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

Abstract

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.

Fluorogenic metabolic probes for direct activity readout of redox enzymes: Selective measurement of human AKR1C2 in living cells

“…For example, enzymes involved in lipid metabolism are likely to have large hydrophobic active sites, which may accommodate aromatic ''nonphysiological'' substrates. In many instances, the metabolic role of an enzyme requires significant substrate plasticity (10,11,34).…”

“…In this instance, a synthetic molecule, bearing distant resemblance to a physiological substrate, would have to function as a competitive substrate. Thus, these approaches depend on significant substrate plasticity of the enzymes of interest (9)(10)(11). Furthermore, with activity comes the issue of selectivity, and as with all nonpeptidic ligand-protein interactions, there are no straightforward design guidelines.…”

Fluorescent Probes for Analysis and Imaging of Monoamine Oxidase Activity

“…In 2005, Sames and co-workers reported the first reaction-based fluorescence sensing scheme for MAO probes, which was based on the (o-aminoethyl)aniline moiety as the reactive site (probe 1, 2; Scheme 4). 23 The enzymatic oxidation of the alkylamine produced the corresponding aldehyde intermediate, which subsequently underwent intramolecular condensation by the arylamine to form the indole ring. This chemical conversion resulted in turn-on fluorescence change, because the (o-aminoethyl) aniline moiety acted as fluorescence quencher through the photo-induced electron-transfer (PET) process 24 but its end-product indole did not act as a PET quencher.…”

Coumarin-Derived Fluorescent Chemosensors

“…18, a sensitive, selective, and fluorogenic probe 131 has been developed for monoamine oxidases (MAO A and B). Nonfluorescent aminocoumarin 131 can be converted to fluorescent pyrrolocoumarin 132 in the presence of MAO A and B (G. Chen et al, 2005). A new fluorogenic transformation based on a quinone reduction/lactonization sequence as shown in Fig.…”