3,4-disulfide maleimide derivative fluorescent molecules based on aggregation-induced emission and preparation method and application thereof

Abstract

The present invention relates to the technical field of fluorescent sensors, and discloses a 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission and its preparation method and application, including adding anhydrous sodium acetate to 4-(3,4-dichloromaleimide) benzaldehyde solution and stirring evenly, dripping 3,5-bis(trifluoromethyl)benzenethiol solution under inert gas protection, removing organic solvent under reduced pressure after stirring reaction, and separating and purifying to obtain yellow solid 3,4-disulfide maleimide derivative fluorescent molecule. The beneficial effects of the present invention are: the fluorescent molecular probe has a strong fluorescence intensity, and the higher the concentration, the stronger the fluorescence emission, with significant AIE characteristics, and the fluorescence intensity quenching after the probe contacts H2S, realizing the detection of H2S, effectively avoiding the ACQ effect; and in the process of detecting H2S, it is not easily interfered by other potential interferents, has high sensitivity, strong selectivity, low cytotoxicity, and has important application value in H2S detection.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent sensors, and in particular to a 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission, and a preparation method and application thereof. Background Art

[0002] Hydrogen sulfide (H2S) is a common toxic gas molecule with a rotten egg smell. Studies have shown that H2S in organisms is the third type of endogenous "gas signal molecule" in addition to CO and NO. The abnormal expression of its concentration is closely related to the occurrence and development of many diseases, such as Huntington's disease, Alzheimer's disease, diabetes, cirrhosis, etc. Therefore, the detection of H2S, especially the detection of H2S in organisms, is of great significance. At present, the detection methods of H2S mainly include spectrophotometry, gas chromatography, electrochemical method and polarography. Although these technologies have many advantages, their disadvantages cannot be ignored, such as high cost, complex sample processing process, and the need to destroy the integrity of the sample during the sample preparation process, which greatly limits the detection application of H2S in organisms.

[0003] In recent years, small molecule fluorescent probes have provided an effective way to detect H2S and other bioactive molecules due to their high sensitivity, good selectivity, high spatial resolution, good biocompatibility and applicability to in situ detection. However, when traditional fluorophores are in a high concentration aggregate state, the interaction between molecules will be enhanced, resulting in energy dissipation through non-radiative energy transitions, resulting in aggregation-induced quenching (ACQ) effect, which affects their luminescence performance. Due to their own hydrophobic characteristics, conventional fluorescent probes may still aggregate in the hydrophobic cavities of certain macromolecules such as proteins even in dilute solutions, causing local probe concentrations to increase and fluorescence quenching through aggregation, which may interfere with the detection results and greatly limit the application of fluorescent probes.

[0004] In contrast to ACQ dyes, aggregation-induced emission (AIE) fluorescent dyes have a rotatable structure or a twisted propeller-shaped conformation. Due to the aggregation, the intramolecular motion is hindered, so that it can emit light with high efficiency, especially when the solution concentration is higher, the fluorescence emission is stronger. Therefore, AIE fluorescent probes are ideal candidates for detecting active species in vivo. For example, the Chinese invention patent document with publication number CN108641711A discloses a H2S aggregation-induced fluorescent probe, a preparation method and its application. The target product is synthesized by two different pathways using 2-azido-9-fluorenone, salicylaldehyde, and salicylaldehyde monohydrazone as raw materials. The target compound can respond to the trace substance hydrogen sulfide in the human body; however, the technology does not mention the toxicity of the probe to cells in the body, and the selectivity and sensitivity in the detection process need to be further improved. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to provide a 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission and a preparation method thereof. The prepared fluorescent molecule has the advantages of good selectivity, high sensitivity and low cytotoxicity when used in H2S detection.

[0006] The present invention solves the above technical problems through the following technical means:

[0007] The first aspect of the present invention provides a 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission, which has the following molecular structure:

[0008]

[0009] Beneficial effects: The present application uses 3,4-disulfide maleimide derivative fluorescent molecules as probes. On the one hand, the probe itself has a strong fluorescence intensity, and the higher the concentration of the probe compound, the stronger the fluorescence emission, showing a significant AIE characteristic. When the probe compound comes into contact with H2S, the fluorescence intensity of the probe is completely quenched, thereby realizing the detection of H2S and effectively avoiding the ACQ effect. On the other hand, the fluorescent molecule shows good selectivity and sensitivity in recognizing H2S. In the process of detecting H2S, it is not easily interfered by other potential interferents such as common active substances such as anions, cations, and biological molecules, and has the advantages of high sensitivity and high selectivity for H2S detection.

