Synthesis and evaluation of N,N-dialkyl enkephalin-based affinity labels for delta opioid receptors

To develop affinity labels for delta opioid receptors based on peptide antagonists, the Phe(4) residues of N,N-dibenzylleucine enkephalin and N,N-diallyl[Aib(2),Aib(3)]leucine enkephalin (ICI-174, 864) were substituted with either Phe(p-NCS) or Phe(p-NHCOCH(2)Br). 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-NH(2)) 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 delta or mu opioid receptors. The delta 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 Phe(4) decreased binding affinity at both delta and mu 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 delta receptors (IC(50) = 34.9 nM) compared to N,N-dibenzylleucine enkephalin (IC(50) = 78.2 nM). The decreases in mu receptor affinities were greater than in delta receptor affinities so that all of the analogues tested exhibited significantly greater delta 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 delta 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 delta opioid receptors.