Discriminative Fluorescence Sensing of Biothiols in Vitro and in Living Cells

Abstract

Simultaneous discriminative sensing of biothiols in vitro and in living cells has remained challenging. Herein, we report a new sulfonamide-based self-quenched fluorescent probe 1 for this purpose with high sensitivity and good selectivity. Treatment of 1 with cysteine (Cys), homocysteine (Hcy), or glutathione (GSH) yields aminoluciferin, 2-cyano-6-aminobenzothiazole homocysteine (CBTHcy), or 2-cyano-6-aminobenzothiazole (CBT), turning "on" the fluorescence at wavelengths of 522, 517, or 490 nm, respectively. Kinetic study indicated that 1 reacts with Cys faster than with Hcy or GSH. With these unique properties of 1, we applied 1 for highly sensitive sensing of Cys, Hcy, and GSH among other 19 natural amino acids (AAs) with good selectivity. Confocal fluorescence microscopic imaging of 1-treated HepG2 cells at two channels (522 ± 8 and 490 ± 8 nm), together with quantitative analysis, indicated that the "turn-on" fluorescence was induced by intracellular Cys-dominating condensation and reduction of 1 but not by intracellular GSH-dominating reduction of 1. This suggests that 1 could be applied for discriminative sensing of intracellular Cys from the abundant GSH. Further development of 1 might bring about an efficient tool for probing cellular functions that relate to biothiols.

Luciferin Regeneration in Firefly Bioluminescence via Proton‐Transfer‐Facilitated Hydrolysis, Condensation and Chiral Inversion

“…Step 2 is the condensation of CHBT and L‐cysteine to form L‐luciferin under non‐enzymatic conditions . This condensation reaction itself has a high second‐order reaction rate constant, which is approximately 70 times higher than that of the well‐defined click reaction . This suggests that CHBT reacts with L‐cysteine to produce L‐luciferin immediately without any enzymes, which is further confirmed by the experimental results showing CHBT and L‐luciferin have similar performances in bioluminescence assay .…”

Fluorescent Probes with Multiple Binding Sites for the Discrimination of Cys, Hcy, and GSH

“…[70] The probe reacted with GSH through sulfonamide cleavage to yield 3 -GSH (CBT). When the probe reacted with Cys or Hcy, the corresponding heterocycles formed.…”

Cysteine‐Mediated Intracellular Building of Luciferin to Enhance Probe Retention and Fluorescence Turn‐On

“…In addition, its green fluorescence1c ( λ em =530 nm) can also be readily utilized for cell imaging, although this has rarely been explored 1c. 2 It has been reported that the reaction between 6‐hydroxyl‐2‐cyanobenzothiazole (CBTOH) and cysteine (Cys) is the last step in the synthesis of luciferin (Scheme ), which features good biocompatibility and fast reaction kinetics (9.19 M −1 s −1 ) under physiological conditions 3. This reaction has been successfully applied for specific labeling of N‐terminal Cys on proteins,3b, 4 controlled self‐assembly in situ to build supramolecules in cells,5 and controlled intracellular synthesis of D ‐luciferin for bioluminescence imaging of enzyme activity in living animals 6.…”