A DCM derivative near-infrared fluorescent molecular probe for specific detection of hypochlorous acid / hypochlorite ions and its preparation method and application

Abstract

A DCM derivative near-infrared fluorescent molecular probe for specific detection of hypochlorous acid / hypochlorite ions and a preparation method and application thereof, utilizing the dicyanomethylene-4H-pyran fluorophore (DCM) having a controllable emission wavelength in the near-infrared region and having good properties such as a large Stokes shift and excellent photostability, using DCM derivatives as fluorescent probes, introducing a cyclic monothioacetal group as a recognition site, the prepared fluorescent molecular probe has the advantages of strong tissue penetration, small light damage and low autofluorescence interference, which is conducive to the realization of HClO / ClO in environmental systems ‑ Detection of endogenous HClO / ClO in living cells ‑ Imaging.

Description

Technical Field

[0001] The present invention relates to a fluorescent molecular probe for detecting hypochlorous acid / hypochlorite ions and a preparation method and application thereof, and in particular to a DCM derivative near-infrared fluorescent molecular probe for specifically detecting hypochlorous acid / hypochlorite ions and a preparation method and application thereof, belonging to the technical field of organic synthesis. Background Art

[0002] Hypochlorous acid (HClO) and its conjugate base (OCl - ) is a common reactive oxygen species (ROS), which is also widely known as an antibacterial agent in daily life, and can also kill pathogens in the innate immune system in organisms. Endogenous HClO is composed of hydrogen peroxide (H2O2) and chloride ions (Cl - ) is produced by myeloperoxidase (MPO) catalytic reaction. However, abnormal hypochlorous acid concentration can lead to a variety of diseases such as cardiovascular disease, arthritis, neurodegeneration and cancer. Therefore, it is very necessary to develop an effective method to achieve high sensitivity and specificity detection of HClO to assist clinical treatment. In recent years, fluorescent molecular probes have been widely used for in vitro / in vivo real-time detection of hypochlorous acid due to their high sensitivity, good biocompatibility and simple detection methods. However, the maximum emission wavelengths of these probes are mostly in the ultraviolet-visible light region (<650nm), which are easily interfered by background fluorescence and have weak tissue penetration, thus limiting their application in biological systems. In contrast, near-infrared (NIR) fluorescent probes (maximum emission wavelengths of 700nm to 900nm) have been vigorously developed and studied due to their strong tissue penetration, low photodamage and low autofluorescence interference. Therefore, the development of near-infrared fluorescent molecular probes with high sensitivity and strong specificity is of great significance for the quantitative detection of HClO in environmental systems and the imaging of HClO in biological systems such as living cells. Summary of the invention

[0003] In view of the problems existing in the current technology, the present invention proposes a DCM derivative near-infrared fluorescent molecular probe for specifically detecting hypochlorous acid / hypochlorite ions with high sensitivity and strong specificity, as well as a preparation method and application thereof, which can be used for quantitative detection of HClO in environmental systems and imaging of HClO in biological systems such as living cells, and has strong tissue penetration, little light damage and low autofluorescence interference.

[0004] The technical means adopted by the present invention to solve the above problems are: a DCM derivative near-infrared fluorescent molecular probe for specific detection of hypochlorous acid / hypochlorite ions, having the following structural formula:

[0005]

[0006] A method for preparing a DCM derivative near-infrared fluorescent molecular probe for specific detection of hypochlorous acid / hypochlorite ions, comprising the following steps:

[0007] (1) Dissolving DCM and 4-acetylbenzaldehyde in acetonitrile, adding piperidine, heating and reflux under the protection of an inert gas such as nitrogen or argon until the reaction is complete, concentrating under reduced pressure, and purifying by column chromatography to obtain a dark red solid product DCMC; wherein the ratio of DCM to 4-acetylbenzaldehyde is 1:1 to 1:2 mmol / mmol, the ratio of DCM to the solvent is 1:8 to 1:15 mmol / mL, and the ratio of DCM to piperidine is 9:1 to 11:1 mmol / mmol;

