An aggregation-induced emission molecular probe, a preparation method and application thereof

Abstract

This invention belongs to the field of bioimaging, specifically relating to an aggregation-induced emission (AIE) molecular probe, its preparation method, and its applications. The AIE molecular probe has the structure shown in formula (I). The AIE molecular probe of this invention has several advantages: 1. It can specifically respond to hyposulfonic acid proteins; 2. It has good solubility and emits almost no fluorescence in aqueous media; 3. The molecule can be used for live cell and in vivo imaging, while it is virtually non-toxic to cells and organisms. When the AIE molecular probe is in an aqueous solution, the molecule is in a dispersed state and does not emit fluorescence. When it encounters hyposulfonic acid proteins, the targeting group on the molecule covalently binds to the hyposulfonic acid, fixing the AIE probe onto the protein, restricting the intramolecular movement of the AIE molecule, leading to aggregation-induced emission, thus achieving response imaging of specific hyposulfonic acid proteins.

Description

Technical Field

[0001] This invention belongs to the field of bioimaging, specifically relating to an aggregation-induced emission molecular probe, its preparation method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Reactive oxygen species (ROS), acting as second messengers, regulate intracellular signal transduction by altering the redox state of target proteins. However, excessive ROS-induced oxidative stress can induce oxidative damage to biomolecules such as proteins, lipids, and nucleic acids. Due to their high nucleophilicity and oxidation sensitivity, thiol proteins are the main targets of ROS-triggered protein oxidative modification. Among them, sulfenic acid (R-SOH), as an early product of protein oxidative stress, is further oxidized by ROS into sulfinic acid and sulfonic acid, causing irreversible oxidative damage to proteins. The products of thiol protein oxidation modification can regulate protein function, activity, and localization, significantly impacting various cellular signaling events, including cell proliferation and differentiation. ROS-induced protein oxidative stress is involved in the etiology of many diseases and in the aging process. Furthermore, reports indicate that abnormal protein oxidative modification is associated with the occurrence and development of cancer and cardiovascular diseases. Therefore, developing imaging methods for detecting sulfenic acid protein oxidative modification is of great significance for studying the regulatory mechanisms of oxidative stress and damage repair mechanisms.

[0004] In recent years, thiol proteins have played important roles in regulating cellular physiological functions and pathological oxidative stress, attracting many researchers to conduct imaging studies on thiol proteins and their oxidation products. Numerous detection methods have been developed to identify and image the oxidation products of thiol proteins. Currently, the main detection methods include spectroscopic methods, indirect methods, and chemiluminescent labeling methods. Spectroscopic methods utilize nuclear magnetic resonance (NMR) and mass spectrometry (MS) to analyze the structure of proteins and their post-translational modifications. However, spectroscopic methods can only detect purified proteins and their oxidation products in vitro, requiring protein extraction before analysis, making the process relatively cumbersome. Indirect methods involve pre-modifying other states of the protein, such as protein thiols and protein sulfonic acids, with reagents, and then converting protein sulfonic acids into stable protein thiols or protein sulfonic acids for further detection. However, this method suffers from false positive signals due to incomplete modification, and the sample undergoes multiple processing steps. Chemical fluorescence labeling is a method that uses chemical reactions to fluorescently label thiol proteins. Fluorescence imaging has advantages such as high sensitivity, high specificity, short response time, simple operation, low cost, and high safety, and has become an attractive protein detection tool.

