Rational design of near-infrared fluorescent probes for superoxide anion radical: Enhancement of self-stability and sensitivity by self-immolative linker

Fluorescent imaging of cellular superoxide anion radical (O2•-) is of great significance to investigate reactive oxygen species-related pathophysiological processes and drug metabolism. However, the application of this technique is far away from maximum partially due to the lack of suitable probes. In this work, we propose a new strategy for design of near-infrared (NIR) O2•- fluorescent probes in which p-cresol is used as a self-immolative linker to conjugate the NIR fluorophore DDAO (9H-1,3-Dichloro-7-hydroxy-9,9-dimethylacridine-2-one) with the O2•--sensing group (i.e., trifluoromethanesulfonate). The introduction of self-immolative linker effectively increases the self-stability of these probes under physiological conditions. Importantly, the electron-withdrawing halogen substituents on the linker greatly enhance the sensitivity of the probes to O2•-. As such, the representative probe DLS4 exhibits high self-stability over a broad range of pHs (5.0–8.5), high selectivity as well as excellent sensitivity to O2•- with a detection limit (LOD) of 7.3 nM and 720-fold fluorescence enhancement upon reaction with O2•-. Moreover, DLS4 enables imaging of O2•- generation in PMA-stimulated RAW 264.7 cells and HeLa cells, and the fluorescence intensities are proportional to the PMA concentrations. In addition, the doxorubicin-induced cytotoxicity of H9c2 cells was also evaluated using DLS4. The present study provides a novel strategy for molecular design of small-molecule O2•- fluorescent probes and the resulting probes show great potential as reliable tools to study the development and progression of O2•--related diseases and drug metabolism in various systems.