[0008] (2) Dissolve DCMC in anhydrous dichloromethane solvent, add 2-mercaptoethanol and methanesulfonic acid, heat and reflux under the protection of inert gas such as nitrogen or argon until the reaction is completed, concentrate under reduced pressure, and purify by column chromatography to obtain the probe DCMC-HClO; the dosage ratio of DCMC to anhydrous dichloromethane is: 1:12~1:16mmol / mL, the dosage ratio of DCMC to 2-mercaptoethanol is: 1:1.8~1:2.5mmol / mmol, and the dosage ratio of DCMC to methanesulfonic acid is: 1.8:1~2.4:1mmol / mL.

[0009] The above-mentioned synthetic route is as follows:

[0010]

[0011] The detection principle of the above-mentioned DCM derivative near-infrared fluorescent molecular probe for specific detection of hypochlorous acid / hypochlorite ions is:

[0012] When the probe DCMC-HClO and HClO / ClO - After the response, the cyclic monothioacetal group will turn into an acetyl group, which will cause the intramolecular charge transfer (ICT) inside the probe to change, resulting in the probe to produce fluorescence at 713nm, ultimately realizing the "Turn-ON" detection of HClO.

[0013]

[0014] The present invention also proposes an application of a DCM derivative near-infrared fluorescent molecular probe for specifically detecting hypochlorous acid / hypochlorite ions, which is used to detect HClO / ClO in an environmental system. - and HClO / ClO in living cells - .

[0015] The beneficial effects of the present invention are:

[0016] 1. The present invention utilizes dicyanomethylene-4H-pyran fluorophore (DCM) which has controllable emission wavelength in the near-infrared region and has good properties such as large Stokes shift and excellent photostability, uses DCM derivatives as fluorescent probes, introduces cyclic monothioacetal groups as recognition sites, and the prepared fluorescent molecular probes have high sensitivity and strong specificity.

[0017] 2. The present invention can synthesize DCM fluorescent molecular probes in only two steps, the raw materials are easily available, the synthesis and post-treatment methods are simple, and the HClO / ClO - The detection has excellent specificity and is not interfered by common anions and reactive oxygen species. - After the response, it emits red fluorescence, and the maximum emission wavelength is in the near-infrared region. It has the advantages of strong tissue penetration, small light damage and low autofluorescence interference, which is conducive to the realization of HClO / ClO in the environmental system. - Detection of endogenous HClO / ClO in living cells - Imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The fluorescence intensity of the fluorescent molecular probe in Example 2 changes with HClO / ClO - Emission spectra of concentration changes;

[0019] Figure 2 The fluorescence intensity and HClO / ClO of the fluorescent molecular probe in Example 2 - Linear relationship of concentration;

[0020] Figure 3 Example 3 Fluorescent molecular probe for HClO / ClO - Selective graphs;

[0021] Figure 4 This is the fluorescence confocal imaging of the fluorescent molecular probe in Example 4 in HeLa cells. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with the accompanying drawings.

[0023] Embodiment 1

[0024] This example is the synthesis of a DCM derivative near-infrared fluorescent molecular probe for specific detection of hypochlorous acid / hypochlorite ions

[0025] Synthesis of compound DCMC

[0026] Take a 50mL round-bottom flask, add DCM (416.44mg, 2mmol) and 4-acetylbenzaldehyde (296.32mg, 2mmol), and add 20mL of acetonitrile to fully dissolve it. Then, piperidine (0.2mmol) is added dropwise to the reaction system, and the reaction system is placed at 50°C for reflux reaction under nitrogen protection. TLC monitors the reaction progress. After the reaction is completed, the reaction solvent is removed under reduced pressure, and a dark red solid product DCMC587.4 mg is obtained by rapid column chromatography, with a yield of 86.8%. 1 H NMR (400MHz, CDCl3) δ8.87(d,J=8.3Hz,1H),7.85(d,J=7.6Hz,2H),7.79(s,1H),7.45(d,J=7.7Hz,2 H),7.36-7.32(m,2H),7.18(d,J=7.4Hz,1H),6.93(t,J=7.8Hz,1H),6.85-6.81(m,2H),2.5(s,3H).

