Torsion-Induced π-Conjugation Offers an Approach to Small Coumarin-Based Color-Changing Fluorescent Sensors of Esterase Activity.

Fluorescent sensors for esterase activity include a diverse array of compounds that utilize intramolecular charge transfer induced by the unmasking of the electron-donating hydroxyl group through esterase action. These sensors exhibit different outcomes, ranging from turn-on fluorescence to fluorescence color changes. In this work, we demonstrate the application of torsion-induced fluorescence changes in designing esterase-dependent chemosensors. We present a method for detecting esterase activity based on the torsion-induced geometrical changes between the ester and carboxylate forms of a fluorophore. Our approach shows that aligning electronic interactions between the C7 heteroatom and the C4 ester substituents within a small coumarin core stabilizes the planar geometry of the coumarin ester, leading to fluorescence emission above 570 nm. Upon esterase-mediated hydrolysis, the coumarin exhibits a blue shift in emission to 460 nm, corresponding to the carboxylate form. This shift occurs due to the nonplanar orientation of the carbonyl relative to the fluorophore. As a result, C4-coumarin ester emissions can be observed in the red fluorescence channel, while carboxylate emissions are detected in the blue/green fluorescence channels. Consequently, we introduce small, highly permeable aryl and acetoxymethyl coumarin esters as indicators of esterase activity and as tools to differentiate between live and dead cells.