A high-affinity fluorescent Zn2+ sensor improved by the suppression of pyridine-pyridone tautomerism and its application in living cells

• 2,2′-Bipyridine-based fluorescent sensor 1 was evaluated as a novel Zn2+ sensor.
• Controlling the occurrence of tautomerism of 1 led to a dramatic increase in the affinity toward Zn2+.
• Good water solubility, a large Stokes shift (140 nm), and a 8.6-fold turn-on response to Zn2+ under physiological conditions were observed.
• The sensor 1 quantitatively detected the Zn2+ levels in living cells.
• We revealed the detection limit of 1 in living cells by using 65Zn2+ radioactive zinc isotope.

A high-affinity and low-molecular-weight fluorescent Zn2+ sensor 1 based on a 2,2′-bipyridine scaffold, which functions as both the chelating moiety for Zn2+ and the fluorophore, was developed and evaluated in biological applications. Controlling the occurrence of tautomerism of the chelating moiety for Zn2+ by introducing an amino group at the 6-position of the pyridine ring dramatically increased the binding affinity toward Zn2+. Fluorescent sensor 1 exhibited a nanomolar-range dissociation constant (Kd = 2.2 nM), a large Stokes shift (140 nm), and an 8.6-fold turn-on response to Zn2+ under physiological conditions. Fluorescence images revealed that fluorescent sensor 1 exhibits good properties with respect to aqueous solubility and cell permeability and can quantitatively detect the Zn2+ levels in living cells. Furthermore, a 65Zn2+ radioactive zinc isotope uptake study revealed the real concentration of accumulated Zn2+ at the detection limit. The novel fluorescent sensor 1 is a promising sensor for use in biological applications.