Self‐Assembled Fluorophore‐Based Probe for Efficient Detection of Endogenous Nitroreductase Activity in <i>Escherichia Coli</i>

Abstract Fluorescent probes are functional molecules whose fluorescent properties are transformed as a response to specific stimuli. Understanding the mechanisms of these transformations is essential for the design of these stimuli‐responsive fluorescent probes. A rational design strategy has been developed to construct stimuli‐responsive supramolecular cluster fluorescent probes. They operate by a new mechanism called self‐assembly induced lactone formation (SAILac) to control the fluorescence properties of SNARF, an asymmetric xanthene fluorophore. Here, to expand SAILac applicability, the structure‐activity relationship of the fluorophore scaffold is studied. SNARF‐OBn( p NO 2 ), designed as nitroreductase‐reactive fluorescent probe based on the SAILac mechanism, is selected as the initial structure. As the result of the structure‐activity relationship studies, a new nitroreductase‐reactive fluorescent probe, Rhodol‐OBn( p NO 2 ), is created, having a superior signal‐to‐noise (S/N) ratio with higher reactivity toward nitroreductase than the original probe. By using Rhodol‐OBn ( p NO 2 ), the activity of endogenous nitroreductase in Escherichia coli is successfully detected.