[0005] However, current detection strategies for imaging thiol protein oxidation modification are all non-responsive, with fluorescent probes always on, resulting in high fluorescence background and the need to clean unreacted fluorescent probes, making the operation process cumbersome and complicated. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide an aggregation-induced emission (AIE) molecular probe, its preparation method, and its applications. The AIE molecular probe of the present invention has several advantages: 1. It can specifically respond to hyposulfonic acid proteins; 2. It has good solubility and emits almost no fluorescence in aqueous media; 3. The molecule can be used for live cell and in vivo imaging, while being virtually non-toxic to cells and organisms. When the AIE molecular probe is in an aqueous solution, the molecules are in a dispersed state and do not emit fluorescence. When it encounters hyposulfonic acid proteins, the targeting groups on the molecule covalently bind to the hyposulfonic acid, immobilizing the AIE probe on the protein, restricting the intramolecular movement of the AIE molecule, leading to aggregation-induced emission, thus achieving specific response imaging to hyposulfonic acid proteins.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0008] In a first aspect, the present invention provides an aggregation-induced emission molecular probe having the structure shown in formula (I);

[0009]

[0010] In a second aspect, the present invention provides a method for preparing an aggregation-induced emission molecular probe as described in the first aspect, comprising:

[0011] The compound with the structure shown in formula (II) was reacted with 1,3-propanesulfonyl lactone to obtain the aggregation-induced emission molecular probe with the structure shown in formula (I).

[0012]

[0013] Preferably, Cs2CO3 is added as a catalyst during the reaction, and the molar ratio of the compound with the structure shown in formula (II), 1,3-propanesulfonyl lactone and Cs2CO3 is 1:9.9-10.1:4.9-5.1, the reaction temperature is 25-35℃, and the reaction time is 40-48h.

[0014] More preferably, the method for preparing the compound with the structure shown in formula (II) includes:

[0015] The compound with the structure shown in formula (III) was first reduced with triphenylphosphine, and the reaction product was reacted with 3,5-dioxanecarboxylic acid to obtain the compound with the structure shown in formula (II).

[0016]

[0017] More preferably, the molar ratio of the compound with the structure shown in formula (III) to triphenylphosphine is 1:2.7-2.8, the reduction reaction is carried out under nitrogen protection at a temperature of 35-45℃ for 4-6 h; the molar ratio of the reaction product of the compound with the structure shown in formula (III) and triphenylphosphine to 3,5-dioxanecarboxylic acid is 1:2.3-2.5, and the reaction time with 3,5-dioxanecarboxylic acid is 1-3 h.

[0018] More preferably, the method for preparing the compound with the structure shown in formula (III) includes:

[0019] The compound with the structure shown in formula (IV) is reacted with malononitrile to produce the compound with the structure shown in formula (III);

[0020]

[0021] In a further preferred embodiment, the molar ratio of the compound with the structure shown in formula (IV) to malononitrile is 1:2.9-3.1.

[0022] A further preferred embodiment of the method for preparing the compound with the structure shown in formula (IV) includes:

[0023] The compound with the structure shown in formula (V) was reacted with (5-bromo-[2]thienyl)-(4-hydroxyphenyl)one to generate the compound with the structure shown in formula (IV);

[0024]

[0025] More preferably, the molar ratio of the compound with the structure shown in formula (V) to (5-bromo-[2]thiophene)-(4-hydroxyphenyl) ketone is 1:1.1-1.3, the reaction temperature is 60-80℃, and the reaction time is 4-6h.

[0026] More preferably, the method for preparing the compound with the structure shown in formula (V) includes:

[0027] The compound with the structure shown in formula (VI) is reacted with pinacol diboronic acid ester to generate the compound with the structure shown in formula (V);

[0028]

[0029] More preferably, the molar ratio of the compound with the structure shown in formula (VI) to pinacol diboronic acid ester is 1:1.8-1.9, and the reaction is carried out by heating and stirring under reflux at 80-85°C overnight.

[0030] More preferably, the method for preparing the compound with the structure shown in formula (VI) includes:

[0031] The compound with the structure shown in formula (VI) was formed by reacting 1,1-bis(4'-hydroxybenzene)-2-(4”-bromobenzene)-2-phenylethylene with 2-azidoethyl 4-toluenesulfonic acid.

[0032] More preferably, the molar ratio of 1,1-di(4'-hydroxybenzene)-2-(4”-bromobenzene)-2-phenylethylene to 2-azidoethyl 4-toluenesulfonic acid is 1:2.4-2.6, the reaction temperature is 75-85℃, and the reaction time is 7-9h.

