Synthesis and Evaluation of Derivatives of Leucine Enkephalin as Potential Affinity Labels for δ Opioid Receptors

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.