Design,Synthesis And Biological Application Of Biothiols Fluorescence Probes

Fluorescent probe has attracted wide attention in various industries because of its advantages such as non-damage,high spatio-temporal resolution and visual in-situ imaging.Especially in the field of biomedicine,fluorescent probes are playing a more and more important role and have become an important focus of scientific research.Small molecular biothiols such as cysteine（Cysteine,Cys）,homocysteine（Homocysteine,Hcy）and reduced glutathione（Glutathione,GSH）,are important substances in complex organisms.When their content in cells is abnormal,it is often closely related to alzheimer’s disease,cardiovascular disease,neurological disease,malignant tumor and other diseases.Therefore,the use of fluorescence probe for real-time monitoring of biothiols levels in cells and the mechanism of its changes in vivo are of great significance for some diseases and evaluating disease progression.The specific research work is as follows:1.The near infrared"turn-on"fluorescent probe 3-1 with chromene skeleton was synthesized by using methylene blue with good biocompatibility and excellent optical properties.The new probe can specifically recognize biothiols and show the fluorescence enhancement signal of 698 nm unndergoing a novel cascade mechanism.In the further study,it was found that probe 3-1 could realize real-time visual imaging of biothiols in various living cancer cells and mice.In addition,we found that the level of biothiols in drug-resistant cancer cells was significantly higher than that in corresponding cancer cells,indicating that biothiols levels may play an important role in drug resistance of cancer cells.2.Based the cascade mechanism discovered in the previous section,we designed and synthesized probe 4-1 by combining 3,3a-dihydrocyclopenta[b]chromen-1（2H）-one with coumarin aldehyde.The probe has good water solubility and can react with Cys at an ultra-fast speed within10 s with a low detection limit（0.49μM）.Using this probe,we successfully stained RAW 264.7 cells and targeted the mitochondria to show obvious fluorescence signals in the orange channel.Probe 4-1 has become a useful model tool because of its unique structure and excellent optical properties,which lays a good foundation for the design of integrated probe for diagnosis and treatment.3.Based on the above work,we found that the level of Cys may be closely related to the development of cancer,while the related studies have shown that oxidative stress is related to the occurrence of cancer and many other diseases.In order to explore the relationship between Cys and oxidizing substance H₂O₂,we synthesized a caged fluorescein probe 5-1 to evaluate complex biological processes in living cells in situ.The co-existence of Cys and H₂O₂ enable the probe to show specific fluorescence changes.Meanwhile,in the presence of luciferase,the simultaneous presence of Cys and H₂O₂ will activate the bioluminescence signal.The probe detected Cys-induced up-regulation of H₂O₂ in living MDA-MB-231 cells for the first time,which laid a foundation for further study on the biological regulation process of balancing the concentration of Cys cells.In addition,the probe also provides evidence for the autooxidation of Cys in vivo.