Determination of the primary structures of heparin- and heparan sulfate-derived oligosaccharides using band-selective homonuclear-decoupled two dimensional 1H NMR experiments

Band-selective homonuclear-decoupled (BASHD) two-dimensional NMR experiments are applied to the assignment of H-1 NMR spectra of oligosaccharides, using as an example a heparin-derived hexasaccharide. The anomeric (H1) region of the H-1 NMR spectrum is band-selected in the F-1 dimension. With the increased resolution that results from less truncation of interferograms in the t(1) dimension, finer digital resolution in the F-1 dimension, and collapse of multiplets to singlets in the F-1 dimension, cross-peaks to the anomeric protons of the two iduronic acid residues, which overlap in normal two-dimensional total correlation spectroscopy (TOCSY) and rotating frame Overhauser enhancement spectroscopy (ROESY) spectra of the hexasaccharide, are resolved in BASHD-TOCSY and BASHD-ROESY spectra, leading to an unequivocal assignment of the H-1 NMR spectrum of the hexasaccharide. Incorporation of the water attenuation by transverse relaxation method for the complete and selective elimination of the water resonance into two-dimensional BASHD experiments makes it possible to observe oligosaccharide resonances at the frequency of the water resonance, as demonstrated with the observation of cross-peaks to resonances at the frequency of the water resonance in BASHD-TOCSY spectra of a second heparin-derived hexasaccharide.