Improved Aromatic Substitution–Rearrangement-Based Ratiometric Fluorescent Cysteine-Specific Probe and Its Application of Real-Time Imaging under Oxidative Stress in Living Zebrafish

Abstract

Biothiols, including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), play a crucial role in many physiological processes. Cys production and metabolism is closely connected with Hcy and GSH; meanwhile, the dynamic antioxidant defenses network by Cys is independent of the GSH system, and Cys can serve as a more effective biomarker of oxidative stress. Hence, it is significant and urgent to develop an efficient method for specific detection of Cys over other biothiols (Hcy/GSH). However, most of the present Cys-specific fluorescent probes distinguished Cys from Hcy through response time, which would suffer from an unavoidable interference from Hcy in long-time detection. In this work, in order to improve the selectivity, we employed an improved aromatic substitution-rearrangement strategy to develop a ratiometric Cys-specific fluorescent probe (Cou-SBD-Cl) based on a new fluorescence resonance energy transfer (FRET) coumarin-sulfonyl benzoxadiazole (Cou-SBD) platform for discrimination of Hcy and GSH. Response of Cou-SBD-Cl to Cys would switch FRET on and generate a new yellow fluorescence emission with a 56.1-fold enhancement of ratio signal and a 99 nm emission shift. The desirable dual-color ratiometric imaging was achieved in living cells and normal zebrafish. In addition, probe Cou-SBD-Cl was also applied to real-time monitor Cys fluctuation in lipopolysaccharide-mediated oxidative stress in zebrafish.

A FRET‐Based Near‐Infrared Fluorescent Probe for Ratiometric Detection of Cysteine in Mitochondria

“…Ratiometric fluorescent probes have been developed to overcome systematic errors by introducing built‐in internal fluorophores and using FRET or through‐bond energy‐transfer (TBET) strategies . The near‐infrared fluorophores have the advantage of unique ratiometric and nearinfrared imaging features such as deep‐tissue penetration, low cellular and tissue fluorescence background, and reduction of probe photobleaching . However, ratiometric fluorescent probes for cysteine detection with near‐infrared emissions are currently limited to a few examples .…”

A Novel Dicyanoisophorone-Based Ratiometric Fluorescent Probe for Selective Detection of Cysteine and Its Bioimaging Application in Living Cells

“…These limits can be addressed by ratiometric fluorescent response, which is more suitable for use in biosystems. However, sofar only a few Cys selective ratiometric fluorescent have been reported [ 25 , 26 , 27 , 28 ].…”

Novel dual-site fluorescent probe for monitoring cysteine and sulfite in living cells

“…[12][13][14][15][16] Various uorescent probes have been designed and reported for the detection of Cys in vitro and in vivo, and have been used to simultaneously distinguish cysteine/ homocysteine, glutathione, and hydrogen sulde in living cells, [17][18][19][20][21][22][23][24][25] and ratiometric uorescent probes have been used to quantify cysteine. [26][27][28][29][30] Our group previously designed and synthesized several uorescent probes for sensitively and selectively detecting Cys over other biothiols in living cells and mice. [31][32][33] However, few of these reported probes could visualize the metabolic processes involving Cys.…”