NMR and molecular modeling studies on two glycopeptides from the carbohydrate-protein linkage region of connective tissue proteoglycans

Complete H-1 and C-13 NMR assignments are reported for two glycopeptides representing the carbohydrate-protein linkage region of connective tissue proteoglycans. These glycopeptides are the octasaccharide hexapeptide, Ser(Glc(p)A beta(1-->3) Gal(p)beta(1-->3)Gal(p)beta(1-->4)Xyl(p)beta)-Gly-Ser-Gly-Ser (Glc(p)A beta(1-->3)Gal(p)beta(1-->3)Gal(p)beta(1-->4)Xyl(p)beta)-Gly (1), and the tetrasaccharide dipeptide, Ser(Glc(p)A beta(1-->3)Gal(p)beta(1-->3)Gal(p)beta(1-->4)Xyl(p)beta)-Gly (2), The vicinal coupling constant data show that the monosaccharide residues adopt C-4(1) chair conformations. Distance geometry/simulated annealing calculations using 2D NOESY derived distance constraints yielded a single family of structures for the tetrasaccharide moiety, with well defined interglycosidic linkage conformations. The phi torsion angles of the glycosidic C1'-O1 bonds showed a strict preference for the -sc range whereas the psi torsion angles (O1-Cn) exhibited dependence upon the interglycosidic linkage position (-ac for beta(1-->3) linkage, +ac for beta(1-->4) linkage). The predominant conformation about the glycopeptide bond is phi = -sc and psi = +ac. The presence of strong d(alpha N) (i, i+1) NOE contacts, and the general absence of d(NN) (i, i+1) contacts (except for a weak Ser-5/Gly-6 d(NN) contact) and the d(beta N) (i, i+1) contacts (except for Ser-1/Gly-2) in the ROESY spectrum, suggest that the backbone for 1 is predominantly in an extended conformation. A comparison of the ROESY data for 1 with those obtained from the unglycosylated hexapeptide (3) of the same sequence suggests that glycosylation has only a marginal influence on the backbone conformation of the hexapeptide.