Design,Synthesis And Pharmaceutical Applications Of Fluorescent Probes Targeting Signaling Molecules In Oxidative Stress

Redox signal response is an important cell signal transduction mechanism.A variety of redox-related substances（free radicals,small molecules,and large molecules）exist in the body to mediate redox signal transduction.In general,intracellular oxidation and re-duction levels are in equilibrium.When this balance is broken with an excess of oxidized species,Oxidative Stress（OS）occurs.OS is an important diagnostic and therapeutic tar-get since it is closely related to the occurrence and progression of many diseases.On one hand,OS occurs at the early stage of some diseases,so related molecules may be used as biomarkers for the early diagnosis of diseases.On the other hand,given the driving role of OS in the occurrence and progression of diseases,antioxidants have become a potential treatment strategy.However,although the pathological significance of OS is well estab-lished,the development of relevant drugs has not been successful.The reason is that OS involves the imbalance of a variety of reactive oxygen and reactive nitrogen metabolites with various chemical reactivity,spatial and temporal distribution,physiological and pathological functions.The lack of accurate detection tools may be one of the factors limiting the study between pathological mechanisms and drugs.As a consequence,fluo-rescent probes for redox-related substances（ROS/RNS/RSS）in living cells have become a research hotspot in recent years.A large number of studies have confirmed the ad-vantages of such probes in spatiotemporal resolution detection of redox molecules.How-ever,their specificity and sensitivity still need to be improved.Therefore,this thesis focused on the specificity and sensitivity of probes,and achieved highly selectivity for superoxide(O₂^·-)and highly sensitive towards peroxyni-trite（ONOO^-）applying the activity-based sensing strategy.（1）Superoxide(O₂^·-)is the primary ROS and its abnormal expression may cause a series of chain reactions,so the specific detection of O₂^·-is helpful to understand the redox state in organisms.In this thesis,a panel of novel fluorescent probes were devel-oped based on extensive screening of the chemical reactivity and theoretical chemical calculations.The 1,2,4,5-tetrazine group is the recognition group of these probes,which specifically reacts with O₂^·-by a single electron transfer（SET）mechanism,and trans-forms to 1,3,4-oxadiazole derivatives compared with commercial probes DHE and DCFHDA,the tetrazine-based probes showed better specificity and stability.The probes can image both exogenous and endogenous O₂^·-sensitively.In addition,the F-Tz4 was applied in high throughput drug screening,and coprostanone was identified as a promis-ing compound for preventing superoxide overload and ameliorating myocardial Ischae-mia-Reperfusion（I/R）injury.（2）Peroxynitrite（ONOO^-）is a secondary product of O₂^·-,and its oxidation capacity is stronger than that of O₂^·-,which is one of the main active species causing biological damages.It is suggested that oxidative nitrification stress is involved in the progression of acute kidney injury（AKI）,and ONOO^-may be an early diagnostic biomarker for AKI.This section aims at designing a highly ONOO^--sensitive probe to image renel tissue with up-regulated ONOO^-,and studying the relationship between AKI and ONOO^-.Firstly,we constructed an evaluation model between probe structure and sensitivity to reasonably estimate the reaction efficiency of the probes designed with computational chemistry.Secondly,a Nile red derivant that can accumulate in renal was screened by analyzing the tissue distribution of common near-infrared fluorophores.On this basis,the probe KNP-1 was designed and synthesized,and the detection limit of KNP-1 was as low as 100 n M,which can realize the early diagnosis of AKI in mice,revealing that ONOO^-can be used as a biomarker for early diagnosis of AKI,and it is at least 24 h earlier than the changes of clinical diagnostic indicators（Serum Creatinin and Blood Urea Nitrogen）.In conclusion,based on the unique chemical reactivity of O₂^·-and ONOO^-,this the-sis realized the highly selective detection of O₂^·-and the highly sensitive detection of ONOO^-through the rational design strategy combining empirical judgment and theoret-ical calculations.This strategy will inspire subsequent probe design;the development of probe detection tools will also promote the relevant pathological mechanism and drug discovery research.