(3, 2)D H, C BIRD-HSQC-TOCSY for NMR structure elucidation of mixtures: application to complex carbohydrates.

Overlap of NMR signals is the major cause of difficulties associated with NMR structure elucidation of molecules contained in complex mixtures. A 2D homonuclear correlation spectroscopy in particular suffers from low dispersion of H chemical shifts; larger dispersion of C chemical shifts is often used to reduce this overlap, while still providing the proton-proton correlation information e.g. in the form of a 2D H, C HSQC-TOCSY experiment. For this methodology to work, C chemical shift must be resolved. In case of C chemical shifts overlap, H chemical shifts can be used to achieve the desired resolution. The proposed (3, 2)D H, C BIRD-HSQC-TOCSY experiment achieves this while preserving singlet character of cross peaks in the F dimension. The required high-resolution in the C dimension is thus retained, while the cross peak overlap occurring in a regular HSQC-TOCSY experiment is eliminated. The method is illustrated on the analysis of a complex carbohydrate mixture obtained by depolymerisation of a fucosylated chondroitin sulfate isolated from the body wall of the sea cucumber Holothuria forskali.