Design And Synthesis Of Fluorescent Probes For Formaldehyde And Superoxide Anion And Their Biological Application

Formaldehyde?FA?is a momentous member of the reactive carbonyl species?RCS?family.Aberrantly elevated FA cross-links proteins and DNA through methylene bridges,which severely impairs their biofunctions and induces the cellular FA stress.Until now,FA has been tightly associated with the initiation and progression of cancer,diabetes,and neurodegenerative disorders,etc.However,the detailed FA-related molecular mechanisms and signaling pathway have not been thoroughly elucidated yet.On the other hand,NADPH oxidase can use NADPH as an electron donor to catalyze O₂ to produce superoxide anion(O₂^·-).O₂^·-is the most important member of reactive oxygen species?ROS?,which can be converted into hydrogen peroxide?H₂O₂?,hydroxyl radical?^·OH?,hypochlorous acid?HClO?,peroxynitrite?ONOO^-?and other ROS through enzymatic or non-enzymatic reactions,and is an important biomarker of oxidative stress.The abnormally high level of O₂^·-can cause oxidative damage to cells,induce cell necrosis or apoptosis,and then induce a variety of diseases,such as stroke,neurodegenerative diseases,and cancer.Therefore,the monitoring of the production,distribution,metabolism and elimination of bioactive small molecules or ions such as formaldehyde and O₂^·-is of great significance to elucidate the involved physiological and pathological mechanisms,as well as to the diagnosis,treatment and drug development of related diseases.Because of its advantages such as ease of operation,non-invasiveness,high spatiotemporal resolution,and real-time monitoring,probe-based fluorescence imaging technology has become an ideal tool for studying the concentrations of bioactive molecules and the related molecular events.To circumvent these issues,in this thesis,we designed and synthesized molecular fluorescent probes with novel structures and excellent properties,and used the probes to monitor the concentrations and biofunctions of formaldehyde and O₂^·-in cell and animal models,revealing the regulatory mechanism and network of formaldehyde and O₂^·-in relevant physiological and pathological processes.This thesis mainly includes the following two parts of research work:1.Imaging of formaldehyde generation during host immune response by an aminoguanidine-based rapid-response fluorescent probeWe use the aminoguanidine unit as a new reaction trigger for the first time,and was conjugated to the tetraphenylethylene fluorophore to generate a unique FA fluorescent probe TPE-AG.TPE-AG achieves a much rapid,highly sensitive and specific fluorescence turn-on response to FA.The results disclose for the first time a high level of FA within macrophages and infected tissues in response to lipopolysaccharide?LPS?stimulation,implicating the possible immunoprotective role of FA against microbial/pathogenic invasion.In this process,FA was produced from the enzymatic demethylation of histones catalyzed by LSD1,which was regulated by both ROS and NO.On the other hand,LSD1 upregulated the expressions of NOX and NOS,and finally elevated the ROS and NO production.These results collectively demonstrated the complex signaling network among ROS,RNS,and RCS during immune response.2.Design and synthesis of two-photon ratiometric fluorescent probes for superoxide anion and its biological application.We designed and synthesized a two-photon ratiometric fluorescent probe TPE-BT-TF,which was fabricated with trifluoromethylsulfonate as the specific response group and tetraphenylethylene-benzothiazole as the fluorescent platform.It was found that the probe can detect O₂^·-with high selectivity and high sensitivity.The initial fluorescence emission of the probe is at 480 nm.After reacting with O₂^·-,the emission wavelength is red-shifted to 580 nm,achieving the ratiometric response to O₂^·-.Using TPE-BT-TF and two-photon confocal microscopy,we successfully achieved ratiometric fluorescence imaging of endogenous O₂^·-produced by rotenone stimulation.The further biological application of the probe TPE-BT-TF is under study.In summary,in this thesis,two molecular fluorescent probes,TPE-AG and TPE-BT-TF,are designed and synthesized for imaging of two bioactive species,formaldehyde and O₂^·-.Both probes have excellent optical properties and good response performance,and can respectively achieve two-photon fluorescence imaging of formaldehyde or O₂^·-in biological samples,providing an efficient in situ imaging tool for the related pathophysiology and molecular mechanism research.