De novo design of an ultrasensitive fluorogenic probe for aminopeptidase N sensing in living system

Aminopeptidase N is a widely expressed zinc-biding metalloprotease which plays important roles in maintaining normal physiological functions and the abnormal activity of APN is highly related chronic inflammatory disease and cancer progression. Thus, the detection and specific tracing of APN activity is of great importance in clinical diagnosis, drug discovery and disease related biological study. To date, enzyme activated fluorescent probe has attracted intense attention due to its high sensitivity and spatial-temporal resolution and biocompatibility in living system. Many researchers have attempted in constructing APN activity based probes and applicated in medical study such as tumor imaging, photodynamic therapy, and surgical navigation. However, these probes were all acquired by incorporating the fluorophore with its natural substrate. Thus, to discovery a new design strategy for APN targeting could provide efficient alternative method for APN probe construction and APN related chemical biology research. By adapting the unnatural amino-acid (2-Aminobutyric acid, Abu) which showed high affinity and specificity toward APN, we herein proposed a new probe design strategy for APN sensing and a near-infrared fluorescent probe TMN-Abu was constructed through this strategy. The in vitro assays showed that TMN-Abu possess high sensitivity toward APN (LOD = 0.57 ng/mL) and high binding affinity toward APN (Km = 19.1 ± 1.84 μM). Most importantly, TMN-Abu was successfully applicated into monitoring endogenous APN activity in living cells, tumor-bearing nude mice and for the first time applicated into diabetic model mice and APN activity dynamic change in biological events such as starvation and EGF stimulation.