Tail-Assisted Excited-State Intramolecular Proton Transfer (<i>ta</i>-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 (<i>ta</i>-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. <i>ta</i>-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 <i>ta</i>-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.