New conformational constraints in isotopically (13C) enriched oligosaccharides

Multidimensional heteronuclear NMR studies have been applied to theresonance assignment and conformational analysis of 13C-enrichedNeu5Acalpha2-3Galbeta1-4Glc. It is demonstrated that three-dimensionalROESY-HSQC experiments provide through-space distance restraints whichcannot be observed with conventional homonuclear 1H techniques due toresonance overlap. In particular, connectivities demonstrating theexistence of the "anti" conformation about the Galbeta1-4Glc glycosidiclinkage are unambiguously observed. It is shown that 13C isotopicenrichment of the trisaccharide at a level >95% enables straightforwardmeasurement of trans-glycosidic 1H-13C and 13C-13C coupling constants and aKarplus-type relation is derived for the latter. In total 15 conformationalrestraints were obtained for the trisaccharide in aqueous solution, all ofwhich were in excellent agreement with theoretical parameters computed froma 5 ns molecular dynamics simulation of the glycan.