[0010] In addition, 3,4-disulfide maleimide derivative fluorescent molecules have strong cell permeability and low cytotoxicity. Using them as probes can realize the detection of H2S in vivo, which has extremely important application value in the detection of H2S in biomedicine and life systems.

[0011] The second aspect of the present invention provides a method for preparing a 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission, comprising the following steps: adding anhydrous sodium acetate to a 4-(3,4-dichloromaleimide)benzaldehyde solution and stirring evenly, then dropwise adding a 3,5-bis(trifluoromethyl)thiophenol solution under the protection of an inert gas, removing the organic solvent under reduced pressure after the stirring reaction is completed, and separating and purifying to obtain a yellow solid 3,4-disulfide maleimide derivative fluorescent molecule.

[0012] Preferably, the molar ratio of the 4-(3,4-dichloromaleimide)benzaldehyde to 3,5-bis(trifluoromethyl)thiophenol is 1:1 to 1:10.

[0013] Preferably, the molar ratio of the 4-(3,4-dichloromaleimide)benzaldehyde to anhydrous sodium acetate is 10:1 to 1:10.

[0014] Preferably, the 4-(3,4-dichloromaleimide)benzaldehyde and 3,5-bis(trifluoromethyl)thiophenol are both dissolved in an organic solvent, and the organic solvent is at least one of tetrahydrofuran, dichloromethane, chloroform and acetone.

[0015] Preferably, the stirring temperature is 0 to 50° C. and the stirring time is 8 to 48 hours.

[0016] Preferably, the separation and purification is carried out by column chromatography.

[0017] Preferably, the reaction equation for the preparation process of the 3,4-disulfide maleimide derivative fluorescent molecule is as follows:

[0018]

[0019] Preferably, the preparation method of 4-(3,4-dichloromaleimide)benzaldehyde comprises: dissolving p-aminobenzaldehyde and 3,4-dichloromaleic anhydride in glacial acetic acid, refluxing at 80-150°C for 12-48h, cooling to room temperature, filtering and collecting yellow precipitates, washing the precipitates with an organic solvent precooled at -4-0°C, and vacuum drying to remove the organic solvent to obtain yellow solid 4-(3,4-dichloromaleimide)benzaldehyde.

[0020] Preferably, the molar ratio of p-aminobenzaldehyde to 3,4-dichloromaleic anhydride is 1.0:1.0-3.0.

[0021] Preferably, the organic solvent is at least one of methanol and dichloromethane.

[0022] Preferably, the preparation method of p-aminobenzaldehyde comprises: dissolving 4-acetylaminobenzaldehyde in methanol, then adding sodium hydroxide aqueous solution, reflux at 60-65°C for 1-5 hours and then cooling to room temperature, then extracting with chlorine dioxide and collecting the organic phase, washing and drying, removing the organic solvent under reduced pressure, and separating and purifying to obtain light yellow solid p-aminobenzaldehyde.

[0023] Preferably, the molar ratio of 4-acetamidobenzaldehyde to sodium hydroxide is 1.0:3.0-5.0.

[0024] Preferably, the crude p-aminobenzaldehyde product is separated and purified by a medium-pressure chromatography column, and a mixture of petroleum ether and dichloromethane in a volume ratio of 2:1 is used as an eluent.

[0025] Preferably, the method for preparing the 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission comprises the following specific steps:

[0026] (1) Preparation of p-aminobenzaldehyde: 4-acetylaminobenzaldehyde is dissolved in methanol, and then a sodium hydroxide aqueous solution is added to make the molar ratio of 4-acetylaminobenzaldehyde to sodium hydroxide be 1.0:3.0-5.0; the mixed solution is refluxed at 60-65° C. for 1-5 hours and then cooled to room temperature, extracted with chlorine dioxide and the organic phase is collected, washed and dried, and then the organic solvent is removed under reduced pressure, and the crude product is separated and purified by a medium pressure chromatography column to obtain light yellow solid p-aminobenzaldehyde;

[0027] (2) Preparation of 4-(3,4-dichloromaleimide)benzaldehyde: dissolving the above-mentioned p-aminobenzaldehyde and 3,4-dichloromaleic anhydride in glacial acetic acid so that the molar ratio of p-aminobenzaldehyde to 3,4-dichloromaleic anhydride is 1.0:1.0-3.0; subjecting the above-mentioned mixed system to reflux reaction at 80-150° C. for 12-48 hours, cooling to room temperature, filtering and collecting yellow precipitates, and then washing the precipitates with methanol and dichloromethane precooled at -4-0° C., and vacuum drying to remove the organic solvent to obtain yellow solid 4-(3,4-dichloromaleimide)benzaldehyde;

