Fluorescent probe for identifying and detecting hypochlorous acid by targeting lysosome as well as preparation method and application of fluorescent probe

Abstract

The invention discloses a fluorescent probe for identifying and detecting hypochlorous acid by targeting lysosome as well as a preparation method and application thereof, and relates to a fluorescent probe as well as a preparation method and application thereof. In order to solve the problems of tedious synthesis steps and long response time of the existing probe for identifying and detecting hypochlorous acid, the preparation method of the fluorescent probe comprises the following steps: reacting 3, 3, 5-trimethoxy-3-cyclohexenone with 1, 3-malononitrile to obtain a compound 1; and 2, reacting the compound 1 with 1-hydroxy-2-naphthaldehyde to obtain the target compound. The fluorescent probe is used for detecting hypochlorous acid, sensing detection that the probe can target lysosome to recognize hypochlorous acid is achieved, and the fluorescent probe is good in selectivity, high in resistance to interference of other metal ions, anions, amino acid and active oxygen and low in detection limit. The method is applied to the technical field of analytical chemistry.

Description

Technical Field

[0001] The present invention relates to a fluorescent probe for targeting lysosomes to recognize and detect hypochlorous acid, a preparation method thereof, and an application thereof. Background Art

[0002] As one of the most important reactive oxygen species in the human body, hypochlorous acid (HOCl) usually has an oxidation-reduction effect and is also a core substance of the immune system. An appropriate amount of HOCl can maintain intracellular homeostasis. However, excessive HOCl often causes damage to tissues and organs. Research shows that diseases such as atherosclerosis, neurodegenerative diseases, and malignant tumors are all related to abnormal HOCl concentrations in the body. In addition, due to its strong oxidizing and bleaching properties, HOCl is often involved in the processes of sterilization, food processing, and sewage treatment. The World Health Organization also stipulates that the HOCl concentration in drinking water cannot exceed 0.5 mg / L. Generally speaking, HOCl plays an important role in daily life. However, abnormal concentrations thereof can also lead to dysfunction of the body. Therefore, developing a practical tool for detecting and tracking HOCl concentrations in the environment and the human body is of great significance for disease prevention and diagnosis.

[0003] So far, the methods commonly used for detecting HOCl include electrochemistry analysis, high performance liquid chromatography, and visible spectrophotometry. However, these methods have disadvantages such as complex sample preparation and cumbersome detection steps. Due to the advantages of easy operation and low cost, fluorescent probes have been widely used in the fields of biochemistry and environmental protection. The reported fluorescent probes usually introduce thioether, dimethyl thiocarbonate, C═C unsaturated double bonds, and some other groups to achieve the purpose of recognizing HOCl. The drawback is that these probes have defects such as difficult synthesis, long response time, and fluorescence instability due to the change in fluorescence intensity under long-term ultraviolet light irradiation, which limits their application in biological and environmental systems. Therefore, there is an urgent need to find a detection tool that can quickly recognize HOCl.

[0004] Isophorone is often used as the parent body of fluorescent probe molecules due to its strong structural modifiability, good cell permeability, and diverse synthesis methods. However, the structural modification steps of the current fluorescent probes for detecting HOCl are relatively complex, which limits their large-scale production. Based on this, designing a fluorescent probe tool with simple synthesis and capable of quickly detecting HOCl is of extremely important significance for environmental protection and disease prevention and diagnosis. Summary of the Invention

[0005] The present invention aims to solve the problems of cumbersome synthesis steps, long response time, and fluorescence instability of the existing probes for recognizing and detecting hypochlorous acid, and provides a fluorescent probe for targeting lysosomes to recognize and detect hypochlorous acid, a preparation method thereof, and an application thereof.

