Synthesis and Characterization of 2-(2′-hydroxy-5′-chlorophenyl)-6-chloro-4(3 H)-Quinazolinone-Based Fluorogenic Probes for Cellular Imaging of Monoamine Oxidases

Monoamine oxidases (MAOs) are essential FAD-dependent enzymes which can efficiently catalyze the oxidative deamination of neurotransmitters and biogenic amines. There are two isoforms, MAOA and MAO B, that are abundant in the liver, gastrointestinal tract, blood platelets, and central nervous systems. These enzymes play an important role in metabolism and neural development by regulating the homeostasis of amine neurotransmitters and peripheral dietary amines. Any excess or deficiency of these enzymes will lead to various neurological and psychiatric disorders such as depression, Parkinson s and Alzheimers s diseases, or even the growth inhibition and progression of tumor. Thus, the development of suitable MAO substrates which can be used for selective and sensitive monitoring of enzyme activity in a complex biological system is of great significance. Recently, fluorescence techniques have attracted considerable attention as simple, effective, and powerful tools for real-time monitoring of protein and enzyme activities in vitro and in vivo. Fluorescent detection is more advantageous compared to colorimetric or radioisotope assay owing to its high sensitivity, relative safety, low cost, and easy handling. To date, several standard chromogenic, radiochemical, and fluorogenic substrates have been successfully employed to identify MAO activity in vitro. However, simple and effective fluorescent probes which can provide a direct and sensitive readout of the MAO activity in living cells are still highly required since most of the existing methods are less sensitive, require a secondary activating enzyme to release the signal for detection, or do not provide live cell fluorescence imaging for the enzyme functions. Herein, we present the rational design and synthesis of a new class of activity-based fluorescent probes for real-time imaging of MAO function in living cells. The general concept for MAO imaging relies on the fact that MAO enzymes catalyze the FAD-dependent oxidation of primary, secondary, and tertiary amines to iminium intermediates, which are further non-enzymatically hydrolyzed to the corresponding aldehydes to facilitate the release of a fluorescent product through a b-elimination process (Scheme 1). In this study, we chose 2-(2’-hydroxy-5’-chlorophenyl)-6-chloro-4ACHTUNGTRENNUNG(3H)-quinazolinone (HPQ) derivatives as fluorescent reporters. The HPQ fluorophores are generally insoluble in water and highly fluorescent in the solid state owing to the