[0028] (3) Preparation of 3,4-disulfide maleimide derivative fluorescent molecules: anhydrous sodium acetate is added to an organic solution of 4-(3,4-dichloromaleimide)benzaldehyde, the molar ratio of 4-(3,4-dichloromaleimide)benzaldehyde to anhydrous sodium acetate is 10:1 to 1:10, and after stirring evenly, 3,5-bis(trifluoromethyl)benzaldehyde solution is added dropwise under the protection of inert gas, so that the molar ratio of 4-(3,4-dichloromaleimide)benzaldehyde to 3,5-bis(trifluoromethyl)benzaldehyde is 1:1 to 1:10; after the addition is completed, the mixed system is stirred at 0 to 50°C for 8 to 48 hours. After the reaction is completed, the organic solvent is removed under reduced pressure, and the yellow solid 3,4-disulfide maleimide derivative fluorescent molecules are obtained by separation and purification.

[0029] Preferably, the reaction equation for the preparation process of the 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission is as follows:

[0030]

[0031] The third aspect of the present invention proposes an application of a 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission in the preparation of a fluorescent probe for detecting H2S.

[0032] Beneficial effects: The 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission in this application has excellent luminescence performance and can be used as a sensitive "on-off" type H2S fluorescent probe. It has extremely important application value in the fields of biosensors, fluorescent probes, fluorescent imaging, organic light-emitting devices, etc.

[0033] Preferably, the aggregation-induced emission-based 3,4-disulfide maleimide derivative fluorescent molecule is used as a H2S fluorescent probe in cell imaging.

[0034] Beneficial effects: The 3,4-disulfide maleimide derivative fluorescent molecules based on aggregation-induced emission in this application are used as "on-off" fluorescent probes in cell imaging, which can efficiently and accurately realize H2S detection in biomedicine and life systems.

[0035] The advantages of the present invention are:

[0036] 1. The present application uses 3,4-disulfide maleimide derivative fluorescent molecules as probes. On the one hand, the probe itself has a strong fluorescence intensity, and the higher the concentration of the probe compound, the stronger the fluorescence emission, showing a significant AIE characteristic. When the probe compound comes into contact with H2S, the fluorescence intensity of the probe is completely quenched, thereby realizing the detection of H2S and effectively avoiding the ACQ effect. On the other hand, the fluorescent molecule shows good selectivity and sensitivity for the recognition of H2S. In the process of detecting H2S, it is not easily interfered by other potential interferents such as anions, cations, biological molecules and other common active substances, and has the advantages of high sensitivity and high selectivity for H2S detection. In addition, the 3,4-disulfide maleimide derivative fluorescent molecules have strong cell permeability and low cytotoxicity. Using them as probes can realize the detection of H2S in vivo, and have extremely important application value in the detection of H2S in biomedicine and life systems.

[0037] 2. The preparation method of the present application is simple, and the prepared 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission has excellent luminescence performance and can be used as a sensitive "on-off" type H2S fluorescent probe, which has extremely important application value in the fields of biosensors, fluorescent probes, fluorescent imaging, organic light-emitting devices, etc.;

[0038] 3. The present application uses aggregation-induced emission-based 3,4-disulfide maleimide derivative fluorescent molecules as "on-off" fluorescent probes for application in cell imaging, which can efficiently and accurately realize H2S detection in biomedical and life systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a fluorescence emission spectrum of the 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission in water with different volume fractions in Example 4 of the present invention.

[0040] Figure 2 This is a fluorescence emission spectrum of different concentrations of NaHS added to the PBS / DMSO solution of the 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission in Example 5 of the present invention.

[0041] Figure 3 This is a linear relationship diagram of the fluorescence intensity of different concentrations of NaHS added to the PBS / DMSO solution of the 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission in Example 5 of the present invention.

[0042] Figure 4This is a bar graph of the change in fluorescence intensity of compound KJ before and after adding NaHS to a PBS / DMSO solution of a 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission in the presence of other interferents in Example 6 of the present invention.

[0043] Figure 5 This is a diagram showing the cytotoxicity of the 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission in Example 7 of the present invention.

