Far‐Red Fluorogenic Probes for Esterase and Lipase Detection

Abstract

Fluorogenic enzyme probes go from a dark to a bright state following hydrolysis and can provide a sensitive, real-time readout of enzyme activity. They are useful for examining enzymatic activity in bacteria, including the human pathogen Mycobacterium tuberculosis. Herein, we describe two fluorogenic esterase probes derived from the far-red fluorophore 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO). These probes offer enhanced optical properties compared to existing esterase probes because the hydrolysis product, DDAO, excites above 600 nm while retaining a good quantum yield (ϕ=0.40). We validated both probes with a panel of commercially available enzymes alongside known resorufin- and fluorescein-derived esterase substrates. Furthermore, we used these probes to reveal esterase activity in protein gel-resolved mycobacterial lysates. These probes represent new tools for esterase detection and characterization and should find use in a variety of applications.

Artificial Fluorogenic Substrates in Microfluidic Devices for Bacterial Diagnostics in Biotechnology

“…The design and use of fluorogenic compounds with fluorescent scaffolds emitting red or blue to violet fluorescence [3,8] opens up the combined use of GFP expression after genetic modification of bacteria [25]. This would give the possibility to determine protein interaction in vivo by multiplexing fluorescence measurements.…”

“…Novel applications with those chemical compounds for bacterial detection [4][5][6], metabolic characterization [2,7,8], drug development [9], preclinical studies [10], and screening for antibiotic resistance [11] are reported.…”

Electronic and Steric Optimization of Fluorogenic Probes for Biomolecular Imaging

“…8 Conjugation of fluorogenic esterase-activated probes to biomolecules can provide detailed spatiotemporal information about biomolecular uptake and localization in live cells. 3a,9 These biomolecule–probe conjugates are, however, typically internalized by endocytic vesicles and can be exposed therein to acidity as low as pH 4.5, 10 making insensitivity to low pH essential to probe function.…”

“…With fluorinated Oregon green, destabilization of the masked substrate was thought to stem from inductive effects resulting in lowered p K a of the conjugate acid of the fluorescein leaving group. 8d Accordingly, design strategies for stable fluorogenic esterase probes have relied heavily on interjecting self-immolative linkers with a higher p K a between the low p K a fluorophore and the site of enzymatic cleavage (Scheme 1). 12 Platforms for such auto-immolative linkers include the acetoxymethyl (AM) ether, 8d,12 quinone methides, 14 and the trimethyl lock.…”

“…8d Accordingly, design strategies for stable fluorogenic esterase probes have relied heavily on interjecting self-immolative linkers with a higher p K a between the low p K a fluorophore and the site of enzymatic cleavage (Scheme 1). 12 Platforms for such auto-immolative linkers include the acetoxymethyl (AM) ether, 8d,12 quinone methides, 14 and the trimethyl lock. 3a,15 The beneficial stability provided by auto-immolative linkers does, however, come at the expense of a longer synthetic route to add atoms that are, ultimately, unnecessary.…”