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Fluorescent probe capable of tracing mitochondria-lysosome interaction in super-resolution mode

A technology of fluorescent probes and mitochondria, which is applied in the field of fluorescent probes, can solve the problems of not being able to provide information on the state changes of organelles, and achieve good cell permeability and excellent effects of non-toxicity

Active Publication Date: 2021-03-05
NANJING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although such probes are good fluorescent materials for visualizing organelles and proteins under the microscope, they only reflect the morphological behavior of individual organelles, and another probe with an emission wavelength is still needed to visualize organelle-organelle interactions
Furthermore, these probes cannot provide information about changes in the state of the organelle

Method used

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  • Fluorescent probe capable of tracing mitochondria-lysosome interaction in super-resolution mode
  • Fluorescent probe capable of tracing mitochondria-lysosome interaction in super-resolution mode
  • Fluorescent probe capable of tracing mitochondria-lysosome interaction in super-resolution mode

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: the preparation of fluorescent probe Coupa:

[0029] References Wu J, Liu W, Zhuang X, et al. Fluorescence turn on of coumarinderivatives by metal cations: a new signaling mechanism based on C=Nisomerization, Org. Lett. 2007, 9, 33-36. Compound 1 was prepared.

[0030] Reference Li X, Wang Y, Matsuura T, Meng J, Synthesis of new spiropyrans and spirooxazines having a heteroaromatic pendant and their photochromic behavior, Heterocycles 1999, 51, 2639-2651. Compound 2 was prepared.

[0031] Compound 1 ((245 mg, 1.0 mmol)) and compound 2 (359 mg, 1.0 mmol) were mixed in 10 ml of acetonitrile. After the solution was stirred at reflux overnight, the solvent was evaporated under reduced pressure. The crude product was separated by gel chromatography with CH as eluent 2 Cl 2 / CH 3 OH (50 / 1, v / v), finally obtain dark blue product Coupa, yield 40%, and use 1 H NMR, 13 C NMR and HR-MS characterized its structure as Figure 1-3 .

[0032]

Embodiment 2

[0033] Embodiment 2: Coupa spectroscopic characterization in vitro

[0034] Use Na 2 S as H 2 S donor and Na 2 SO 3 as SO 2 The donor mimics the microenvironment of mitochondrial reactive sulfur species (RSS) and detects fluorescence spectra excited at 405nm and 560nm. Prepare 3 mL of PBS / DMSO=10 / 1 probe solution with a concentration of 10 μM, and add 10 equivalents of active sulfur species Na 2 S and Na 2 SO 3 , to detect the spectral behavior of the probe solution. The result is as Figure 4 a(Na 2 S) and Figure 4 b(Na 2 SO 3 ), the results showed that under the two RSS conditions, the fluorescence intensity of the merocyanine system (560nm excitation, 650nm emission) decreased significantly, while the fluorescence intensity of the coumarin system (405nm excitation, 480nm emission) did not change. Considering the microenvironment of the mitochondrial active sulfur species (RSS), the fluorescence intensity of the fluorescent probe cyanine system of the present inv...

Embodiment 3

[0036] The fluorescence spectrum of Coupa under the different viscosity of embodiment 3

[0037] Using 0-80% (V / V) glycerol binary solution in methanol to simulate different viscosities, investigate the fluorescence spectrum of Coupa under excitation at 405nm and 560nm. First, prepare 3 mL of glycerol / methanol binary solutions with different ratios, then add 10 μM probes to them, vortex and mix thoroughly, and then use a fluorescence spectrometer to excite at 405 nm and 560 nm respectively to obtain fluorescence spectra. like Figure 5 As shown, the results show that at 405nm ( Figure 5 a) and 560nm ( Figure 5 b) Under excitation, the fluorescence emission increases with the viscosity, indicating that the fluorescence intensity is positively correlated with the viscosity.

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Abstract

The invention discloses a micromolecular fluorescent probe with high permeability, biocompatibility and viscosity response, which is used for monitoring the interaction of mitochondria and lysosome inliving cells. The fluorescent probe can monitor mitochondria and lysosome at the same time by emitting red light on the lysosome and emitting blue light on the mitochondria, and related fluorescenceintensity changes of the two colors are related to the interaction process of the mitochondria and lysosome in mitochondrial autophagy. A new choice is provided for dynamically monitoring the interaction of the mitochondrial and lysosome in mitochondrial autophagy.

Description

technical field [0001] The invention belongs to the field of super-resolution imaging, and relates to a fluorescent probe that can be used to trace the interaction between mitochondria and lysosome in living cells. Background technique [0002] Mitochondria-lysosome interactions, including mitochondria-lysosome fusion (i.e., mitophagy) and mitochondria-lysosome contact (MLC), are important processes in maintaining cellular homeostasis in eukaryotic cells, and their interactions are also associated with Neurodegenerative diseases and cancer are associated. Under the existing technology, mitochondria and lysosomes are usually labeled with two different probes or fluorescent proteins for observing the interaction between mitochondria and lysosomes under super-resolution imaging. A number of synthetic fluorescent probes have been developed to prolong photobleaching resistance, reduce phototoxicity, and reduce background signal for imaging morphology of subcellular organelles. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D405/06C09K11/06G01N21/64
CPCC07D405/06C09K11/06G01N21/6428G01N2021/6439C09K2211/1029C09K2211/1088
Inventor 方红宝何卫江陈韵聪张玉明郭子建
Owner NANJING UNIV
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