2D NOESY Simulations of Amide Protons in Acetamido Sugars

Because of the flexibility of most small peptides, quantitative simulation of amide proton nuclear Overhauser effects (NOE) by relaxation matrix methods which ignore internal motion are not thought to be generally useful. But NOE simulations for the carbon-bound protons of some complex oligosaccharides carried out as a function of geometry have produced an excellent fit to experimental data suggesting that these carbohydrates adopt relatively rigid conformations. Therefore we have devised a method for simulation of amide proton NOE and applied it to the amide protons of GlcNAc in some Lewis blood group oligosaccharides which have been proposed to adopt rigid single conformations. The quantitative simulation of amide proton NOE is complicated by chemical exchange with solvent, by the effects of rapid quadrupolar relaxation of ¹⁴N, and by dipolar relaxation of ¹⁴N. The effects of chemical exchange can be eliminated by choice of solvent pH and temperature, and quadrupolar effects can be shown to be small for this case. The influence of ¹⁴N dipolar relaxation is significant but can be readily calculated and incorporated into existing schemes for relaxation matrix computation of NOE in carbohydrates. NOESY spectra are reported in H₂O for two Lewis blood groups oligosaccharides, lacto-N-difucohexaose-1 and lacto-N-fucopentaose-1, with solvent suppression pulse sequences which have been used for peptides. Crosspeaks whose magnitudes depend on glycosidic dihedral angles are observed between the amide proton of GlcNAc and protons of the adjacent galactose residue. These values can be matched by simulations with the same model geometry deduced for these oligosaccharides from carbon-bound proton NOE supporting the rigid model hypothesis. Crosspeaks observed for the amide protons with protons in the same GlcNAc residue are sensitive to the orientation of the amide plane, and we propose slightly different dihedral angles for these two oligosaccharides. Vacuum ultraviolet circular dichroism spectra are reported on these human milk oligosaccharides which bear on the difference in the amide orientation in GlcNAc.