We report the determination of the solution structure and dynamics of 13C-enriched Neu5Acα2−3Galβ1−4(Fucα1−3)GlcNAc (sialyl Lewisx), 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 th...