Self-Assembled Fluorophore-Based Probe for Efficient Detection of Endogenous Nitroreductase Activity in Escherichia Coli

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(pNO₂), 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(pNO₂), is created, having a superior signal-to-noise (S/N) ratio with higher reactivity toward nitroreductase than the original probe. By using Rhodol-OBn (pNO₂), the activity of endogenous nitroreductase in Escherichia coli is successfully detected.