The solution conformation of sialyl-α(2→6)-lactose studied by modern NMR techniques and Monte Carlo simulations

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Abstract
We present a comprehensive strategy for detailed characterization of the solution conformations of oligosaccharides by NMR spectroscopy and force-field calculations. Our experimental strategy generates a number of interglycosidic spatial constraints that is sufficiently large to allow us to determine glycosidic linkage conformations with a precision heretofore unachievable. In addition to the commonly used {1H,1H} NOE contacts between aliphatic protons, our constraints are: (a) homonuclear NOEs of hydroxyl protons in H2O to other protons in the oligosaccharide, (b) heteronuclear {1H,13C} NOEs, (c) isotope effects of O1H/O2H hydroxyl groups on13C chemical shifts, and (d) long-range heteronuclear scalar coupling across glycosidic bonds. We have used this approach to study the trisaccharide sialyl-α(2→6)-lactose in aqueous solution. The experimentally determined geometrical constraints were compared to results obtained from force-field calculations based on Metropolis Monte Carlo simulations. The molecule was found to exist in 2 families of conformers. The preferred conformations of the α(2→6)-linkage of the trisaccharide are best described by an equilibrium of 2 conformers with Φ angles at −60° or 180° and of the 3 staggered rotamers of the Ω angle with a predominantgt conformer. Three intramolecular hydrogen bonds, involving the hydroxyl protons on C8 and C7 of the sialic acid residue and on C3 of the reducing-end glucose residue, contribute significantly to the conformational stability of the trisaccharide in aqueous solution.

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