Tail-Assisted Excited-State Intramolecular Proton Transfer (ta-ESIPT) Fluorophores: A Universal Ratiometric Platform for Hydration-Sensitive Biomolecular Imaging and Sensing

Excited-state intramolecular proton transfer (ESIPT) fluorophores are valuable for ratiometric bioimaging due to their microenvironmental sensitivity, but traditional enol–keto systems suffer from poor biocompatibility and reduced efficiency in polar, protic environments. Here, we introduce a tail-assisted ESIPT (ta-ESIPT) strategy in which proton transfer occurs from an amide donor to an amino nitrogen acceptor. This mechanism applies to biocompatible charge-transfer fluorophores, such as naphthalimide, coumarin, NBD, and acedan. ta-ESIPT fluorophores exhibit broad environmental stability and a hydration-gated response─proton transfer is inhibited in aqueous environments but restored in nonaqueous microenvironments, yielding ratiometric red-shifted emission. This property enables the precise visualization of biomolecular interactions. By conjugating ta-ESIPT fluorophores with protein ligands, we achieve precise, ratiometric imaging of targets like SNAP-tag, hCA, avidin, and HaloTag in live cells, with fluorescence signals that directly correlate with binding affinities. This correlation enables real-time monitoring of protein interactions and evaluation of inhibitors while minimizing nonspecific interference in the complex cellular environment, ensuring dynamic and accurate protein recognition.