Secondary H/D isotope effect on hydrogen-bonded hydroxyl groups as a tool for recognizing distance constraints in conformational analysis of oligosaccharides

An 'isotopomer-selected NOE' (ISNOE) method for the unequivocal identification of mutually hydrogen-bond-linked hydroxyl groups is described. It relies on the fact that the OH group's signal patterns obtained for a partially deuterated sample originate from both isotopomers of the 'partner' hydroxyl, whereas a NOE for this group can originate from cross-relaxation with the protio isotopomer of this hydroxyl only. Hence, the isotopically shifted component of this group's signal does not appear in a ROE difference spectrum obtained with selective excitation of the 'partner' hydroxyl. This method is also applicable in those cases when only one of two mutually hydrogen-bonded groups exhibits resolvable isotope shifts. Furthermore, it is shown that isotope shifts may occur even for pairs of OH groups that are not mutually hydrogen-bonded, if these participate in hydrogen bonds with other hydroxyls and thereby affect conformational equilibria. The ISNOE experiment enables one to distinguish between these two sources of isotope shifts. Since the O . . . O distance for hydrogen-bonded hydroxyls in sugars is known to lie between 2.7 and 3.0 Angstrom, the hydrogen bonds established by ISNOE can be used in conformational analysis as reliable, motionally non-averaged distance constraints for the conformations containing these bonds.