THE SOLUTION CONFORMATION OF SIALYL-ALPHA(2-]6)-LACTOSE STUDIED BY MODERN NMR TECHNIQUES AND MONTE-CARLO SIMULATIONS

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 {H-1,H-1} NOE contacts between aliphatic protons, our constraints are: (a) homonuclear NOEs of hydroxyl protons in H2O to other protons in the oligosaccharide, (b) heteronuclear {H-1,C-13} NOEs, (c) isotope effects of (OH)-H-1/(OH)-H-2 hydroxyl groups on C-13 chemical shifts, and (d) long-range heteronuclear scalar couplings across glycosidic bonds. We have used this approach to study the trisaccharide sialyl-alpha(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 alpha(2 --> 6)-linkage of the trisaccharide are best described by an equilibrium of 2 conformers with PHI-angles at -60-degrees or 180-degrees and of the 3 staggered rotamers of the OMEGA-angle with a predominant gt 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.