Mitochondria-targeting Multifunctional Fluorescent Probes For Hypochlorous Acid: The Designed Syntheses And Applications

Hypochlorite(HOCl)and mitochondrial status are related to the pathogenesis and treatment strategies of many diseases.Therefore,real-time detection of HOCl and mitochondrial status is of great significance to the development of biomedical research.Among many detection methods,fluorescence detection method is the best choice for detecting HOCl and mitochondrial status in organisms because of its high sensitivity,good selectivity,wide linear range,non biological invasion,low toxicity,and real-time detection.Among many fluorescent detection materials,organic small-molecule fluorescent probe is an ideal tool for the detection in organisms because of its advantages of locating a variety of subcellular organelles,low cost,no pretreatment,not affected by external electromagnetic field,long-distance luminescence,and so on.Therefore,it is of great significance for the development of analytical chemistry and biomedical research to develop organic small-molecule fluorescent probes and apply them to the detection of intracellular HOCl and mitochondrial status.In addition,the design,synthesis,and spectral analysis of organic small-molecule fluorescent probes also play a very important role in the development of organic chemistry and molecular photochemistry.In recent years,more and more researchers have been engaged in the research and development of fluorescent probes to detect HOCl and mitochondrial status,which has become a hot topic in international research.Among them,the design and synthesis of HOCl fluorescent probes targeting mitochondria and realizing multiple functions in real time is a very meaningful work.These probes can detect HOCl and other analytes in real time,and reveal the physiological and pathological process of mitochondria at the same time,so as to provide a multi-channel analysis method for the detection and treatment of many diseases.However,the research of this kind of probes is still a difficult problem in the field of organic small-molecule fluorescent probes.In this paper,we proposed a new method to construct multifunctional HOCl probes targeting mitochondria.The research direction of this paper is the design,synthesis,and application of novel multifunctional HOCl fluorescent probes targeting mitochondria.Its advantages mainly include: the novel probe structure designed by us can realize the splicing of different fluorophores,which provides a very useful organic structure for the detection of multi-analytes including HOCl in the future;the probe designed by us not only has mitochondria-targeting capacity,but also has mitochondria-immobilized ability to some extent.Compared with the reported mitochondria-targeting HOCl probes,this immobilization allows the probe to detect intracellular HOCl concentration more accurately;the developed probe can also use the signals of other fluorophores to reveal complex mitochondrial physiological and pathological processes.Besides,the ratiometric detection of HOCl and the real-time detection of multi-analytes containing HOCl were fulfilled.In this paper,our main work including the following three parts:(1)We have designed and synthesized a class of mitochondria-targeting HOCl probes with double fluorophores,which provides a series of practical structural construction units for future multifunctional HOCl probes targeting mitochondria.Through the synthesis of probe NCIR,it is proved that this kind of structure can react with additional recognition group to construct multifunctional probes.We also proved that the different splicing structures of this series have HOCl response ability through the reaction of these probes with HOCl solutions,and verified the mechanism of the reaction of these probes with HOCl.We also proved that imidazolium salt can be used as mitochondria-targeting groups in cell,just the same as triphenylphosphine salt and pyridine salt.(2)The detection capacities of probe CIR(coumarin-imidazolium salt-rhodamine)were studied in details.CIR further proved that our probe structure has high selectivity and sensitivity for HOCl.CIR can also exist in depolarized mitochondria,and its fluorescence intensity in cells is not affected by mitochondrial status,which enables it to track the changes of mitochondrial morphology in real time.We used CIR to simultaneously detect the changes of HOCl and mitochondrial morphology in MCF-7cells for the first time.This experiment also confirmed for the first time that HOCl could change the morphology of mitochondria in MCF-7 cells.CIR can also detect the low concentration of basal HOCl produced in RAW264.7 cells.We also used it to confirm for the first time that the mitochondrial morphology of RAW264.7 cells changed little with the increase of intracellular HOCl concentration before and after the induction of inflammation model.(3)The detection capacities of probe NCIR(NBD-coumarin-imidazolium saltrhodamine)were studied in details.NCIR proved that the structure designed by us has the ability to analyze multi-analytes.NCIR can simultaneously detect intracellular thiols,HOCl,and mitochondrial status.Like CIR,NCIR has high selectivity and sensitivity for HOCl.In addition,NCIR can also detect cysteine/homocysteine with high selectivity and sensitivity.NCIR can also exist in depolarized mitochondria,but different from CIR,NCIR has a strong fluorescence response to mitochondrial status,which makes it difficult for NCIR to real-time detect mitochondrial morphology.Instead,NCIR can distinguish MCF-7 cells from HBL-100 cells by the changes of fluorescence intensity,and simultaneously detect the changes of mitochondrial membrane potential and the opening of mitochondrial permeability transition pore(m PTP).This paper presents our unique understanding of the design strategy of organic smallmolecule probes,and provides a reference for the design and synthesis of multifunctional probes in the future.At the same time,we successfully used imidazolium salt as a mitochondria-targeting group for the first time,which provides an additional idea for the design of mitochondria-targeting probes in the future.In addition,there are few fluorescence analysis methods that can detect the opening of m PTP.NCIR can detect the opening of m PTP in cells,which provides a new idea for the development of this kind of probes in the future.