Synthesis of enkephalin-based affinity labels for delta opioid receptors

Affinity labels (irreversible ligands) are useful pharmacological tools to study receptor structure and function. As part of a program to prepare affinity labels based on opioid peptides, we developed a general synthetic strategy to introduce different electrophilic labeling moieties (i.e. isothiocyanate and bromoacetamide) via a p-amino functionality on a phenylalanine residue. The affinity labelling groups were incorporated into the Phe⁴ position of the delta opioid receptor antagonists ICI-174,864 (1) and N,N-dibenzyl leucine enkephalin (2). Using an orthogonal Boc/Fmoc protection strategy, the common peptide precursors were assembled in solution, the affinity labels introduced and the peptides deprotected. In cloned δ receptors expressed in CHO cells (3), only the isothiocyanate derivative of N,N-dibenzyl leucine enkephalin exhibited wash-resistant inhibition of [³H]DPDPE binding.