Fluorescent Zinc Sensor with Minimized Proton-Induced Interferences: Photophysical Mechanism for Fluorescence Turn-On Response and Detection of Endogenous Free Zinc Ions

A new fluorescent zinc sensor (HNBO–DPA) consisting
of 2-(2′-hydroxy-3′-naphthyl)­benzoxazole
(HNBO) chromophore and a di­(2-picolyl)­amine (DPA) metal chelator has
been prepared and examined for zinc bioimaging. The probe exhibits
zinc-induced fluorescence turn-on without any spectral shifts. Its
crystal structure reveals that HNBO–DPA binds a zinc ion in
a pentacoordinative fashion through the DPA and HNBO moieties. Steady-state
photophysical studies establish zinc-induced deprotonation of the
HNBO group. Nanosecond and femtosecond laser flash photolysis and
electrochemical measurements provide evidence for zinc-induced modulation
of photoinduced electron transfer (PeT) from DPA to HNBO. Thus, the
zinc-responsive fluorescence turn-on is attributed to suppression
of PeT exerted by deprotonation of HNBO and occupation of the electron
pair of DPA, a conclusion that is further supported by density functional
theory and time-dependent density functional theory (DFT/TD-DFT) calculations.
Under physiological conditions (pH 7.0), the probe displays a 44-fold
fluorescence turn-on in response to zinc ions with a Kd value of 12 pM. The fluorescent response of the probe
to zinc ions is conserved over a broad pH range with its excellent
selectivity for zinc ions among biologically relevant metal ions.
In particular, its sensing ability is not altered by divalent transition
metal ions such as Fe­(II), Cu­(II), Cd­(II), and Hg­(II). Cell experiments
using HNBO–DPA show its suitability for monitoring intracellular
zinc ions. We have also demonstrated applicability of the probe to
visualize intact zinc ions released from cells that undergo apoptosis.
More interestingly, zinc-rich pools in zebrafish embryos are traced
with HNBO–DPA during early developmental stages. The results
obtained from the in vitro and in vivo imaging studies demonstrate the practical usefulness of the probe
to detect zinc ions.