Abstract
As part of an effort to develop peptide-based affinity labels for opioid receptors, [Leu5]enkephalin (LeuEnk) and DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr), potent agonists for δ receptors, were selected as the parent peptides for further modification. The affinity label derivatives were prepared using standard Fmoc solid-phase peptide synthesis in conjunction with Fmoc-Phe(p-NHAlloc) (Fmoc: 9-flourenylmethoxycarbonyl;) and selective modification of the p-amino group on this residue. The electrophilic isothiocyanate and bromoacetamide groups were introduced into the para position of Phe4; the corresponding free amine-containing peptides were also prepared for comparison. The pure peptides were evaluated in radioligand binding assays using Chinese hamster ovary (CHO) cells expressing δ and μ opioid receptors. Modification of Phe4 in LeuEnk and DTLET significantly decreased δ-receptor binding affinity (40 to >2,000-fold). Among the synthesized analogues, [Phe(p-NH2) 4]DTLET showed the highest δ-receptor binding affinity (IC 50 = 39 nM) and enhanced selectivity for δ receptors compared to DTLET while other derivatives exhibited much lower δ receptor affinity. The differences in affinities between the two series of analogues and between the derivatives of LeuEnk and N,N-dibenzyl[Leu5]Enk reported previously suggest subtle differences in interactions of Phe4 with δ receptors depending on other modifications in the sequences.