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

Abstract

The conformation is described of the sialylalpha(2-> 3/6)N-acetyllactosamine structural element, frequently occurring in glycoproteins. NOE spectroscopy of NeuAcalpah(2-> 3) Galbeta(1-> 4)GlcNAcbeta(1-> N)Asn and NeuAcalpha-(2-> 6)Galbeta(1-> 4)GlcNAcbeta(1-> N)Asn is presented and for each glycosidic linkage, except for the alpha(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 beta(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 alpha(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 onyl a single conformation can be excluded. The NOE spectrum of the compound with the alpha(2-> 6)-linkage indicates a gt orientation for the Gal C-6 hydroxymethyl group. On this basis, the HSEA energy calculations for the alpha(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 NeuAcalpha(2->6)Galbeta(1->4)GlcNAcbeta structural element. This provides an explanation for the shift effects induced by alpha(2->6) attachment of NeuAc to the N-acetyllactosamine unit.