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The metabolism of glycosphingolipids by the malaria-causing parasite Plasmodium falciparum plays an important role in the progression of the disease. We report a new and highly sensitive method to monitor the uptake of glycosphingolipids in infected red blood cells (iRBCs). A tetramethylrhodamine-labeled glycosphingolipid (GM1TMR) was used as a substrate. Uptake was demonstrated by fluorescence microscopy. The iRBCs were lysed with a 15% solution of saponin and washed with phosphate buffered saline to release intact parasites. The parasites were further lysed and the resulting homogenates were analyzed by capillary electrophoresis with laser-induced fluorescence detection. The lysate from erythrocytes infected at 1% parasitemia generated a signal 20 standard deviations larger than uninfected erythrocytes, which suggests that relatively low infection levels can be studied with this technique. 10.1021/ac1021776

Glycosphingolipids are found on all vertebrate cells and constitute major cell surface determinants on all nerve cells, where they contribute to cellular diversity and function. We report a method for the analysis of glycosphingolipid metabolism in single cells. The ganglioside [G.sub.M1] was tagged with the fluorescent dye tetramethylrhodamine. This labeled compound was taken up and metabolized by a culture of pituitary tumor (AtT-20) cells. After 50 h, the cells were formalin fixed. Cells were aspirated into a fused-silica capillary and lysed, and components were separated by capillary electrophoresis with a laser-induced fluorescence detector. All metabolic products that retained the fluorescent dye could be detected at the low-zeptomole level. A total of 54 AtT-20 cells were individually analyzed using this procedure. The electrophoretic profiles were remarkably reproducible, which facilitated identification of components based on the migration time of fluorescently labeled standards. Eleven components were detected, and the average peak height of these components spanned more than 2 orders of magnitude, so that trace metabolites can be detected in the presence of abundant components. The most highly abundant components generated 10% relative standard deviation in normalized abundance. The average cell took up roughly 2 amol ([10.sup.6] copies) of the labeled substrate. This method allows determination of cell-to-cell diversity and regulation of glycosphingolipid metabolism.

A semiquantitative study comparing NBD-labeled carbohydrates with carbohydrates labeled with other commonly used dyes is described. It is confirmed that NBD-X dye is an ideal labeling tool in the screening or monitoring of the binding interactions between promising ligands and immobilized protein receptors.

This work presents new frontal affinity chromatography (FAC) methodologies for high-throughput screening of compound libraries, designed to increase screening rates and improve sensitivity and ruggedness in performance. A FAC column constructed around the enzyme N-acetylglucosaminyltransferase V (GnT-V) was implemented in the identification of potential enzyme inhibitors from two libraries of trisaccharides. Effluent from the FAC column was fractionated, sequentially processed via LC/MS, and referenced to a similar analysis through a control FAC column lacking the enzyme. The resulting multidimensional data sets were compared across corresponding sample and control fractions to identify binders, in a semiautomated approach. A strong binder in the protonated form at m/z 795 was identified from the first library of 81 compounds, exhibiting an estimated [K.sub.d] value of 0.3 [micro]M. Other binders yielded [K.sub.d] values ranging from 0.35 to 3.35 [micro]M. To demonstrate the improvement in performance of this FAC-LC/MS approach over the conventional online FAC/MS approach, 15 compounds from this library were blended with a second library of 1000 synthetic trisaccharides and screened against GnT-V. All ligands in the 15-compound set were identified in this larger screen, and no ligands of greater affinity than compound 1 were found. Our results show that FAC-LC/MS is a reliable method for screening large compound libraries directly and useful for large-scale ligand discovery initiatives.

Deoxygenated uridine 5'-diphosphogalactos derivatives were evaluated as donors for retaining galactosyltransferas.

Acyclic 1-chloro-1(ethylthio) derivatives were synthesized by the reaction of acetyl chloride and boron trifluoride diethyl etherate with D-glucose, D-galactose and D-mannose peracetylated ethyl dithioacetals. The reaction resulted in the cyclization of beta-D-galactofuranosides and the glycosylation of carbohydrate acceptors. Furthermore, cyclizations led to the formation of O,S-acetals which react with mercuric salt and methanol to produce disaccharides with D-furanosyl residues.

