Synthesis And Application Of Reversible Fluorescence Probe Of Superoxide Anion Radical In Cell Organelles

Hepatic ischemia-reperfusion（HIRI）is an unavoidable operation in liver surgery,such as liver transplantation and liver cancer resection,and may lead to complications such as postoperative inflammatory response,tissue dysfunction,and even liver failure.Ischemia-reperfusion（IR）injury is closely related to oxidative stress.Abnormal changes in reactive oxygen species（ROS）levels or impaired metabolism are important causes of oxidative stress.The first ROS molecule formed after single electron reduction is the superoxide anion radical(O₂^·-),which serves as a precursor for other ROS.O₂^·-is an important marker of oxidative stress that plays an important role in the mechanism of IR injury.It is difficult to achieve dynamic,reversible,and real-time detection ofO₂^·-due to the dynamic concentration levels ofO₂^·-in different subcellular regions and their functions.Therefore,the development of tools to accurately and reversibly indicateO₂^·--mediated changes within specific subcellular organelles will help to study the pathogenic mechanism of IR injury and elucidate IR injury-related signaling pathways.Fluorescence imaging is a powerful tool for high-resolution imaging of subcellular and organelle-specific reactive substances in living cells to study organelle-specific processes with high biocompatibility,high spatial and temporal resolution,and in situ real-time tracking.However,most subcellular organelle ROS fluorescent probes are reaction-based fluorescent probes that achieve fluorescence modulation by oxidative bond breaking based on active site chemistry,but these probes can detect changes in target levels with only one reaction,and there is still a lack of relevant tools to study long-term dynamic changes in ROS during pathological processes.As a result,this thesis designed and synthesized two fluorescent probes forO₂^·-real-time,dynamic,reversible fluorescence imaging of the endoplasmic reticulum and cell membrane,elucidated theO₂^·--regulated protein signaling pathway during HIRI,and successfully applied it to fluorescence imaging-guided surgery.The following is the research for this thesis:1.We designed and synthesized a novel organic small molecule fluorescent probe（DPC）for the detection of endoplasmic reticulum（ER）O₂^·-in real time,dynamically,and reversibly.DPC was synthesized from long alkyl chain sulfonate and caffeic acid by the amide reaction,in which the amphiphilic dodecyl chain sulfonate serves as the targeting group of the ER and specifically recognizes.The DPC is capable of instantaneous,dynamic,and reversible detection ofO₂^·-in the concentration range of 0–20μM.DPC is capable of dynamic and reversible detection of ER O₂^·-concentration changes in living cells and in vivo and provides real-time,reversible fluorescence imaging of changes inO₂^·-concentration levels.With DPC,we successfully observed a significant increase in ER O₂^·-levels of HIRI cells and mice,indicating that ER O₂^·-could serve as a potential marker for the diagnosis of HIRI and be involved in HIRI-related signal transduction.In addition,we used DPC to achieve fluorescence imaging of surgically excised HIRI lesion sites and to elucidate HIRI-related signaling pathways,providing a new direction for the early diagnosis of HIRI.2.We developed Cell membrane-O₂^·-as a cell membrane-targeted fluorescent probe for the dynamic,reversible,and transient detection ofO₂^·-.Cell membrane-O₂^·-was showed to be composed of the targeting group of cell membrane andO₂^·--specific recognition group.Cell membrane-O₂^·-could detectO₂^·-in a dynamic and reversible manner,and its fluorescence intensity was unaffected by other ROS,reactive nitrogen species（RNS）,or p H.Using high-resolution fluorescence imaging,we successfully detectedO₂^·-on cell membranes.