Zinc Sensing by a Janus-Faced Fluorophore and its Application in the Cellular Microenvironment

Abstract

In this work, we have investigated the metal sensing capabilities of dimeric fused heterocycles, PQ-BCN and PQN-BCN, bearing a pyrrole-pyridine chelating unit. PQ-BCN displayed selectivity towards Zn²⁺ in micromolar levels by steady-state absorbance and fluorescence-assisted screening experiments. The binding stoichiometry of the complex was determined to be a 1 : 2 probe-to-metal ratio. PQN-BCN, however, did not show any preferences towards any metal. NMR titration experiments provided insights into the differing behaviors of PQ-BCN and PQN-BCN, highlighting the role of pyrrolic NH in Zn²⁺ coordination. Different binding affinities obtained using absorbance and fluorescence titrations indicated that the first Zn²⁺ binds strongly in the ground state, but the second Zn²⁺ coordination contributes significantly to fluorescence enhancement. Computational experiments revealed the preference for PQ-BCN compared to PQN-BCN, possibly originating from the difference in the Mulliken charges of the respective chelating nitrogens. A lifetime study on the PQ-BCN-Zn²⁺ complex gave a bi-exponential fit with an enhancement of its slower component upon interaction with Zn²⁺ compared to the free probe. It is the slower component that significantly contributes to the enhancement of fluorescence. Cellular studies were further performed with PQ-BCN using MOC-2 (oral cancer cells), revealing the probe‘s non-toxic nature and its capability to monitor zinc in cells in the presence/absence of zinc transporter ZnPT. Visualization of zinc was also successful without its external supplementation when cells were under peroxide-induced oxidative stress, thereby demonstrating the innate capability of the probe to detect zinc.