Rational Design Of Reaction-based Fluorescence Probes For ONOO~- Related Disease Biological Imaging

Based on the unique advantages,such as simple operation,high sensitivity,visualization and nondestructive detection of samples,fluorescence imaging has important application value and been extensively used in the analysis of material composition,life process research and medical diagnosis.Especially,it has important application value in the detecting,positioning and tracing of biomolecular markers,as well as the investigation of biomolecular functions.At present,designing and synthetizing fluorescent probes responding specifically to biomolecular markers in living organisms is still an important topic for chemists and life scientists.Peroxynitrite（ONOO^-）,as a highly active substance in the organism,is closely related to physiological diseases including arthritis,diabetes,cancer,cardiovascular diseases and neurodegenerative diseases.At the same time,related studies have shown that ONOO^-will also play a positive role in life,such as signal transduction through nitrotyrosine residues and resistance to pathogens in the immune response.Therefore,real-time detection of the concentration change and distribution of the biomolecule ONOO^-in the living body is of great significance to the research of biology and life medicine.However,ONOO^-has the characteristics of short half-life and low concentration.It is highly desirable to develop fluorescent probes with high sensitivity,good selectivity and fast response to ONOO^-for the study of complex life systems.However,there is a great challenge to achieve this goal.Therefore,based on the above,this work was reasonably designed to synthesize a series of activation type highly sensitive,rapid responsive fluorescence probes which were successfully applied to the specific fluorescence detection and monitoring ONOO^-in different diseases.Furthermore,the link between ONOO^-and related diseases has been explored,providing a simple and effective assessment for the screening of drugs,early diagnosis and treatment.Here are five parts in the dissertation and the main contents are presented as follows:Chapter 1 is the introduction section.This part introduces the basic theory of ONOO^-fluorescent probe,including the composition of fluorescence,recognition mechanism,molecular design principle,the importance and necessity of ONOO^-detection.Moreover,recent development of ONOO^-fluorescence probes has been summarized.Finally,based on the current research status and development trend,the starting point and research ideas of this thesis are proposed.Chapter 2,a peroxynitrite activatable two-photon fluorescence probe BN-PN has been designed and synthesized for the imaging of ONOO^-in mice liver.The probe specifically reacted with peroxynitrite to furnish 140-fold fluorescence increase in vitro,which elucidated a high sensitivity for ONOO^-.Thus,subtle changes of ONOO^-levels in live cells can be sensitively imaged with this probe by two-photon microscopy.The probe also denoted the overproduction of ONOO^-in APAP-induced liver injury,and proved that administration with NAC can effectively alleviate DILI and reduce ONOO^-production in mouse liver.Furthermore,the probe demonstrated the rapid rise of ONOO^-level in the liver of DILI mice administrated with alcohol,it shows that drinking alcohol during medication will further lead to liver injury and increase the degree of liver deterioration.In one word,a two-photon fluorescence probe BN-PN for ONOO^-produced in the process of liver injury has been synthesized,which provides a new method for drug screening of liver injury and is conducive to the development of new drugs and drug screening.Chapter 3,a highly sensitive NIR fluorescence probe DDAO-PN has been designed and synthesized.It has been successfully applied on in vivo detection of ONOO^-in inflammation progress,the probe respondeds to ONOO^-with a significant NIR fluorescence enhancement at 657 nm（84-fold）within 30 s in solution.Intracellular imaging of exogenous ONOO^-with the probe demonstrated 68-fold fluorescence increase（F/F₀）.Impressively,the probe can in vivo detect ONOO^-fluxes in LPS-induced rear leg inflammation with 4.0-fold fluorescence increase and LPS-induced peritonitis with8.0-fold fluorescence increase,respectively.The remarkable fluorescence enhancement and quick response enabled real-time tracking of in vivo ONOO^-with large signal-to-noise（S/N）ratio.These results clearly denoted that DDAO-PN was able to be a NIR fluorescence probe for the in vivo detection and high-fidelity imaging of ONOO^-with a high sensitivity,and will boost the researchers of inflammation-related diseases.It provides a new method for the study of the relationship between inflammation and ONOO^-.Chapter 4,a high sensitivity AIE and ESIPT mechanism-regulated infrared fluorescent probes DPPO-PN has been designed and synthesized.It was used to detect ONOO^-in the process of rheumatoid arthritis.The in vitro experiment shows that the probe responses to the ONOO^-within 30 s,and the fluorescence was significantly enhanced at632 nm and increased by 160-fold.In the cell experiment,the probe fluorescence increased by 2.7-fold and 2.5-fold respectively.The excellent fluorescence properties of probes were used in LPS/CFA drug induced rheumatoid arthritis RA model mice.Within 5 min,the probe is applied to the two RA models of LPS/CFA and the fluorescence increased by7.0-fold and 2.8-fold,respectively.The results indicated that better fluorescence imaging of RA model mice could be possible by using this probe.Consequently,this probe could be used as an important detection tool for RA model mice,making the early diagnosis of RA possible.In general,the probe not only has better detection on ONOO^-,but also the development process of RA as an assessment means.Chapter 5,three novel CO fluorescent donors were designed and synthesized.In this donor,1-methyldihydroindole-2,3-dione not only acts as the recognition domain of ONOO^-,but also as the fluorescence quenching group of flavonol fluorophore.The probe FB-PN-3 not only has good water solubility,but also has specific reaction and high sensitivity toward ONOO^-.It can produce 175 folds fluorescence enhancement（600 nm）in vitro and is not affected by other reactive oxygen species and reactive nitrogen.The probe FB-PN-3 has good photostability.After rapid reaction with ONOO^-,it will release fluorophores,and then realize the controlled release of CO under visible light irradiation.At the same time,the probe FB-PN-3 had a good fluorescence imaging of the live cells with exogenous ONOO^-.Therefore,probe FB-PN-3 was a new type of ONOO^-activated CO donor with controlled CO release.It is expected that in the process of CO gas therapy in the future,the relationship between ONOO^-and CO therapy in the pathological process of inflammation and tumor will be established,so as to provide a method for the treatment of life by CO.