ANALYSIS OF RESIDUE TYPES IN CURDLAN SULFATE BY NUCLEAR-MAGNETIC-RESONANCE

The degree of substitution at each position in curdlan sulfate (CRDS), a polysaccharide having a linear (1 --> 3)-linked beta-D-glucose backbone and activity against human immunodeficiency virus in vitro, has been determined by proton and carbon-13 NMR spectroscopy. Complete H-1 NMR assignment of CRDS was carried out by using a combination of two-dimensional double-quantum-filtered correlated spectroscopy and two-dimensional homonuclear Hartmann-Hahn spectroscopy. The latter was especially useful for identifying H-1 signals of the heavily substituted polysaccharide, which shows severely overlapped signals in its one-dimensional spectrum. Nine anomeric proton signals were identified, and four substitution patterns could be determined with reference to the proton chemical shifts of curdlan itself. It was revealed that the C-6 position of CRDS is completely sulfated and about one-third of the C-2 hydroxyls are also substituted, while the degree of substitution at the C-4 position is much smaller. Two-dimensional nuclear Overhauser enhancement spectroscopy of CRDS showed some sequential NOE connectivities between H-1 of one residue and H-3 of the neighboring residue. Carbon-13 NMR resonances were also completely assigned by using a two-dimensional C-13-H-1 heteronuclear correlation method. Crosschecks for these assignments were given by an isotope effect experiment; i.e., the chemical shift values of C-13 nuclei linked to free hydroxyl groups in the polysaccharide dissolved in H2O were 6 to 8 Hz lower than those observed in D2O Solution, while the chemical shift difference was negligibly small for sulfated C-13 nuclei. The positions of sulfation in each glucose unit of CRDS derived from C-13 NMR analyses are consistent with those from H-1 NMR.