Design And Synthesis Of Fluorescent Probes For Imaging Of Manganese In Live Cells

Manganese(Mn²⁺) is one of the several essential metals for organisms. It is crucial for numerous metabolic functions, as a cofactor for a wide variety of enzymes, such as transferases, hydrolases, lyases, arginase, and superoxide dismutase and so on. The abnormal changes of Mn²⁺ will affect the normal physiological metabolic activities. Enormous evidence suggests Mn²⁺ deficiency may lead to growth retardation, osteoporosis and movement disorders. Conversely, excess of Mn²⁺ causes neurological damage even disease such as a Parkinson's disease. The level of manganese ions is different in various areas of cells, and the relevant functions are aslo differ. However, the Mn²⁺ homeostatic mechanism and concentration change in different subcellular regions and its association with the chemical biology process is still unclear. Therefore, we have an urgent need to develop methods to accurately detect the manganese ion concentration in different subcellular structures.Fluorescence imaging method has been widely used in biology, chemisty and medicine in recent years due to the advantages of high selectivity and sensitivity, fast response time and simple operation. Currently, fluorescence imaging to detect Mn²⁺ in organelles is rarely reported. Therefore, it is urgent to develop new fluorescence imaging probes to detect the changes of Mn²⁺ concentration with precise targeting ability, as well as high sensitivity and selectivity. It will provide ideal tools to reveal the relationship of Mn²⁺ with biological processes and occurrence and development of diseases.Based on the reason above, we designed a series of new Mn²⁺ fluorescence sensors, which were used in the cells and vivo imaging analysis successfully. Specific work of this paper was as follows:First, we designed a new near-infrared mitochondrial-targeting fluorescent probe termed Cy-MitoMn for imaging of Mn²⁺ by covalent conjugation of a bis-cyclohexyl-pyridine substituted macrocyclic ligand to a near-infrared fluorophore cyanine. The near-infrared fluorescence of Cy-MitoMn was quenched due to the mechanism of photon-induced electron transfer?PET?. With adding Mn²⁺, the PET process was blocked effectively by forming the Mn²⁺-ligand complex, leading the recovery of the near-infrared fluorescence of cyanine. The fluorescence intensity at 784 nm became stronger with Mn²⁺ concentration increasing. Cy-MitoMn showed the linear fluorescence change over a board range of Mn²⁺ concentration from 0 ?M to 2.4 ?M, The detection limit was calculated to be 12 nM. The dissociation constant?Kd? of Cy-MitoMn and Mn²⁺ was 2.48x10-6 M. Experimental results showed that the probe was capable of detecting visually Mn²⁺ concentration changes in the mitochondria of the human liver cells. Moreover, it was the first example to achieve Mn²⁺ fluorescence imaging analysis in vivo. In addition, by utilizing Cy-MitoMn and another H2O2 sensor developed in our group, we verify that Mn²⁺ could induce production of H2O2 and effectively remit the apoptosis in liver cells. The probe would provide a ideal imaging method to explore the biological processes related mitochondrial Mn²⁺ and the association of manganese ions with mitochondrial oxidative stress.Second, we designed a novel fluorescent probe termed BDP-CytoMn that can locate in the cytoplasm and detect Mn²⁺ by covalent conjugation of the Mn²⁺ ligand to a fluorophore BODIPY. Similarly, the fluorescence of BODIPY was quenched due to PET process. After addition of Mn²⁺, the fluorescence of BODIPY was recovered due to blocking of PET process. The probe can respond quickly to the changes of Mn²⁺ concentration.