Structure‐Guided Design of a Selective Fluorogenic Probe for Monitoring Butyrylcholinesterase Activity in Living Cells

Butyrylcholinesterase (BuChE) is an important modulator of cholinergic signaling whose activity increases in several pathological contexts, including neurodegeneration and metabolic dysfunction. However, selective chemical tools for monitoring BuChE activity in complex biological systems remain limited, largely due to its high structural similarity to acetylcholinesterase (AChE) and the deeply buried nature of its catalytic site. Here, we report a series of activatable fluorescent probes based on a 1,8‐naphthalimide scaffold for selective BuChE sensing. Probe performance was systematically optimized by varying ester substituents (cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl) at the recognition site and by incorporating self‐immolative linkers to spatially decouple the fluorophore from the enzymatic trigger. Among the probes developed, the cyclopropyl ester derivative JCB‐3 exhibited superior performance, displaying high selectivity for BuChE over acetylcholinesterase (AChE), a low detection limit of 0.84 U/L, and a rapid fluorescence turn‐on response within 20 min. Molecular docking studies supported the favorable binding of JCB‐3 within the BuChE catalytic pocket. In live‐cell imaging experiments, JCB‐3 demonstrated excellent cell permeability, low cytotoxicity, and robust activation by endogenous BuChE. Together, these findings establish JCB‐3 as a selective chemical probe for imaging BuChE activity and provide mechanistic insight into steric factors that govern cholinesterase selectivity.