Synthesis, radiolabeling and receptor binding of [3H][(1S,2R)ACPC2]endomorphin-2.

Previously, we have shown that substitution of Pro(2) for cis-2-aminocyclopentanecarboxylic acid, ACPC in endomorphin-2 results in an analogue with greatly augmented proteolytic stability, high mu-opioid receptor affinity and selectivity. We now report the synthesis and biochemical characterization of [(3)H][(1S,2R)ACPC(2)]endomorphin-2 with a specific activity of 1.41 TBq/mmol (38.17 Ci/mmol). Specific binding of [(3)H][(1S,2R)ACPC(2)]endomorphin-2 was saturable and of high affinity with an equilibrium dissociation constant, K(d)=1.80+/-0.21nM and receptor density, B(max)=345+/-27 fmol x mg protein(-1) at 25 degrees C in rat brain membranes. Similar affinity values were obtained in kinetic and displacement assays. Both Na(+) and Gpp(NH)p decreased the affinity proving the agonist character of the radioligand. [(3)H][(1S,2R)ACPC(2)]endomorphin-2 retained the mu-specificity of the parent peptide. The new radioligand will be a useful tool to map the topographical requirements of mu-opioid peptide binding due to its high affinity, selectivity and enzymatic stability.