Preparation method and application of fluorescent probe for efficiently detecting peroxynitroso group

Abstract

The invention aims to disclose a preparation method and application of a fluorescent probe for efficiently detecting peroxynitroso. Peroxynitrite groups are one of active reactive oxygen and active nitrogen species, and have a wide range of attention due to high oxidation and nitration capabilities. The peroxynitrite is used as an important physiological activation factor and signal molecule, and can regulate the dynamic redox dynamic balance in a living body. However, when the content of the peroxynitrite is too high, the peroxynitrite can damage protein, nucleic acid and amino acid in cells, so that serious diseases such as neurodegenerative diseases and the like are caused in organisms. Fluorescence sensing and fluorescence imaging can mark and track molecules with wide biological activity, and have good temporal and spatial resolution. Therefore, researchers develop a plurality of small molecular fluorescent probes for detecting and characterizing nitrite peroxide. Coumarin has unique fluorescence, and the fluorescence characteristic of coumarin depends on the structure of coumarin to a great extent, so that the modification of coumarin becomes a basis for constructing an efficient fluorescent probe. Design, synthesis and characterization of a novel coumarin-based fluorescent probe are introduced, and the novel coumarin-based fluorescent probe is used for efficient detection of peroxynitrite.

Description

Technical Field

[0001] The present invention relates to the field of chemical medicine, and in particular to a preparation method and application of a fluorescent probe for efficiently detecting peroxynitrite. Background Art

[0002] The synthesis of coumarin is partly based on classical methods such as Pechmann reaction or Knoevenagel condensation. In the past decade, coumarin derivatives have been a hot topic of research. Researchers have synthesized different coumarin derivatives, greatly expanding the structural combination of existing coumarins. There is a subtle relationship between the structure of coumarin derivatives and their optical properties, and their fluorescence properties depend to a large extent on their structure. Coumarin has a conformationally restricted cinnamate form and therefore has high photostability, but the fluorescence quantum yield of coumarin itself is low. By appropriately substituting to enhance intramolecular charge transfer (ICT), coumarin derivatives can emit strong fluorescence in the blue-green light region. At the same time, the maximum absorption wavelength and emission wave are significantly shifted. The present invention discloses a coumarin-based fluorescent probe for the efficient detection of peroxynitrite. This project is supported by the Qinghai Province Applied Basic Research Project (2024-ZJ-701). Summary of the invention

[0003] Based on the current status of the above-mentioned prior art research, the present invention provides a method for preparing a fluorescent probe for efficiently detecting peroxynitrite and its use. The fluorescent probe shown in the present invention has a good response to peroxynitrite and can be used for the efficient detection of peroxynitrite.

[0004] Specifically, the present invention provides a fluorescent probe, the structure of which is shown in the following formula (I):

[0005]

[0006] The present invention designs a coumarin-based fluorescent probe that interacts with ONOO - After the reaction, the fluorescence intensity decreased. It showed good excitation wavelength and emission wavelength, with certain anti-interference and low detection limit. At the same time, within the normal human pH range, ONOO - Has higher sensitivity.

[0007] The specific preparation method of the fluorescent probe of the present invention is as follows: Weigh malononitrile (132.04 mg, 2 equiv.), hexahydropyridine (340.6 mg, 4 equiv.) and 50 mL of anhydrous ethanol respectively, mix under Ar gas protection and stir at room temperature for 0.5 h. Weigh the compound 2-oxo-7-(pyrrolidin-1-yl)-2H-benzopyran-3-carboxaldehyde (243.26 mg, 1 mmol, 1 equiv.), and add it to the previously mixed reaction solution. Stir at room temperature for 6 h. After the solvent is removed by rotary evaporation, it is purified using a silica gel column (PE:EA=3:1). After purification, the solvent is dried by rotary evaporation to obtain compound (I). Yield: 43%. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 This is a schematic diagram of the change in fluorescence intensity of ONOO- over time in Example 1 in PBS:DMSO=10:1.

[0009] Figure 2 This is a schematic diagram of the fluorescence intensity of ONOO- detected in PBS:DMSO=10:1 at different pH values ​​in Example 1.

[0010] Figure 3 This is the interference term test of probe h in PBS:DMSO=10:1 of Example 1. 1: blank, 2: Asp, 3: Glu, 4: Ala, 5: Cys, 6: Gly, 7: Ile, 8: Leu, 9: Phe, 10: Pro 11: Ser, 12: Thr, 13: Val, 14: His, 15: Lys. Example 1

[0011] 2-((2-oxo-7-(pyrrolidin-1-yl)-2H-chromen-3-yl)methylene)malononitrile: 1 H NMR(400MHz,Chloroform-d)δ8.07(s,1H),6.35(s,1H),3.63(s,2H),3.47(s,6H).

[0012] Test Example 1: Detection of ONOO in Example 1 - The time of fluorescence intensity test experiment.

[0013] Experimental method: ONOO was measured using a Thermo Scientific Lumina Fluorescence Spectrometer. - The fluorescence intensity changes over time after (10 μM) and Example 1 (10 μM) were mixed in PBS buffer solution.

[0014] Experimental results: With Example 1 and ONOO - The fluorescence intensity is decreasing. The fluorescence intensity decreases rapidly between 1-8 minutes, and decreases slowly between 8-13 minutes, and basically stops decreasing after 18 minutes. The fluorescence intensity at λ = 495nm decreases from F t =1min=1120 down to F t =18min=383. This indicates that Example 1 can accurately and quickly detect ONOO-.

[0015] Test Example 2: Detection of ONOO in Example 1 - Experimental method for the pH test of fluorescence intensity: ONOO - The change in fluorescence intensity after (10 μM) and Example 1 (10 μM) are fully reacted in PBS buffer solutions of different pH values ​​(pH=4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9).

[0016] Experimental results: When pH = 4-7, the fluorescence intensity at λ = 495nm shows a rapid downward trend and reaches the lowest at pH = 7; when pH = 7-9, the fluorescence intensity at λ = 495nm shows a slow increasing trend. - After the reaction, the fluorescence intensity decreases. The greater the decrease, the higher the sensitivity of Example 1 in measuring peroxynitrite. The fluorescence intensity of Example 1 is low in the physiological environment of the human body at pH = 7.4, indicating that it can be well applied to the detection of ONOO- in the human environment.

[0017] Experimental Example 3: Interference test of fluorescence intensity of ONOO- in Example 1 Experimental method: ONOO - (10 μM), Example 1 (10 μM) and different interference items (10 μM) after sufficient reaction in PBS buffer solution.

[0018] Experimental results: It can be seen that in the presence of other interfering substances, the fluorescence intensity is almost the same as the fluorescence intensity when there is no interfering substance, indicating that Example 1 has good anti-interference ability.

Claims

1. The present invention discloses a method for preparing a fluorescent probe for efficiently detecting peroxynitrite and its use. The fluorescent probe for detecting peroxynitrite of the present invention has a structure as shown in the following formula (I). The fluorescent probe of the present invention has a good response to peroxynitrite and can be used for the efficient detection of peroxynitrite: （Ⅰ）。 2. A fluorescent probe of peroxynitrite, characterized in that: Its structure is shown in the following formula (I): （Ⅰ）。 3. The fluorescent probe according to claim 1, characterized in that The compound is shown in (I) below: （Ⅰ）。 4. A new use of the fluorescent probe as claimed in claim 1, characterized in that: The fluorescent probe has a good response to peroxynitrite and can be used for the efficient detection of peroxynitrite.

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