An effective strategy for structural elucidation of oligosaccharides through NMR spectroscopy combined with peracetylation using doubly 13C-labeled acetyl groups

The use of NMR spectroscopy for the elucidation of larger carbohydrate structures isolated from natural sources is principally limited by severe overlap of H-1 signals, poor sensitivity when experiments involve C-13 nuclei, and difficulties in conclusively establishing linkage positions. Peracetylation of oligosaccharides with doubly C-13-labeled acetyl groups provides several major advantages for their structural elucidation when combined with specifically tailored NMR pulse sequences. The 2.5-4.7 Hz J-coupling constants between acetyl carbonyl-C-13 nuclei and protons of the sugar ring at the sites of acetylation enables these sites to be readily assigned. By inference, glycosidic linkage positions on monosaccharides can be unambiguously determined. This can be used in lieu of permethylation analysis, yet does not require degradation of oligosaccharides. Spectral dispersion in the directly detected (1H) dimension is increased -2.6-2.7-fold due to the downfield shifting of sugar-ring protons at the positions of acetylation. Peracetylation also introduces three new frequency dimensions for NMR studies, namely the (CO)-C-13, C-13(Me), and H-1(Me) frequencies of the acetyl groups. These frequencies can be correlated to sugar protons, either independently or in combination, in alternative 2-, 3-, or 4-D experiments. The use of Hartmann-Hahn coherence transfer combined with zero-quantum dephasing periods permits purely absorptive in-phase multiplets to be extracted and enables accurate scalar couplings between ring protons to be measured, even in multidimensional experiments. Results are illustrated on a nonasaccharide-alditol derived from N-linked glycoproteins and on some smaller structures containing sialic acids and N-acetylhexosamines. Methods for small-scale sample acetylation using the superacylation catalyst, 4-dimethylaminopyridine, are described. A brief historical perspective pertinent to the fundamental contributions of Dr. R.U. Lemieux to the field of carbohydrate NMR is also presented.