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 1H NMR spectra of oligosaccharides, using as an example a heparin-derived hexasaccharide. The anomeric (H1) region of the 1H 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 1H 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.