A fluorescent molecular probe for detecting hydroxyl radicals and its preparation method and application

Abstract

A fluorescent molecular probe for detecting hydroxyl free radicals, and a preparation method and application thereof. The fluorescent molecular probe has the following structural formula: 3,5-dimethylbenzaldehyde, 2-aminothiophenol, 2-methylpyridinium salt and piperidine are used as raw materials, the synthesis steps are simple, the raw materials are easily available, the detection method is simple, and the cost is low. The prepared fluorescent molecular probe has low biological tissue light absorption or self-light intensity, strong biological tissue penetration ability, high imaging signal-to-noise ratio and resolution, and good water solubility, which is more conducive to the monitoring of hydroxyl free radicals in living cells.

Description

Technical Field

[0001] The invention relates to a fluorescent molecular probe for detecting hydroxyl free radicals and a preparation method and application thereof, belonging to the technical field of organic small molecule fluorescent probes and biosensor technology. Background Art

[0002] Reactive oxygen species (ROS) mainly include hydroxyl radicals (·OH), singlet oxygen ( 1 O2), superoxide anion (O2 ·- ), hydrogen peroxide (H2O2), hypochlorous acid (HClO) and peroxyl radicals (·OOR), which have been shown to play important roles not only in the immune, nervous and cardiovascular systems, but also in a variety of physiological and pathological processes. Among them, hydroxyl radicals are one of the strongest oxidants in aqueous solution. They can produce chain reactions with various organic compounds through electron transfer, electrophilic addition, dehydrogenation reactions and other pathways, and ultimately degrade them into harmless substances such as CO2 and H2O. In vivo, hydroxyl radicals are also one of the most harmful cellular ROS, which can cause serious damage to biological macromolecules such as nucleic acids, carbohydrates, lipids and proteins. Therefore, the development of highly selective and sensitive methods to monitor the generation, distribution and metabolism of hydroxyl radicals is of great significance for further understanding of their biological effects.

[0003] However, due to the short lifespan, high reactivity and low concentration of hydroxyl radicals in cells, although electron spin resonance (ESR) spectroscopy, high performance liquid chromatography, spectrophotometry and electrochemical sensing have been developed for their in vitro detection, they are not suitable for monitoring hydroxyl radicals in vivo. In contrast, fluorescence imaging has excellent sensitivity and selectivity, easy operation, non-invasive characteristics and real-time sensing capabilities. Therefore, it is of great significance to develop new fluorescent molecular probes that can specifically identify hydroxyl radicals. Summary of the invention

[0004] In view of the problem that the above-mentioned method is not applicable to detecting hydroxyl free radicals in vivo, the present invention proposes a fluorescent molecular probe for detecting hydroxyl free radicals and a preparation method and application thereof, which can be used for detecting hydroxyl free radicals in living cells.

[0005] The technical means adopted by the present invention to solve the above problems is: a fluorescent molecular probe for detecting hydroxyl radicals, the structural formula of which is as follows:

[0006]

[0007] A method for preparing a fluorescent molecular probe for detecting hydroxyl radicals, comprising the following steps:

[0008] (1) dissolving 3,5-dimethylbenzaldehyde, 2-aminothiophenol, 30% H2O2 and 37% HCl in anhydrous alcoholic solution such as anhydrous methanol and anhydrous ethanol, stirring at room temperature until the reaction is complete, pouring in pure water, adjusting the pH to near neutral, filtering, collecting and washing the filter cake, and vacuum drying to obtain a light yellow product BTM; wherein the amount ratio of 3,5-dimethylbenzaldehyde to 2-aminothiophenol is: 1:1-1:2 mmol / mmol, the amount ratio of 3,5-dimethylbenzaldehyde to the anhydrous alcoholic solution is: 1:8-1:15 mmol / mL, the amount ratio of 3,5-dimethylbenzaldehyde to 30% H2O2 is: 1:0.8-1:1.2 mmol / mL, and the amount ratio of 3,5-dimethylbenzaldehyde to 37% HCl is: 1.5:1-2.5:1 mmol / mL;

[0009] (2) Dissolving BTM, 2-methylpyridinium salt and piperidine in an anhydrous alcoholic solution, heating under reflux at 90° C. until the reaction is completed, removing the solvent by rotary evaporation, and purifying by column chromatography to obtain a red fluorescent molecular probe BTMP; wherein the dosage ratio of BTM to 2-methylpyridinium salt is 1:1.2-1:2 mmol / mmol, the dosage ratio of BTM to piperidine is 1:1.8-1:2.5 mmol / mmol, and the dosage ratio of BTM to anhydrous alcoholic solution is 1:8-1:20 mmol / mL.

[0010] The reaction route is as follows:

[0011]

[0012] The invention discloses an application of a fluorescent molecular probe for detecting hydroxyl radicals, which is used for quantitative detection of hydroxyl radicals in solution and imaging of endogenous hydroxyl radicals in living cells.

