The Construction And Biosensing Study Of Arylboronate-based Fluorescent Probe For Peroxynitrite

Recently,fluorescent probes have been used widely in the fields of quantitative analysis,life process research,medical diagnosis and so on,because of its superiorities of simple operation,high sensitivity,real-time and nondestructive detection.In particular,it plays an important role in the localization and tracking of biological active ions and molecules.At present,the design and synthesis of fluorescent probes that can produce specific fluorescence response to biological active objects has become a frontier subject for chemists and life scientists.Fluorescent probes have also become one of the most important tools in the field of life science.Peroxynitrite?ONOO^-?,a highly reactive oxidant and an efficient nitrating agent in living systems.It has been implicated in a variety of disease states,including Alzheimer's disease,arthritis,cancer,autoimmune and inflammatory diseases,and other disorders.However,evidence also revealed that ONOO^- plays a positive role in either signal transduction via nitrating tyrosine residues or immunogenic response against invading pathogens.Therefore,methods for detection of cellular ONOO^- are of considerable importance for both disease diagnosis and exploration of its various pathophysiological properties.The fluorescent probe technique is well suited to meet the need for mapping the spatial and temporal distribution of biomolecules within living cells due to its sensitivity,visualization,and nondestructive detection.Thus,accurate detection of ONOO^- with high sensitivity and selectivity is imperative for elucidating its functions in health or disease states.In this thesis,we have designed and synthesized a series of arylboronate-based fluorescent probes.These probes have been successfully applied to detect ONOO^- with excellent selectivity over a series of biologically relevant reactive oxygen species.1.In this work,based on a protected-deprotected strategy and a covalent-assembly strategy,we designed and synthesized of a new arylboronate-based fluorescent probe 2-1,and demonstrate its potential for detecting ONOO^- with fast response?within five seconds?and high selectivity over a series of ROS including H₂O₂.Notably,a dramatic fluorescence enhancement of 28-fold at 486 nm and and the detection limit was measured to be 48 nM.With the probe,the imaging of the exogenous and stimulation-induced ONOO^- in RAW264.7 murine macrophages has successfully been realized.2.In this work,we designed and synthesized a new fluorescent probe 3-1,which could detect ONOO^- visualized in endothelial cells after oxygen-glucose deprivation?OGD?.This probe could detect ONOO^- with a big fluorescence off-on ratio?155-fold?,fast response rate?50 s?,high sensitivity?detection limit: 0.83 nM?,and excellent selectivity over a seriesof biologically relevant reactive oxygen species as well as anions,cations,and biothiols.The sensing mechanism has proved to be the ONOO^--triggered covalent assembly of a bright iminocourmarin fluorophore.With the probe,the stimulation-induced ONOO^- in RAW264.7 murine macrophages as well as the endogenous ONOO^- in endothelial cells after oxygen-glucose deprivation?OGD?has been successfully visualized.3.Ratiometric probes can eliminate the adverse effects induced by probe concentration,probe environment and excitation intensity through a built-in calibration of two emission bands,thereby providing an ideal method to quantitively detdct biomolecules.We designed and synthesized a new ratiometric fluorescent probe 4-1 for peroxynitrite detection based on ICT mechanism.The studies show that 4-1 displays a ratiometric response at 440 nm and 396 nm to ONOO^- that is caused by an oxidation process.Moreover,the fluorescence intensity ratio?I396/I440?of 4-1 is much more enhanced?12-fold?,and it displays a high selectivity toward ONOO^- over other ROSs.Finally,4-1 was successfully applied to ratiometric detection of exogenous and endogenous ONOO^- in living cells.4.The excited-state intramolecular proton transfer?ESIPT?mechanism can brings a large Stocks shift.A fluorescent “off-on” probe 5-1 with the excited-state intramolecular proton transfer?ESIPT?fluorescence switching mechanism was designed and synthesized based on the oxidation reaction of arylboronate.The probe was characterized by nuclear magnetic resonance and mass spectrometry,and its sensing properties for H₂O₂ were also investigated by UV-vis and fluorescence spectra.Compared with ONOO^-,the results indicated that the probe displayed a better sensitivity and selectivity for H₂O₂.