NOVEL SYNTHESIS, PURIFICATION, AND CHARACTERIZATION OF THE CYCLODIPEPTIDES CYCLO-(L-PHE-L-PRO) AND CYCLO-(L-TYR-L-PRO) FOR APPLICATION AGAINST ECTOPARASITES IN ANIMAL PRODUCTION

The growing demand for sustainable pest control practices in animal production necessitates alternative biotechnological processes for managing ectoparasites that compromise animal health and productivity. Traditional chemical management faces significant pressure due to the rapid evolution of drug resistance. In this context, cyclodipeptides (CDPs) emerge as promising and versatile candidates for the development of new natural biopesticides. Recent studies indicate that this class of molecules, characterized by a diketopiperazine ring, possesses a versatile structure, featuring conformational rigidity, specific intermolecular interactions and significant acaricidal properties. In this way, this study aims to synthesize, purify, and characterize two cyclodipeptides named cyclo-(L-Phe-L-Pro) and cyclo-(L-Tyr-L-Pro) to combat ectoparasites that affect cattle and poultry. Linear precursor sequences were assembled using standard Fmoc-based Solid-Phase Peptide Synthesis (SPPS) strategy, employing an Oxyma Pure/DIC coupling system. The synthesis began with resins pre-loaded with the respective aromatic amino acids (L-phenylalanine or L-tyrosine) for the subsequent coupling of L-proline. Intramolecular ring closure to form the CDPs occurred concomitantly during the final Fmoc deprotection step of the proline residue, using 20% piperazine in DMF/ethanol, which promoted spontaneous cyclization with the C-terminus and the subsequent cyclic peptide release from the resin support. The target cyclodipeptides are purified via Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC, Shimadzu Prominence) and lyophilized, with molecular characterization confirmed by Electrospray Ionization Mass Spectrometry (ESI-MS, Shimadzu LCMS-8045) in positive detection mode. Therefore, standard SPPS promoted successful obtaining of the CDPs molecules, which will be necessary for the future in vitro and in vivo studies of new bioinsecticides and bioacaricides for poultry and cattle production.