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 anta gonists, the Phe(4) residues of N,N-dibenzylleucine enkephalin and N,N-dial lyl[Aib(2),Aib(3)]leucine enkephalin (ICI-174,864) were substituted with ei ther Phe(p-NCS) or Phe(p-NHCOCH2Br). A general synthetic method was develop ed for the conversion of small peptide substrates into potential affinity l abels. The target peptides were synthesized using Phe(p-NH2) and a Boc/Fmoc orthogonal protection strategy which allowed for late functional group con version 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 triflu oromethanesulfonate (TMS-OTf). The target peptides were evaluated in radiol igand binding experiments in Chinese hamster ovary (CHO) cells expressing d elta or mu opioid receptors. The delta receptor affinities of the N,N-diben zylleucine enkephalin analogues were 2.5-10-fold higher than those for the corresponding ICI-174,864 analogues. In general, substitution at the para p osition 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 (IC50 = 34.9 nM) compared to N,N-dibenzylleuci ne enkephalin (IC50 = 78.2 nM). The decreases in mu receptor affinities wer e greater than in delta receptor affinities so that all of the analogues te sted exhibited significantly greater delta receptor selectivity than the un substituted parent peptides. Of the target peptides tested, only N,N-dibenz yl[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 a s the electrophilic affinity labeling moiety. As a result of this study, en kephalin analogue 2 emerges as a potential affinity label useful for the fu rther study of delta opioid receptors.