[0006] The structural formula of a fluorescent probe for targeting lysosomes to recognize and detect hypochlorous acid in the present invention is as follows:

[0007]

[0008] The preparation method of the fluorescent probe for targeting lysosomes to recognize and detect hypochlorous acid in the present invention includes the following steps:

[0009] I. 3,3,5-Trimethoxy-3-cyclohexenone reacts with malononitrile to obtain compound 1:

[0010] Dissolve 3,5,5-trimethoxy-3-cyclohexenone and malononitrile in ethanol, then add piperidine, heat under reflux, the heating temperature is 75 - 85 °C, react for 10 - 12 h, detect the reaction with a TCL plate. After the reaction is complete, cool to room temperature, add deionized water to the solution to precipitate, filter the precipitate, wash, and dry to obtain compound 1;

[0011] Among them, the molar ratio of 3,5,5-trimethoxy-3-cyclohexenone, malononitrile and piperidine is 1.0 - 1.05:1.0 - 1.5:0.05 - 0.10, preferably 1:1.2:0.08; the molar volume ratio of 3,5,5-trimethoxy-3-cyclohexenone to ethanol is 1.0 - 1.1 mmol:1.8 - 2.2 mL;

[0012] II. React compound 1 with 1-hydroxy-2-naphthaldehyde to obtain the target compound:

[0013] Add compound 1, 1-hydroxy-2-naphthaldehyde and piperidine into a round-bottom flask containing acetonitrile, heat at 75 - 85 °C, react for 2 - 4 h, after the reaction, cool to room temperature, add deionized water to cool, filter the precipitate, wash, and dry to obtain the target compound; among them, the molar ratio of compound 1, 1-hydroxy-2-naphthaldehyde and piperidine is 1.0 - 1.05:1.0 - 1.1:0.05 - 0.10, preferably 1:1:0.06; the molar volume ratio of compound 1 to acetonitrile is 1.0 - 1.1 mmol:15 - 20 mL, preferably 1 mmol:15 mL.

[0014] A fluorescent probe for targeting lysosomes to recognize and detect hypochlorous acid in the present invention is applied to sense and detect hypochlorous acid in an aqueous environment system. The said sensing detection includes fluorescence detection, ultraviolet detection, visual qualitative detection or test paper detection. It is also used for fluorescence imaging detection of hypochlorous acid in cell lysosomes.

[0015] The preparation reaction formula of the fluorescent probe for targeting lysosomes to recognize and detect hypochlorous acid in the present invention is as follows:

[0016]

[0017] In the reaction formula, 1 is Compound 1, and QW-Lyso is a fluorescent probe for targeting lysosomes to recognize and detect hypochlorous acid.

[0018] Advantages of the present invention:

[0019] 1) The synthesis of the probe only requires two steps, and the raw materials are economical, and the post-treatment process is relatively simple; 2) The present invention realizes the sensing detection of the probe for targeting lysosomes to recognize hypochlorous acid, and has good selectivity, strong anti-interference ability against other metal ions, anions, amino acids and reactive oxygen species, and a low detection limit. 3) In addition, significant fluorescence color changes can be observed under ultraviolet light, and the recognition of hypochlorous acid can be achieved through different fluorescence color regions, which is a fluorescent probe with a chromogenic sensing function. 4) This probe for recognizing hypochlorous acid has the characteristics of rapid response, strong fluorescence signal and good fluorescence stability, and can realize rapid and real-time detection of hypochlorous acid. In addition, the present invention can also be applied to the fluorescence imaging recognition of hypochlorous acid in lysosomes in cells. Therefore, the present invention is a simple, rapid and sensitive hypochlorous acid detection reagent, and has broad application prospects in the field of water sample environment and life system. Description of the Drawings

[0020] Figure 1 It is a fluorescence selectivity spectrogram;

[0021] Figure 2 It is the influence of coexisting anions on the determination of hypochlorous acid;

[0022] Figure 3 It is the influence of coexisting cations on the determination of hypochlorous acid;

[0023] Figure 4 It is the fluorescence spectral response diagram of the probe QW-Lyso (concentration: 1×10-5 mol / L) to different concentrations of hypochlorous acid in C 2 H 5 OH / PBS (5 / 5, v / v, 10 mM PBS, pH = 7.0);

[0024] Figure 5 It is the fluorescence emission diagram of the probe QW-Lyso after adding hypochlorous acid under ultraviolet light at 365 nm;

[0025] Figure 6 It is the naked-eye visual detection of hypochlorous acid by the method of dipping the test paper;

[0026] Figure 7 It is the response time of the probe QW-Lyso to the fluorescence recognition of hypochlorous acid;

