Mechanisms of ESIPT regulation and selective Cu²⁺-triggered fluorescence quenching in a flavonol probe

Recently, a novel flavonol fluorescent probe NSHF has been reported, which can specifically recognize copper ions (Cu²⁺) through a fluorescence "on-off" response. However, its underlying mechanism, particularly the excited-state intramolecular proton transfer (ESIPT) process and the origin of Cu²⁺-selective fluorescence quenching, remains unclear. Herein, systematic theoretical calculations were carried out to elucidate the microscopic mechanism. First, the effects of photoexcitation on the intramolecular hydrogen bond (IHB) and ESIPT energy barrier of NSHF were analyzed, and the differences between gas phase and acetonitrile were compared. Second, the photophysical characteristics of NSHF and NSHF+Cu²⁺ were investigated to reveal the intrinsic cause of the experimental fluorescence changes, and the solvent effects on the absorption/emission behaviors were also clarified. Finally, the related properties of NSHF and its complexes with Cu²⁺, Ca²⁺, Mg²⁺, and Zn²⁺ were compared to illustrate the mechanism of specific Cu²⁺ recognition by NSHF. The results demonstrate that photoexcitation strengthens the IHB of NSHF, promotes ESIPT, and leads to keto-form fluorescence emission. Compared with the gas phase, acetonitrile weakens the IHB, increases the ESIPT energy barrier, and induces red-shifts in both absorption and fluorescence spectra. NSHF exhibits localized emission character with strong fluorescence intensity. Upon coordination with Ca²⁺, Mg²⁺, and Zn²⁺, the complexes retain localized emission character with only slight variations in fluorescence intensity. In contrast, Cu²⁺ coordination completely prohibits the radiative transition and induces intense charge-transfer (CT) emission, which promotes non-radiative energy dissipation and results in fluorescence quenching. This study clarifies the ESIPT mechanism of NSHF and its specific fluorescence response toward Cu²⁺, providing a theoretical basis for understanding the sensing mechanisms of flavonol-based probes.