Construction And Biological Application Of HClO/ONOO? Fluorescent Probe

A number of studies have indicated that professional phagocytes(mainly neutrophils and macrophages)can secretea large amount of reactive oxygen species(ROS)into phagosomes formed by invagination of the cell membrane,resulting in the destruction and elimination of ingested pathogens,such as virus,bacteria and toxins.While ROS produced by phagocytes plays a key role in innate immunity,the excessively produced they will also be released outside the phagocytes and attack normal cells and tissues,causing the impairment of biological macromolecules such as proteins and DNA in normal cells and tissues,triggering a variety of diseases(for instance inflammation,cancer,pulmonary fibrosis and neurodegenerative diseases).In addition,the level of reactive oxygen species in diseased cells of these diseases will also be significantly increased.For example,the concentration of reactive oxygen species in cancer cells is about ten times that of normal cells.Therefore,active oxides are not only closely related to the pathogenesis of various diseases,but are also potential markers of various diseases.Among many reactive oxygen species,hypochlorous acid(HCl O)and peroxynitrite(ONOO~-)have higher oxidizing properties and can directly damage almost all types of biomolecules through oxidation,nitrification,halogenation and deamination.Therefore,they are more closely related to diseases such as inflammation and cancer,and are an important object of current biomedical research.In various cell biology tools,fluorescent probes have significant advantages in detecting cellular oxidative stress caused by reactive oxygen species due to their sensitivity,non-invasiveness,non-radiation,real-time monitoring and other characteristics.In addition,red and near-infrared fluorescent probes have shown great application prospects in chemical biology research and clinical diagnosis,due to red and near-infrared light(600?900 nm)of the strong tissue penetrability,low light damage,and less disturbed by the autofluorescence of biomolecules.Therefore,the development of red or near-infrared fluorescent probes capable of real-time and sensitive monitoring of endogenous HCl O or ONOO~-in phagocytes and diseased cells is of great significance for assessing the occurrence and progress of related diseases.The main purpose of this thesis is to start with molecular structure design,using silicon-oxazine andpyro red fluorescent dyes as fluorophores,develops a series of HCl O and ONOO~-fluorescent probes with high sensitivity,high selectivity and long emission wavelength,and explore their biological applications in disease diagnosis at the cellular and living level.The research content mainly includes the following three aspects:In chapter 2,a series of novel near-infrared Si-oxazine fluorescent dyes Si O1-3 was synthesized by a simple three-step reaction,and the fluorescence emission wavelength was extended to the near-infrared region(about 750 nm).Furthermore,Si O1-3 was reduced by sodium borohydride to obtain the reduction product"Hydrosiloxazine HSi O1-3".Studies have shown that HSi O3 can react with HCl O and ONOO~-to generate highly fluorescent Si O3 with high chemical stability and light stability.In vitro tests show that the probe HSi O3 has good selectivity and fast response to HCl O and ONOO~-,and the detection limits are up to 5.4 n M and 5.0 n M,respectively.Cell and in vivo experiments has proved that HSi O3 can detect endogenous and exogenous HCl O/ONOO~-in living cells and mouse abdominal cavity.This probe has been successfully used for HCl O/ONOO~-in mouse idiopathic pulmonary fibrosis caused by bleomycin in fluorescence imaging,and provides an excellent diagnostic tool for the occurrence and progression of pulmonary fibrosis.In chapter 3,by condensing 2-hydrazinopyridine and 2-hydrazinobenzothiazole on the aldehyde group of 9-aldehyde pyiro red(Py-CHO),two"Off?On"type HCl O fluorescent probes based on the mechanism of C=N isomerization were synthesized(Py HP and Py HZ).Studies have shown that both Py HP and Py HZ can react with HCl O to generate highly fluorescent triazole derivatives.Due to the electron withdrawing effect of the triazole group on the fluorophore(Piro Red),the fluorescence response wavelength of this type of probe is about 50 nm red-shifted from Piro Red itself,reaching about 630nm.Both Py HP and Py HZ are sensitive(the detection limits are 0.9 n M and 5.4 n M,respectively),fast(both the reaction is completed within 15 seconds),and specifically react with HCl O to produce bright fluorescence(?200 and 318 fold fluorescence enhancement,respectively).Among them,Py HP has been successfully used for fluorescence monitoring of endogenous HCl O in mouse peritonitis tissues and the distinction between cancer cells and normal cells.In chapter 4,using 9-aldehyde pyrrole(Py-CHO)as the starting material,a near-infrared(NIR)fluorescent probe Py OX with aldoxime unit as HCl O as the recognition site was designed and synthesized.The probe can quickly react with HCl O to generate a highly fluorescent"Py CNO".The nitrile oxide group on the 9th position of the product can be effectively electronically conjugated with the piro red structure,so that the maximum emission wavelength extends to the near-infrared region,reaching about 680nm.The probe Py OX has a fast"Off?On"fluorescence response to HCl O and high sensitivity(detection limit:2.4 n M).Based on the significant difference in HCl O levels in cancer cells and normal cells,this probe has been successfully used for the selective differentiation of cancer cells and normal cells,as well as fluorescence diagnosis and fluorescence-guided surgical resection of mouse tumors.