Rational design of halogen-substituted near-infrared fluorescent probes for butyrylcholinesterase: from drug screening to imaging and therapeutic evaluation in Alzheimer's disease.

Abstract

Monitoring butyrylcholinesterase (BuChE) activity is crucial for tracking Alzheimer's disease (AD) progression and evaluating therapeutics; however, high-performance near-infrared (NIR) probes with rapid response and clear design principles remain scarce. Here, we report a series of dicyanoisophorone-based NIR fluorescent probes engineered via a "halogen effect" to systematically tune reactivity toward BuChE. Through spectroscopic screening, the CF3-substituted probe DCNC7 emerged as the optimal candidate, benefiting from the strong electron-withdrawing and hydrophobic nature of the trifluoromethyl group, which enhances binding affinity and catalytic recognition. DCNC7 exhibits over 120-fold NIR fluorescence enhancement upon the BuChE reaction, with fast kinetics (∼15 min), high sensitivity (detection limit 0.0206 U/L), and excellent selectivity. We validated DCNC7 for in situ imaging of BuChE in AD mouse cells and brain tissues, enabling both identification of natural inhibitors and longitudinal assessment of AD progression. Moreover, the screened inhibitor was evaluated for its suppressive effect on BuChE activity in brain tissue and its therapeutic efficacy in vivo. Collectively, DCNC7 offers a reliable tool for AD monitoring, drug screening, and efficacy evaluation, and the halogen effect-based design strategy provides a generalizable route to develop rapid-response, high-affinity enzyme probes for complex disease models.