[0027] Figure 8For the fluorescent recognition of exogenous hypochlorous acid by the probe QW-Lyso in living HepG2 cells;

[0028] Figure 9 For the fluorescent recognition of endogenous hypochlorous acid by the probe QW-Lyso in living A549 cells;

[0029] Figure 10 For the fluorescent recognition of exogenous hypochlorous acid by the probe QW-Lyso in living zebrafish;

[0030] Figure 11 For the fluorescent recognition of endogenous hypochlorous acid by the probe QW-Lyso in living zebrafish;

[0031] Figure 12 For the fluorescent recognition of hypochlorous acid targeted to intracellular lysosomes by the probe QW-Lyso. Specific embodiments

[0032] The technical solution of the present invention is not limited to the following specific embodiments listed, and also includes any combination between the specific embodiments.

[0033] Specific embodiment 1: The structural formula of a fluorescent probe for targeting lysosomes to recognize and detect hypochlorous acid in this embodiment is as follows:

[0034]

[0035] Specific embodiment 2: The preparation method of a fluorescent probe for targeting lysosomes to recognize and detect hypochlorous acid in this embodiment is as follows:

[0036] 1. 3,3,5-Trimethoxy-3-cyclohexenone reacts with malononitrile to obtain compound 1:

[0037] Dissolve 3,5,5-trimethoxy-3-cyclohexenone and malononitrile in ethanol, then add piperidine, heat under reflux, the heating temperature is 75 - 85 °C, react for 10 - 12 h, detect the reaction with a TLC plate. After the reaction is complete, cool to room temperature and add deionized water to the solution to precipitate. Filter the precipitate, wash, and dry to obtain compound 1;

[0038] Among them, the molar ratio of 3,5,5-trimethoxy-3-cyclohexenone, malononitrile to piperidine is 1.0 - 1.05:1.0 - 1.5:0.05 - 0.10; the molar volume ratio of 3,5,5-trimethoxy-3-cyclohexenone to ethanol is 1.0 - 1.1 mmol:1.8 - 2.2 mL;

[0039] 2. React compound 1 with 1-hydroxy-2-naphthaldehyde to obtain the target compound:

[0040] Compound 1, 1-hydroxy-2-naphthaldehyde and piperidine were added to a round-bottom flask containing acetonitrile, and the heating temperature was 75-85 °C. After reacting for 2-4 h, it was cooled to room temperature, added with deionized water to cool, and the precipitate was filtered, washed, and dried to obtain the target compound; the molar ratio of Compound 1, 1-hydroxy-2-naphthaldehyde and piperidine was 1.0-1.05:1.0-1.1:0.05-0.10; the molar volume ratio of Compound 1 to acetonitrile was 1.0-1.1 mmol:15-20 mL.

[0041] Specific Embodiment 3: The difference between this embodiment and Specific Embodiment 2 is that: in Steps 1 and 2, the washing was carried out 5 times with deionized water. Others are the same as Specific Embodiment 2.

[0042] Specific Embodiment 4: The difference between this embodiment and Specific Embodiment 2 or 3 is that: the molar ratio of 3,5,5-trimethoxy-3-cyclohexenone, 1,3-propanedinitrile and piperidine is 1:1.2:0.08. Others are the same as Specific Embodiment 2 or 3.

[0043] Specific Embodiment 5: The difference between this embodiment and one of Specific Embodiments 2 to 4 is that: the molar volume ratio of 3,5,5-trimethoxy-3-cyclohexenone to ethanol is 1.0 mmol:2.15 mL. Others are the same as one of Specific Embodiments 2 to 4.

[0044] Specific Embodiment 6: The difference between this embodiment and one of Specific Embodiments 2 to 5 is that: the molar ratio of Compound 1, 1-hydroxy-2-naphthaldehyde and piperidine is 1:1:0.06. Others are the same as one of Specific Embodiments 2 to 5.

[0045] Specific Embodiment 7: The difference between this embodiment and one of Specific Embodiments 2 to 6 is that: the molar volume ratio of Compound 1 to acetonitrile is 1 mmol:15 mL. Others are the same as one of Specific Embodiments 2 to 6.

