Glycosidic linkage flexibility: The ψ torsion angle has a bimodal distribution in α-L-Rhap-(1→2)-α-L-Rhap-OMe as deduced from 13C NMR spin relaxation.

The molecular dynamics (MD) computer simulation technique is powerful for the investigation of conformational equilibrium properties of biomolecules. In particular, free energy surfaces of the torsion angles (those degrees of freedom from which the geometry mostly depends) allow one to access conformational states, as well as kinetic information, i.e., if the transitions between conformational states occur by simple jumps between wells or if conformational regions close to these states also are populated. The information obtained from MD simulations may depend substantially on the force field employed, and thus, a validation procedure is essential. NMR relaxation data are expected to be highly sensitive to the details of the torsional free energy surface. As a case-study, we consider the disaccharide α-l-Rhap-(1 → 2)-α-l-Rhap-OMe that features only two important torsion angles, ϕ and ψ, which define the interglycosidic orientation of the sugar residues relative to each other, governed mainly by the exo-anomeric effect and steric interactions, respectively. In water, a ψ- state is preferred, whereas in DMSO, it is a ψ+ state, suggesting inherent flexibility at the torsion angle. MD simulations indicated that bistable potentials describe the conformational region well. To test whether a unimodal distribution suffices or if a bimodal distribution better represents molecular conformational preferences, we performed an alchemical morphing of the torsional free energy surface and computed T1, T2, and NOE13C NMR relaxation data that were compared to experimental data. All three NMR observables are substantially affected by the morphing procedure, and the results strongly support a bimodal Boltzmann equilibrium density with a major and a minor conformational state bisected at ψ ≈ 0°, in accord with MD simulations in an explicit solvent.