Synthesis and evaluation of n,n-dialkyl enkephalin-based affinity labels for δ opioid receptors

Abstract

To develop affinity labels for δ opioid receptors based on peptide antagonists, the Phe4 residues of N,N-dibenzylleucine enkephalin and N,N-diallyl[Aib2,Aib3]leucine enkephalin (ICI-174,864) were substituted with either Phe(p-NCS) or Phe(p-NHCOCH2Br). A general synthetic method was developed for the conversion of small peptide substrates into potential affinity labels. The target peptides were synthesized using Phe(p-NH2) and a Boc/Fmoc orthogonal protection strategy which allowed for late functional group conversion of a p-amine group in the peptides to the desired affinity labeling moieties. A key step in the synthesis was the selective deprotection of a Boc group in the presence of a tert-butyl ester using trimethylsilyl trifluoromethanesulfonate (TMS-OTf). The target peptides were evaluated in radioligand binding experiments in Chinese hamster ovary (CHO) cells expressing δ or μ opioid receptors. The δ receptor affinities of the N,N-dibenzylleucine enkephalin analogues were 2.5-10-fold higher than those for the corresponding ICI-174,864 analogues. In general, substitution at the para position of Phe4 decreased binding affinity at both δ and μ receptors in standard radioligand binding assays; the one exception was N,N-dibenzyl[Phe(p-NCS)4]leucine enkephalin (2) which exhibited a 2-fold increase in affinity for δ receptors (IC50 = 34.9 nM) compared to N,N-dibenzylleucine enkephalin (IC50 = 78.2 nM). The decreases in μ receptor affinities were greater than in δ receptor affinities so that all of the analogues tested exhibited significantly greater δ receptor selectivity than the unsubstituted parent peptides. Of the target peptides tested, only N,N-dibenzyl[Phe(p-NCS)4]leucine enkephalin (2) exhibited wash-resistant inhibition of radioligand binding to δ receptors. To our knowledge, 2 represents the first peptide-based affinity label to utilize an isothiocyanate group as the electrophilic affinity labeling moiety. As a result of this study, enkephalin analogue 2 emerges as a potential affinity label useful for the further study of δ opioid receptors.