[0013] The fluorescent molecular probe BTMP for detecting hydroxyl radicals provided by the present invention has no fluorescence, but the product after responding to hydroxyl radicals has red fluorescence and has an excited-state intramolecular proton transfer (ESIPT) effect. A fluorescence detector can be used to achieve specific detection of hydroxyl radicals. The detection principle is as follows:

[0014]

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

[0016] 1. The fluorescent molecular probe provided by the present invention can specifically interact with hydroxyl radicals to produce changes in the fluorescence spectrum. During the detection process, it is not interfered by other active oxygen species in the biological system, thereby achieving quantitative and specific identification of hydroxyl radicals.

[0017] 2. The fluorescent molecular probe provided by the present invention has simple synthesis steps, readily available raw materials, simple detection methods, and low costs. The prepared fluorescent molecular probe has low biological tissue light absorption or self-light intensity, strong biological tissue penetration ability, high imaging signal-to-noise ratio and resolution, and good water solubility, which is more conducive to the monitoring of hydroxyl radicals in living cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Example 2 Emission spectrum of the fluorescence intensity of the fluorescent molecular probe as it changes with the concentration of hydroxyl radicals;

[0019] Figure 2 Example 2 Linear relationship between the fluorescence intensity of the fluorescent molecular probe and the concentration of hydroxyl radicals;

[0020] Figure 3 Example 3 Selectivity diagram of fluorescent molecular probe for hydroxyl radicals;

[0021] Figure 4 Example 4 Fluorescence confocal imaging of fluorescent molecular probes in Hela cells, wherein: a) cells were treated with the probe for 20 minutes and then with LPS; b) cells were treated with the probe for 20 minutes and then with PBS (0.01 M, pH = 7.4) buffer solution. DETAILED DESCRIPTION

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

[0023] Embodiment 1

[0024] Synthesis of compound BTM

[0025] 3,5-dimethylbenzaldehyde (268.36 mg, 2.0 mmol) was added to a 50 mL round-bottom flask and dissolved with 20 mL of anhydrous ethanol. 2-aminothiophenol (300 mg, 2.4 mmol), 30% H2O2 (2 ml) and 37% HCl (1 ml) were added to the reaction system and stirred at room temperature for reaction. TLC was used to monitor the reaction until it was complete. The reaction system was poured into pure water and the pH was adjusted to near neutral with NaOH solution. A large amount of precipitation was observed. The filter cake was filtered, collected and washed, and vacuum dried to obtain 446.86 mg of a light yellow product with a yield of 88.2%. 1 H NMR (500MHz, CDCl3): δ9.88 (s, 1H), 8.36 (s, 1H), 8.18 (d, J = 8.2Hz, 1H), 8.06 (s,1H),8.02(d,J=8.0Hz,1H),7.77(s,1H),7.53-7.50(m,2H),2.42(s,3H).

[0026] Synthesis of Compound BTMP

[0027] Compound BTM (253.32 mg, 1.0 mmol) and 10 mL of ethanol were added to a 25 mL round-bottom flask. After the mixture was completely dissolved, 2-methylpyridinium salt (352.61 mg, 1.5 mmol) and 0.5 mmol of piperidine were added and the mixture was heated under reflux at 90° C. The mixture was monitored by TLC until completion, and the mixture was cooled at room temperature and then the solvent was removed by rotary evaporation. The mixture was purified by column chromatography to obtain 278.02 mg of a red product with a yield of 59.1%. 1 H NMR (500MHz, DMSO-d6): δ8.84(d,J=7.5Hz,1H),8.18(d,J=7.0Hz,1H),8.10(t,J=8.0Hz,1H),8.02(d,J=8.0Hz,1H),7.85(s,1H),7.66-7.63(m, 2H),7.53(t,J=6.5Hz,1H),7.51(t,J=7.0Hz,1H),7.29(d,J=7.6Hz,1H),7.26(s,1H),6.59-6.57(m,2H),4.35(s,3H),2.44(s,3H).HRMS-ESI(C 22 H 19 IN2S)m / z:calc.for[MI] + :343.1263; found,343.1405.

[0028] Embodiment 2

[0029] This example is a fluorescence response experiment of a fluorescent molecular probe to hydroxyl radicals.

[0030] This example uses mild EDTA-Fe 2+ The complex undergoes Fenton reaction in water and biological systems to produce ·OH. 2+ The complexes were mixed in a PBS buffer solution (0.01M, pH=7.4), and then H2O2 was added to make the final concentrations of OH 0, 2, 5, 10, 15, 20, 25, 50, and 100 μM, respectively, and the final concentration of the probe was 10 μM. After the system to be tested was reacted at room temperature for 20 minutes to fully respond, fluorescence detection was performed. The results are shown in Figure 1 As shown in the figure, it can be seen that the probe itself has no fluorescence at 667nm. As the concentration of ·OH increases, the fluorescence gradually increases, and in the concentration range of 0 to 15μM, it has a good linear relationship with the fluorescence intensity at 667nm (R 2 =0.99448)(see Figure 2), the standard calculation method (S / N=3) was used to obtain the lowest detection limit (LOD) of ·OH in the solution as 51.2 nM, indicating that the probe can achieve a good response to ·OH in the solution.

