Context Effects on Human Milk Oligosaccharide Linkage Conformation and Dynamics Revealed by <i>MA’AT</i> Analysis

An emerging NMR method, <i>MA'AT</i> analysis, has been applied to investigate context effects on the conformational properties of several human milk oligosaccharides (HMOs). The <i>MA'AT</i> model of the β-(1→4) linkage in the disaccharide, methyl β-lactoside (MeL), was compared to those obtained for the same linkage in the HMO trisaccharides, methyl 2'-fucosyllactoside (Me2'FL) and methyl 3-fucosyllactoside (Me3FL), and in the tetrasaccharide, methyl 2',3-difucosyllactoside (Me2',3DFL). <i>MA'AT</i> analysis revealed significant context effects on the mean values and circular standard deviations (CSDs) of the psi (ψ) torsion angles in these linkages. α-Fucosylation at both O2'Gal and O3Glc of MeL to give Me2',3DFL significantly constrained librational motion about ψ (70% reduction in the CSD) and shifted its mean value by ∼18°. α-Fucosylation at the O3Glc of MeL to give Me3FL constrained ψ more than α-fucosylation at the O2Gal to give Me2'FL. These effects can be explained by the expected solution conformation of Me3FL, which closely resembles the Lewis^x trisaccharide. Comparisons of <i>MA'AT</i> models of ψ to those obtained by 1 μs aqueous molecular dynamics simulation (GLYCAM06) revealed identical trends, that is, <i>MA'AT</i> analysis was able to recapitulate molecular behavior in solution that was heretofore only available from MD simulation. The results highlight the capabilities of <i>MA'AT</i> analysis to determine probability distributions of molecular torsion angles in solution as well as degrees of librational averaging of these angles.