New conformational constraints in isotopically (13C) enriched oligosaccharides

Multidimensional heteronuclear NMR studies have been applied to the resonance assignment and conformational analysis of C-13-enriched NeuSAc alpha 2-3Gal beta 1-4Glc. It is demonstrated that three-dimensional ROESY-HSQC experiments provide through-space distance restraints which cannot be observed with conventional homonuclear H-1 techniques due to resonance overlap. In particular, connectivities demonstrating the existence of the "anti" conformation about the Gal beta 1-->4Glc glycosidic linkage are unambiguously observed, It is shown that C-13 isotopic enrichment of the trisaccharide at a level >95% enables straightforward measurement of trans-glycosidic H-1-C-13 and C-13-C-13 coupling constants and a Karplus-type relation is derived for the latter, In total 15 conformational restraints were obtained for the trisaccharide in aqueous solution, all of which were in excellent agreement with theoretical parameters computed from a 5 ns molecular dynamics simulation of the glycan.