Synthesis of monocyclic and bicyclic peptides: A study of lactam-bridged beta-endorphin analogues

Conformational restriction is important in the study of biologically active peptides, and can alter the receptor binding properties of ligands such as peptide hormones. We have been interested in investigating the receptor binding and activating properties of human β-endorphin analogues that are conformationally constrained by side-chain to side-chain lactam bridges linking Lysi and Aspi+4 residue pairs. Based on previous studies of β-endorphin analogues, which demonstrated the significance of an amphiphilic α-helical region at the C-terminus, we designed three new analogues of β-endorphin. Two analogues were monocyclic and one was dicyclic with side-chain to side-chain lactam bridges placed in this amphiphilic region to constrain and stabilize the α-helical conformation.; Lactam-bridged peptides were synthesized and cyclized using previously known solid-phase conditions. Circular dichroism spectra of these peptides showed that all of the side-chain cyclized peptides were more helical than human β-endorphin in aqueous and TFE solutions. In the smooth muscle assays, where opioid activities are mediated by μ-receptors, β-endorphin and cyclo(16–20)(Lys16,Asp20)β-endorphin (EPc2) were more potent agonists than cyclo(21–25)(Lys21,Asp 25)β-endorphin (EPc1) and cyclo(16–20)(21–25) (Lys 16,21, Asp20,25)β-endorphin (EPbc). In radioligand binding experiments to study the displacement of [3H]-naloxone from μ-receptors, β-endorphin and monocyclic EPc1 showed strong agonist activity whereas monocyclic EPc2 and dicyclic EPbc behaved as partial agonists at these receptors. These results suggest that the lactam-bridge constraints placed in these peptides have altered the receptor binding affinities and signal transmission properties of β-endorphin.; To synthesize conformationally restricted biologically active peptides successfully, especially peptides having side-chain to side-chain lactam bridges placed in overlapping positions in the linear peptide sequence, we undertook the development of a new synthetic approach for the preparation of fully protected bicyclic peptides suitable for segment condensations. In a model study towards this goal, we used a combination of solid-phase and solution-phase synthesis to prepare a bicyclic heptapeptide that is known to stabilize the α-helix. Two different segment condensations 6 + 1 and 4 + 3 were explored. A Cobalt(III) protecting group at the carboxy terminus was employed, which allowed an extra degree of orthogonality to the protection scheme in the peptide synthesis. Both syntheses were successful, and was found that the 6 + 1 condensation gave better yields with fewer reaction steps, whereas the 4 + 3 segment condensation resulted in lower racemization at the amino acid involved in the condensation, which is the first amino acid attached to Rink acid resin, and is susceptible to racemization using the 6 + 1 condensation approach. Now because the bicyclic peptide segment can be successfully synthesized in quantitative yields, it can be inserted into biologically active peptides to improve receptor-binding and can also be used to develop simple model peptides to study protein folding.