Design of Optical Switches as Metabolic Indicators:  New Fluorogenic Probes for Monoamine Oxidases (MAO A and B)

This study describes the design of sensitive, selective, and fluorogenic reporter substrates for monoamine oxidase (MAO) enzymes. This was achieved by an iterative effort, guided by PET and TICT photophysical concepts, which led to the development of irreversible redox switches based on a facile oxidation-cyclization reporting mechanism. Specifically, enzymatic oxidation of the ethylamino group in probe 9 proceeded via a putative aldehyde intermediate, which subsequently underwent spontaneous and intramolecular condensation with the aniline amino group furnishing an indole product in an irreversible fashion. This overall change resulted in a significant change in the emission intensity. When expressed in terms of brightness, the origins of this emission switch may be rationalized by the changes in quantum yield and absorbance strength. The fluorescence readout directly correlated with the kinetics of the oxidative step (i.e., reporting mechanism was fast, the intermediate aldehyde was not detected). Probe 9 is a good substrate for MAO B (Km = 510 +/- 40 muM, kcat = 21 min-1) with the kinetic parameters comparable to physiological substrates. This probe not only allows for direct and continuous measurement of MAO activity in mitochondria and tissue homogenates, but more importantly sets the stage for future studies in intact cells and organs.