Studies on Design,Synthesis and Applications of Glutathione-Specific Fluorescent Probes

Author:Xu Zhi Qiang

Supervisor:liu sheng hua yin jun


Degree Year:2019





As a special functional dye,organic fluorescent dyes can be used as molecular probes and fluorescence imaging techniques to realize real-time non-destructive detection of biological analytes and dynamic,non-invasive visual tracking of biological processes due to its high sensitivity,high spatial resolution,good biocompatibilities and real-time imaging capabilities.It is widely used in biomolecular detection,fluorescence imaging in vivo,early cancer diagnosis and drug metabolism tracing,and has become an important observation tool in the field of chemical biology and biomedical engineering.With the development of fluorescence technology,the construction of fluorescent probes with high specificity and high sensitivity to detect biological analytes and visualize biological processes has made long.term progress.However,most fluorescent probes usually have only one channel of fluorescence signal during the fluorescence signal acquisition process and the band channel is limited,which is easy to encounter interference of photobleaching,energy scattering and self-absorption caused by the dye itself stability and signal bandwidth.Therefore,how to:use fluorescent signals reasonably and effectively to improve the detection and imaging performance of fluorescent probes and solve the problems encountered in the practical application of fluorescent dyes has become a hot spot topic in current research.Glutathione is a tripeptide compound containing active thiol groups and plays an important physiological role in many physiological processes in biological systems.Intracellular glutathione is an important biomarker for human health,and its abnormal content is associated with various diseases such as cancer,AIDS,neurodegenerative diseases,delayed growth,liver damage and cardiovascular disease.Therefore,it is a challenging and innovative work to develop multi-purpose probes with single fluorescence signal or multi-channel fluorescent probes using glutathione as the detection analyte.In response to this research concept,we constructed three probe molecules with different fluorescence emission behaviors by using glutathione as the detection analyte and connecting mitochondria and/or lysosomal targeting functional groups at the same time,and studied their potential applications.The main research contents are showed as follows:In chapter 1,we briefly outlined an overview of current research status and development of fluorescent probes using glutathione as a detection analyte by different reaction recognition sites.In chapter 2,a double-targeting GSH-specific near-infrared fluorescent probe Cy-S-Np has been developed based on a cationic cyanine IR-780 dye coupled with a morpholine-coating naphthalimide,and a thioether as a linking group.Studies on UV-Vis absorption and fluorescence spectra showed that the probe has high selectivity to glutathione in the near-infrared fluorescence region and is not interfered by other amino acids.The results of cell experiments indicated that the probe could be applied to the fluorescence imaging of exogenous and endogenous glutathione,and could label mitochondria and lysosome glutathione in the subcellular organelles to realize the design strategy of’one-probe and double-targeting’.Moreover,the mouse imaging confirmed that this cyanine-based probe could be capable of monitoring glutathione in vivo.In chapter 3,a two-channel fluorescent probe,Lyso-NpSSD,has been designed and constructed with naphthalimide derivative as a green fluorescence emitting fluorophore and dansulfonamide derivative as an orange fluorescence emitting fluorophore linked by sulfonamide as a double fluorophore connecting group for the detection of glutathione.Studies on UV-Vis absorption and fluorescence spectra showed demonstrated that the probe can respond to glutathione with high selectivity and is not interfered by other amino acids.Cellular experiments showed that the probe can be applied to two-color fluorescence imaging of glutathione in living cells.In addition,interestingly,subcellular organelles co-localization experiments indicated that the fluorescence signals of the probes in two different channels were able to simultaneously and specifically label lysosomes in living cells.In chapter 4,near-infrared fluorophore cyanine dye IR-780 was introduced instead of orange fluorescence emission fluorophore dansyl-sulfonamide derivative to expand the emission band distance between the cyanine IR-780 and the naphthalimide derivative,and a dual-channel fluorescence-on probe CyP-SNp has been developed for glutathione,which contained the naphthalimide derivative as the visible fluorophore and the cyanine dye IR-780 as the near-infrared fluorophore bridged by sulfonamide as a double fluorophore linking group and thiol-reactive sulfonamide moiety.Studies on UV-Vis absorption and fluorescence spectra showed that the probe can respond to glutathione with high specificity in both visible and near-infrared channels.More importantly,the green fluorescence in the visible channel and the red fluorescence in the near-infrared channel could simultaneously and specifically mark the mitochondria in living cells,so as to realize the spatiotemporal and synchronous fluorescence imaging of lysosomal GSH at the same subcellular organelle in two completely different fluorescence channels.In addition,we also revealed the mechanism of operation of this probe through a variety of spectroscopy techniques.In chapter 5,the study has confirmed that the probe CyP-SNp could be applied to two-channel cell imaging.In view of its good two-photon property of naphthalimide fluorophore in the visible channel and the cyanine dye IR-780 fluorophore in the near-infrared channel with near-infrared emission property,we then investigated whether it could also be used for dual-channel fluorescence imaging in living tissue.The imaging results of mouse liver tissue slices showed that the probe had two-photon imaging ability and near-infrared imaging ability,and could be applied to realize two-channel tissue fluorescence imaging of glutathione in mouse liver tissue with imaging depths of up to 200 μm in visible channel and imaging depths of up to 120 μm in near-infrared channel.In addition,the probe could also be used to monitor glutathione in vivo by taking advantage of the near-infrared emission.In chapter 6,the summary of the research content of this paper and the outlook of future work were given.