[0044] Figure 6 This is a fluorescence imaging diagram of H2S in Hela cells using the 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission in Example 8 of the present invention. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0047] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0048] Example 1

[0049] In a first aspect, the present embodiment provides a 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission, which has the following molecular structure:

[0050]

[0051] The second aspect of this embodiment provides a method for preparing a 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission, comprising the following steps:

[0052] (1) Preparation of p-aminobenzaldehyde: In a 250 mL round-bottom flask, 4-acetylaminobenzaldehyde (3.5 g, 21.5 mmol) was dissolved in 140 mL methanol, and then 50 mL of sodium hydroxide (3.43 g, 85.7 mmol) aqueous solution was added to the above solution; the above mixed solution was refluxed at 65°C for 2 h, cooled to room temperature after the reaction, 100 mL of water was added to the system and extracted with dichloromethane (50 mL x 4), the organic phase was collected and washed with saturated saline solution, and then dried with anhydrous sodium sulfate, and the organic solvent was removed under reduced pressure under vacuum; then a medium pressure chromatography column was used to separate and purify the crude product using petroleum ether and dichloromethane in a volume ratio of 2:1 as eluent to obtain a light yellow solid product. The prepared product was characterized by hydrogen nuclear magnetic spectrum, and the results were as follows:

[0053] 1 H NMR (400MHz, CDCl3, δppm): 9.67 (s, 1H), 7.61 (d, J = 8.6 Hz, 2H), 6.75 (d, J = 8.6 Hz, 2H), 5.71 (s, 2H).

[0054] The obtained product is p-aminobenzaldehyde, and the yield is 75%.

[0055] (2) Preparation of 4-(3,4-dichloromaleimide)benzaldehyde: The above-prepared p-aminobenzaldehyde (1 g, 8.26 mmol) and 3,4-dichloromaleic anhydride (1.37 g, 8.26 mmol) were dissolved in 30 mL of glacial acetic acid, and the mixture was refluxed at 120°C for 12 h. After the reaction was completed, it was cooled to room temperature, filtered with a Buchner funnel and the yellow precipitate was collected. The precipitate was then washed with methanol and dichloromethane precooled in a -4°C refrigerator, and the organic solvent was removed by vacuum drying to obtain a yellow solid product. The prepared product was characterized by H NMR spectrum, and the results were as follows:

[0056] 1 H NMR (400MHz, CDCl3, δppm): 10.02 (s, 1H), 7.97 (d, J = 8.4Hz, 2H), 7.59ppm (d, J = 8.4Hz, 2H).

[0057] The obtained product is 4-(3,4-dichloromaleimido)benzaldehyde, and the yield is 85%.

[0058] (3) Preparation of 3,4-disulfide maleimide derivative fluorescent molecules: At room temperature, anhydrous sodium acetate solid (205 mg, 2.5 mmol) was added to a 10 mL anhydrous tetrahydrofuran solution containing the compound 4-(3,4-dichloromaleimide)benzaldehyde (269 mg, 1 mmol). After stirring for 10 minutes, 1 mL of tetrahydrofuran solution containing 2.2 mmol 3,5-bis(trifluoromethyl)benzenethiol was slowly added to the above solution under nitrogen protection. After the addition was completed, the above mixed system was reacted at room temperature for 12 hours. After the reaction was completed, the organic solvent was removed under reduced pressure under vacuum conditions, and a yellow solid was separated and purified. The prepared product was characterized by hydrogen and carbon nuclear magnetic spectra, and the results were as follows:

[0059] 1 H NMR (400MHz, d6-DMSO, δppm): 10.07 (s, 1H), 8.12 (d, J = 8.0Hz, 2H), 7.94 (s, 2H), 7.76 (s, 4H), 7.71ppm (d, J = 8.0Hz, 2H).

[0060] 13 C NMR (600MHz, d6-DMSO, δppm): 192.8, 166.3, 137.0, 135.48, 132.86, 131.38, 131.0, 130.8, 130.7, 130.2, 126.7, 124.5, 121.8, 121.6. HR-MS (ESI): m / z=689.0003.

[0061] The obtained product is a 3,4-disulfide maleimide derivative fluorescent molecule (represented by KJ) with a yield of 80%.

