Dermorphin and [Lys7]dermorphin, selective μ-opioid receptor ligands originating from amphibian skin, have been modified with various electrophiles in either the 'message' or 'address' sequences as potential peptide-based affinity labels for μ-receptors. Introduction of the electrophilic isothiocyanate and bromoacetamide groups on the para position of Phe3 and Phe5 was accomplished by incorporating Fmoc-Phe(p-NHAlloc) into the peptide followed by selective deprotection and modification. The corresponding amine-containing peptides were also prepared. The pure peptides were evaluated in radioligand binding experiments using Chinese hamster ovary (CHO) cells expressing μ- and δ-opioid receptors. In dermorphin, introduction of the electrophilic groups in the 'message' domain lowered the binding affinity by > 1000-fold; only [Phe(p-NH2)3]dermorphin retained nanomolar affinity for μ-receptors. Modifications in the 'address' region of both dermorphin and [Lys7]dermorphin were relatively well tolerated. In particular, [Phe(p-NH2)5, Lys7]dermorphin showed similar affinity to dermorphin, with almost 2-fold higher selectivity for μ-receptors. [Phe(p-NHCOCH2Br)5]- and [Phe(p-NHCOCH2Br)5, Lys7]dermorphin exhibited relatively high affinity (IC50 = 27.7 and 15.1 nM, respectively) for μ-receptors. However, neither of these peptides inhibited [3H]DAMGO binding in a wash-resistant manner.
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