Research On Novel Mitochondrial Targeting Probes For The Detection Of Nitroreductase And SO₂ Derivatives

With the progress of the times and the rapid growth of life science and technology,organic small molecule fluorescent probe as the representative of the emerging biochemical sensors,which are used more and more widely in the fields of environmental monitoring,life science and medicine research.Organic small molecule fluorescent probe imaging technology makes the application of biological imaging,environmental quality testing,clinical medical diagnosis on the original basis of a step up.Through the in situ lossless fluorescence labeling of cells,tissues and even living organisms,people can more clearly,quickly and accurately observe the metabolic process and physiological role of the target in biological system.Mitochondria are important organelles in cells,called "energy plants" for cell operation,which are the main sites for energy supply,aerobic respiration.It is closely related to cell growth and regulation of cell cycle,and is involved in cell differentiation,signal transduction and apoptosis.Therefore,it is of great importance to develop organic small molecule fluorescent probes with mitochondrial targeting properties and detecting substances that are closely related to mitochondrial function.Hence,we synthesized the nitroreductase probe BICP(benzoindocyanine probe)and the detection of sulfur dioxide derivative probe P1 with mitochondrial targeting function,respectively,based on intramolecular charge transfer(ICT)mechanism.Our main works are as follows:(1)In the second Chapter,we use benzoindole cation as mitochondrial targeting group and pull electron group,which are introduced into fluorophore to become benzoindocyanine fluorophore(BICF)based on intramoleculular charge transfer(ICT)mechanism with strong fluorescence.In view of the fluorescence quenching properties of 4-nitrobenzyl bromide,the substitution of 4-nitrobenzyl bromide on the hydroxyl groups of BICF resulted in the intermolecular charge transfer process being blocked by the fluorescent-free mitochondrial targeting probe BICP.The nitro reductase selectively reduces the nitro group of 4-nitrobenzyl bromide to the amino group and undergoes a rearrangement reaction,so that the fluorescence is restored to detect the mitochondrial nitro reductase.The high selectivity,high sensitivity(detection limit:1.9 ng mL-1)and high reliability ratio(?83 times fluorescence enhancement)detection of nitroreductase in simulated biological environment solution and living cells were realized by ICT machanism.In addition,BICP was successfully applied to detect the mitochondria-specific activities of type I and II NTR in tumour cells.Low cytotoxicity and good biocompatibility of this probe has also been demonstrated.The results show oxygen-independent type I NTR was expressed in tumor cells even in situations when the cells were incubated under normoxic conditions,while under hypoxic conditions,the expression of NTR was increased substantially.Moreover,BICP is able to indicate the NTR activities in dynamic correlation to the concentrations of NTR.These findings reveal BICP can provide a promising molecular tool for elucidating mitochondria-specific NTR activity for varying applications in NTR-based or mitochondrial biology.(2)In the third Chapter of this thesis,we use 4-chloro-7-diethylamino coumarin aldehyde as the fluorophore precursor,and introduce the benzoindole in the 3-position aldehyde to the conjugated system,and use it as an electron-withdrawing group(with the 7-position electron donor-diethylamino group to form a "push-pull"electron system)and mitochondrial targeting groups,a fluorescent probe P1 with long wavelength emission was designed and synthesized.The probe is based on the intramolecular charge transfer mechanism(ICT)-ratio detection of mitochondrial HSO3-/SO32-small molecule fluorescent probe.The fluorescence emission wavelength is 675 nm due to the ICT effect and the large conjugated "push-pull" electron system.When the HSO3-/SO32-nucleophilic addition double bond makes the ICT effect of the system blocked,the conjugation system decreases.The fluorescence emission blue shift to 550 nm,the ability to HSO3-/SO32-to achieve the ratio of fluorescence signal detection.Spectral experiments examined that probe P1 had high selectivity and high sensitivity to HSO3-/SO32-(detection limit was 0.16?mol/L).The cytotoxicity test showed that the biocompatibility of probe P1 was good.From the co-localization experiments we can see that the probe has excellent mitochondrial localization performance.The.addition of NaHSO3 and 2,4-dinitrobenzenesulfonamide indicates that the probe is capable of imaging the mitochondrial exogenous and endogenous HSO3-/SO32-.