A facile turn‐on chemiluminescence probe for sensitive imaging on aminopeptidase N activity

Abstract

Aminopeptidase N, as a target for drug discovery, shows marked relationships with many diseases, especially liver injury and cancer. Here, we explored a chemiluminescence (CL) probe for sensing APN by tethering the APN‐specific substrate group to the ortho‐acrylated phenoxy‐dioxetane scaffold. In this way, two CL probes (APN‐CL and BAPN‐CL) were designed with noncapped leucine and butoxy‐carbonyl capped leucine as the protecting group to preserve the chemiexcitation energy. The uncovered leucine was demonstrated to be essential for detection of APN activity by comparing the CL intensity of two CL probes. Probe APN‐CL was turned on upon APN cleavage, resulting in a high chemiluminescent emission, whereas the chemiexcitation energy of probe BAPN‐CL was still restrained even with the high‐level APN. The result was further elucidated by molecular docking simulations. Probe APN‐CL exhibited a fast response and high sensitivity with a detection limit of 0.068 U/L, and an excellent specificity for the discrimination of APN from biological ions, small molecules, and other proteases commonly found in living system. By virtue of good stability and cell viability, probe APN‐CL imaged abnormal levels of APN in tumour cells and tumour‐bearing mice. Moreover, this probe APN‐CL could be easily used to evaluate APN inhibitors and APN levels in plasma samples from 20 patients. Overall, as a facile and cost‐effective probe, APN‐CL will be a promising alternative in the early diagnosis of pathologies and for cost‐effective screening of inhibitors.

Recent Advances of Aminopeptidases‐Responsive Small‐Molecular Probes for Bioimaging

“…[64] In recent years, many APN-responsive bioluminescence/ chemiluminescence probes have been developed, and their structures are shown in Figure 12. [64][65][66][67] In 2014, Li et al developed a series of traditionally designed bioluminescent imaging probes by coupling D-amino luciferin to the Nterminus of aminopeptidase's recognition amino acids, such as phenylalanine (Phe), tyrosine (Tyr), Alanine (Ala), and leucine (leu) [64] Among these amino acids, Ala is preferably used as the caged group for their probes designed for APN and got the best performance in all cage groups chosen. Probe 36 exhibited favorable solubility and demonstrated superior performance in both enzyme activity assessment and cell activity evaluations, with a 483-fold bioluminescent signal increase after incubation with APN and removing the cage group, significantly more than the other candidates.…”

“…Another chemiluminescent probe (Probe 36) was developed by Cao and Lu et al in 2022. [67] The Probe 39 was simply designed by linking an APN-specific leucine group to an orthoacylated phenoxy-dioxetane scaffold. This work proved not only Ala can be utilized as an APN recognizing group, but leucine could be as well.…”

“…In recent years, many APN‐responsive bioluminescence/chemiluminescence probes have been developed, and their structures are shown in Figure 12 [64–67] . In 2014, Li et al.…”

“…Another chemiluminescent probe (Probe 36) was developed by Cao and Lu et al. in 2022 [67] . The Probe 39 was simply designed by linking an APN‐specific leucine group to an ortho‐acylated phenoxy‐dioxetane scaffold.…”