Conformational analysis of an α-galactosyl trisaccharide epitope involved in hyperacute rejection upon xenotransplantation

α-Galactosyl epitopes are carbohydrate structures bearing an α-Gal-(1→3)-Gal terminus (α-Gal epitopes). The interaction of these epitopes on the surface of animal cells with anti α-Gal antibodies in human serum is believed to be the main cause in antibody-mediated hyperacute rejection in xenotransplantation. In this paper, conformational analysis of an N-linked α-d-Galp-(1→3)-β-d-Galp-(1→4)-β-d-Glcp trisaccharide epitope was conducted in terms of each monosaccharide residue conformation, primary hydroxymethyl group configuration, and interglycosidic conformations. Selective 2D J-δ INEPT experiments have been carried out at three different temperatures to evaluate three-bond, long-range 13C–1H coupling constants for the crucial α-(1→3) linkage. The NMR experimental data were complemented by theoretical calculations. The flexibility and dynamics of the trisaccharide have been studied by Metropolis Monte Carlo simulations. Ensemble-averaged three-bond, long-range 13C–1H coupling constants and nuclear Overhauser effects were in good agreement with the experimental data. The α-(1→3) glycosidic linkage has shown a restricted flexibility as indicated by NMR spectroscopy and molecular modeling.