Electrophile-Free, Self-Immolative Linker-Enabled Near-Infrared Ratiometric Fluorescent Probe for High-Fidelity, Nonperturbative Imaging of Acetylcholinesterase (AChE) Dynamics in Epileptic Mice

Fluorescence monitoring of acetylcholinesterase (AChE) levels is critical for evaluating neurotransmitter regulation; however, nonspecific background hydrolysis, poor targeting fidelity, and single-channel readout severely compromise quantitative accuracy. Conventional electrophilic leaving groups (quinone methides and aldehydes) deplete intracellular nucleophiles (cysteine or glutathione), thereby inducing oxidative stress and disrupting redox homeostasis. Herein, a series of near-infrared (NIR) ratiometric fluorescent probes (Cy667, Cy701, and Cy777) have been rationally designed that integrate a polarity-reversing nucleophilic self-immolative linker, a specific AChE recognition unit, and an ammonium-assisted AChE-targeting moiety. The well-regulated self-immolative nucleophilic cleavage completely eliminates undesired side reactions and electrophilic byproduct interference while maintaining intracellular redox homeostasis without exhausting glutathione (GSH). The optimal probe, Cy701, exhibits exceptional selectivity (>100-fold over other hydrolases) and sensitivity (LOD = 4.9 mU/mL) toward AChE, enabling a ratiometric signal log(F636/F832) in the NIR region. Molecular docking calculations verify that Cy701 has an excellent binding affinity for AChE, which enables real-time visualization of endogenous AChE in PC12 and HepG2 cells. Notably, Cy701 displays superior blood–brain barrier (BBB) permeability and favorable intracellular retention. It has been robustly implemented for in vivo NIR ratiometric imaging of AChE dynamics in acute and chronic epileptic mouse models, unambiguously discriminating pathological states from healthy conditions with a signal-to-background ratio (SBR) of 10.0 (P < 0.001). This work constitutes a universal design paradigm for high-fidelity, nonperturbative enzyme-activatable fluorescent probes and offers a robust scaffold for accurate AChE monitoring and in-depth neurobiological dissection of epilepsy.