Synthesis and opioid activity of conformationally constrained dynorphin A analogues. 2. Conformational constraint in...

Several cyclic lactam analogues of Dyn A-(1-13)NH2 were prepared in order to reduce the conformational flexibility in different regions of the native linear peptide. Cyclo[D-Asp(i),Dap(i+3)]Dyn A-(1-13)NH2 (Dap = alpha,beta-diaminopropionic acid) analogues were designed on the basis of molecular modeling using AMBER, which suggested that this constraint may be compatible with an alpha-helix. The cyclic portion of these constrained analogues spanned from residues 3 to 9, a region proposed by Schwyzer (Biochemistry 1986, 25, 4281) to adopt a helical conformation at kappa receptor sites. Analogues containing Dab (alpha,gamma-diaminobutyric acid) or Orn in position i + 3 were also synthesized to examine the effects of larger ring size. The cyclic peptides exhibited marked differences in binding affinities for kappa, mu, and delta receptors and in opioid activity in the guinea pig ileum (GPI). Cyclo[D-Asp6,Dap9]Dyn A-(1-13)NH2 showed both high kappa receptor affinity and potent agonist activity in the GPI, while cyclo[D-Asp3,Dap6]Dyn A-(1-13)NH2 exhibited very weak binding affinity at all opioid receptors as well as very weak opioid activity in the GPI. Cyclo[D-Asp5,Dap8]Dyn A-(1-13)NH2 showed moderate binding affinity for kappa receptors and was the most kappa selective ligand in this study, but this peptide exhibited very weak agonist activity in the GPI assay. Compared to the corresponding linear peptides, all of the cyclic peptides exhibited decreased mu receptor affinity, while kappa receptor affinity was retained or improved. Therefore the corresponding linear peptides were generally mu selective while the cyclic constrained peptides demonstrated slight selectivity for kappa vs mu receptors or were nonselective. Increasing the ring size by incorporating Dab or Orn in positions 6, 8, or 9 did not significantly affect the binding affinity for the three opioid receptor types nor the opioid activity observed in the GPI. Circular dichroism spectra of the cyclo[D-Asp(i),Dap(i+3)] derivatives in 80% trifluoroethanol at 25 and 5 degrees C suggested differences in the stability of a helical structure when the constraint was incorporated near the N-terminus vs in the middle of the peptide.