[0027] Synthesis of Compound DCMC-HClO

[0028] Take a 50mL round-bottom flask, add compound DCMC (338.36mg, 1mmol) to it, and add 15mL of anhydrous dichloromethane to fully dissolve it, add 2-mercaptoethanol (156.26mg, 2mmol) and 500μL of methanesulfonic acid to the reaction system. Under argon protection, the reaction system is heated to reflux for reaction. TLC monitors the reaction progress. After the reaction is completed, the organic solvent is removed by vacuum distillation, and the probe DCMC-HClO 132.30mg is obtained by column chromatography purification, and the yield is 33.2%. 1 H NMR (400MHz, CDCl3) δ7.67(d,J=7.8Hz,2H),7.39(t,J=7.6Hz,1H),7.31(d,J=8.3Hz,1H),7.23(d,J=7.6Hz,2H),7.17(d,J=7.4Hz,1H),6 .93(t,J=7.8Hz,1H),6.83-6.80(m,2H),5.66(s,1H),3.69(d,J=7.2Hz,2H),2.66(d,J=7.4Hz,2H),1.83(s,3H).HRMS-ESI(m / z)calc.for C 24 H 18 N2O2S:398.1089; found, [M+H] + 399.1317.

[0029] Embodiment 2

[0030] This example shows the fluorescence intensity of the fluorescent molecular probe and the relationship between HClO / ClO - Changes in concentration

[0031] The fluorescent molecular probe prepared in Example 1 was prepared into a mother solution with a concentration of 1 mM in DMSO solution, 20 μL was taken and dissolved in 980 μL of acetonitrile and PBS buffer solution (0.01 M, pH = 7.4) in the test system, and 20 μL of ClO with concentrations of 50 μM, 100 μM, 150 μM, 200 μM, 300 μM, 500 μM, 800 μM, 1000 μM, 2000 μM were added. - The concentration of the probe in the final detection system was 10 μM, and ClO - The concentrations of 0μM, 0.5μM, 1μM, 1.5μM, 2μM, 3μM, 5μM, 8μM, 10μM, and 20μM were respectively. After incubation at room temperature for 20 min to fully respond, the fluorescence spectra were analyzed using a fluorescence spectrophotometer. Figure 1 .pass Figure 1 It can be seen that with the increase of ClO - With the increase of concentration, the fluorescence intensity (I 713 nm ) gradually increases. Figure 2 is the fluorescence intensity of the fluorescent molecular probe at 713 nm (I 713 nm ) and ClO - The linear relationship between the concentration of Figure 2 It can be concluded that there is a good linear relationship in the range of 0-3μM (R 2 =0.99270), the detection limit was calculated to be 2.5 nM.

[0032] Embodiment 3

[0033] This example is a fluorescent molecular probe for HClO / ClO - Selectivity of response

[0034] The fluorescent molecular probe prepared in Example 1 was prepared into a 1 mM stock solution in DMSO solution. 20 μL of the stock solution was dissolved in 980 μL of acetonitrile and 980 μL of PBS buffer solution (0.01 M, pH = 7.4) in the test system. 20 μL of 250 μM ClO was added. - mother solution, 20 μL of 1000 μM anion (Br - , Cl - 、F - ,I - , S 2- ) stock solution, 20 μL of 1000 μM reactive oxygen species (H2O2,1 O2, OH, NO and O 2- ), so that in the final detection system, the concentration of the probe is 10 μM, ClO - The concentration of anions (Br - , Cl - 、F - ,I - , S 2- ) and reactive oxygen species (H2O2, 1 O2, OH, NO and O 2- ) was 10 μM. After incubation at room temperature for 20 min to fully respond, the fluorescence spectrum was analyzed using a fluorescence spectrophotometer. Figure 3 As shown. Figure 3 It can be seen that the probe is only sensitive to ClO - There is an obvious response, while other ions remain unchanged.

