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The growing number of synthetic glycopeptides required an in-house method for the analysis of the final products. A reversed-phase high-performance liquid chromatographic protocol was developed to verify the presence of N-acetylglucosamine and N-acetylgalactosamine in synthetic glycopeptides after hydrolysis and derivatization with 4-dimethylaminoazobenzene-4'-sulphonyl chloride. Different sugar and glycoamino acid standards were used to separate the two carbohydrate moieties, and the location of the derivatized 2-aminoglucose was established by the use of radiolabeled sugar. The utility of the approach is demonstrated by the amino acid analysis of N- and O-glycosylated synthetic peptides and the method could provide an alternative for sugar analysis of glycoproteins.
Pseudomonas aeruginosa, a common respiratory tract colonizer and pathogen, adheres to injured tracheal cells and to tracheobronchial mucin. These phenomena suggest that there are specific receptors for this organism in the respiratory tract. The receptor on injured tracheal cells contains n-acetylneuraminic acid as the principal sugar, but the structure of the receptor in mucin has not been described. Using a microtiter plate assay to study bacterial adherence to mucin, we have partially characterized the mucin receptor for P. aeruginosa. The receptor for both nonmucoid and mucoid strains is sensitive to periodate oxidation, suggesting that it is carbohydrate in nature, and the amino sugars n-acetylglucosamine and n-acetylneuraminic acid inhibited the adherence of both types of strains. Nonmucoid strains were more sensitive to inhibition by n-acetylneuraminic acid than to inhibition by n-acetylglucosamine, but the mucoid strains varied in their sensitivities to inhibition by each amino sugar. Preincubation of mucin with heat-inactivated influenza A virus (which binds to neuraminic acid) significantly reduced the adherence of P. aeruginosa. Treatment of mucin with Clostridium perfringens neuraminidase also reduced bacterial adherence significantly. Treatment of mucin with pronase did not affect adherence. Our results suggest that n-acetylglucosamine and n-acetylneuraminic acid are important constituents of the binding sites for P. aeruginosa on human tracheobronchial mucin.
The archaeon Pyrococcus furiosus is a strictly anaerobic heterotroph that grows optimally at 100 degrees C by the fermentation of carbohydrates. It is known to contain high concentrations of novel intracellular solutes such as beta-mannosylglycerate and di-myo-inositol 1,1'-phosphate (DIP) (L. O. Martins and H. Santos, Appl. Environ. Microbiol. 61:3299-3303, 1995). Here, 31P nuclear magnetic resonance (NMR) spectroscopy was used to show that this organism also accumulates another type of phospho compound, as revealed by a major multiplet signal in the pyrophosphate region. The compounds were purified from cell extracts of P. furiosus by anion-exchange and gel filtration chromatographic procedures and were structurally analyzed by 1H, 13C, and 31P NMR spectroscopy. They were identified as two uridylated amino sugars, UDP N-acetylglucosamine and UDP N-acetylgalactosamine. Unambiguous characterizations and complete assignments of 1H and 13C resonances from such sugars have not been previously reported. In vitro 31P NMR spectroscopic analyses showed that, in contrast to DIP, which is maintained at a constant intracellular concentration (approximately 32 mM) throughout the growth phase of P. furiosus, the UDP amino sugars accumulated (to approximately 14 mM) only during the late log phase. The possible biochemical roles of these compounds in P. furiosus are discussed.
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3-(4-Carboxybenzoyl)-2-quinolinecarboxaldehyde has been utilized as a precolumn derivatization agent for various amino sugars. Constituents of various biological mixtures can be converted to highly fluorescent isoindole derivatives, separated by high-performance capillary electrophoresis and determined at attomole (10(-18) mol) levels by a laser-induced fluorescence detector. This method has been applied to the analysis of monosaccharides and acid-hydrolyzed polysaccharides. Carbohydrate moieties derived from a glycoprotein were also tagged and determined.
Thirteen Habs-serotype strains could be classified into 11 groups depending on the characteristic patterns of 2-amino sugar composition; strains of serotypes 2, 5, and 7 had the same pattern, and each other serotype strain had its own distinctive pattern. In our classification, fucosamine and quinovosamine were of importance. We found that fucosamine from strain P14 (Habs serotype 1) was of the D configuration only, unlike DL-fucosamine from strain N10 (Habs serotype 11).
A simple and highly sensitive spectrophotometric method for the determination of glucosamine and its analogous amino sugars was established based on fading of the palladium(II)-o-hydroxyhydroquinonephthalein-hexadecyltrimethylammonium complex. In the determination of glucosamine, Beer's law is obeyed in the range of 0.02 - 0.18 microg ml(-1), with an effective molar absorptivity at 630 nm and the relative standard deviation being 8.4 x 10(5) dm3 mol(-1) cm(-1) and 1.08% (n = 10). This method is about 70-times more sensitive than the Elson-Morgan method. The method was successfully applied to the assay of glucosamine in actual samples.
The synthesis of several new sugar amino acid derivatives of 2-acetamido-2-deoxy-D-glucose, bearing a C-glycosyl functionality as building blocks for the design and synthesis of natural glycoconjugates mimetics, is described. These compounds were prepared from the readily accessible per-benzylated amino C-allyl glucopyranosyl compounds, with TMSOTf/Ac(2)O-mediated selective acetolysis of the 6-O-benzyl group as the key step.
