Structural analysis of sialic acid-containing carbohydrates by the reductive-cleavage method

The applicability of the reductive-cleavage method to the structural analysis of sialic acid-containing carbohydrates was investigated using fully methylated methyl alpha- and beta-N-acetylneuraminic acid (1 and 2, respectively). Both compounds were fully stable to reductive cleavage in the presence of borane dimethylsulfide and boron trifluoride etherate but were slowly degraded in the presence of triethylsilane and promoters such as trimethylsilyl trifluoromethanesulfonate, boron trifluoride etherate, or a mixture of trimethylsilyl methanesulfonate and boron trifluoride etherate. However, after selective reduction of the ester groups of 1 and 2 with sodium borohydride, the products (3 and 4, respectively) underwent rapid reductive cleavage to yield the expected anhydroalditols, which were characterized as their acetates (8 and 11) by GLC-MS. The major product was also characterized as its benzoate (9) by 1H NMR spectroscopy. The usefulness of this analytical method was demonstrated using 3'-sialyllactose, i.e., alpha-Neu5Ac-(2-->3)-beta-D-Galp-(1-->4)-D-Glc. The methylated and ester-reduced trisaccharide (13) was reductively cleaved with Me2S.BH3-BF3.ET2O and the products were acetylated. Analysis by GLC-MS revealed the products expected from terminal (nonreducing) Neu5Ac (8 and 11), 3-linked Galp (16), and 4-linked Glcp (15) residues in relative molar ratios of 1.2:1.0:1.0, respectively. However, direct reductive cleavage of the fully methylated trisaccharide (12) yielded the fully methylated disaccharide-anhydroalditol derivative alpha-Neu5Ac-(2-->3)-1,5An-D-Gal (14) and the product (15) derived from 4-linked Glcp residues. These experiments therefore established both the composition and the sequence of the trisaccharide.