Design And Synthesis Of Novel Fluorescent Probes And Their Application In Cellular Oxidative Stress Imaging

In recent years,fluorescent probes have become an important tool for obtaining microstructural images of biological cells and tissues as well as for revealing physiological activities and mechanisms in living organisms due to the advantages of simple operation,high sensitivity,real-time in situ detection and multicolor imaging in combination with fluorescent imaging techniques.Cellular oxidative stress is one of the important causes of many diseases in living organisms,and the study of the relevant molecular mechanisms involved in the regulation of oxidative stress will be of great scientific importance for the diagnosis and treatment of diseases.Fluorescent probes have been widely used to analyze molecular imaging studies related to oxidative stress processes.However,most of the probes are limited by the emission wavelength and cannot be used in the imaging of deep tissue,the aggregation of fluorophores leads to the reduction of fluorescence luminescence efficiency and the single function cannot meet the simultaneous monitoring of multiple events of physiological activities.Therefore,in this thesis,several novel fluorescent probes were constructed rationally for bioimaging and sensing analysis of oxidative stress-related active molecules,as follows:（1）Iron（Fe）is one of the essential trace elements that participate in the regulation of various physiological processes in the body,especially biological oxidation processes.The imbalance of Fe levels in cells causes cellular dysfunction and further leads to the development of several diseases.In Chapter 2,a novel ICT-based mechanism was successfully constructed for the detection of Fe²⁺using a two-photon and near-infrared fluorescent probe,DCM-Fe,which exhibits excellent selectivity,outstanding sensitivity,fast response,and the advantage of naked-eye recognition for the detection of Fe²⁺.More importantly,the probe was successfully applied for the first time to monitor Fe²⁺in live cells in real time by two-photon microscopy fluorescence imaging,which has the potential of application for detecting the physiological mechanisms of Fe²⁺during oxidative stress.（2）Hg²⁺-induced oxidative stress burst is considered to be an important pathogenic mechanism for Hg toxicity.Therefore,the development of fluorescent probes for efficient and specific monitoring of Hg²⁺-induced oxidative stress levels is of great importance.In Chapter 3,a novel Hg²⁺-activated monitoring of peroxynitrite（ONOO^–）levels near-infrared emission fluorescent probe,NIR-HO,was rationally designed.The probe exhibited rapid response,excellent specificity and high sensitivity for the detection of Hg²⁺and ONOO^–in vitro.Cell imaging showed that Hg²⁺-induced oxidative stress contributed to the increased concentration of ONOO^–.In addition,GSH,NAC and EDTA were used as potential detoxifying agents to alleviate Hg²⁺toxicity and the probe was successfully used for imaging Hg²⁺and ONOO^–in vivo.The probes provide a simple and effective optical tool for visualizing Hg²⁺-induced oxidative stress processes in the physiological environment.（3）Excessive inhalation of sulfur dioxide（SO₂）can lead to physical damage,and the mechanism is closely related to oxidative stress.On the other hand,SO₂,as a potential gas signaling small molecule,plays an important role in the process of mitochondrial oxidative stress.Accurate monitoring of changes in SO₂ levels in mitochondria during heat shock can help to study the physiological mechanisms of related diseases.In Chapter 4,a fluorescent probe MITO-TPE was developed for mitochondrial SO₂ imaging based on the AIE mechanism.The probe showed good selectivity,high sensitivity and fast response time in response to SO₂,as well as achieved real-time imaging of SO₂ levels in mitochondria of living cells and zebrafish.In addition,for the first time,an abnormal elevation of SO₂-induced superoxide anion radical（O₂˙^–）was found to further induce the occurrence of mitochondrial oxidative stress.More importantly,the probe enabled the first imaging of the imbalance of SO₂levels in mitochondria during heat shock.The probe provides an effective analytical tool for exploring SO₂ biological activity.（4）Cancer is one of the most important diseases that threaten human life and health.In the past few years,although various antitumor drugs have been identified to induce oxidative stress in tumor cells further leading to apoptosis,direct visualization evidence of the oxidative stress process is still lacking.In Chapter 5,a novel fluorescent probe RSS-HCl O was developed based on coumarin-semicarbazone and NBD-Cl as fluorophore and recognition group,enabling the detection of Cys/Hcy,GSH and HCl O in three fluorescent channels.The probe alone has almost no fluorescence emission due to the intramolecular PET process.The fluorescence of the green and red channels increased significantly after the reaction of the probe with Cys/Hcy;for GSH,the fluorescence of the red channel increased,while the fluorescence of the green channel did not change significantly;in contrast,in the presence of HCl O,the fluorescence increased only in the blue channel.In addition,the probe exhibited lower detection limits and shorter reaction times for the three thiols and HCl O.This multifunctional fluorescent probe can be used to differentiate the three biological thiols and HCl O in cells and zebrafish.The probe can be used as a potential and effective tool to reveal pharmacological processes during cancer therapy.（5）Liver disease caused by liver injury has become a serious threat to human health.Direct visual evidence of lysosomal dysfunction during liver injury is still lacking.In Chapter 6,a dual-site fluorescent probe RDNA-1 was rationally designed for tracking adenosine triphosphate（ATP）and hydrogen sulfide（H₂S）multisites in lysosomes based on rhodamine 6G and 1,8-naphthylimine fluorophores.The probe showed excellent selectivity,high sensitivity and well discrimination detection ability for ATP and H₂S in vitro.Cellular imaging showed that the probe can specifically image lysosomal ATP and H₂S.The probe can also be used to detect changes in ATP and H₂S levels during liver injury and that H₂S has a reparative function on liver-injured cells.Importantly,the probe has also been successfully used for in vivo imaging of zebrafish.Thus,the probe provides a new approach to study the physiological mechanisms of liver injury,which is useful for the early diagnosis and treatment of liver-related diseases.（6）Lipid droplets and lysosomes are two important organelles in cells that are involved in a variety of life activities including oxidative stress.Dysfunction of lipid droplets and lysosomes is closely related to the development of many diseases.In addition,after lipid droplets are wrapped by autophagosomes,the lipid compounds in lipid droplets are transported to lysosomes,where they are hydrolyzed by proteases to free fatty acids,which provide the required fatty acids and energy to cells during stress.In this chapter,a multifunctional probe LD-Lyso is rationally designed by introducing a p H-activated"on-off"structure in the indole portion,which has the potential to target lipid droplets for imaging in neutral or alkaline environments with high log P values and fluorescence emission dependent on the polar environment of the solution.When the probe was located in an acidic environment,the structure opened the loop and the probe had increased emission in the near-infrared emission region at 730 nm,thus offering the possibility of lysosomal imaging.This multifunctional fluorescent probe provides a valuable research tool for studying lipid autophagic processes and related life activities.