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Leave-on near-infrared lysosome probe

A lysosome and near-infrared technology, used in the synthesis and application of fluorescent probes, can solve the problems of fluorophore quenching, high cellular phototoxicity, and susceptibility to biological autoluminescence interference, and achieve low cytotoxicity and light stability good sex effect

Inactive Publication Date: 2015-09-02
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this lysosomal localization probe is alkaline in lysosomes, and the pH value in lysosomes will continue to increase during long-term incubation with this probe. In addition, due to the PET effect, it cannot The fluorescence of the fluorophore is completely quenched, therefore, these probes often exhibit high non-specific background fluorescence in cells
Furthermore, traditional fluorescent probes have disadvantages such as short emission wavelength, weak tissue penetration, susceptibility to bioluminescent interference, high phototoxicity to cells, and poor photostability. Lysosome-localized fluorescent probe with good sex and long-wavelength emission

Method used

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  • Leave-on near-infrared lysosome probe
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  • Leave-on near-infrared lysosome probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Synthesis of malachite green-based probe targeting lysosome

[0034] Compound b: Dissolve 6.1g of compound a, 9.8g of butyl bromobutyrate and 15g of potassium carbonate in 250ml of acetone, stir at room temperature for 12 hours, filter out potassium carbonate after the reaction, spin the filtrate to dryness, dissolve in 500ml of ethyl acetate, Washed with water (150ml×3), dried over anhydrous sodium sulfate, and spin-dried to obtain 10.6g white solid b, yield 89.4% Characterization data: 1H NMR (CDCl3, 400MHz): δ=9.88(s, 1H), 7.68(d, J=6.0Hz, 2H), 6.96(d, J=5.6Hz, 2H), 4.22(q, J=6.8Hz, 2H), 3.95(t, J=5.6Hz, 2H), 2.31(t, J= 7.6Hz, 2H), 2.12(m, J=7.2Hz, 2H), 1.31(t, J=4.8Hz, 3H); 13CNMR(CDCl3, 100MHz):

[0035] 190.1, 172.0, 165.2, 130.4, 114.3, 71.3, 59.6, 30.1, 25.4, 13.7

[0036]Compound d: Dissolve 2g of compound b, 2.57g of compound c and 6g of anhydrous zinc chloride in 120ml of absolute ethanol, heat to reflux for 24 hours under the protection of argon, spin dry ...

Embodiment 2

[0040] Prepare 10uM probe solutions in different solvents (dichloromethane, methanol, tetrahydrofuran, dimethyl sulfoxide, dioxane, acetone, acetonitrile, N,N-dimethylformamide) for UV-visible spectrophotometer and Fluorescence spectrophotometer test.

[0041] The results showed that the absorption of the probe shifted slightly at 620nm in different solvents, but the fluctuation was not large ( figure 1 ); there is no fluorescence in different solvents, and the ultraviolet absorption and fluorescence emission of the probe do not change with the change of polarity.

Embodiment 3

[0043] Prepare 1uM probe solutions with different viscosities (methanol and ethylene glycol 1:0-0:1, V / V, ethylene glycol and glycerol system 1:0-0:1, V / V) for fluorescence spectrophotometry gauge test.

[0044] The results show that when the concentration of methanol is large, that is, the viscosity is small, the probe has no fluorescence, and with the continuous increase of the solution viscosity, the fluorescence of the probe increases extremely, and the fluorescence intensity increases by 2500 times ( figure 2 ).

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Abstract

The invention relates to synthesis and an application of a leave-on near-infrared lysosome probe, belonging to the technical field of biology. A synthetic method of a malachite green lysosome targeted fluorescent probe comprises the following steps: synthesizing substituted aldehyde by adopting p-hydroxy benzaldehyde and ethyl 4-bromobutyrate in acetone by taking potassium carbonate as an acid-binding agent, synthesizing ethyl butyrate modified malachite by adopting the substituted aldehyde and N,N-dimethylaniline in ethanol by taking ZnC12 as a catalyst, hydrolyzing the malachite in acetone by taking a potassium hydroxide solution as a catalyst, and oxidizing butyrate derivate malachite in ethyl acetate by taking 2,3-dichloro-5,6-dicyan p-benzoquinone as an oxidizing agent, so as to obtain the malachite green lysosome targeted fluorescent probe. Intracellular experiments prove that the malachite green gyro type near-infrared targeted lysosome probe has good membrane permeability and can be specifically activated by lysosome; no fluorescence exists outside the lysosome; and therefore, leave-on lysosome marking purposes are achieved.

Description

technical field [0001] The present invention relates to the synthesis and application of a lysosome-targeted fluorescent probe, in particular to the application of a fluorescent probe containing malachite green gyro-type molecular probes that are sensitive to viscosity in lysosomes and activated by fluorescence. Belongs to the field of biotechnology. Background technique [0002] Lysosome is an organelle surrounded by a single-layer membrane structure in eukaryotic cells. The interior is acidic, the pH is between 4.0 and 6.0, and it contains about 50 different degrading enzymes. Therefore, lysosomes are called cells. recycling center. Under normal circumstances, lysosomes can decompose aging cell debris and useless biomolecules into small biomolecules that can be used by cells for reuse by cells. However, when a certain enzyme in the lysosome is missing or dysfunctional, macromolecules cannot be decomposed and accumulate in the lysosome, which increases the viscosity of th...

Claims

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Application Information

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IPC IPC(8): C09K11/06C07D295/096C12Q1/02G01N21/64
Inventor 朱麟勇张大生夏亮林秋宁包春燕
Owner EAST CHINA UNIV OF SCI & TECH
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