A Radical-Generating Probe to Release Free Fluorophores and Identify Artemisinin-Sensitive Cancer Cells

The smart light-up probes have been extensively developed to image various enzymes and other bioactive molecules. Upon activation, these probes result in light-up fluorophores that exist in a protein-bound or a free form. The difference between these two forms has not yet been reported. Here, we present a pair of smart light-up probes that generate a protein-bound fluorophore and a free fluorophore upon activation by heme. Probe 8 generated a radical-attached fluorophore that predominantly existed in the free form, while probe 10 generated an α,β-unsaturated ketone-attached fluorophore that showed extensive labeling of proteins. In live-cell imaging, probe 8 showed greater fluorescence intensity than probe 10 when low concentrations (0.1–5 μM) of the probes were used, but probe 8 was less fluorescent than probe 10 when the concentrations of the probes were high (10 μM). Finally, probe 8 was used to reflect the activation level of the endoperoxide bond in cancer cells and to effectively distinguish ART-sensitive cancer cells from ART-insensitive ones.