Precipitated Fluorophore-Based Molecular Probe for In Situ Imaging of Aminopeptidase N in Living Cells and Tumors

Aminopeptidase N (APN) is capable of cleaving N-terminal amino acids from peptides with alanine in the N-terminal position and plays a key role in the growth, migration, and metastasis of cancer. However, reliable in situ information is hard to be obtained with the current APN-responsive molecular probes because the released fluorophores are cytoplasmic soluble and thus rapidly depart from the enzymatic reaction sites and spread out all over the cytoplasm. Here, we report a de novo precipitated fluorophore, HBPQ, which is completely insoluble in water and shows strong yellow solid emission when excited with a 405 nm laser. Owing to the controllable solid fluorescence of HBPQ by the protection–deprotection of phenolic hydroxyl, we further utilized HBPQ to design an APN-responsive fluorogenic probe (HBPQ-A) for the imaging of intracellular APN. Importantly, HBPQ-A can not only perform in situ imaging of APN in different organelles (e.g., lysosomes, mitochondria, endoplasmic reticula, and so forth) but also display a stable and indiffusible fluorescent signal for reliable mapping of the distribution of APN in living cells. In addition, through real-time imaging of APN in 4T1 tumors, we found that the fluorescent signal with high fidelity generated by HBPQ-A could remain constant even after 12 h, which further confirmed its diffusion-resistant ability and long-term reliable imaging ability. We believe that the precipitated fluorophore may have great potential for long-term in situ imaging.