthamine is isolated from Galanthus nivalis and is used in this optical pure form in the therapy of Alzheimers disease. Recent efforts for a total synthesis of unichiral (-)-galanthamine is connected with the need for a fast and reliable assay for the determination of the optical purity of the end product, as well as for optimizing and controlling the final steps in total synthesis particularly the asymmetric transformation of narwedine. In this paper the enantiomeric resolution of these compounds is reported employing a capillary electrophoretic system with beta-cyclodextrin derived chiral selectors. With the proposed system a number of galanthamine and narwedine derived analogous compounds could be separated, including 1-bromo- and N-alkyl-substituted compounds.

Electrophoresis on a microfluidic chip for analysis of fluorescence‐labeled human rhinovirus

We report the analysis of human rhinovirus serotype 2 (HRV2) on a commercially available lab‐on‐a‐chip instrument. Due to lack of sufficient native fluorescence, the proteinaceous capsid of HRV2 was labeled with Cy5 for detection by the red laser (λex 630 nm) implemented in the instrument. On the microdevice, electrophoresis of the labeled virus was possible in a BGE without stabilizing detergents, which is in contrast to conventional CE; moreover, analysis times were drastically shortened to the few 10 s range. Resolution of the sample constituents (virions, a contaminant present in all virus preparations, and excess dye) was improved upon adaptation of the separation conditions, mainly by adjusting the SDS concentration of the BGE. Purity of fractions from size‐exclusion chromatography after labeling of virus was assessed, and affinity complex formation of the labeled virus with various recombinant very‐low‐density lipoprotein receptor derivatives differing in the number of concatenated V3 ligand binding repeats was monitored. Virus analysis on microchip devices is of particular interest for experiments with infectious material because of easy containment and disposal of samples. Thus, the employment of microchip devices in routine analysis of viruses appears to be exceptionally attractive.

Rhinovirus-stabilizing activity of artificial VLDL-receptor variants defines a new mechanism for virus neutralization by soluble receptors

Members of the low‐density lipoprotein receptor family possess various numbers of ligand binding repeats that non‐equally contribute to binding of minor group human rhinoviruses. Using an artificial concatemer of five copies of repeat 3 of the human very‐low density lipoprotein receptor, we demonstrate protection of HRV2 against low‐pH mediated uncoating and inhibition of penetration of an RNA‐specific fluorescent dye into the intact virion. This indicates that the recombinant receptor inhibits viral breathing and irreversible conformational modifications of the capsid that precede RNA release, providing a new mechanism for rhinovirus neutralization by soluble receptor molecules.

Enantioseparation of derivatized amino acids by capillary isoelectric focusing using cyclodextrin complexation.

Enantioseparation of dansylated as well as 6‐aminoquinolyl‐N‐hydroxysuccinimidylcarbamate (AQC)‐derivatized amino acids by means of capillary isoelectric focusing using various cyclodextrin derivatives is demonstrated. Separation is based on the enantioselective shift of the isoelectric points upon complexation with the chiral selectors. The zwitterionic, diastereomeric analyte‐cyclodextrin complexes exhibited differences in the pI values up to more than 0.25 pI units. Enantioresolution was achieved for a number of derivatized amino acids and various selectors added to the carrier ampholyte solution. The hydroxypropyl‐β‐cyclodextrin proved to be the best selector for this purpose. Enantioseparation as dependent on the selector concentration was evaluated in a range between 5 and 30 mM. Separation could be attained down to selector concentrations corresponding to a degree of complexation as low as 30%. The peaks appear according to the degree of complexation between the positions adopted without and with full complexation. The kinetics of complex formation and dissociation was fast enough in most instances to produce single peaks, even with complexation degrees near 0.5 and significant pI shifts. Peak widths were slightly enlarged in these instances. The method offers excellent perspectives for preparative applications.

Capillary electrophoresis of boron cluster compounds in aqueous and nonaqueous solvents.

The electrophoretic properties of boron cluster compounds were determined in water, methanol and ACN as solvents of the BGE and discussed based on the principles of ion migration. Two types of boron cluster compounds were investigated. One type consisted of derivatives of the nido‐7,8‐dicarbaundecaborate cluster, the other types are derivatized cobalt bis(dicarbollide) ions (COSANs) whose central cobalt atom is sandwiched by two 7,8‐dicarbaundecaborate clusters. The BGE in all solvents was acetate/acetic acid buffer with pH 4.75 in water, 9.7 in methanol and 22.3 in ACN, respectively, at different ionic strength between 5 and 30 mM. The dependence of the mobility on ionic strength could not be explained by the theory of Debye, Hückel and Onsager, but good agreement was found upon considering an ion size parameter. Limiting mobilities were derived by curve fitting, and by the aid of the solvent viscosities the hydrodynamic radii of the analyte anions were calculated. They are between 0.25 and 0.48 nm, and were nearly independent of the solvent. Electrophoresis of the analytes in a BGE consisting of 6 mM perchloric acid in ACN allows the conclusion that the present boron cluster compounds behave as stronger acids than perchloric acid.