Nuclear magnetic resonance spectroscopy of peracetylated oligosaccharides having 13C-labeled carbonyl groups in lieu of permethylation analysis for establishing linkage substitutions of sugars

Abstract

Peracetylation of free hydroxyl groups in model saccharides with [ 13C-carbonyl]acetic anhydride resulted in additional splittings of sugar ring proton signals in NMR spectra, due to 3-bond J couplings between each acetyl carbonyl carbon and a sugar ring proton at that position. Quantification of 144 of these 3-bond coupling constants in different saccharide structures showed a range between 2.5 and 4.7 Hz, whereas all possible 4-bond couplings between sugar ring protons and acetyl carbonyl carbons were within linewidth (<0.5 Hz). Therefore, further splitting of sugar ring proton signals in the range of 2.5–4.7 Hz upon acetylation with a [ 13C-carbonyl]acetyl group identifies that position as (formerly) having a free hydroxyl group. This performs the same basic function as permethylation analysis, but does not require hydrolysis of glycosidic linkages. Additionally, proton-detected 2D heteronuclear multiple bond correlation (HMBC) experiments or proton-detected heteronuclear correlation spectroscopy (hetCOSY) enabled ring proton–carbonyl- 13C 3-bond J connectivities to be correlated with high sensitivity. Modified NMR pulse sequences are reported that include frequency selective decoupling schemes to enable coupling constants to be determined from 2D data. The tailored pulse sequences resulted in higher spectral resolution and sensitivity for [ 13C-carbonyl]–ring proton correlations.