URONIC ACID/AMINO SUGAR LINKAGE AND 1-D NMR OF 2 SAMPLES

University Of Georgia, Athens GA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Glycosyl Linkage Analysis For glycosyl linkage analysis, the sample is methylated by a modification of the method of Hakomori, followed by pre-reduction by superdeuteride. Then the sample is repermethylated by the NaOH method, depolymerized, reduced, and acetylated;and the resultant partially methylated alditol acetates (PMAAs) are analyzed by gas chromatography-mass spectrometry (GC-MS) as described by York et al (1985) Methods Enzymol. 118:3-40. Briefly, an aliquot is taken from the sample after lyophilizing and dissolved in 0.4 mL DMSO, and 0.6 mL potassium dimsylate (3.6 M) in DMSO is added. After stirring for 7 hours at room temperature, the reaction mixture is cooled to 0 [unreadable]C, excess methyl iodide (0.7 mL) is added, and the tube sealed. Incubation is then continued overnight at room temperature. Following sample workup, the methylated material is reduced by super-deuteride, followed by re-permethylation using NaOH base and methyl iodide to make sure that the permethylation is complete. Then the permethylated sample is hydrolyzed using 2 M trifluoroacetic acid (2 h in sealed tube at 100 [unreadable]C), reduced with NaBD4, and acetylated using acetic anhydride/pyridine. The resulting PMAAs are analyzed on a Hewlett Packard 5890 GC interfaced to a 5970 MSD (mass selective detector, electron impact ionization mode);the separation of uronic acid is performed on a 30 m Supelco 2330 bonded phase fused silica capillary column. The resulting PMAAs are also analyzed on a Hewlett Packard 5890 GC interfaced to a 5970 MSD (mass selective detector, electron impact ionization mode);the separation of aminosugar is performed on a 30 m EC-1 bonded phase fused silica capillary column.