[0031] Embodiment 3

[0032] This example is a selective experiment of fluorescent molecular probes on hydroxyl radicals

[0033] In order to explore the selectivity of fluorescent molecular probes for different reactive oxygen species, we selected a variety of well-known reactive oxygen species: H2O2, 1 O2, O2 - 、ROO·、NO·、ONOO - , ClO - The solution was prepared into an aqueous solution and added to the probe solution, with the concentrations of 30 μM, 10 μM for the probe, and 10 μM for ·OH. By measuring the response of the probe fluorescence intensity to different reactive oxygen compounds and comparing it with the ·OH control group, it was found that the probe only had a significant fluorescence enhancement at 667 nm for ·OH, and had a weak fluorescence change for other tested small molecules (see Figure 3 ). It was confirmed that the probe has excellent selectivity for ·OH.

[0034] Embodiment 4

[0035] This example is an imaging experiment of endogenous hydroxyl radicals in living cells using fluorescent molecular probes.

[0036] Hela cells were seeded into 96-well cell culture plates and cultured overnight at 37°C and 5% CO2. The cells were divided into two groups, both of which were first incubated with the probe (20 μM) for 20 min. The experimental group was treated with 10 μg / mL LPS (which stimulates the generation of ROS including hydroxyl free radicals), while the control group was treated with PBS buffer solution (0.01 M, pH = 7.4). After 60 min, cell imaging was performed. The results are shown in Figure 4 As shown in the figure, it can be seen that the experimental group ( Figure 4 a) showed obvious red fluorescence, while the control group ( Figure 4 b) There is no obvious red fluorescence, indicating that the probe can respond to endogenous hydroxyl radicals in living cells.

[0037] 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 fluorescent molecular probe for detecting hydroxyl radicals, characterized in that: The structural formula is as follows:

2. A method for preparing a fluorescent molecular probe for detecting hydroxyl radicals according to claim 1, characterized in that: Here are the steps: (1) 3,5-dimethylbenzaldehyde, 2-aminothiophenol, H2O2 and HCl are dissolved in an anhydrous alcohol solution, stirred at room temperature until the reaction is complete, poured into pure water, the pH is adjusted to neutral, purified and dried to obtain a light yellow product BTM, wherein the structural formula of BTM is: (2) BTM, 2-methylpyridinium salt and piperidine are dissolved in an anhydrous alcohol solution, heated under reflux until the reaction is complete, and purified to obtain a red fluorescent molecular probe BTMP, wherein the structural formula of BTMP is:

3. The method for preparing a fluorescent molecular probe for detecting hydroxyl radicals according to claim 2, characterized in that: In the step (1), the dosage ratio of 3,5-dimethylbenzaldehyde to 2-aminothiophenol is 1:1-1:2 mmol / mmol, the dosage ratio of 3,5-dimethylbenzaldehyde to anhydrous alcoholic solution is 1:8-1:15 mmol / mL, the dosage ratio of 3,5-dimethylbenzaldehyde to H2O2 is 1:0.8-1:1.2 mmol / mL, wherein the concentration of H2O2 is 30%, and the dosage ratio of 3,5-dimethylbenzaldehyde to HCl is 1.5:1-2.5:1 mmol / mL, wherein the concentration of HCl is 37%.

4. The method for preparing a fluorescent molecular probe for detecting hydroxyl radicals according to claim 2, characterized in that: In the step (2), the usage ratio of BTM to 2-methylpyridinium salt is 1:1.2-1:2 mmol / mmol, the usage ratio of BTM to piperidine is 1:1.8-1:2.5 mmol / mmol, and the usage ratio of BTM to anhydrous alcoholic solution is 1:8-1:20 mmol / mL.

5. The method for preparing a fluorescent molecular probe for detecting hydroxyl radicals according to claim 2, characterized in that: The anhydrous alcohol solution is anhydrous methanol or anhydrous ethanol.

6. The method for preparing a fluorescent molecular probe for detecting hydroxyl radicals according to claim 2, characterized in that: In the step (1), the purification and drying method is: suction filtration, collecting and washing the filter cake, and then vacuum drying.

7. The method for preparing a fluorescent molecular probe for detecting hydroxyl radicals according to claim 2, characterized in that: In the step (2), the heating reflux temperature is 90°C.

8. The method for preparing a fluorescent molecular probe for detecting hydroxyl radicals according to claim 2, characterized in that: In the step (2), the purification method is: removing the solvent by rotary evaporation, and then purifying by column chromatography.

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