Designing Multi-Functional Fluorescent Probes For Visualization Of The Redox Signals And Viscosity In Living Cells

Multifunctional single fluorescent probes are widely applied in the field of bioimaging research because of their advantages of multiple responsive sites and differentiated real-time monitoring.They are considered as one of the efficient tools for biological researches due to their ability of imaging interrelated active substances in a biological process by multi-fluorescence channel.Accordingly this paper focuses on the design of nucleus-and mitochondria-targeting multifunctional fluorescent probes for visually monitoring multiple redox signals and viscosity in living cells.The details are as follows:（1）Although much effort has been placed in designing multifunctional fluorescent probes,there is no report on the design of nuclear-targeting multifunctional fluorescent probes.At present,nuclear dyes are designed by generally using cationic groups such as pyridine,indole,and benzothiazole salts as key components,which are capable of binding with DNA phosphate groups via electrostatic interaction,However,grafting the above cationic groups is also the main strategy for designing mitochondrial-targeted probes based on their electrostatic interaction with the negatively charged mitochondrial inner membrane.These facts invite inevitably the key scientific issue of how to convert probes that contain the above cationic groups from mitochondrial-to nucleus-targeting through chemical structure modification,which simultaneously allows that the nuclear-targeting probes exhibit multi-channel response to multiple redox signals.Previously,our group designed a mitochondrial-targeting multifunctional probe COU-PM based on bridging diethyl-aminocoumarin and 4-pyridyl acrylonitrile.Based on this work,we designed its derivatives COUD-PM and COUD-PB through inserting an additional double bond（Figure 1）with the aim of improving its flexibility to achieve strong binding with the small groove of DNA.We confirmed by fluorescence co-localization experiments that COUD-PM and COUD-PB could localize to the nucleus quickly and efficiently,and exhibit a turn-on red fluorescence（λem=695 and 700 nm,respectively）based on the fact that their binding with DNA restrains their twisted intramolecular charge transfer（TICT）process.Our data indicate that the switch of COU-PM from mitochondria-to nucleus-targeting can be achieved only by inserting an additional double bond into its conjugated system.The binding constants of COUD-PM and COUD-PB with ct DNA are 3.1×10⁶ and 5.4×10⁵ M^-1,respectively.Through molecular docking software analysis,it is confirmed that they could effectively bind to small groove of DNA through weak interactions such as electrostatic force and hydrogen bond.Additionally,the probes COUD-PM and COUD-PB were successfully applied to visualize cell cycle and nuclear morphological changes during cisplatin-induced apoptosis of Hep G2 cells.Compared with COUD-PM,COUD-PB is characterized by the presence of an additional phenylboronic acid pinacol ester,allowing that it has the advantage of more response sites.Furthermore,it was found that COUD-PB features a large two-photon absorption cross section（σ=1050 GM）.Accordingly,we use COUD-PB two-photon imaging of multiple redox signals in the nucleus:sulfur dioxide（SO₂）,peroxynitrite anion（ONOO^-）and hydrogen peroxide（H₂O₂）.Specifically,nucleophilic attack of SO₂at the double bond to generate an adduct COUD-PB-SO₂,which emits green fluorescence（λem=500 nm）.ONOO^-can selectively oxidize and break the double bond,generating COUD-PB-ONOO^-with the emission of yellow fluorescence（λem=550 nm）.COUD-PB-H₂O₂ is yielded based on the oxidative cleavage of borate units by H₂O₂,which emits orange fluorescence（λem=615 nm）.Together,by virtue of the excellentspectral separation（≥50 nm）,the probe is capable of visualizing the change of nuclear morphology（700 nm）,SO₂（500 nm）,ONOO^-（550 nm）and H₂O₂（615 nm）in a fashion of hitting four birds with one stone.（2）As one of the important biothiols,cysteine（Cys）plays an important role in maintaining cellualr redox homeostasis.Meanwhile,viscosity is one of the elements that constitute the cell microenvironment.Abnormal viscosity will significantly affect cell function.To construct a bifunctional fluorescent probe for simultaneous monitoring of Cys and viscosity in mitochondria,we used quinoline-vinyl-N,N-dimethylaminoaniline dye QA as fluorophore and acrylate as Cys response group to engineer the probe QA-PA（Figure 2）.The probe exhibits bright red fluorescence（λem=670 nm）response to increased viscosity which restricts the molecular rotation.The Michael addition of Cys to the acrylate unit of the probe,followed by intramolecular cyclization of seven-membered heterocycles and 1,6-elimination,generates the fluorophore QA with the emission of yellow fluorescence（λem=550 nm）.The probe was successfully applied to visualize the changes of mitochondrial Cys levels and viscosity in yellow and red dual channels,respectively.