4-dimethylamino-1,8-naphthalimide as a turn on fluorophore for site selective and real time probing of the short-lived hydroxyl radical

Overproduction of reactive oxygen species (ROS) contributing to oxidative stress is characteristic of many diseases. Measuring ROS is quite challenging even in simple medium. This is particularly the case for highly reactive and short-lived ROS, even when indirect fluorescence probes are used. Here, a new approach is reported to measure the generation of the hydroxyl radical (HO•), one of the most reactive ROS. The discovery involves the demethylation of 4-dimethylamino-1,8-naphthalimide (NMI) fluorescence probe by HO• resulting in a significant increase in quantum yield by approximately 25-fold. This reaction was selective for HO• compared to other relevant ROS. By grafting NMI close to the Cu-binding domain of amyloid-beta (Aβ16), real-time fluorescence detection of HO• produced by Aβ16 bound Cu through the Fenton-type reaction was made possible. In a more complex environment with scavengers present, the HO• produced in the bulk was undetectable, indicating the short lifespan of HO• in the presence of other molecules. This study serves as a proof of concept demonstrating that NMI reaction-based HO• detection could be a valuable and versatile tool for investigating the role of HO• in real-time within defined locations in biologically relevant complex systems.