[0046] Specific Embodiment 8: A fluorescent probe for targeting lysosome recognition and detection of hypochlorous acid is applied to sense and detect hypochlorous acid in an aqueous environment system.

[0047] Specific Embodiment 9: The difference between this embodiment and Specific Embodiment 8 is that: the said sensing and detection include fluorescence detection, ultraviolet detection, visual qualitative detection or test paper detection. Others are the same as Specific Embodiment 8.

[0048] Specific Embodiment 10: A fluorescent probe for targeting lysosome recognition and detection of hypochlorous acid is applied to perform fluorescence imaging detection of hypochlorous acid in cell lysosomes.

[0049] The following is a detailed description of the embodiments of the present invention. The following embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0050] Example 1

[0051] Preparation of the target compound, the hypochlorous acid fluorescent probe QW-Lyso.

[0052] (1) Synthesis of Compound 1:

[0053]

[0054] Dissolve 3,5,5-trimethyl-3-cyclohexenone (3.8790 g, 28 mmol) in 60 mL C 2 H 5 OH, and then successively add malononitrile (1.28 g, 35.4 mmol) and piperidine (0.192 g, 2.24 mmol). Heat under reflux, with the heating temperature being 80 °C, and react for 12 h. Monitor the reaction process by TLC. After the reaction is complete, cool the mixture to room temperature, pour it into cold water, filter the precipitate, wash the obtained filter cake several times with water, and dry it to obtain Compound 1, a coffee-colored solid, with a yield of 48.47%.

[0055] (2) Synthesis of the target compound:

[0056]

[0057] Add Compound 1 (0.1863 g, 1 mmol) and 1-hydroxy-2-naphthaldehyde (0.1722 g, 1 mmol) to 15 ml of acetonitrile, and dropwise add piperidine (0.0052 g, 0.06 mmol) thereto. Heat under reflux, with the heating temperature being 80 °C, and react for 3 h. Detect the reaction with a TLC plate. After the reaction is complete, cool to room temperature, filter the precipitate, wash it 5 times with water, and dry it to obtain the target compound QW-Lyso, with a yield of 54.2%.

[0058] Example 2

[0059] Application of the hypochlorous acid fluorescent probe QW-Lyso

[0060] Dissolve the hypochlorous acid fluorescent probe QW-Lyso synthesized in Example 1 in ethanol to prepare a C -5 mol / L solution of C 2 H 5 OH / H 2 O (2 / 3, v / v), and use a spectrophotometer to detect its fluorescence selectivity for different ions (anions, cations, and reactive oxygen species). Figure 1It is a fluorescence selectivity spectrogram. Figure 1 As can be seen from Figure 1 , when the probe itself is excited at a wavelength of 459 nm, there is almost no fluorescence emission. After adding different detection ions, the fluorescence intensity change is detected by a fluorescence spectrophotometer, and the results are as Figure 1 shown. After adding hypochlorous acid, the maximum fluorescence emission wavelength is observed at 550 nm, and the fluorescence intensity is significantly enhanced.

[0061] To further determine that it does not affect the selectivity for hypochlorous acid in the coexistence with other ions, a competitive fluorescence experiment was carried out, and the results Figure 2 、 Figure 3 are shown. Fluorescence spectral analysis shows that it does not affect the recognition of hypochlorous acid by the probe in the coexistence with other ions.

[0062] Different concentrations of hypochlorous acid are added to the solution, and the change in its fluorescence intensity is detected. As Figure 4 shown by fluorescence spectral analysis, in the range where the concentration of added hypochlorous acid is from 0 to 9.5×10 -5 mol / L, as the concentration of added hypochlorous acid increases, the maximum emission wavelength undergoes a blue shift, from 600 nm to 550 nm. At the same time, the change in fluorescence intensity has a good linear relationship curve with the added concentration, realizing the quantitative detection of hypochlorous acid.

[0063] Figure 5 It is the fluorescence color change (from magenta fluorescence to orange fluorescence respectively) after adding 5×10 -5 mol / L hypochlorous acid to 1×10 -5 mol / L probe solution and placing it under ultraviolet light at 365 nm, realizing the visual qualitative detection of hypochlorous acid.

