Chemistry of the carboxylic acid of dihydrofluorescein in oxidation and its application to fluorogenic ROS sensing

• Published in: Free radical research 2021-04, Vol.55 (4), p.461-468
• Main Authors: Le, Hoa Thi, Nguyen, Dinh Phi Long, Shin, Hyo Seob, Jung, Woong, Kang, Chulhun, Kim, Tae Woo
• Format: Article
• Language: English
• Subjects: Carboxylic Acids - chemistry; Carboxylic Acids - metabolism; Fluoresceins - chemistry; Fluoresceins - metabolism; Fluorescence; Fluorescent Dyes - chemistry; Fluorescent Dyes - metabolism; Hep G2 Cells; Humans; hydrogen peroxide; Molecular Structure; Optical Imaging; Oxidation-Reduction; oxidative stress; reactive oxygen species (ROS); Reactive Oxygen Species - chemistry; Reactive Oxygen Species - metabolism; Tumor Cells, Cultured
• ISSN: 1071-5762; 1029-2470
• DOI: 10.1080/10715762.2021.1944622

The conjugation site of dihydrofluorescein (H 2 F) is important for the rational design of H 2 F-based reactive oxygen species (ROS) sensors. Despite the prevalence of H 2 F analogs detecting cellular ROS, the role of the carboxylic acid of H 2 F in oxidation is still unclear. To get insight into the conjugation site of H 2 F, we synthesized H 2 F diacetate (2) and its amide derivative (3). The absorption and emission spectra of deacetylated 2 and 3 in the presence of H 2 O 2 /hematin showed that the carboxylic acid of H 2 F plays a crucial role in the oxidation of H 2 F. NMR and HPLC analysis of the oxidation product of deacetylated 3 showed a quantitative and fast generation of non-fluorescent spirolactam (F-Lactam). As regards these observations, we untouched the carboxylic acid at the 3rd position and designed an H 2 F-based ROS sensor (7) that conjugated the lipophilic chain at the 5th position instead. A series of confocal microscopic experiments of 7 demonstrated that 7 prefers the ER location and that ROS are elevated in the cells by ER stress inducers.