Rational design of fluorescent probes: Improving hydrophilicity, ratiometric and NIR trapping of endogenous leucine aminopeptidase

In situ trapping endogenous leucine aminopeptidase (LAP) in organism is of great significance, but current fluorescent probes for LAP are susceptible to complex experimental conditions because of lacking internal efficient standard. Here, we present HCHMC-S-Leu, a novel near-infrared (NIR) ratiometric fluorescent probe for high effective tracking of endogenous LAP activity via intramolecular S→N rearrangement. The study mainly focuses on how to exactly modulate the electronic effects and extend the emission wavelength shift for realizing an ideal ratiometric detection of LAP, as well as solving the intrinsic problems of hydrophilicity and short emission wavelength range. By incorporating a dipeptide (cysteinyl-leucine: Cys-Leu) into a fluorescent chloro-hydroxyl-merocyanine (CHMC) scaffold with rational design, a novel fluorescent probe HCHMC-S-Leu is screened out. As demonstrated, the new probe exhibits high effective feedback on LAP activity with various advantages, such as favorable hydrophilicity, ideal ratiometric detection of LAP, and NIR emission. Furthermore, imaging of endogenous LAP in living cancer cells and zebrafish models with HCHMC-S-Leu is also successful. This scaffold of NIR ratiometric fluorescent probe represents a new powerful tool for the study of LAP in living systems.