Multi-dimensional imaging of endogenous leucine aminopeptidase via fast response fluorescent read-out probe

Abnormal expression of leucine aminopeptidase (LAP) in living system is associated with various diseases. But so far, there is no detailed imaging analysis of LAP distribution in living cells and organisms with fluorescent probes. In this work, we present a fast-response fluorescent read-out probe (NBD-S-Leu) for imaging LAP in living cancer cells and zebrafish models from multi-dimensional. NBD-S-Leu was constructed by incorporating a dipeptide (cysteinyl-leucine: Cys-Leu) into a commercially available fluorescent 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) scaffold with rational design. Upon exposure to LAP, the amide bond in dipeptides will be cleaved, and subsequently, an intramolecular S -> N rearrangement will be occurred, resulting in a dramatic fluorescence light-up and realize the chemoselective detection of LAP. The sensing mechanism was fully supported by TD-DFT calculations and HPLC-HRMS analysis. NBD-S-Leu performs well and features excellent water solubility, fast-response (similar to 4 min) to LAP, low cytotoxicity, as well as good tissue-permeable. Furthermore, NBD-S-Leu has been effectively utilized to image endogenous LAP activity in various types of living cells and zebrafish models. Impressively, with the help of 2.5-dimensional (2.5 D) and 3-dimensional (3 D) confocal imaging, the endogenous LAP distribution in different living cells and zebrafish organs were clearly observed from more than one angle for the first time owing to the high signal-to-noise ratio. Collectively, this fluorescent read-out system does not only provide an alternative method for accurate detection of LAP activity but also can help LAP-related pathological analysis in living organisms and screening inhibitors.

Automated summary

The study presents a fast-response fluorescent read-out probe, NBD-S-Leu, for imaging LAP in living cancer cells and zebrafish models, featuring excellent water solubility, fast response, low cytotoxicity, and tissue permeability. Through 2.5D and 3D confocal imaging, the endogenous LAP distribution in different cells and zebrafish organs was clearly observed for the first time. The fluorescent read-out system not only provides an alternative method for accurate detection of LAP activity but also helps LAP-related pathological analysis and inhibitor screening in living organisms.