Novel Fluorescent Probes Based On Detection Of Cell Microenvironment And SO₂ Design And Bioimaging

Over the last few decades,significant progress has been made in the development of fluorescent probes that are widely used to monitor active species（e.g.enzymes,reactive oxygen/sulfur/nitrogen）and the cellular microenvironment（e.g.p H,viscosity,polarity）that affect cellular function in living organisms.Mitophagy,an important organelle activity that controls the quality and quantity of mitochondria,is accompanied by changes in the cellular microenvironment,such as lower p H,increased viscosity,and reduced polarity.Additionally,SO₂,important reactive sulfur,plays a crucial role in regulating vasodilation,anti-hypertension,and maintaining intracellular redox homeostasis.Furthermore,SO₂also affects the redox homeostasis of lysosomes and decreases the activity and number of lysosomes.Therefore,based on the vital physiological functions of the cellular microenvironment and SO₂,the development of fluorescent probes for monitoring mitophagy and specifically identifying SO₂in lysosomes would be beneficial for understanding the physiopathology relevant to mitophagy and lysosomal SO₂.Here,two novel fluorescent probes with excellent performance were constructed by rational design for visualizing p H/viscosity changes during mitophagy and p H-mediated lysosomal change in SO₂levels,respectively.The studies are as follows:1.A novel multifunctional fluorescent probe SP-DCN was designed and synthesized by cross-coupling polarity-insensitive bis-cyanoisophorone fluorescent dye（DCN）with p H-sensitive spiropyran（SP）via double bonding.By simultaneously responding to p H and viscosity within two different channels of red and green,SP-DCN visualizes mitophagy in a two-color mode.DCN and double bonds as sites that control transient intramolecular charge transfer（TICT）mechanism sensitive response to viscosity.At low viscosity,SP-DCN has only a weak emission at 580 nm.Once viscosity increases,the fluorescence of the green channel at 580 nm is enhanced due to the inhibition of double bond rotation.On the other hand,SP undergoes a reversible helical cyclization reaction at different p H values.The ring opening of SP under alkaline conditions and the presence of oxygen negative ions lead to enhanced intramolecular charge transfer（ICT）between indole and benzene rings,resulting in enhanced fluorescence of the red channel at 666 nm.Therefore,SP-DCN allows the detection of viscosity and p H changes at 580 nm and 666 nm,respectively,with good photostability,selectivity,and biocompatibility.Moreover,the cell imaging results showed that SP-DCN monitors cell p H and viscosity fluctuations sensitively.SP-DCN effectively accumulates in mitochondria and successfully visualizes the changes in p H and viscosity during mitophagy in A549 cells induced by starvation,drugs,and hypoxia in a dual-color mode.2.Benzopyranosonium is a fluorescent dye with a high molar absorbance coefficient,excellent solubility,and biocompatibility.Its 4-position is easily attacked by the nucleophilic reagent owing to the presence of positively charged oxygen atoms.Therefore,benzopyranosonium may be an ideal site for achieving an ultrafast sulfite response.In addition,2-position of benzopyranosonium is susceptible to the influence of acid and base and is prone to ring-opening under basic conditions.Due to the presence of effective photo-induced electron transfer（PET）quenching between the molecules,the ring-opening interconversion isomerizes to form trans-chalcone without fluorescence,reducing the effective attack of HSO₃^-.However,this ring-opening tends to occur at larger p H values.Therefore,benzothiazole,a strong electron-withdrawing group,attached to the benzopyranosonium 2-position is more capable of driving the sensitivity of benzopyranosonium to acids or bases.Here,a novel fluorescent probe BP-BT for p H-mediated SO₂was developed.It was shown that the presence of protons greatly influences the response characteristics of BP-BT with HSO₃^-,and the fluorescent signal at 420 nm was output only at low p H（p H<6.5）and in the presence of HSO₃^-.And the F_{420 nm}/F₀was found to be maximum at p H=5.0,which is as much as 51 times higher than that under alkaline conditions.In addition,BP-BT has good biocompatibility,photostability,and selectivity.Finally,BP-BT was successfully applied to monitor the expression levels of endogenous and exogenous sulfites in lysosomes.