Abstract

The Phe¹ cyclic tetrapeptide Phe-c[d-Cys-Phe-d-Pen]NH₂(Et) (JH-54) has been shown previously to exhibit high affinity and selectivity for the μ-opioid receptor. To examine the role of the Phe¹ residue in the unexpected high affinity of this peptide, 11 analogs of JH-54 have been synthesized and evaluated for opioid ligand binding and for efficacy using the [³⁵S]GTPγS assay. Alteration of the bridging groups between the d-Cys² andd-Pen⁴ residues of JH-54 from dithioether to disulfide revealed the importance of the relative position of the aromatic rings of the first and third residues in determining μ- and δ-affinities. The one carbon distance between the α carbon and phenyl ring in the N-terminal residue was critical. Additional steric bulk in the N-terminal Phe¹ residue was accommodated without large reductions in affinity in two naphthyl analogs, but not with 3,3-(diphenyl)alanine. Conformational restriction of the Cα-Cβ and/or Cβ-Cγ bonds had little effect on affinities in two peptides with 2-amino-2-carboxytetralin in position 1, but it abolished activity in an isoquinoline analog and differentially altered activity in four phenylproline¹-containing peptides. Most surprisingly, replacement of the Phe¹ aromatic ring with cyclohexyl resulted in a peptide of moderate affinity (K_i = 32.5 nM) and potency (EC₅₀ = 58.8 nM). Thus, the tyrosylpara-hydroxyl substituent and even aromaticity in the N-terminal amino acid of these tetrapeptides are shown to be important, but not critical, features for μ-opioid receptor affinity, agonist potency, and efficacy.