SOLUTION STRUCTURE OF A SYNTHETIC N-GLYCOSYLATED CYCLIC HEXAPEPTIDE DETERMINED BY NMR-SPECTROSCOPY AND MD CALCULATIONS

The synthesis and conformational analysis by NMR spectroscopy and MD calculations of the N-glycosylated cyclic hexapeptide cyclo(-D-Pro-Phe-Ala-[N-2-acetamido-2-desoxy-beta-D-glucopyranosyl)]Gln-Phe-Phe-) (I) and the cyclic hexapeptide precursor cyclo(-D-Pro-Phe-Ala-Glu(OtBu)-Phe-Phe-) (II) were carried out to study the influence of N-glycosylation on conformation of peptides. For both compounds, all of the distance constraints derived from 2D NOE measurements could not be satisfied by one conformation. Therefore, second conformers interconverting fast compared to the NMR time scale are assumed. The two conformations differ in the beta-turn structure between Ala3 and Phe6 (beta-II- or beta-I-turns, respectively). The beta-II'-turn about amino acids D-Pro1 and Phe2 is highly conserved in both MD simulations. The conformations were refined by using restrained MD simulations in vacuo and in water. Additional MD simulations with application of time-dependent distance constraints provide further information about the internal flexibility of I. The conformational equilibrium could be confirmed; several conformational changes were detected evidenced by a large number of torsion angle fluctuations during the time scale of the simulation. Both proposed backbone conformers were significantly populated. The averaging over coupling constants and NOE data reveal the high flexibility of the structure and the good agreement with experimental data for both I and II. The N-glycosylation does not affect the conformation or the overall shape of the peptide backbone or side chains. It has no influence on the hydrogen-binding pattern or on the fast dynamical equilibrium of the molecule.