Design Of Nucleus-and Mitochondria-targeting Fluorescent Probes Based On The Cationic Skeletons

As the regulatory center of eukaryotic cells,the nucleus plays an irreplaceable role in cell growth,differentiation and metabolism.Nucleus dysfunction can lead to apoptosis,necrosis and is closely related to the development of various diseases.Therefore,it is of great significance for life science research to design nucleus-targeting fluorescent probes to realize accurate visualization of nucleus state.At present,the design of nucleus-targeting probes usually uses cationic groups（such as pyridinium salts,quinolone salts and benzothiazole salts）as key components to achieve effective binding to DNA through electrostatic interactions with DNA phosphate groups.However,due to the high negative potential of mitochondrial inner membrane,grafting cationic groups is also an important strategy to construct mitochondria-targeting probes.Therefore,a key scientific problem in the field of nucleus-targeting probe design is how to realize the switch of cationic groups from mitochondria-to nucleus-targeting via chemical structure modification.Solving the above problem is very important for developing highly specific nucleus-targeting probes.Viscosity is one of the important microenvironment parameters in cells,which affects the transport,diffusion,signal transmission and other processes between biomolecules in cells.Normal viscosity is the prerequisite for nucleus function,while abnormal viscosity will affect the integrity and expression of genes,which lead to cell dysfunction and the development of inflammation.Therefore,designing fluorescent probes for mapping nucleus viscosity is of great significance to explore its pathophysiological roles.H₂O₂ is the most stable reactive oxygen species.H₂O₂ is a“double-edged sword”.Specifically,low concentration of H₂O₂ plays an important role in regulating cell growth,proliferation and differentiation,while high concentration of H₂O₂ will lead to oxidative stress,which induces the development of cancer and other related diseases.Mitochondria,as an important organelle of eukaryotic cells,its function depends on the control of redox homeostasis.Therefore,designing fluorescent probes to monitor the changes of H₂O₂ concentration in mitochondria is of great significance to explore its pathophysiological roles.Boronate is the most widely used response group in the design of H₂O₂ fluorescent probes.It proceeds via nucleophilic attack of H₂O₂,Bayer-Villiger oxidation-like rearrangement and hydrolysis,therby releasing free hydroxyl groups.However,the peroxynitrite anion（ONOO^-）reacts with boronate through the same mechanism,and its reaction rate is five orders of magnitude faster than that of H₂O₂.The above facts invite inevitably the question of how to design boronate-based fluorescent probes with high selectivity toward H₂O₂over ONOO^-.In this thesis,we focused on the above two key scientific issues and carried out the following works:（1）Hydroxystyrene pyridine salt（HSP）is not only a classical two-photon fluorophore featuring a push-pull electronic structure and an intramolecular charge transfer process but also an outstanding mitochondria-targeting agent based on its pyridine salt cation group.Accordingly,we take HSP as an example,we designed its hydroxyl,methoxy and fluorine atoms substituted derivatives,including DHSP,MHSP,DMSP and FHSP（Figure 1）,try to achieve the conversion of HSP from mitochondria-to nucleus-targeting through simple chemical structure modification.We found that only introcuction of an additional o-hydroxyl group into HSP transforms its mitochondria-to nucleus-targeting.We confirmed by the time-dependent fluorescence co-localization experiments that DHSP with o-hydroxyl has excellent nucleus-targeting ability,and localizes effectively in the nucleus within 5 minutes.The binding constant with DNA was determined to be 1.92×10⁶ M^-1 by the fluorescence titration experiments,which is close to that of the conventional nucleus dye DAPI(10⁶ M^-1),indicating that DHSP is a strong binding agent with DNA.Molecular docking calculation shows that DHSP can effectively bind to DNA through the electrostatic interaction between its pyridine salt cation and the phosphate anion of DNA and the hydrogen bond interaction between its o-hydroxyl and cytosine and adenine of DNA.The circular dichroism experiments suggest that DHSP binds with DNA in the minor groove.The probe was also successfully applied in visualizing the morphological changes of nucleus characteristics in the process of cell cycle and apoptosis,and mapping in vivo obvious condensation and deep staining of the nucleus during cerebral ischemia-reperfusion injury in mice.（2）The designed nucleus-targeting probe DHSP in the above chapter is the molecular rotor between the push（o-dihydroxybenzene as an electron donor）and pull（methylpyridinium salt as an electron acceptor）parts around a single bond,thereby featuring a twisted intramolecular charge transfer process.Considering that viscosity can block the molecular rotation,we reasoned that the probe should be also a turn-on nucleus viscosity probe（Figure 2）.Indeed,the probe exhibited a turn-on response to viscosity at 540 nm.In addition,the probe was successfully applied in visualizing both the increase of nucleus viscosity induced by nystatin,LPS,starvation and hydrogen peroxide（H₂O₂）,and the decrease of nucleus viscosity induced by excessive intake of heavy metals.（3）We bridged coumarin and benzopyranium salt skeleton as a near-infrared fluorophore BC-OH to design a mitochondria-targeting H₂O₂ fluorescent probe BCB by using pinacol phenylborate ester as the response group（Figure 3）.The probe can map the fluctuation of H₂O₂ levels by virtue of its different activity of electrophilic points（site 1-3）without the interference of ONOO^-,H₂S and SO₂.Nucleophilic attack of H2O2 at the boronate（site 1）releases the free fluorophore BC-OH,exhibiting a turn-on fluorescence response at 670 nm.Nucleophilic attack of ONOO^-at the benzopyranyl onium salt with high electrophilic activity（site 2）,followed by oxidative cleavage of the carbon-carbon bond to generate HCA with an emission wavelength of 457 nm.Whereas,nucleophilic attack of hydrogen sulfide（H₂S）and sulphur dioxide（SO₂）at the electrophilic site 3 of the probe affords the adducts（BCB-SH and BCB-SO₂）which features an emission wavelength of 573 nm.Additionally,the probe allowed the visualization of endogenous and exogenous production of H₂O₂ in mitochondria,the detection of increasing H₂O₂ levels in a Parkinson’s disease cell model,and the intravital imaging of H₂O₂ in zebrafish.