Dispersing the crowd: Adopting ¹³ C direct detection for glycans

As a direct consequence of technological advancements, the interest in direct detection of low-gamma/low-sensitivity heteronuclei for NMR experiments has been revived. Until recently, experimental development of ¹³C/¹⁵N detected experiments has been focused on protein NMR. In the present report, we extend the use of ¹³C-detected experiments to structural studies of glycans in natural abundance. The narrow ¹H and wider ¹³C signal dispersion make glycans ideal candidates for heteronuclear detection. We show that ¹³C-detected HSQC offers a ten-fold increase in ¹³C dimension resolution compared to the analogous ¹H-detected HSQC, when the experiments are acquired for the same amount of time. The enhanced resolution comes at the expense of 2 to 3-fold loss in SNR; however, the observed signal loss is a fraction of the theoretical 8-fold difference expected between experiments. Further, we show that by combining a ¹H constant time element (CT), SMILE data reconstruction and ¹³C-direct detection, complete resonance assignments of highly degenerate glycan signals are possible. Finally, we demonstrate the potential of our strategy to aid in the assignment of complex glycans, by using a novel ¹³C-detected version of the CT-HSQC-TOCSY experiment performed on sialyl Lewis X pentasaccharide model system.

Keywords: (13)C direct detection; Carbohydrates; Cellobiose; Constant time; Glycans; Low-gamma nuclei; Natural abundance; Oligosaccharide; Pure shift; Resolution; SMILE; Sialic acid; Sialyl Lewis X; TOCSY.