Conformational analysis of the sialylα(2→3/6)N-acetyllactosamine structural element occurring in glycoproteins, by two-dimensional NOE 1H-NMR spectroscopy in combination with energy calculations by hard-sphere exo-anomeric and molecular mechanics force-field with hydrogen-bonding potential

The conformation is described of the sialylα(2→3/6)N-acetyllactosamine structural element, frequently occurring in glycoproteins. NOE spectroscopy of NeuAcα(2→3)Galβ(1→4)GlcNAcβ(1→N)Asn and NeuAcα(2→6)Galβ(1→4)GlcNAcβ(1→N)Asn is presented and for each glycosidic linkage, except for the α(2→6)-linkage, a number of interglycosidic NOEs are measured. The analysis of these effects is performed using a full relaxation matrix. Analysis of intraresidue NOEs provides a calibration of the calculation method. Hard-sphere exo-anomeric (HSEA) energy calculations indicate a single conformation for the β(1→4)-linkage in both compounds, both being consistent with the NOE data. HSEA and molecular-mechanics force-field with hydrogen-bonding potential energy calculations both indicate the existence of three preferred conformations for the α(2→3)-linkage. The analysis of the NOE spectra are consistent with a distribution over two or three of these conformations; by combination with the energy diagram for this linkage the existence of only a single conformation can be excluded. The NOE spectrum of the compound with the α(2→6)-linkage indicates a gt orientation for the Gal C-6 hydroxymethyl group. On this basis, the HSEA energy calculations for the α(2→6)-linkage indicate an extended low-energy surface with a number of preferred conformations. The absence of NOEs across this linkage is interpreted in terms of a non-rigid, but overall folded conformation of the NeuAcα(2→6)Galβ(1→4)GlcNAcβ structural element. This provides an explanation for the shift effects induced by α(2→6) attachment of NeuAc to the N-acetyllactosamine unit.