Synthesis Of Superoxide Anion Fluorescent Probes And Its Bioimaging Application

Reactive oxygen species（ROS）is a general term for a class of oxygen-containing reactive substances,including superoxide radical anion(O₂^·-),singlet oxygen and peroxynitrite anion（ONOO^-）,hydrogen peroxide（H₂O₂）,hydroxyl radical（·OH）,etc.In order to maintain the normal REDOX homeostasis of cells,the production and clearance of intracellular ROS are precisely regulated by a series of antioxidant defense systems.Steady-state concentrations of ROS act as second messenger molecules that regulate various physiological processes in cells.Superoxide radical anion(O₂^·-)is the first reactive oxygen species（ROS）produced by electron acquisition from oxygen,which can be converted to other ROS by enzymatic or non-enzymatic means.It has the characteristics of high reactivity,short half-life and low steady-state concentration.Low concentrations of O₂^·-act as messenger molecules to regulate various physiological processes in cells.However,high concentrations of O₂^·-can act as toxins to oxidise lipids,DNA and proteins,leading to oxidative stress in cells,which is closely related to the occurrence and development of diseases such as cancer,acute stroke and atherosclerosis.Fluorescent probes have been widely used in the detection of related biomolecules due to their rapid response,good visualization effect,and ability to detect cells in situ.Therefore,it is of great significance to construct fluorescent probes to detect fluctuations in O₂^·-levels.In this paper,three kinds of superoxide anion based nucleophilic fluorescent probes were designed and synthesized with trifluorometasulfonate as the corresponding site,and their performance was studied in biological systems.Firstly,using p-tosyl as ER specific group,coumarin group with excellent optical properties and effective intramolecular charge transfer（ICT）structure as fluorophore,and using the nucleophilic addition reaction between superoxide anion and trifluoromethyl sulfonate,we constructed a fluorescent probe ER-Tf for monitoring the change of intracellular superoxide anion concentration.The probe ER-Tf itself showed almost no fluorescence when excited at 405 nm wavelength.With increasing O₂^·-concentration,the fluorescence intensity at 462 nm increased continuously.The probe is sensitive to superoxide anion,has high selectivity,low detection limit,good biocompatibility and low toxicity,and can realize real-time detection of superoxide anion concentration changes in biological systems.In addition,ER-Tf was successfully used to track changes in superoxide anion concentration in endoplasmic reticulum stimulated by rotenone.Secondly,fluorescein fluorophore was used as the energy acceptor,coumarin fluorophore as the energy donor,piperazine was used as the connecting arm to connect the two fluorophores,trifluoromethyl sulfonate was used as the superoxide anion reaction site,p-tosyl sulfonamide was used as the endoplasmic reticulum targeting group,and the change of electron donating ability of fluorescein fluorophore after trifluoromethyl sulfonate was hydrolyzed to hydroxyl group was used.An ER specific ratiometric fluorescent probe,ER-CRh,was designed and synthesized for ratio imaging of superoxide anions in living cells.In the spectral test,the blue fluorescence at 481 nm slowly decreased with the addition of superoxide anion,while a new emission band appeared at 556 nm and gradually increased.In addition,ER-CRh showed good selectivity for superoxide anion,and could be used for ratio imaging of the changes of intracellular superoxide anion level after endoplasmic reticulum stress caused by tunicamycin and homocysteine,and the changes of superoxide anion level in the hippocampus of mice with depression induced by lipopolysaccharide.Thirdly,using trifluoromethyl sulfonate as the superoxide anion response site,a fluorescent probe QC based on ICT mechanism was constructed for detecting the changes of superoxide anion concentration in cancerous cells.In the spectral test,at 480 nm wavelength excitation,probe QC itself shows almost no fluorescence,and the fluorescence intensity increases continuously at 550 nm with increasing O₂^·-concentration.The intensity of the probe emission peak showed a good linear relationship with the change of superoxide anion concentration.The bioimaging results show that QC can be applied to the changes of superoxide anion concentration induced by rotenone and PMA in biological systems.