[0035] Embodiment 4

[0036] This example is a fluorescent molecular probe for the detection of exogenous HClO / ClO in living cells. - Imaging studies

[0037] 10% fetal bovine serum was added to the DMEM medium, and HeLa cells were cultured in the medium at 37°C and 5% CO2. After 12 hours, the cell culture medium was removed from the wells and the cells were washed three times with D-Hanks. A portion of the cells (control group) was treated with 20 μM DCMC-HClO for 30 minutes. For the experimental group, HClO (50 μM) was added to the culture medium, and then the cells were washed with PBS to remove the remaining HClO, and incubated with the probe DCMC-HClO (20 μM) in 1.0 mL of fresh culture medium for 30 minutes before fluorescence imaging. The results are shown in Figure 2. Figure 4 As shown, the experimental group ( Figure 4 In a), obvious fluorescence signals can be seen, while in the control group ( Figure 4 b) Almost no fluorescence is observed, indicating that DCMC-HClO can be used to image exogenous hypochlorous acid / hypochlorite ions in living cells.

[0038] The above embodiments are only used to illustrate the present invention, rather than to limit the present invention. Technicians in the relevant technical field may make various changes or modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions should also fall within the protection scope of the present invention, and the protection scope of the present invention should be defined by the claims.

Claims

1. A DCM derivative near-infrared fluorescent molecular probe for specific detection of hypochlorous acid / hypochlorite ions, characterized in that: Has the following structural formula:

2. A method for preparing a DCM derivative near-infrared fluorescent molecular probe for specific detection of hypochlorous acid / hypochlorite ions according to claim 1, characterized in that: (1) DCM and 4-acetylbenzaldehyde are dissolved in acetonitrile, and piperidine is added. The mixture is heated under reflux under inert gas protection until the reaction is complete. After purification, the compound DCMC is obtained, wherein the structural formulas of DCM and DCMC are: (2) DCMC is dissolved in anhydrous dichloromethane, and 2-mercaptoethanol and methanesulfonic acid are added. The mixture is heated under reflux under inert gas protection until the reaction is complete. After purification, the probe DCMC-HClO is obtained.

3. The method for preparing the DCM derivative near-infrared fluorescent molecular probe for specific detection of hypochlorous acid / hypochlorite ions as claimed in claim 2, characterized in that: The inert gas is nitrogen or argon.

4. The method for preparing the DCM derivative near-infrared fluorescent molecular probe for specific detection of hypochlorous acid / hypochlorite ions as claimed in claim 2, characterized in that: The purification method is to perform column chromatography purification after concentration under reduced pressure.

5. The method for preparing the DCM derivative near-infrared fluorescent molecular probe for specific detection of hypochlorous acid / hypochlorite ions as claimed in claim 2, characterized in that: In step (1), the usage ratio of DCM to 4-acetylbenzaldehyde is 1:1-1:2 mmol / mmol, the usage ratio of DCM to solvent is 1:8-1:15 mmol / mL, and the usage ratio of DCM to piperidine is 9:1-11:1 mmol / mmol.

6. The method for preparing the DCM derivative near-infrared fluorescent molecular probe for specific detection of hypochlorous acid / hypochlorite ions as claimed in claim 2, characterized in that: In step (2), the ratio of DCMC to anhydrous dichloromethane is 1:12-1:16 mmol / mL, the ratio of DCMC to 2-mercaptoethanol is 1:1.8-1:2.5 mmol / mmol, and the ratio of DCMC to methanesulfonic acid is 1.8:1-2.4:1 mmol / mL.

7. An application of the DCM derivative near-infrared fluorescent molecular probe for specific detection of hypochlorous acid / hypochlorite ions as claimed in claim 1, characterized in that: The fluorescent molecular probe is used to detect HClO / ClO in the environmental system - .

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