Rational design of environment-sensitive fluorescent probes for butyrylcholinesterase and their application in biological imaging.

Butyrylcholinesterase (BChE) is closely associated with Alzheimer's disease (AD), with its expression significantly elevated in the brains of AD patients. This enzyme has emerged as a potential biomarker for monitoring AD progression. Therefore, developing a reliable chemical tool to detect BChE both in vitro and in vivo is of considerable interest. In this study, we rationally designed a series of environmentally sensitive fluorescent probes targeting BChE, based on a highly potent, selective, and reversible BChE inhibitor. Among these, the most promising probe ESP4 demonstrated an ultrafast response to BChE with exceptional sensitivity (LOD = 1.81 nM), enabling high-precision detection of BChE. Additionally, ESP4 maintained robust inhibitory activity against BChE in the low nanomolar range (IC50 = 71.78 ± 1.90 nM). Notably, ESP4 exhibited excellent selectivity for BChE, showing no interference from other biological species, including acetylcholinesterase (AChE). The probe also accurately measured the IC50 of tacrine (7.83 nM), a standard BChE inhibitor, demonstrating its reliability in evaluating BChE inhibition. Due to its high sensitivity, rapid response, and superior selectivity, ESP4 enabled real-time imaging of BChE in biological systems such as cells, zebrafish, and AD mouse models. Collectively, these findings highlighted ESP4 as a valuable tool for BChE detection, contributing to a deeper understanding of the physiological role of BChE in health and disease.