Design,Synthesis And Biological Application Of Novel NIR Mitochondria-Targeted Viscosity And ROS Probes

Intracellular viscosity governs essentially all the diffusion mediated processes,including mass transportation,signal transduction,biomolecule interactions,diffusion of metabolites,and electron transport.As a vital organelles in eukaryotic cells,mitochondria is characterized by specific viscosity,which in turn,plays essential roles in their functions such as ATP generation.It is recognized that anomalous mitochondrial viscosity is related to neurodegenerative diseases,diabetes,and cell malignancy.Therefore,intracellular viscosity represents a potential biomarker for different diseases.Due to the heterogeneous nature of intracellular viscosity,it is essential to develop organelle-specific probes for monitoring viscosity or its dynamics in varying biological processes such as apoptosis and mitophagy.In this thesis,we designed and synthesized a novel NIR fluorescent probe NMV to detect the change of viscosity in mitochondria.The structure of probe was fully characterized by ¹H NMR,¹³C NMR and high-resolution mass spectra（HRMS）.The fluorescence intensity of the probe exhibited an excellent linear relationship with viscosity value,and fluorescence is quite stable in different pH solvents at the same viscosity.Besides,bioactive species,such as metal ions,amino acids,reductive species,and oxidizing species,do not interfere with the detection of viscosity.Based on the low biotoxicity,good light stability and low biological background of the probe,we successfully applied it to the fluorescence imaging viscosity in living cell.The results of cell imaging co-localization experiments showed that the probe can be specifically located in the mitochondria of the cell due to the positive charge of pyridine salt of the probe structure.The results showed that the viscosity of cervical cancer cells（HeLa）,was significantly increased by starvation.The intracellular viscosity of HeLa also increased significantly under the stimulation of nystatin.The above experimental results indicate that the probe is expected to be applied in imaging viscosity changes of related diseases in vivo,and provide an excellent analytical means for elucidating the mechanism of disease development.In order to further investigate the effect of active oxygen concentration changes in mitochondria on viscosity,based on the dye generated in situ strategy,we designed and synthesized a near infrared fluorescent probe NMS to detect superoxide anions in mitochondria.After the sulfonic acid ester in the probe responds to the superoxide anion,the phenol hydroxyl group is released,followed by the molecular cyclization reaction to generate the coumarin parent nucleus in situ.The probe produces near-infrared fluorescence due to the synergistic effect of the electron-donating thiazine and electron-withdrawing pyridine salt,which enhanced the degree of intramolecular charge transfer.The positive charge of pyridine salt enables the probe to specifically locate in the mitochondria,while the benzyl chloride make the probe anchor the mitochondria,due the addition reaction of benzyl chloride and thiol protein rich in the mitochondria,so as to enabling accurate detection of superoxide anions in the mitochondria.