Design Of Dual-modified Fluorescent Skeletons Based On Xanthene Compound And Bioimaging Applications

With the rapid development of fluorescence imaging technology,fluorescent molecular probes have been widely used in the detection of various biomarkers due to their advantages of high spatial and temporal resolution,good specificity,high sensitivity,non-invasive and in situ detection,etc.They have become indispensable imaging tools in the field of biological analysis.In recent years,more and more dual-responsive fluorescent molecular probes have been developed to explore biomolecular signaling pathways,intermolecular interactions,and improve the accuracy of disease diagnosis.In this paper,three novel fluorophores with dual-modifiable sites were synthesized based on xanthene compound(Dy OH).They are successfully applied to construct dual-responsive fluorescent probes for studying the signaling pathways of biomolecules in different biological models and the morphological changes of lysosomes during cell death.The specific work content is as follows:(1)Hexosaminidases(Hexs)as an exoglycosidase participate in the hydrolysis of non-reducing end of glycoconjugates in organisms.Hexs activity is significantly changed in many diseases such as hereditary neurodegenerative diseases,kidney function damage,cancer,etc.It is particularly important to construct a fluorescent probe to detect the activity of Hexs for studying its physiological role.A fluorescent probe Hex-1 was synthesized based on fluorophore scaffold Dy OH.Hex-1 exhibited good water solubility,large stokes shift and low toxicity,and it also could rapidly and sensitively detect Hexs.Furthermore,Hex-1 was successfully applied to localize the distribution of endogenous Hexs,and evaluate the fluctuation of Hexs activity during drug-induced kidney injury.(2)Three novel fluorophores with dual-modifiable sites(N atom and-OH group)were synthesized based on Dy OH.The nitrogen atom and hydroxyl group could be rapidly protonated and deprotonated under different p H conditions,which endow the fluorophore with different optical properties.Taking the fluorophore QOH as an example,QOH emitted strong fluorescence at 560 nm under alkaline solution and its emission wavelength was red-shifted to 720 nm with a large Stokes shift(120 nm)under acidic conditions.The effect of the introduction of substituent at different modification sites and the properties of substituent on the optical properties of QOH were fully discussed by theoretical calculation.(3)Fluorescent probes QM-R and Q-SH for detecting H₂O₂ and H₂S were synthesized based on the nitrogen atom and hydroxyl group of QOH,respectively.QM-R emitted strong near-infrared fluorescence at 720 nm because quinoline salt enhanced the intramolecular"push-pull"electronic effect.After reacting with H₂O₂,the fluorescence emission of QM-R was significantly blue-shifted to 560 nm owing to the disappearance of quinoline salt.Q-SH had photoinduced electron transfer effect that quenched the fluorescence due to the strong electron-withdrawing group(2,4-dinitrophenyl).Upon addition of H₂S,Q-SH was hydrolyzed and released the fluorophore QOH that exhibited a significant enhancement of the fluorescence at 560nm,realizing the detection of H₂S.Based on the above fluorescent signals,QM-R/Q-SH had successfully applied in monitoring the change of exogenous and endogenous H₂O₂/H₂S level in living cells.(4)Hydrogen sulfide(H₂S)is a typical gas signal molecule that participates in various pathological and pathological processes.The reducibility of H₂S plays a crucial role in resistance to oxidative stress.It is of great significance to design a fluorescent probe for accurately monitoring the dynamics of H₂S during oxidative stress.A fluorescent probe QM-RSH was synthesized based on QOH,which could sequentially respond to H₂O₂ and H₂S.The NIR fluorescence of QM-RSH was quenched with the addition of H₂O₂.In the presence of H₂O₂,QM-RSH exhibited high selectivity and sensitivity for detecting H₂S.Base on the"on-off-on"signal change,QM-RSH can be successfully applied to accurately monitor the fluctuation of H₂S level during oxidative stress in Hep G2 cells and inflammatory zebrafish without interference from other physiological processes.(5)Cell death plays an important role in body development,maintenance of tissue function and homeostasis.Different types of cell death could cause disparate physiological responses,such as bradyarrhythmias and immune response.It is very important to establish a fluorescent molecular probe that distinguish the types of cell death for studying the pathology.A p H-activated fluorescent probe PNE-Lyso was successfully developed based on the fluorophore POH.PNE-Lyso could rapidly,sensitively and reversibly detect p H change in vitro.Under the simulated lysosome environment(p H=5.5),PNE-Lyso could sensitively detect Hexs with ratio fluorescence signal.In addition,PNE-Lyso could specifically target lysosomes due to its eosinophilic.Thus,PNE-Lyso successfully distinguished apoptotic cells from necrotic cells,and investigated the process of drug-induced cell death through tracking the change of lysosome morphology.