The O-cyanomethyl ethers of carbohydrates act as intermediates for the production of sugar amines, carboxylic acids, amides and amidine salts. The technique of carbohydrate functionalization leads to a family of analogs for the study of carbohydrate binding proteins. The addition of amino acids to O-aminoethyl and O-carboxymethyl carbohydrates produces glycopeptides. The reaction enables linkage of the carbohydrate moiety through a hydroxyl group to the C- or the N-terminus of a peptide.

Matrix-assisted laser desorption ionization mass spectrometry has been developed for direct mass analysis of enzymatic reaction products of oligosaccharides in human blood serum without the use of extraction or chromatographic separation. Molecular labeling of the substrate is used to achieve beth the detection sensitivity and selectivity required in rapid analysis of reaction products in serum. It is found that tetramethylrhodamine (TMR)-labeled oligosaccharides provide 100-fold sensitivity enhancement over the corresponding underivatized oligosaccharides. In order to selectively retain the TMR-labeled molecules on the sample probe while washing away contaminants in a serum sample, a sample/matrix preparation method is developed. This technique provides detection sensitivity of labeled oligosaccharides in the range of hundreds of femtomoles per microliter. The mass measurement accuracy is better than 0.01% when a linear time-of-flight mass spectrometer is used. The application of the technique is illustrated for the subpicomole detection and quantitation of the conversion of the disaccharlde [Alpha]Fuc(1 [yields] 2)[Beta]Gal-TMR to the blood group B active trisaccharide [Alpha]Fuc(1 [yields] 2)[[Alpha]GaI(1 [yields] 3)][Beta]Gal-TMR, catalyzed by the blood group B galactosyltransferase present in human serum.

Research has been conducted on the Mitsunobu reaction-based methodology. Results indicate that it produces a variety of 3-O substituted analogues with complete regio- and chemoselectivity.

Carbohydrate oxime formation is catalyzed with up to 20-fold increases in overall reaction rate at 100 mM aniline. The application of the method has provided access to complex glycoconjugates.

The synthesis and screening of two beta-DGalp-(1-3)-beta-D-GlcpN (lactor-N-biose) disaccharide libraries are reported. Compounds bearing a hydrophobic substituent on the NH group showed the highest affinity for the lectin.

The synthesis of the (super 13)C-labeled substrate-analogue inhibitor TU-514 and the subsequent refinement of the solution structure of the Aquifex aeolicus LpxC-TU-514 complex using residual polar couplings are reported. A revaluation of the catalytic role of an active site histidine, H253, on the basis of both its pK(sub a) as determined by NMR titration and pH-dependent kinetic analyses is discussed.

Several p-hydroxybenzyl-derived protecting groups have been found to be removed in near quantitative yield either thermally or by mild oxidation. When the phenoxy oxygen is protected as its acetate ester or silylethoxymethoxy acetal, the resulting p-substituted benzyl ethers are compatible with many of the standard manipulations of oligosaccharide synthesis. Also, these groups are orthogonal to both benzyl and p-methoxybenzyl ethers.

The growth in number of recombinant glycosyltranferases has resulted to the increase in the use of such enzymes in chemical-enzymatic syntheses of oligosaccharides. As a result, the enzymatic step to include the preparation of oligosaccharide analogues is adversely affected. The disruption occurs when glycosyltransferases allow the transfer of unnatural sugar to the natural substrate oligosaccharide or transfer natural sugar to an unusual substrate.

A simple and rapid chemical synthesis of sugar nucleotides involves a direct displacement of an anomeric leaving group by a nucleoside diphosphate. The anomeric mixtures of these sugar nucleotides are in a form suitable for glycosyltransferase-catalyzed oligosaccharide synthesis. In this synthesis, enzymes choose the correct anomer as the donor substrate, and wrong anomers of the sugar nucleotide do not inhibit the activity of the enzymes.

The use of glycosyltransferases in the synthesis of oligosaccharide analogues is described.