One- and Two-Dimensional 1H-NMR Characterization of Two Series of Sulfated Disaccharides Prepared from Chondroitin Sulfate and Heparan Sulfate/Heparin by Bacterial Eliminase Digestion1

The 1H-NMR spectra of eight unsaturated disaccharides obtained by bacterial eliminase digestion of chondroitin sulfate and of heparan sulfate/heparin were recorded in order to construct an NMR data base of sulfated oligosaccharides and to investigate the effects of sulfation on the proton chemical shifts. These shifts were assigned by two-dimensional HOHAHA (homonuclear Hartmann-Hahn) and COSY (correlation spectroscopy) methods. The results indicated the following. (1) Two sets of proton signals were observed, corresponding to the alpha and beta anomers of these disaccharides, except those containing N-sulfated GlcN (2-deoxy-2-amino-D-glucose), in which only one set of signals appeared, corresponding to the alpha anomer. (2) Signals of protons bound to an O-sulfated carbon atom and those bound to the immediately neighboring carbon atoms were shifted downfield by 0.4-0.7 and 0.07-0.3 ppm, respectively. (3) For the disaccharides containing the N-sulfated GlcN, the signals of the protons bound to C-2 and C-3 were shifted upfield by 0.6 and 0.15 ppm, respectively, but that of C-1 was shifted downfield by 0.25 ppm when compared with those of the corresponding N-acetylated disaccharides. (4) For the chondroitin sulfate disaccharides sulfated on the C-4 position of GalNAc (2-deoxy-2-N-acetylamino-D-galactose) or the C-2 position of delta GlcA (D-gluco-4-ene-pyranosyluronic acid), the signal of the H-3 proton of delta GlcA or the H-4 proton of GalNAc was shifted upfield by 0.1-0.15 ppm, indicating the steric interaction of the two sugar components. (5) These effects of sulfation on chemical shifts are additive.