Two trinucleating ligands N(CH2-m-C6H4-CH(2)tacn)(3) (L1, tacn = 1,4,7-triazacyclononane) and 1,3,5-C6H3(o-C6H4-CH2N(CH(2)py)(2))(3) (L2, py = pyridine) were synthesized, and their tricopper(II) complexes [(CuCl)(3)L1]Cl-3 (1) and [(CuCl)(3)L-2]Cl-3 (2) were investigated as catalysts for the hydrolysis of 4-nitrophenyl phosphate (NPP). Structural analysis revealed that the three Cu(II) ions in L1 are adjacent to each other, while those in L2 lack such features due to the steric hindrance of dipicolylamine (dpa) units. Hydrolysis results indicated that vicinal metal ions within same ligand backbone have high levels of cooperation for substrate recognition and thus high initial catalytic efficiency, but the binding of inorganic phosphate as the hydrolysis product at the tricopper site is a major disadvantage. Significantly, complex [((CuCl)-Cl-II)(3)(HPO4)L1](PF6) (3) was isolated from NPP hydrolysis with 1 and was characterized by single crystal X-ray diffraction. In this structure, the triply bridging phosphate at the tricopper site disorders in two opposite orientations. These results, in collaboration with high-resolution mass spectrometry studies, indicated that NPP was first coordinated at the tricopper site, and was subsequently attacked by a water molecule from the underneath of the tricopper plane, a reaction model that supports the proposed mechanism of biological phosphate hydrolysis at the trimetallic active site of pyrophosphatases. (C) 2015 Elsevier B.V. All rights reserved.