In an attempt to identify potential peptide-based affinity labels for opioid receptors, endomorphin-2 (Tyr-Pro-Phe-PheNH2), a potent and selective endogenous ligand for μ-opioid receptors, was chosen as the parent peptide for modification. The tetrapeptide analogs were prepared using standard Fmoc-solid phase peptide synthesis in conjunction with incorporation of Fmoc-Phe(p-NHAIIoc) and modification of the p-amino group. The electrophilic groups isothiocyanate and bromoacetamide were introduced into the para position on either Phe3 or Phe4; the corresponding free amine-containing peptides were also prepared for comparison. The peptides bearing an affinity label group and their free amine analogs were evaluated in a radioligand-binding assay using Chinese hamster ovary (CHO) cells expressing μ- and δ-opioid receptors. Modification on Phe4 was better tolerated than on Phe3 for μ-receptor binding. Among the analogs tested, [Phe(p-NH2)4]endomorphin-2 showed the highest affinity (IC50 = 37 nM) for μ-receptors. The Phe(p-NHCOCH2Br)4 analog displayed the highest μ-receptor affinity (IC50 = 158 nM) among the peptides containing an affinity label group. Most of the compounds exhibited negligible binding affinity for δ-receptors, similar to the parent peptide.
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