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4-N substituted anthracene pyridone fluorescent dye and preparation method and application thereof

An anthrapyridone and fluorescent dye technology, applied in the field of fluorescent dyes, can solve the problems of real shape destruction of cells or biological tissues, carcinogenicity, toxicity, etc.

Inactive Publication Date: 2014-04-02
DALIAN UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, ethidium bromide (EB), propidium iodide (PI), etc. need to use methods such as cell fixation to disintegrate the cell membrane before they can be applied to the fluorescent labeling of biological samples, but this method is often harmful to cells or organisms. The true shape of the tissue is disruptive
At the same time, acridine and phenanthridine dyes such as EB have great toxicity and carcinogenic effects.
[0004] In addition, the excitation wavelength of some fluorescent dyes is in the ultraviolet region. Due to the high energy of ultraviolet light, it will cause damage to biological components such as nucleic acids and proteins in cells; at the same time, long-term light excitation will also cause photobleaching of fluorescent dyes.
These have limited the application of such dyes in fluorescence imaging
In addition, when ultraviolet light is used as the excitation wavelength, due to the absorption of the biological sample itself in this range, it is difficult for ultraviolet light to penetrate the interior of the biological tissue sample. At the same time, some biomolecules in the biological sample will produce a strong fluorescent background when excited by ultraviolet light. , making fluorescence detection difficult

Method used

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  • 4-N substituted anthracene pyridone fluorescent dye and preparation method and application thereof
  • 4-N substituted anthracene pyridone fluorescent dye and preparation method and application thereof
  • 4-N substituted anthracene pyridone fluorescent dye and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. Synthesis of dye A and live cell fluorescence imaging

[0038] (1) Synthesis of Dye A

[0039]

[0040] 4-Bromo-N-methylthrapyridone (13g, 38.2mmol), ethylenediamine (5mL, 83.3mmol), potassium carbonate (5.3g, 38.2mmol) and anhydrous copper sulfate (1.0g 6.3mmol) were dissolved in In 150mL of ethylene glycol methyl ether, reflux reaction for 20h, the raw material 4-bromo-N-methyl anthrapyridone detected by TLC, filtered to remove insoluble matter, the filtrate was evaporated under reduced pressure to obtain a dark red solid, the crude product was washed with ethyl acetate Washing (3×50 mL) and drying gave 11.0 g of the target product with a yield of 89%.

[0041] 1 H NMR (400MHz, DMSO) δ10.47(t, J=9.6Hz, 1H, NH), 8.55(d, J=7.9Hz, 1H, ArH), 8.36(d, J=7.7Hz, 1H, ArH) , 8.23(t, J=6.0Hz, 1H, NH), 7.94(d, J=9.7Hz, 1H, ArH), 7.87-7.70(m, 3H, ArH, CH), 7.55(d, J=9.7Hz , 1H, ArH), 5.69(s, 1H, CH), 5.35(s, 1H, CH), 3.77(s, 3H, CH 3 ), 3.66-3.55 (m, 2H, CH 2 )...

Embodiment 2

[0044] Example 2. Synthesis of dye B and live cell fluorescence imaging

[0045] (1) Synthesis of Dye B

[0046]

[0047] The synthesis method of dye B is similar to that of dye A, and the main raw materials used are 4-bromo-N-methylthrapyridone and ethanolamine. The crude product is separated by silica gel column, yield: 83%

[0048] 1 H NMR (400MHz, DMSO) δ10.53(s, 1H, NH), 8.53(d, J=3.2Hz, 1H,), 8.37(d, J=5.6Hz, 1H, ArH), 7.76-7.94(m , 4H, ArH, CH), 7.41(d, J=3.2Hz, 1H), 5.02(s, 1H), 3.74(m, 5H), 3.51(s, 2H).MS(TOF MS ES+) calculated for[ C 19 h 17 N 2 o 3 ] + : 321.1234, measured: 321.1238.

[0049] (2) Observe the staining of compound B on HeLa living cells under a confocal laser scanning microscope:

[0050] In 2 mL of cell culture medium, compound B was added to the cultured HeLa cells so that the final concentration was 5 μM. Incubate for 30 minutes at 37°C, 5% CO2 in a cell incubator. Then, 0.01 M PBS was shaken and rinsed for 5 minutes × 3, and then the ...

Embodiment 3

[0051] Example 3. Synthesis of dye C and live cell fluorescence imaging

[0052] (1) Synthesis of Dye C

[0053]

[0054] The synthesis method of dye C is similar to that of dye A, and the main raw materials used are 4-bromo-N-methylthrapyridone and N,N-dimethylpropylenediamine. The crude product was separated on a silica gel column, yield: 60%.

[0055] 1 H NMR (400MHz, CDCl3) δ10.53(s, 1H, NH), 8.52(d, J=6.8Hz, 1H, ArH), 8.24(d, J=7.8Hz, 1H, ArH), 7.82-7.62( m, 4H, ArH, CH), 7.30 (d, 1H, ArH), 3.88 (s, 3H, CH3), 3.52 (dd, J=12.5, 6.8Hz, 2H, CH2), 2.53 (t, J=7.0 Hz, 2H, CH2), 2.33 (s, 6H, CH3), 2.01 (dd, J=14.1, 7.0Hz, 2H, CH2). MS (TOF MS ES+) calculated for [C22H24N3O2] + : 362.1863, measured: 362.1869.

[0056] (2) Observe the staining of compound C on living cell HeLa under a confocal laser scanning microscope:

[0057] In 2 mL of cell culture medium, compound C was added to the cultured HeLa cells so that the final concentration was 5 μM. Incubate for 30 minute...

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Abstract

The invention discloses a 4-N substituted anthracene pyridone fluorescent dye and a preparation method and application thereof. The compound has the structural general formula I, wherein in the general formula I, n is an integer from 1-10; and R is selected from hydrogen atom, methyl, hydroxyl, amino group, dimethylamino group, trimethyl ammonium halide group and guanidine hydrochloride group. The 4-N substituted anthracene pyridone fluorescent dye disclosed by the invention is simple to synthesize, and has good light stability, relatively long emission wavelength and good cellular membrane permeability. Therefore, the invention also aims to provide application of the 4-N substituted anthracene pyridone fluorescent dye in biological dyeing, and the 4-N substituted anthracene pyridone fluorescent dye can be used for dyeing fixed cells, living cells and biological tissues.

Description

technical field [0001] The present invention relates to a new class of fluorescent dyes in the field of fine chemicals, their preparation methods and applications, in particular to the application of a class of dyes with an anthrapyridone structure, their preparation methods, and the use of the fluorescent dyes, their conjugates or Application of its composition in biological dyeing. Background technique [0002] As a functional indicator, fluorescent dyes have been widely used in various fields of science and technology, especially their practical applications in life sciences, clinical medical diagnosis, fluorescent immunoassay, etc., which have attracted widespread attention from scientists all over the world. In the field of cell biology, fluorescent dyes are used for the position and content of intracellular components and other changes, and the identification and classification of cells also rely on flow cytometry with fluorescence technology as the core. [0003] At ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C09B5/14C09K11/06G01N21/64C12Q1/02
Inventor 彭孝军强新新樊江莉王静云宋锋玲孙世国
Owner DALIAN UNIV OF TECH