NMR analysis of conformationally dependent (n)J(C, H) and (n)J(C, C) in the trisaccharide α-L-Rhap-(1 → 2)[α-L-Rhap-(1 → 3)]-α-L-Rhap-OMe and a site-specifically labeled isotopologue thereof.

An array of NMR spectroscopy experiments have been carried out to obtain conformationally dependent (1)H,(13)C- and (13)C,(13)C-spin-spin coupling constants in the trisaccharide α-L-Rhap-(1 → 2)[α-L-Rhap-(1 → 3)]-α-L-Rhap-OMe. The trisaccharide was synthesized with (13)C site-specific labeling at C2' and C2″, i.e. in the rhamnosyl groups in order to alleviate (1)H spectral overlap. This facilitated the measurement of a key trans-glycosidic proton-proton cross-relaxation rate using 1D (1)H,(1)H-T-ROESY experiments as well as a (3)J(C, H) coupling employing 1D (1)H,(13)C-long-range experiments, devoid of potential interference from additional J coupling. By means of both the natural abundance compound and the (13)C-labeled sample 2D (1)H,(13)C-J-HMBC and (1)H,(13)C-HSQC-HECADE NMR experiments, total line-shape analysis of (1)H NMR spectra and 1D (13)C NMR experiments were employed to extract (3)J(C, H) , (2)J(C, H), (3)J(C, C), and (1)J(C, C) coupling constants. The (13)C site-specific labeling facilitates straightforward determination of (n)J(C, C) as the splitting of the (13)C natural abundance resonances. This study resulted in eight conformationally dependent coupling constants for the trisaccharide and illustrates the use of (13)C site-specific labeling as a valuable approach that extends the 1D and 2D NMR methods in current use to attain both hetero- and homonuclear spin-spin coupling constants that subsequently can be utilized for conformational analysis.