[0062] The reaction equation of the above preparation process is as follows:

[0063]

[0064] Example 2

[0065] This embodiment provides a method for preparing a 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission, comprising the following steps:

[0066] (1) Preparation of p-aminobenzaldehyde: In a 250 mL round-bottom flask, 4-acetylaminobenzaldehyde (3.5 g, 21.5 mmol) was dissolved in 140 mL of methanol, and then 50 mL of sodium hydroxide (2.57 g, 64.5 mmol) aqueous solution was added to the above solution; the above mixed solution was refluxed at 63° C. for 1 h, and after the reaction was completed, it was cooled to room temperature, 100 mL of water was added to the system and extracted with dichloromethane (50 mL x 4), the organic phase was collected and washed with saturated saline solution, and then dried over anhydrous sodium sulfate, and the organic solvent was removed under reduced pressure under vacuum; then, the crude product was separated and purified using a medium-pressure chromatography column with petroleum ether and dichloromethane in a volume ratio of 2:1 as eluent to obtain a light yellow solid product p-aminobenzaldehyde with a yield of 64%.

[0067] (2) Preparation of 4-(3,4-dichloromaleimide)benzaldehyde: The p-aminobenzaldehyde (1 g, 8.26 mmol) prepared above and 3,4-dichloromaleic anhydride (2.74 g, 16.5 mmol) were dissolved in 30 mL of glacial acetic acid, and the mixture was refluxed at 80°C for 48 h. After the reaction was completed, the mixture was cooled to room temperature and filtered with a Buchner funnel to collect the yellow precipitate. The precipitate was then washed with methanol and dichloromethane precooled in a -4°C refrigerator, and the organic solvent was removed by vacuum drying to obtain a yellow solid product 4-(3,4-dichloromaleimide)benzaldehyde with a yield of 70%.

[0068] (3) Preparation of 3,4-disulfide maleimide derivative fluorescent molecules: At room temperature, anhydrous sodium acetate solid (205 mg, 2.5 mmol) was added to a 10 mL anhydrous tetrahydrofuran solution containing the compound 4-(3,4-dichloromaleimide)benzaldehyde (269 mg, 1 mmol). After stirring for 10 minutes, 1 mL of a tetrahydrofuran solution containing 5 mmol of 3,5-bis(trifluoromethyl)benzenethiol was slowly added dropwise to the above solution under nitrogen protection. After the addition was completed, the above mixed system was reacted at 10°C for 48 hours. After the reaction was completed, the organic solvent was removed under reduced pressure under vacuum conditions, and the yellow solid product 3,4-disulfide maleimide derivative fluorescent molecule (represented by KJ) was separated and purified, with a yield of 78%.

[0069] The reaction equation of the above preparation process is as follows:

[0070]

[0071] Example 3

[0072] This embodiment provides a method for preparing a 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission, comprising the following steps:

[0073] (1) Preparation of p-aminobenzaldehyde: In a 250 mL round-bottom flask, 4-acetylaminobenzaldehyde (3.5 g, 21.5 mmol) was dissolved in 140 mL of methanol, and then 50 mL of sodium hydroxide (4.28 g, 107.5 mmol) aqueous solution was added to the above solution; the above mixed solution was refluxed at 60° C. for 5 h, and after the reaction was completed, it was cooled to room temperature, 100 mL of water was added to the system and extracted with dichloromethane (50 mL x 4), the organic phase was collected and washed with saturated saline solution, and then dried over anhydrous sodium sulfate, and the organic solvent was removed under reduced pressure under vacuum; then, a medium-pressure chromatography column was used to separate and purify the crude product using petroleum ether and dichloromethane in a volume ratio of 2:1 as eluent to obtain a light yellow solid product p-aminobenzaldehyde with a yield of 72%.

[0074] (2) Preparation of 4-(3,4-dichloromaleimide)benzaldehyde: The p-aminobenzaldehyde (1 g, 8.26 mmol) and 3,4-dichloromaleic anhydride (4.11 g, 24.8 mmol) prepared above were dissolved in 30 mL of glacial acetic acid, and the mixture was refluxed at 150° C. for 25 h. After the reaction was completed, the mixture was cooled to room temperature and filtered with a Buchner funnel to collect the yellow precipitate. The precipitate was then washed with methanol and dichloromethane precooled in a -4° C. refrigerator, and the organic solvent was removed by vacuum drying to obtain a yellow solid product 4-(3,4-dichloromaleimide)benzaldehyde with a yield of 80%.

