Glycopeptide-Membrane Interactions:  Glycosyl Enkephalin Analogues Adopt Turn Conformations by NMR and CD in Amphipathic Media

Four enkephalin analogues (Tyr-D-Thr-Gly-Phe-Leu-Ser-CONH2, 1, and the related O-linked glycopeptides bearing the monosaccharide β-glucose, 2, the disaccharide β-maltose, 3, and the trisaccharide β-maltotriose, 4) were synthesized, purified by HPLC, and biophysical studies were conducted to examine their interactions with membrane model systems. Glycopeptide 2 has been previously reported to penetrate the blood-brain barrier (BBB), and produce potent analgesia superior to morphine in mice (J. Med. Chem. 2000, 43, 2586−90 and J. Pharm. Exp. Ther. 2001, 299, 967−972). The parent peptide and its three glycopeptide derivatives were studied in aqueous solution and in the presence of micelles using 2-D NMR, CD, and molecular mechanics (Monte Carlo studies). Consistent with previous conformational studies on cyclic opioid agonist glycopeptides, it was seen that glycosylation did not significantly perturb the peptide backbone in aqueous solution, but all four compounds strongly associated with 5−30 mM SDS or DPC micelles, and underwent profound membrane-induced conformational changes. Interaction was also observed with POPC:POPE:cholesterol lipid vesicles (LUV) in equilibrium dialysis experiments. Although the peptide backbones of 1−4 possessed random coil structures in water, in the presence of the lipid phase they each formed a nearly identical pair of structures, all with a stable β-turn motif at the C-terminus. Use of spin labels (Mn2+ and 5-DOXYL-stearic acid) allowed for the determination of the position and orientation of the compounds relative to the surface of the micelle.