Role of metal ion in specific recognition of pyrophosphate ion under physiological conditions and hydrolysis of the phosphoester linkage by alkaline phosphatase.

Complexes synthesized from Zn(II), Cu(II), and Cd(II), using a dipicolyl amine derivative (L), showed unique specificity toward pyrophosphate ion (PPi or P4O7(4-)) among all other common anionic analytes, including different biologically significant phosphate ion (PO4(3-), H2PO4(2-)) or phosphate-ion-based nucleotides, such as AMP, ADP, ATP, and CTP. However, the relative affinities of PPi toward these three metal complexes were found to vary and follow the order K(a)(L.Zn-PPi) > are given in units of (a)(L.Cu-PPi) ≥ K(a)(L.Cd-PPi). Luminescence responses of the receptor L were substantial on binding to Zn(2+) and Cd(2+), while relatively a much smaller luminescence response was observed in the presence of Cu(2+). Luminescence responses of L.M-PPi (M is Zn(2+), Cd(2+), and Cu(2+)) were further modified on binding to the PPi ion. This could be utilized for quantitative detection of PPi in physiological condition as well as for developing a real time "turn-on" (for L.Zn and L.Cu) and "turn-off" (for L.Cd) fluorescence assay for evaluating the enzymatic activity of alkaline phosphatase (ALP). Experimental results revealed how the subtle differences in the binding affinities between PPi and M in L.M (M is Zn(2+), Cd(2+), and Cu(2+)), could influence the cleavage of the phosphoester linkage in PPi by ALP. The DFT calculations further revealed that the hydrolytic cleavage of the metal ion coordinated phosphoester bond is kinetically faster than that for free PPi and thus, rationalized the observed difference in the cleavage of the phosphoester bond by an important mammalian enzyme such as ALP in the presence of different metal complexes.