[0075] (3) Preparation of 3,4-disulfide maleimide derivative fluorescent molecules: At room temperature, anhydrous sodium acetate solid (205 mg, 2.5 mmol) was added to a 10 mL anhydrous tetrahydrofuran solution containing the compound 4-(3,4-dichloromaleimide)benzaldehyde (269 mg, 1 mmol). After stirring for 10 minutes, 1 mL of a tetrahydrofuran solution containing 10 mmol of 3,5-bis(trifluoromethyl)benzenethiol was slowly added dropwise to the above solution under nitrogen protection. After the addition was completed, the above mixed system was reacted at 50°C for 8 h. After the reaction was completed, the organic solvent was removed under reduced pressure under vacuum conditions, and the yellow solid product 3,4-disulfide maleimide derivative fluorescent molecule (represented by KJ) was separated and purified, with a yield of 75%.

[0076] The reaction equation of the above preparation process is as follows:

[0077]

[0078] Example 4

[0079] In this embodiment, the AIE characteristics of the 3,4-disulfide maleimide derivative fluorescent molecule prepared above are studied. The specific method includes: preparing 11 centrifuge tubes, taking 3 ml of the 3,4-disulfide maleimide derivative fluorescent molecule (KJ) prepared in Example 1 with a concentration of 10 μM with a pipette and adding them to different centrifuge tubes respectively, and then adding DMSO and deionized water respectively, so that the volume fraction of deionized water to DMSO in each centrifuge tube is 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% respectively, and after mixing evenly, the fluorescence emission spectrum is tested in a fluorescence emission spectrometer with a fluorescence excitation wavelength of 425 nm and a slit of 10 / 10 nm. The test results are shown as follows: Figure 1 shown.

[0080] from Figure 1 It can be seen that when the volume fraction of water is less than 50%, compound KJ has almost no fluorescence. This is because DMSO is a benign solvent for KJ. When its volume fraction is large, KJ is in a state of low concentration in the test solution. When the volume fraction of water reaches 50%, as the volume fraction of water gradually increases, the fluorescence intensity of compound KJ also increases significantly. Especially when the volume fraction of water reaches 80% to 90%, its fluorescence intensity reaches the strongest, indicating that compound KJ has significant AIE characteristics. This is because when the volume fraction of water is high, water is a poor solvent for KJ, KJ is in a highly aggregated state in DMSO, the rotation of the thiol bond is inhibited, and the conjugated plane of the structure increases, so the fluorescence intensity also increases; when the volume of water is higher than 90%, its fluorescence intensity decreases slightly, which is caused by molecular aggregation and precipitation, but compound KJ still has significant AIE characteristics.

[0081] Example 5

[0082] This embodiment provides an application of a 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission, specifically the fluorescence responsiveness detection of KJ to H2S. NaHS hydrolyzes to produce H2S in the solution, and the concentration of H2S is positively correlated with the concentration of NaHS. By adding different concentrations of NaHS to the KJ solution, H2S solutions of different concentrations can be obtained. The specific method includes: mixing PBS and DMSO in a volume ratio of 9:1 to obtain a PBS / DMSO mixed solution, and dissolving the 3,4-disulfide maleimide derivative fluorescent molecule prepared in Example 1 with the PBS / DMSO mixed solution to obtain a KJ solution with a concentration of 10 μM. The above KJ solution was added to different cuvettes, and different amounts of NaHS solution were added thereto to make the NaHS concentrations in the solutions 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 25 μM, respectively. After being fully shaken and mixed at room temperature, fluorescence spectra were scanned in a fluorescence emission spectrometer with a fluorescence excitation wavelength of 425 nm and a slit of 10 / 10 nm. The fluorescence spectra of different concentrations of NaHS are shown in the following figure. Figure 2 shown.

[0083] from Figure 2 It can be seen that when the NaHS concentration in the solution is 0 μM, compound KJ itself has a significant fluorescence intensity; as the NaHS concentration continues to increase, the fluorescence intensity of compound KJ at 540nm is significantly quenched; when the NaHS concentration reaches 25 μM, its fluorescence is almost completely quenched. Under a 365nm ultraviolet lamp, it can be observed that after the continuous addition of NaHS, the fluorescence of the KJ solution changes from yellow light to quenching. The above phenomenon shows that compound KJ exhibits an "on-off" type fluorescence response to H2S.

[0084] The linear relationship between the concentrations of NaHS in the above experiment, which are 0, 2, 4, 6, 8, 10, 12, 14, 16, and 18 μM and the corresponding fluorescence intensity at 540 nm is as follows: Figure 3 As shown, the linear fitting equation y =

[0085] 1915.2364-88.0318x(R 2 =0.998), the detection limit was calculated by the formula LOD=3σ / k, where k is the slope and σ is the background noise, and the detection limit was calculated to be 75 nM.

