Bacterial polysaccharides are comprised of a variety of monosaccharides, L-rhamnose (6-deoxy-L-mannose) being one of them. This sugar is often part of alpha-(1 -> 2)- and/or alpha-(1 -> 3)-linkages and we have therefore studied the disaccharide alpha-L-Rhap-(1 -> 2)-alpha-L-Rhap-OMe to obtain information on conformational preferences at this glycosidic linkage. The target disaccharide was synthesized with C-13 site-specific labeling at C1' and at C2', i.e., in the terminal group. 2D H-1, C-13-HSQC-HECADE and H-1, C-13-J-HMBC NMR experiments, 1D C-13 and H-1 NMR spectra together with total line-shape analysis were used to extract conformationally dependent hetero- and homonuclear spin-spin coupling constants. This resulted in the determination of (2)JC(2',H1'), (3)J(C1',C1), (3)J(C1',C3), (3)J(C2',C2), (2)J(C1',C2), (1)JC(1',C2'), and (1)J(C1',H1'). These data together with previously determined J(CH) and H-1, H-1 NOEs result in fourteen conformationally dependent NMR parameters that are available for analysis of glycosidic linkage flexibility and conformational preferences. A 100 ns molecular dynamics (MD) simulation of the disaccharide with explicit water molecules as solvent showed a major conformational state at phi(H) approximate to 40 degrees and psi(H) approximate to -35 degrees, consistent with experimental NMR data. In addition, MD simulations were carried out also for alpha-L-Rhap-(1 -> 3)-alpha-L-Rhap-OMe and a rhamnan hexasaccharide. The gathered information on the oligosaccharides was used to address conformational preferences for a larger structure, a 2- and 3-linked nonasaccharide, with implications for the 3D structure of rhamnan polysaccharides, which should be regarded as flexible polymers.