Stable-isotope-assisted NMR studies on ¹³C-enriched sialyl Lewis(X) in solution and bound to E-selectin

We report the determination of the solution structure and dynamics of ¹³C-enriched Neu5Acα23Galβ1-4(Fucα1-3)GlcNAc (sialyl Lewis(x)), together with its bound-state conformation in association with E-selectin. The availability of ¹³C-enriched material enabled both the measurement of trans-glycosidic ¹³C-¹³C coupling constants as conformational probes in the tetrasaccharide and the application of three-dimensional nuclear Overhauser effect ¹³C-¹H heteronuclear single-quantum correlation experiments for the measurement of intramolecular transferred nuclear Overhauser effects (TRNOEs) characterizing the bound-state conformation of the ligand. The additional dispersion afforded by the third (¹³C) dimension permitted straightforward identification and quantitation of these TRNOEs, and several TRNOEs were identified that had not previously been reported. The bound-state conformation of the ligand, determined by use of a heteronuclear full-relaxation matrix analysis of the TRNOE data, differs significantly from that reported in previous studies.