Proton NMR Conformational Analysis of Cyclic β-Casomorphin Analogues of the Type Tyr-cyclo[-Nω-D-Orn-Xaa-Yaa-Gly-]

A conformational study of the cyclic β-casomorphin-5 analogues H-Tyr-cyclo-D-Orn-2-Nal-Pro-Gly-] ( 1 ) (μ-selective agonist; 2-Nal = 2-naphthylalanine), H-Tyr-cyclo-D-Orn-2-Nal-D-Pro-Gly-] ( 2 ) (mixed μ agonist/δ antagonist) and H-Tyr-cyclo-D-Orn-Phe-D-Pro-Gly-] ( 3 ) (highly potent μ and δ agonist) has been carried out using ¹H NMR spectroscopy. A complete assignment of the proton resonances of the three pentapeptides has been achieved. Compound 1 was shown to exist in two conformations, a major one (90%) characterized by a cis amide bond between 2-Nal³ and Pro⁴, and a minor one (10%) showing cis amide bonds both between D-Orn² and 2-Nal³ and between 2-Nal³ and Pro⁴. Peptides 2 and 3 each showed only one conformer with all-trans peptide bonds in both cases. Temperature dependence studies of the amide proton chemical shifts indicated the existence of several intramolecular hydrogen bonds in the case of compounds 2 and 3 but not in the case of peptide 1. The backbone conformations of 2 and 3 were found to be similar, both being characterized by two consecutive γ turns around the D-Pro⁴ and D-Orn² residues, respectively, and by a D-Orn²-CO←HN^δ-D-Orn² hydrogen bond. Altogether, the overall backbone conformation and the preferred side chain conformation were found to be roughly similar for the three title peptides. For all three compounds a close proximity between the aromatic moiety of the 3-position residue (2-Nal or Phe) and the D(or L)-Pro⁴ residue was established on the basis of ROESY experiments. The examination of low energy conformations obtained in molecular modelling studies by taking into account the various experimentally found NMR parameters (NOEs, vicinal H,H coupling constants, torsion angles, H-bonds) led to proposals of the solution conformation for each peptide. These conformations are in close agreement with a pharmacophore model for μ opioid receptor binding compounds.