The degree of substitution at each position in curdlan sulfate (CRDS), a polysaccharide having a linear (1 + 3)-linked @-glucose backbone and activity against human immunodeficiency virus in vitro, has been determined by proton and carbon-13 NMR spectroscopy. Complete 'H 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 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-l of one residue and H-3 of the neighboring residue. Carbon-13 NMR resonances were also completely assigned by using a two-dimensional 'sC-'H heteronuclear correlation method.
Crosschecks for these assignments were given by an isotope effect experiment; i.e., the chemical shift values of 13C nuclei linked to free hydroxyl groups in the polysaccharide dissolved in H,O were 6 to 8 Hz lower than those observed in D,O solution, while the chemical shift difference was negligibly small for sulfated 13C nuclei. The positions of sulfation in each glucose unit of CRDS derived from 13C NMR analyses are consistent with those from 'H NMR.