Fluorogenic and Chromogenic Detection of Carboxypeptidase Y with a Nonpeptide-Based Small-Molecule Probe

Carboxypeptidase Y (CPY) is a vital enzyme with many well-known biological significances, such as determination of amino acid sequences and biosynthesis of polypeptides. Herein we developed a nonpeptide-based fluorescent probe to sense CPY activity accurately and successfully applied it to the inhibitory study. Based on the organic synthesis and comprehensive characterization, the refined probe CPY-P3 displayed excellent specificity towards CPY and exhibited over 400-fold enhancement of fluorescence intensity upon the hydrolysis of CPY. Compared with the commonly used peptide-based fluorescent substrate (Suc-IIW-AMC), CPY-P3 showed about 12-fold higher binding affinity and comparable catalytical efficiency for CPY. Moreover, CPY-P3 was successfully applied in CPY inhibitor characterization. To our knowledge, CPY-P3 is the first nonpeptide-based small-molecule fluorescent probe for the time-course CPY activity detection. Meanwhile, the generation of kelly color makes the detection directly visible without any complicated device. In all, this small-molecule probe should be a potentially useful tool in understanding the roles of CPY in protein sequencing and engineering, as well as the drug discovery of CPY-related diseases. (C) 2018 Elsevier B.V. All rights reserved.