Purification of Cyclic Tetrapeptides: cyclo[Pro-Sar-Phe-D-Trp] and cyclo[Pro-Sar-Phe-D-(4F)Phe]

Abstract

Opioid addiction is a national epidemic; in 2017, 67.8% of drug overdoses involved opioids.1 To combat this addiction crisis, scientists are seeking to develop therapies that would reduce opioid-seeking tendencies. Previous research by Brice-Tutt et al.2 demonstrated the peptide cyclo[Pro-Sar-Phe-D-Phe] blocks drug-induced and stress-induced morphine seeking behavior in mouse models, making it a promising lead compound. Our research focuses on determining the structure activity relationship of this scaffold at residue four. A series of peptide analogs was synthesized, and the present study reports on the purification of two such analogs, cyclo[Pro-Sar-Phe-D-Trp] and cyclo[Pro-Sar-Phe-D-(4F)Phe]. Normal-phase and reverse-phase flash chromatography techniques were used to isolate pure fractions of the peptides. Each peptide’s purity and peptide identity were confirmed using liquid chromatography and mass spectrometry. Results indicate an oxidation process occurring with cyclo[Pro-Sar-Phe-D-Trp] that could be affecting purity and should be investigated further. Fractions of cyclo[Pro-Sar-Phe-D-Trp] and cyclo[Pro-Sar-Phe-D-(4F)Phe] were 98.7% and 98.0% pure, respectively. These peptides will be sent to the University of Florida for in vivo testing. From these results, structure-activity analysis can be conducted to better determine the importance of position four in receptor affinity and activity