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

Abstract

The conjugation site of dihydrofluorescein (H₂F) is important for the rational design of H₂F-based reactive oxygen species (ROS) sensors. Despite the prevalence of H₂F analogs detecting cellular ROS, the role of the carboxylic acid of H₂F in oxidation is still unclear. To get insight into the conjugation site of H₂F, we synthesized H₂F diacetate (2) and its amide derivative (3). The absorption and emission spectra of deacetylated 2 and 3 in the presence of H₂O₂/hematin showed that the carboxylic acid of H₂F plays a crucial role in the oxidation of H₂F. 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 H₂F-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.

Keywords:

• Fluorescence
• reactive oxygen species (ROS)
• hydrogen peroxide
• oxidative stress

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT [2017R1D1A1B03034972 for TWK, 2019H1D3A1A02071159 for HTL, and 2017R1A2A2A05069805 for CK]. This work was supported by the BK21 plus program “AgeTech-Service Convergence Major” through the National Research Foundation (NRF) funded by the Ministry of Education of Korea [5120200313836 for DPLN].