Spectroscopic investigations on dermorphin and its [L‐Ala2] analog

Dermorphin, DM, (H-Tyr-d-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) and its [l-Ala2]-analog, LDM, have been investigated in aqueous and DMSO solutions by means of 1H- and 13C-n.m.r., CD and u.v. with the aim of establishing possible differences in the conformational preferences of the two peptides. A study of the pH dependence of 13C chemical shifts resulted in the determination of the pKa of the α-amino group (6.97 0.02 for DM and 7.13 0.02 for LDM). A significantly higher value (7.75) is obtained for LDM from the pH dependence of CD spectra. This difference is attributed to the presence of associates at the high concentrations required for the 13C-n.m.r. measurements. The average pKa's of the two phenol groups have also been obtained and the values determined from u.v. absorption spectra (9.79 for DM, 9.85 for LDM) are in fair agreement with those determined from the CD spectra (9.94 for DM and 10.11 for LDM). An analysis of titration shifts in 13C-n.m.r. spectra indicates that the α-amino group is not involved in interactions stabilizing folded conformers in aqueous solution. Temperature coefficients and exchange times of the NH protons suggest that both peptides exist in DMSO-d6 as a mixture of rapidly interconverting conformers. A model is proposed in terms of an extended β-sheet conformation stabilized by intermolecular association at the high concentrations required for the n.m.r. measurements. CD spectra are dominated by contributions of the aromatic chromophores and cannot be used as a source of information about the main chain conformation. However, differences between the spectra of the two peptides indicate that the conformational freedom of one tyrosine side chain (probably Tyr1) is slightly more restricted in DM than in LDM.