Unprecedented Photoinduced‐Electron‐Transfer Probe with a Turn‐ON Chemiluminescence Mode‐of‐Action

PeT-based fluorescent probes were demonstrated to be powerful tools for detection and imaging, owing to their significant fluorescence enhancement in response to specific targets. While numerous examples of fluorescence-based PeT have been frequently reported, there is not even a single example of a PeT probe that operates via a chemiluminescence mode. Here we report the first PeT-based turn-on probe that acts via a chemiluminescent operation mode. We designed, synthesized, and evaluated a novel chemiluminescent probe, featuring a PeT-based turn-on mechanism. The probe consists of a phenoxy-1,2-dioxetane, linked to an azide unit that acts as a PeT quencher. Upon cycloaddition of a strained cycloalkyne with the azide, a triazole-dioxetane is formed, which undergoes relatively slow chemiexcitation, resulting in a measurement window with an exceptionally high signal-to-noise ratio (over 5000-fold). The PeT-dioxetane probe could effectively detect and image two model proteins labeled with strained cycloalkyne units (Myc-DBCO and Max-DBCO) through either NHS or maleimide conjugations. Comparative analysis shows that our PeT-based chemiluminescent probe significantly outperforms a commercially available fluorescent analog. We anticipate that the insights gained from this study will facilitate the development of additional chemiluminescent probes utilizing various PeT-quenching pathways.