ANALYSIS BY THE REDUCTIVE-CLEAVAGE METHOD OF A POLYSACCHARIDE CONTAINING 2-ACETAMIDO-2,6-DIDEOXY-D AND -L-GALACTOPYRANOSYL RESIDUES

Modifications of the reductive-cleavage method for the analysis of 2-acetamido sugars were tested using a polysaccharide containing alpha-linked 2-acetamido-2,6-dideoxy-L-galactopyranosyl (FucpNAc) residues, beta-linked D-FucpNAc residues and 2-linked D-glucopyranosyl residues. Reductive cleavage of the fully methylated O-antigenic polysaccharide of Pseudomonas aeruginosa ATCC 33358 in the presence of triethylsilane and trimethylsilyl trifluoromethanesulfonate, followed by quenching with methanol and subsequent acetylation, unexpectedly resulted in nearly complete cleavage of all glycosidic linkages to yield 2-O-acetyl-1,5-anhydro-3,4,6-tri-O-methyl-D-glucitol (1a) and methyl 3-O-acetyl-2,6-dideoxy-4-O-methyl-2-(N-methylacetam and -L-galactopyranosides (3a and 4a) as the major products. When the reductive-cleavage reaction was quenched with (S)-2-butanol, the major FucNAc derivatives were the diastereomeric (S)-2-butyl glycosides 15a and 17a, confirming the presence of enantiomeric FucNAc residues in the repeating unit of the polysaccharide. However, compounds 15a and 17a were not detected in equimolar proportions, presumably as a consequence of diastereoselectivity in the reaction of the chiral alcohol with the respective intermediate oxazolinium ions. Reductive cleavage of the fully methylated polysaccharide in the presence of a mixture of trimethylsilyl methanesulfonate and boron trifluoride etherate, followed by quenching with methanol, resulted in incomplete cleavage, giving rise to three disaccharide derivatives whose sequences overlap that of the trisaccharide repeating unit in the polysacharide. The lack of selectivity for cleavage at beta-D-FucNAc residues suggests that the alpha-L-FucNAc residues underwent anomerization prior to transglycosidation.