We obtained four DNCP--NalA conjugates (1-4); the conjugation of DNCP (35) and DNCP (36) was unsuccessful. DNCP--NalA conjugates (1-4) were pharmacologically characterized via radioligand binding and functional cAMP assays to investigate their affinity, potency and efficacy at the mouse KOR. The DNCP--NalA conjugates exhibited affinities in the low nanomolar range, with DNCP--NalA(1) being the strongest binder at KOR with a Ki value of 3.9 nM, as compared to -NalA, which has an affinity Ki of 72 nM. All four DNCP--NalA conjugates were full agonists at KOR (Emax = 89-101%), while -NalA alone was only a partial agonist with an EC50 of 130 nM and Emax of 61%. The most potent conjugates were DNCP--NalA(1) and (4) with EC50 values of 2.0 nM and 1.0 nM, respectively. DNCP--NalA(2) and DNCP--NalA(3) showed agonist activities at KOR with EC50 values of 7.5 and 14 nM in cAMP assay, respectively, while their Ki values were 31 and 24 nM in radioligand binding assay, respectively. Receptor reserve may account for this discrepancy between potency and affinity values of DNCP--NalA(2) and DNCP--NalA(4) which can be observed in functional GPCR assays with opioid receptors30. The higher potency and efficacy of the conjugates at KOR compared to -NalA can be attributed to interactions of the macrocycles with the ECL2 region as described later.