[0086] Example 6

[0087] This embodiment provides an application of a 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission, specifically, a competitive experiment using other interfering substances to explore the specific detection of KJ for H2S, and the specific method includes: mixing PBS and DMSO in a volume ratio of 9:1 to obtain a PBS / DMSO mixed solution, dissolving the 3,4-disulfide maleimide derivative fluorescent molecule prepared in Example 1 in the PBS / DMSO mixed solution to obtain a KJ solution with a concentration of 10 μM. The above KJ solution is added to different cuvettes, and then other potential interfering substances, such as anion solution (F - , Cl - Br - ,I - PO4 3- 、NO2 - 、NO3 - 、HCO3 - 、CO3 2- 、HSO3 - 、SO3 2- 、HSO4 - 、SO4 2- , A C O - ), cationic solution (Na + , K + Mg 2+ 、Zn 2+ , Ca 2+ ), biomolecules (·OH, 1 O2, H2O2, ONOO - , TBHP, Vc, GSH, Cys, H2S), after being fully shaken and mixed at room temperature, the fluorescence spectrum was scanned in a fluorescence emission spectrometer with a fluorescence excitation wavelength of 425nm and a slit of 10 / 10nm; then a 25μM NaHS solution was added and the fluorescence spectrum was scanned again. The bar graph of the change in the fluorescence intensity of compound KJ before and after the addition of NaHS in each group under the coexistence of interfering substances is shown in the figure below. Figure 4 shown.

[0088] from Figure 4 It can be seen that when potential interferents are added to the KJ solution, there is almost no obvious fluorescence change; and when NaHS is added to the KJ solution containing different potential interferents, the fluorescence is almost completely quenched, indicating that the fluorescence response induced by H2S is not affected by other interferents. Therefore, the 3,4-disulfide maleimide derivative fluorescent molecule KJ based on aggregation-induced emission has excellent selectivity and anti-interference for H2S detection among various competitive analytes.

[0089] Example 7

[0090] The present embodiment provides an application of a 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission. The cytotoxicity of KJ prepared in Example 1 was studied by MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide) colorimetry. The specific method includes: dissolving the 3,4-disulfide maleimide derivative fluorescent molecule prepared in Example 1 with a DMSO solution to obtain a KJ mother solution, and then taking different amounts of the KJ mother solution and adding it to a cell culture medium to obtain a culture medium containing a fluorescent molecule KJ with different concentration gradients (0, 20, 40, 60, 80, 100 μM) for later use. Human uterine squamous carcinoma SiHa cells in the logarithmic growth phase were inoculated in a 96-well microplate with a cell inoculation density of 5000 cells / well and incubated in a cell culture incubator at 37°C and 5% CO2. After the cells adhered to the wall for 16 hours, the old culture medium was discarded and culture solutions containing different concentration gradients of fluorescent molecules KJ (0, 20, 40, 60, 80, 100 μM) were added respectively. The untreated cells were used as the control group, and 6 parallel wells were set up at the same time. After 24 hours of continuous culture, the culture solution containing the fluorescent molecule KJ was removed, and then 20 μL of MTT with a concentration of 5.0 mg / mL was added to each well. After being placed in the incubator for 4 hours, the old solution was discarded, and 150 μL of DMSO was added. After being placed on a shaker for 10 minutes, the absorbance of each well at 490 nm was detected on an enzyme-linked immunosorbent assay, and the cell survival rate of each experimental group was calculated according to the cell survival rate calculation formula. The cell survival rate calculation formula is as follows:

[0091] SR=(A / A0)*100%

[0092] Among them, SR is the cell survival rate, A is the absorbance value of the experimental group, and A0 is the absorbance value of the blank control group.

[0093] The results of SiHa cell survival rate under different concentrations of compound KJ are as follows Figure 5 As shown. Figure 5 It can be seen that with the increase of the concentration of compound KJ, the cell survival rate decreased, but even when the concentration of KJ was as high as 100 μM, the cell survival rate still remained above 80%, which shows that compound KJ has very low cytotoxicity.

[0094] Example 8

[0095] The present embodiment provides an application of a 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission, specifically for the fluorescence imaging of H2S in SiHa cells. The specific method includes: using the 3,4-disulfide maleimide derivative fluorescent molecule prepared in Example 1 with a cell culture medium to prepare a KJ solution with a concentration of 10 μM. Add it to the pre-cultured SiHa cells, incubate in a cell culture incubator at 37°C and 5% CO2 for 30 minutes, wash the cells three times with cold PBS, and remove the KJ solution that has not entered the cells; then add 30 μM NaHS solution and continue incubating for 30 minutes. After washing twice with cold PBS, immediately use a confocal fluorescence microscope to observe the intracellular fluorescence imaging. The excitation light wavelength is 405 nm, and the receiving wavelength is 420-600 nm. The fluorescence imaging of KJ for H2S in SiHa cells is shown in the figure below. Figure 6 shown.

