NEW CONFORMATIONAL CONSTRAINTS IN ISOTOPICALLY (C-13) ENRICHED OLIGOSACCHARIDES

Multidimensional heteronuclear NMR studies have been applied to the re sonance assignment and conformational analysis of C-13-enriched NeuSAc alpha 2-3Gal beta 1-4Glc. It is demonstrated that three-dimensional R OESY-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 ex istence of the ''anti'' conformation about the Gal beta 1-->4Glc glyco sidic linkage are unambiguously observed, It is shown that C-13 isotop ic enrichment of the trisaccharide at a level >95% enables straightfor ward measurement of trans-glycosidic H-1-C-13 and C-13-C-13 coupling c onstants and a Karplus-type relation is derived for the latter, In tot al 15 conformational restraints were obtained for the trisaccharide in aqueous solution, all of which were in excellent agreement with theor etical parameters computed from a 5 ns molecular dynamics simulation o f the glycan.