Studies on the conformational flexibility of α-l-rhamnose-containing oligosaccharides using 13C-site-specific labeling, NMR spectroscopy and molecular simulations: implications for the three-dimensional structure of bacterial rhamnan polysaccharides

Abstract

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 α-(1 → 2)- and/or α-(1 → 3)-linkages and we have therefore studied the disaccharide α-L-Rhap-(1 → 2)-α-L-Rhap-OMe to obtain information on conformational preferences at this glycosidic linkage. The target disaccharide was synthesized with ¹³C site-specific labeling at C1′ and at C2′, i.e., in the terminal group. 2D ¹H,¹³C-HSQC-HECADE and ¹H,¹³C-J-HMBC NMR experiments, 1D ¹³C and ¹H 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 ²J_C2′,H1′, ³J_C1′,C1, ³J_C1′,C3, ³J_C2′,C2, ²J_C1′,C2, ¹J_C1′,C2′, and ¹J_C1′,H1′. These data together with previously determined J_CH and ¹H,¹H 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 ϕ_H ≈ 40° and ψ_H ≈ −35°, consistent with experimental NMR data. In addition, MD simulations were carried out also for α-L-Rhap-(1 → 3)-α-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.