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A fluorescent probe that can be used for imaging malondialdehyde in living cells and its preparation method

A fluorescent probe and malondialdehyde technology, which is applied in the field of fluorescent dyes, can solve the problems of low detection limit, inability to use imaging, and cumbersome pretreatment of detection samples, so as to avoid serious self-absorption, short response time, and excellent selectivity. Effect

Active Publication Date: 2019-02-26
WUHAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, there are many detection methods for malondialdehyde, and the most widely used clinically is the 2-thiobarbituric acid method. The product generated by condensation is detected by ultraviolet light, which has the advantages of low detection limit and high sensitivity, and the disadvantages are cumbersome operation, poor specificity, and poor repeatability (see Anal.Biochem.1979,95,351-358 and Free Radical Biol.Med.1990,9,515- 540)
In order to overcome the shortcomings of this detection method, a variety of detection methods have been developed, including liquid chromatography, electrophoresis, Raman spectroscopy, mass spectrometry, etc. The defect of these methods lies in the tedious pretreatment of the detection samples, These pretreatment processes often require strong acidic or / and heating reaction conditions, so the detection objects are basically body fluids such as urine, serum, etc., which cannot be used for imaging in living cells under physiological conditions

Method used

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  • A fluorescent probe that can be used for imaging malondialdehyde in living cells and its preparation method
  • A fluorescent probe that can be used for imaging malondialdehyde in living cells and its preparation method
  • A fluorescent probe that can be used for imaging malondialdehyde in living cells and its preparation method

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Experimental program
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Effect test

Embodiment 1

[0032] The synthetic route of fluorescent probes MDAP-1, MDAP-2, and MDAP-3 that can be used for imaging malondialdehyde in living cells is shown in the following reaction formula:

[0033]

[0034] ①The intermediates (3a, 3b, 3c) were synthesized by Buchwald-Harwig amino coupling method

[0035] A. Synthesis of Compound 3a

[0036] Weigh 300 mg of 6-bromo-2-propyl-1H-benzo[de]isoquinoline-1,3(2H)-dione (compound 1, 2.0 mmol) (see J.Org. Chem.2013,78,3980-3988) into a 50 ml two-necked flask, then successively added 370 mg of methyl 5-amino-2-nitrobenzoate (compound 2a, 1.89mmol) (the synthesis of compound 2a see Tetrahedron. Lett.2005, 46, 7477-7481), 42 mg Pd 2 (dba) 3 (0.046mmol) and 66 mg (±)-2,2'-bis-(diphenylphosphino)-1,1'-binaphthyl (BINAP), stirred and mixed, degassed, and added 18 ml of freshly distilled Toluene, heated to boiling, reacted overnight under reflux conditions, and cooled to room temperature. After the solvent was removed under reduced pressure, i...

Embodiment 2

[0053] Example 2 Fluorescence response of fluorescent probes MDAP-1, MDAP-2, MDAP-3 and malondialdehyde (MDA)

[0054] Prepare the stock solution (100mM) of fluorescent probes MDAP-1, MDAP-2 and MDAP-3 in DMSO, take a small amount and add it to the EP tube, dilute it with PBS buffer solution (10mM, pH 7.4), add a certain amount of MDA solution, Then dilute with DMSO and PBS to a final concentration of probe of 10 μM and a final concentration of MDA of 1 mM (solvent composition at this time: DMSO:PBS=1:9, volume ratio, pH 7.4). The fluorescent response of the EP tubes was measured after incubation at 37°C for four hours. Taking the maximum excitation wavelength as 370nm, the emission wavelengths of the three compounds are all around 550-553nm, and the Stokes shifts are all around 180nm, which is quite large compared to ordinary fluorescent probes. It can effectively avoid the interference of excitation light on emission, which is very beneficial for cell imaging; the fluoresce...

Embodiment 3

[0055] Embodiment 3 The correlation between the amount of fluorescent probe MDAP-1 and malondialdehyde (MDA)

[0056] Take a small amount of the DMSO stock solution of MDAP-1 in an EP tube, dilute it with PBS buffer (10mM, pH 7.4), add a certain amount of MDA solution, and then dilute it with PBS and DMSO until the final concentration of the probe is 10μM. The concentrations were 0, 2, 5, 10, 20, 50, 100, 200, 400, 600, 800, 1000 μM, respectively. Incubated at 37°C for 4 hours, and detected the fluorescence intensity of the solution (excitation wavelength: 370nm; emission wavelength: 553nm. The fluorescence diagram of MDAP-1-MDA titration is shown in figure 2 ; The relationship between the fluorescence intensity and MDA is shown in image 3 . Solvent composition: DMSO:PBS=1:9, volume ratio, pH 7.4). The results showed that the fluorescence intensity of MDAP-1 increased with the increase of the concentration, and within the concentration range of 0-200μM, the fluorescence i...

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Abstract

The invention discloses a fluorescence probe used for malonaldehyde imaging inside living cells and a preparation method thereof and belongs to the technical field of biological fluorescence analysis. The fluorescence probe is obtained in the following way: benzoyl hydrazine (containing substituted benzoyl hydrazine) as the effective part of the probe and malonaldehyde and 1,8-naphthalimide as a fluorescence reporter group are connected through a certain linking group (such as imino group). The fluorescence probe can effectively detect malonaldehyde in a physiological environment, and endogenous and exogenous malonaldehyde bioimaging in living cells is successfully realized. Malonaldehyde bioimaging in living cells is realized for the first time in the invention. The optimal probe MDAP-1 has high sensitivity (the lowest detection limit reaches 0.6micron), strong fluorescence response and short response time, has excellent selectivity to malonaldehyde, but has no fluorescence response to metal ions, oxidation active products and active carbonyl compounds, etc.

Description

technical field [0001] The invention relates to a fluorescent dye used in the technical field of biological fluorescence analysis, in particular to a fluorescent probe which can be used for imaging malondialdehyde in living cells and a preparation method thereof. Background technique [0002] Fluorescent probes have the advantages of high spatio-temporal resolution, easy operation, and little interference with life processes. In the research of cell biology, fluorescence spectroscopy is often used to track the position and changes of specific components (targets) in cells, thereby revealing the specific physiological / pathological functions of biological targets. At present, there are quite a lot of commercial fluorescent probes for a specific biological target, such as the dye Hoechst 33342 that can be used for nuclear imaging. Since there are many biological targets in cells, the research of fluorescent probes for specific targets is currently a hot field. [0003] Oxidat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C09K11/06C07D221/14G01N21/64A61K49/00
Inventor 吴松
Owner WUHAN UNIV
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