[0064] Figure 6 When the test paper is placed in a solution containing the probe QW-Lyso (10 μM) and different concentrations of hypochlorous acid (0 - 1100 μM), it is taken out and dried in the air after being soaked for two hours. When the dried test paper is placed under ultraviolet light at 365 nm, as the concentration of hypochlorous acid increases, the test paper shows a visible color change from light pink to gray. This phenomenon indicates that the probe QW-Lyso can be loaded on the test paper for fluorescence colorimetric detection of hypochlorous acid.

[0065] Figure 7Time response of the probe QW-Lyso for targeted recognition of hypochlorous acid. The fluorescence intensity of QW-Lyso (50 μM) at the maximum emission wavelength (Em = 600 nm) was measured as a function of time, and the fluorescence intensity of the probe solution of QW-Lyso (50 μM) with the addition of hypochlorous acid (250 μM) at the maximum emission wavelength (Em = 550 nm) was also measured as a function of time. The results showed that the fluorescence response time of QW-Lyso to hypochlorous acid was less than 5 s, and the fluorescence intensity remained basically constant within 50 minutes, indicating that QW-Lyso has the characteristics of rapid response and good stability for targeted recognition of hypochlorous acid.

[0066] Figure 8 For the fluorescence recognition of exogenous hypochlorous acid in cells by the probe QW-Lyso. HepG2 cells were selected as the research object. The cells were first cultured with hypochlorous acid (50 μM) for 30 min, washed 3 times with PBS, and then fixed with 4% paraformaldehyde. After 5 min, the paraformaldehyde was washed away with PBS, and the cells were further co-incubated with the probe QW-Lyso (10 μM) for 30 min, then washed three times with PBS, and finally imaged under a laser confocal microscope. The fluorescence imaging results showed that fluorescence signals were only observed in the experimental group where the probe QW-Lyso was co-incubated with hypochlorous acid, indicating that the probe QW-Lyso can perform fluorescence imaging recognition of intracellular hypochlorous acid.

[0067] Figure 9 For the fluorescence recognition of endogenous hypochlorous acid in cells by the probe QW-Lyso. A549 cells were selected as the research object. The cells were first cultured with lipopolysaccharide LPS (50 μM) for 30 min, washed 3 times with PBS, and then fixed with 4% paraformaldehyde. After 5 min, the paraformaldehyde was washed away with PBS, and the cells were further co-incubated with the probe QW-Lyso (10 μM) for 30 min, then washed three times with PBS, and finally imaged under a laser confocal microscope. The fluorescence imaging results showed that fluorescence signals were only observed in the experimental group where the probe QW-Lyso was co-incubated with hypochlorous acid, indicating that the probe QW-Lyso can perform fluorescence imaging recognition of intracellular hypochlorous acid.

[0068] Figure 10 For the fluorescence recognition of exogenous hypochlorous acid in zebrafish by the probe QW-Lyso. Zebrafish were selected as the research object. The zebrafish were first cultured with hypochlorous acid (50 μM) for 2 h, then the hypochlorous acid was washed away with PBS, and the zebrafish were further co-incubated with the probe QW-Lyso (10 μM) for 2 h, then washed three times with PBS, anesthetized with MS222, and imaged under a laser confocal microscope. The fluorescence imaging results showed that fluorescence signals were only observed in the experimental group where the probe QW-Lyso was co-incubated with hypochlorous acid, indicating that the probe QW-Lyso can perform fluorescence imaging recognition of hypochlorous acid in zebrafish.

[0069] Figure 11 For the fluorescent recognition of endogenous hypochlorous acid by the probe QW-Lyso in zebrafish. Zebrafish were selected as the research object. After culturing zebrafish with lipopolysaccharide LPS (50 μM) for 2 h, LPS was washed away with PBS, and then further co-incubated with the probe QW-Lyso (10 μM) for 2 h and then rinsed three times with PBS, and anesthetized with MS222 and placed under a laser confocal microscope for imaging. The fluorescence imaging results showed that fluorescence signals could only be observed in the experimental group where the probe QW-Lyso was co-incubated with hypochlorous acid, indicating that the probe QW-Lyso could perform fluorescent imaging recognition of endogenous hypochlorous acid in zebrafish.

