Studies on the structure and dynamics of N-terminal sequences of dermorphin and their analogs by means of solid state NMR spectroscopy and XRD

Deltorphin I (Tyr-d-Ala-Phe-Asp-Val-Val-Gly-NH_2) and dermorphin (Tyr-d-Ala-Phe- -Gly-Tyr-Pro-Ser-NH_2) are natural opioid peptides that have been isolated from the skin of South American frogs [1]. The presence of d-amino acid is crucial for their biological activity. The synthetic analogs of given heptapeptides containing l-alanine are not analgesics [2]. Analysis of the influence of stereochemistry on molecular packing, dynamics and biological functions of neuropeptides is still important for receptor studies and practical applications (e.g. design of new selective pain killers). Presented research is focused on the structure and dynamics of two N-terminal sequences of dermorphin: tripeptide Tyr-d-Ala-Phe 1, tetrapeptide Tyr-D-Ala-Phe-Gly 2, and their analogs with l-alanine: Tyr-Ala-Phe 3 and Tyr-Ala-Phe-Gly 4, using solid state NMR and X-ray diffraction. This study clearly demonstrates that 1 and 2 crystallized under different conditions to form exclusively one structure [3, 4]. In contrast, tripeptide and tetrapeptide with l-Ala in the sequence very easily form different crystal modifications. Tyr-Ala-Phe 3 crystallizes into two forms: 3a and 3b [5], while Tyr-Ala-Phe-Gly 4 gives three modifications: 4a, 4b and 4c [4]. It seems that one of the factors, which can be important in the preorganization mechanism anticipating the formation of crystals, is the intramolecular CH-đ interaction between aromatic rings of tyrosine and/or phenylalanine and the methyl group of alanine. Such interaction is possible only for d-Ala residue. For l-Ala in the peptide sequence, the methyl group is aligned on the opposite side with respect at least to one of the aromatic groups. It can be further speculated that such internal CH-π contacts can also occur during the interaction of ligand–receptor, making the message sequence of opioid peptides more rigid and finally selective. By employing different NMR experiments (e.g. PISEMA MAS and PILGRIM) it was proven that the main skeleton of analyzed peptides is rigid, whereas significant differences in the molecular motion of the aromatic residues were observed [4, 6]. Solid state 2H NMR spectroscopy of samples with deuterium labeled aromatic rings: Tyrd4-d-Ala-Phe 5, Tyr-d-Ala-Phed5 6, Tyrd4-Ala-Phe 7, Tyr-Ala-Phe^d5 8 was used to analyze the geometry and time scale of the molecular motion. At ambient temperature, the tyrosine ring of sample 5 is rigid and in the sample 6 the phenylalanine ring undergoes a "π -flip". The tyrosine rings of form I of 7 and 8 are static, while the phenylalanine rings of form II of 7 and 8 undergo a fast regime exchange [6]. Variable temperature 2H measurements proved that the tyrosine and phenylalanine rings of two forms of compounds 7 and 8 became more mobile with increasing temperature. In contrast, the aromatic rings of samples 5 and 6 preserve their dynamics regime (static tyrosine and "π -flip" phenylalanine) in a large range of temperatures [6]. The analysis of 13C, 15N labeled tetrapeptide Tyr-D-Ala-Phe-Gly 2’-phospholipid membrane interactions suggests that peptide 2’ is aligned on the surface of the membrane (RFDR MAS) and the sandwich-like π -CH_3-π arrangement of the pharmacophore is preserved (DARR) [7]