Two alditol acetate methods for the gas chromatographic (GC) analysis of neutral and amino sugars were compared. Following sodium borohydride reduction, one method uses methylimidazole as an acetylation catalyst without prior removal of water or borate salts and the other method uses sodium acetate after removal of borate and water. Depending on the acetylation conditions, muramic acid produced different derivatives. With methylimidazole, reliable derivatization of muramic acid was not possible, although other sugars derivatized reliably. With sodium acetate, all sugars tested were reproducibly derivatized. The utility of the sodium acetate method is shown by the trace GC-mass spectrometric analysis of muramic acid and rhamnose derived from bacterial peptidoglycan-polysaccharide complexes in mammalian tissue.
Glycosylpyrazoles are efficiently formed by reaction of saccharide hydrazones with pentan-2,4-dione (acetylacetone), but in aqueous buffer, pyrazole derivatives of amino sugars couple with a further equivalent of acetylacetone affording high yields of ketoenamines. These ketoenamines were considerably more stable than the ketoenamines formed from 2-amino-2-deoxy aldoses that have been described as intermediates in the classical Elson-Morgan reaction. Moreover, high yields of perketoenamine derivatives were achieved with oligosaccharides derived from hydrolysis of chitosan. The removal of the ketoenamine moieties to regenerate the free amine was readily accomplished with aqueous hydrazine.
A new procedure for quantitating the amount of N-acetyl-D-mannosamine (ManNAc) or ManNAc-6-phosphate produced by 2'-epimerase activities involved in sialic acid metabolism has been developed. The ManNAc generated by the action of N-acetyl-D-glucosamine (GlcNAc) and UDP-GlcNAc 2'-epimerases is condensed with pyruvate through the action of N-acetylneuraminate lyase and the sialic acid released is measured by the thiobarbituric acid assay. For the analysis of prokaryotic GlcNAc-6-phosphate 2'-epimerase, ManNAc-6-phosphate can also be evaluated by this coupled assay after dephosphorylation of the sugar phosphate. This system provides a sensitive, rapid, reproducible, specific and simple procedure (feasible with commercial reagents) for measuring amino sugar 2'-epimerases from eukaryotic and prokaryotic sources. The technique reported here permitted us to detect UDP-GlcNAc 2'-epimerase and GlcNAc 2'-epimerase in mammalian cell extracts and GlcNAc-6-phosphate 2'-epimerase in bacterial extracts.
In vitro adherence of Candida albicans to human vaginal epithelial cells was studied, aimed at identifying the surface components involved in this binding. The inhibitory effect of yeast cell wall components and their constituents on the adherence of yeasts to epithelial cells was tested. Only chitin, its hydrolysate derivative and N-acetylglucosamine, the constituent of chitin, acted as inhibitors. Binding was also inhibited by the amino sugars glucosamine and mannosamine, while none of the other sugars tested (methylated or nonmethylated) exhibited such an effect. These data suggest that the amino groups of sugars are responsible for the inhibition of adherence of this eucaryotic microorganism to epithelial cells.
The metabolism of amino sugars in extracts of mouse cells was studied by the use of 2-acetamido-2-deoxy-alpha-D-glucopyranose, 2-acetamido-2-deoxy-alpha-D-manno-pyranose, and N-acetylneuraminic acid as tracers with or without added 0.5mM 2-deoxy-2-(2-fluoroacetamido)-alpha-D-glucopyranosyl pyrophosphate) and cytidine 5'-(N-acetylneuraminyl monophosphate) was inhibited by the fluoro sugar.
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Disaccharide anthracyclines analogues have been shown to exhibit different antitumour activity as compared with parents compounds doxorubicin and daunomycin. Here we report the crystal structure of the disaccharide analog MAR70 complexed with the DNA hexamer d(CGATCG). The structure has been solved at 1.54A resolution and is similar to previous crystallized anthracycline-DNA complexes with both sugar rings of the disaccharide chain lying in the DNA minor groove. Comparison with the structure of MEN10755 another disaccharide anthracycline co-crystallized with the same DNA hexamer suggests a correlation between the position of the amino sugar on the disaccharide chain and the conformation of this moiety when binding to DNA. This is discussed with respect to the influence on drug activity and on the possible interaction with other cellular targets.
Complete gas-liquid chromatographic separation of O-methyloxime acetates (syn and anti isomers) prepared from eight neutral sugars, three hexosamines, and muramic acid has been obtained, using a fused-silica Carbowax 20M capillary column. A single hydrolytic step for carbohydrate-containing biological material (less than or equal to 2.5 X 10(-3) M sugar solution in 4 N trifluoroacetic acid at 125 degrees C for 1 h) has been developed, and results have been compared with those obtained with standard hydrolytic conditions in order to ensure complete release of amino sugars from glycoproteins, together with minimum losses of neutral sugars. The combination of this acid hydrolysis with the above improved derivatization procedure for the gas-liquid chromatographic analysis has led to a simple, rapid, and sensitive analytical method, which has been successfully tested on three glycoproteins (fetuin, mucin, and peroxidase) and two plant cell-wall polysaccharide fractions (soluble fibers from carrots and soybeans).