Monitoring inorganic pyrophosphatase activity with a fluorescent dizinc(II) complex of a macrocycle bearing one dansylamidoethyl antenna

The dizinc(II) complexes of L were used for recognition of anions by fluorescence spectroscopy (L being a heteroditopic hexaazamacrocycle with two diethylenetriamine coordination heads with a 2-methylpyridyl and a dansylamido ethyl arms, and m-xylyl spacers). The protonation of L and stability constants of its zinc(II) complexes were determined in aqueous solution, at 298.2±0.1 K and I = 0.10±0.01 M in KNO3. At 2:1 Zn2+/L ratio, the dinuclear complexes plainly dominate. The ligand alone does not display fluorescence change upon increasing the pH, but in the presence of Zn2+ the emission reaches a maximum at pH about 7.5, at which 95% of the ligand is in the dinuclear complex form. The emission appears concomitantly with the [Zn2H-1L]3+ species formation, which supports that the latter complex corresponds to metal-promoted deprotonation of the dansylamide NH. The [Zn2H-1L]3+ complexes were used for the recognition of phosphate and polyphosphate anions in aqueous solution buffered at pH 7.5 with 2 mM PIPPS, at 298.2 K. The binding of anions causes a decrease of the emission. The association constants determination revealed that HPPi3– is the strongest bound anion (log Kapp = 5.57), followed by HATP3– (two times weaker), and the remaining anions display lower binding constants having the HPO42– the weakest uptake by the receptor. The observed selectivity of the [Zn2H-1L]3+ receptor for PPi in relation to HPO42–, and the fact that the formation of [Zn2H-1L]3+ complex is not disturbed by the presence of Mg2+ allowed monitoring the PPi hydrolysis by inorganic pyrophosphatase in real-time.