[0096] Figure 6 The two images on the left are fluorescence images, the two images on the right are bright field images of cells, and the middle image corresponds to the Merge image of the two images on the same row; the three images in the upper row are fluorescence images of SiHa cells without the addition of NaHS solution, and the three images in the lower row are fluorescence images of SiHa cells after the addition of NaHS solution. It can be seen that cells treated with compound KJ alone have obvious yellow fluorescence, and when H2S is added, the yellow fluorescence in the cells is significantly quenched, indicating that the 3,4-disulfide maleimide derivative fluorescent molecule KJ based on aggregation-induced emission has good cell membrane permeability; at the same time, the fluorescent molecule KJ can detect H2S in living cells.

[0097] The implementation principle of the present application is as follows: the present application uses 3,4-disulfide maleimide derivative fluorescent molecules as probes. On the one hand, the probe itself has a strong fluorescence intensity, and the higher the concentration of the probe compound, the stronger the fluorescence emission, showing a significant AIE characteristic. When the probe compound comes into contact with H2S, the fluorescence intensity of the probe is completely quenched, thereby realizing the detection of H2S and effectively avoiding the ACQ effect; on the other hand, the fluorescent molecule shows good selectivity and sensitivity in the recognition of H2S, and is not easily interfered by other potential interferents such as anions, cations, biological molecules and other common active substances in the process of detecting H2S, and has the advantages of high sensitivity and high selectivity for H2S detection; in addition, the 3,4-disulfide maleimide derivative fluorescent molecules have strong cell permeability and low cytotoxicity. Using them as probes can realize the detection of H2S in organisms, and has extremely important application value in the detection of H2S in biomedicine and life systems.

[0098] The preparation method of the present application is simple, and the prepared 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission has excellent luminescence performance and can be used as a sensitive "on-off" type H2S fluorescent probe. It has extremely important application value in the fields of biosensors, fluorescent probes, fluorescent imaging, organic light-emitting devices, etc.

[0099] In the present application, 3,4-disulfide maleimide derivative fluorescent molecules based on aggregation-induced emission are used as "on-off" fluorescent probes in cell imaging, which can efficiently and accurately realize H2S detection in biomedicine and life systems.

[0100] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission, characterized in that: It has the following molecular structure:

2. The method for preparing a 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission as claimed in claim 1, characterized in that: The following steps are involved: Anhydrous sodium acetate is added to a 4-(3,4-dichloromaleimide)benzaldehyde solution and stirred evenly, and then a 3,5-bis(trifluoromethyl)thiophenol solution is added dropwise under the protection of an inert gas. After the stirring reaction is completed, the organic solvent is removed under reduced pressure, and a yellow solid 3,4-disulfide maleimide derivative fluorescent molecule is obtained by separation and purification; the molar ratio of the 4-(3,4-dichloromaleimide)benzaldehyde to the 3,5-bis(trifluoromethyl)thiophenol is 1:2.2 to 1:10; and the molar ratio of the 4-(3,4-dichloromaleimide)benzaldehyde to anhydrous sodium acetate is 1:2.

5.

3. The method for preparing a 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission according to claim 2, characterized in that: The 4-(3,4-dichloromaleimide)benzaldehyde and 3,5-bis(trifluoromethyl)thiophenol are both dissolved in an organic solvent, and the organic solvent is at least one of tetrahydrofuran, dichloromethane, chloroform and acetone.

4. The method for preparing a 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission according to claim 2, characterized in that: The reaction temperature is 0-50°C, and the reaction time is 8-48h.

5. The method for preparing the 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission according to claim 2, characterized in that: Column chromatography was used for separation and purification.

6. The method for preparing a 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission according to claim 2, characterized in that: The reaction equation for the preparation process of the 3,4-disulfide maleimide derivative fluorescent molecule is as follows:

7. Use of the 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission as claimed in claim 1 in the preparation of a fluorescent probe for detecting H2S.

8. The use of the 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission according to claim 7, characterized in that: The aggregation-induced emission-based 3,4-disulfide maleimide derivative fluorescent molecules are used as H2S fluorescent probes in cell imaging.

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