Getting to know the nitrogen next door: HNMBC measurements of amino sugars

Long-range ¹H-¹⁵N correlations detected by the heteronuclear multiple-bond correlation (HMBC) experiment are explored for the characterization of amino sugars. The gradient-enhanced HMBC, IMPACT-HMBC, and a modified pulse sequence with the ¹J-filters removed, IMPACT-HNMBC, are compared for sensitivity and resolution. ¹⁵N chemical shifts and long-range proton correlations are reported using the IMPACT-HNMBC experiment for N-acetyl-glucosamine, N-acetyl-galactosamine, and for a series of glucosamine analogs with an N-sulfo substitution, unmodified amino group, and 6-O-sulfonation. As is common with sugars, for all the compounds examined both anomeric forms are present in solution. For each compound studied, the ¹⁵N chemical shifts of the α anomer are downfield of the β form. For the N-acetylated sugars, the β anomer has a unique long-range ¹⁵N correlation to the anomeric proton not observed for the α anomer. Though N-sulfonation results in a significant change in the ¹⁵N chemical shift of the glucosamine analogs, 6-O sulfo substitution has no significant effect on the local environment of the amino nitrogen. For N-acetylated sugars in D₂O solution, peaks in the ¹⁵N projection of the HMBC spectrum appear as triplets as a result of J-modulation due to ²H-¹⁵N coupling.