N-cyclo-[Leu5]enkephalin: a rational approach for the synthesis of conformationally restricted cyclic pentapeptides

The enkephalins are neuropeptides belonging to the class of endogenous opioids. The conformationally restricted analog, N-cyclo-[Leu5]enkephalin, was recently synthesized. Since the synthesis of cyclic pentapeptides which lack proline and contain amino acids with bulky side chains is problematic, the synthesis, purification, and analytical characterization of N-cyclo-[Leu5]enkephalin is described in detail. This conformationally restricted cyclic pentapeptide was prepared from H-Gly-Phe-Leu-[O-(2,6-dichlorobenzyl)-Tyr]-Gly-NHNH2, which was synthesized by the solid-phase method of peptide synthesis. Cyclization was accomplished through an azide intermediate at high dilution, using high-pressure liquid chromatography to monitor the reaction. The desired cyclic monomer was isolated and purified by semipreparative HPLC. The structure of the purified cyclic product was confirmed by multiple chemical techniques including amino acid analysis, lack of an amino terminus (as assessed by reaction with ninhydrin and Edman reagent), and mass spectroscopy.