Precision Design of Fluorogenic Probes via Orthogonal Tuning of Binding and Photophysics for Isoform-Selective ALDH2 Imaging

Fluorogenic probes that report enzyme activity are essential for studying biological functions. However, designing them for targets with low catalytic turnover and narrow substrate specificity remains a significant challenge. Here, we present a precision design framework that separates the requirements for sensitivity and selectivity by integrating molecular docking, quantum chemical modeling of fluorogenic mechanisms, and targeted fine-tuning of the probe structures. As a proof of concept, we developed A5, a fluorogenic substrate for aldehyde dehydrogenase 2 (ALDH2) that exhibits high isoform selectivity and a >240-fold signal enhancement over the standard NADH assay. A5 enables quantitative imaging of ALDH2 activity across multiple biological scales─in blood samples, live cells, and intact mouse brains─and supports the identification of small-molecule activators with therapeutic potential in an Alzheimer's disease model. This work establishes a modular strategy for creating activity-based probes tailored to challenging enzymatic targets, with broad applications in precision imaging, drug discovery, and mechanistic biochemistry.