[0070] Figure 12 For the fluorescent recognition of hypochlorous acid targeted to intracellular lysosomes by the probe QW-Lyso. A549 cells were selected as the research object. The co-localization studies of QW-Lyso (10 μM) and hypochlorous acid with commercial probes targeting lysosomes, mitochondria, and nuclei were compared, and a high degree of overlap of the probe QW-Lyso targeting lysosomes was observed by a laser confocal fluorescence microscope.

Claims

1. A fluorescent probe for detecting hypochlorous acid targeting lysosomes, characterized in that: The structural formula of the fluorescent probe for targeting lysosomes to recognize and detect hypochlorous acid is as follows:

2. The method for preparing a fluorescent probe for detecting hypochlorous acid by targeting lysosomes according to claim 1, characterized in that: The preparation method is:

1. 3,3,5-trimethoxy-3-cyclohexenone reacts with 1,3-malononitrile to obtain compound 1: Dissolve 3,5,5-trimethoxy-3-cyclohexenone and 1,3-propanedinitrile in ethanol, add piperidine, heat to reflux, the heating temperature is 75-85°C, react for 10-12h, and detect the reaction with a TCL plate. After the reaction is complete, cool to room temperature and add deionized water to the solution to precipitate. Filter the precipitate, wash, and dry to obtain compound 1; The molar ratio of 3,5,5-trimethoxy-3-cyclohexenone, 1,3-malononitrile and piperidine is 1.0-1.05:1.0-1.5:0.05-0.10; the molar volume ratio of 3,5,5-trimethoxy-3-cyclohexenone and ethanol is 1.0-1.1 mmol:1.8-2.2 mL; 2. Compound 1 is reacted with 1-hydroxy-2-naphthaldehyde to obtain the target compound: Add compound 1, 1-hydroxy-2-naphthaldehyde and piperidine to a round-bottom flask filled with acetonitrile, heat to 75-85°C, react for 2-4 hours, cool to room temperature, add deionized water to cool, filter the precipitate, wash, and dry to obtain the target compound; wherein the molar ratio of compound 1, 1-hydroxy-2-naphthaldehyde and piperidine is 1.0-1.05:1.0-1.1:0.05-0.10; the molar volume ratio of compound 1 to acetonitrile is 1.0-1.1 mmol:15-20 mL.

3. The method for preparing a fluorescent probe for targeting lysosomes to recognize and detect hypochlorous acid according to claim 2, characterized in that: The washing in steps 1 and 2 was performed 5 times with deionized water.

4. The method for preparing a fluorescent probe for targeting lysosomes to recognize and detect hypochlorous acid according to claim 2, characterized in that: The molar ratio of 3,5,5-trimethoxy-3-cyclohexenone, 1,3-malononitrile and piperidine is 1:1.2:0.

08.

5. The method for preparing a fluorescent probe for targeting lysosomes to recognize and detect hypochlorous acid according to claim 2, characterized in that: The molar volume ratio of 3,5,5-trimethoxy-3-cyclohexenone to ethanol is 1.0 mmol:2.15 mL.

6. The method for preparing a fluorescent probe for targeting lysosomes to recognize and detect hypochlorous acid according to claim 2, characterized in that: The molar ratio of compound 1, 1-hydroxy-2-naphthaldehyde and piperidine is 1:1:0.

06.

7. The method for preparing a fluorescent probe for targeting lysosomes to recognize and detect hypochlorous acid according to claim 2, characterized in that: The molar volume ratio of compound 1 to acetonitrile is 1 mmol:15 mL.

8. The fluorescent probe for targeting lysosomes to recognize and detect hypochlorous acid as claimed in claim 1 is used for sensing and detecting hypochlorous acid in a water environment system.

9. The use according to claim 8, characterized in that The sensing detection includes fluorescence detection, ultraviolet detection, visual qualitative detection or test paper detection.

10. The fluorescent probe for targeting lysosomes to recognize and detect hypochlorous acid as claimed in claim 1 is used for fluorescent imaging detection of hypochlorous acid in cell lysosomes.

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