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, [Leu 5 ]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 Phe 4 ; 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 Phe 4 in LeuEnk and DTLET significantly decreased δ‐receptor binding affinity (40 to >2,000‐fold). Among the synthesized analogues, [Phe(p‐NH 2 ) 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[Leu 5 ]Enk reported previously suggest subtle differences in interactions of Phe 4 with δ receptors depending on other modifications in the sequences. © 2003 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 71: 552–557, 2003