[0033] Thirdly, the present invention provides the application of aggregation-induced emission molecular probes as described in the first aspect in the oxidative imaging of thiol proteins.

[0034] The beneficial effects achieved by one or more technical solutions of the present invention are as follows:

[0035] This invention relates to an AIE molecular probe based on tetraphenylethylene, which has the characteristics of good solubility, specific binding to hyposulfonate proteins, and low fluorescence background. When the target protein is present, the AIE probe emits strong fluorescence. When the target protein is not present, the probe has good solubility in biological media due to the hydrophilic group attached to it, and does not emit fluorescence, thus realizing OFF-On response imaging of hyposulfonate proteins.

[0036] The AIE molecular probe synthesis method of the present invention is simple, the material is composed of organic molecules, does not introduce toxic components, does not have the problem of toxic reagent residues, and has high biosafety. Attached Figure Description

[0037] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0038] Figure 1 The mass spectrum of the compound CHD-TPEAI shown in formula (I) is shown below.

[0039] Figure 2 The NMR spectrum of the compound CHD-TPEAI shown in formula (I) is shown below.

[0040] Figure 3 The UV absorption spectrum of the molecular probe CHD-TPEAI prepared in Example 1 is shown below.

[0041] Figure 4 The images show the fluorescence spectra of the molecular probe CHD-TPEAI after incubation with different analytes in Example 2. Group 1: CHD-TPEAI; Group 2: CHD-TPEAI + β-lactoglobulin; Group 3: CHD-TPEAI + H2O2; Group 4: CHD-TPEAI + β-lactoglobulin + H2O2.

[0042] Figure 5 The fluorescence response of the molecular probe CHD-TPEAI in Example 2 to other small and large molecules;

[0043] Figure 6 The results of the CHD-TPEAI cytotoxicity experiment in Example 3 are as follows: (a) Cell survival rate of HeLa cells after incubation with different concentrations of CHD-TPEAI material; (b) Cell survival rate of HeLa cells after incubation with CHD-TPEAI material for different times.

[0044] Figure 7The image shows a fluorescence confocal image of the probe CHD-TPEAI in response to hyposulfonic acid protein in cells in Example 3: blue represents the nuclear dye Hoechst; red represents compound CHD-TPEAI of formula (I); and the image is an overlay of the two.

[0045] Figure 8 This is a small animal in vivo imaging image of the probe CHD-TPEAI responding to hyposulfonic acid protein in mouse tumors at different times in Example 3. Detailed Implementation

[0046] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0047] In the following embodiments, the aggregation-induced emission molecular probe with the structure shown in Formula (I) is named CHD-TPEAI. The compound with the structure shown in Formula (II) is designated as Compound 5. The compound with the structure shown in Formula (III) is designated as Compound 4. The compound with the structure shown in Formula (IV) is designated as Compound 3. The compound with the structure shown in Formula (V) is designated as Compound 2. The compound with the structure shown in Formula (VI) is designated as Compound 1.

[0048] Example 1

[0049] Synthesis of CHD-TPEAI

[0050] (1) 1,1-Di(4'-hydroxybenzene)-2-(4”-bromobenzene)-2-phenylethylene, 2-azidoethyl 4-toluenesulfonic acid, and Cs₂CO₃ were dissolved in DMF solution in a molar ratio of 1:2.5:2.5, and the mixture was heated and stirred at 80 °C for 8 hours. After the reaction was completed, the mixture was cooled to room temperature and extracted with EA and H₂O. The organic phase was collected and dried over anhydrous Na₂SO₄. The crude product was purified by column chromatography to give compound 1.

[0051] (2) Compound 1, pinacol diborate, [1,1'-bis(diphenylphosphine)ferrocene]palladium chloride, and KOAc were dissolved in 1,4-dioxane at a molar ratio of 1:1.85:0.046:4. The mixture was heated and refluxed overnight at 80°C with stirring under nitrogen protection. After cooling to room temperature, the mixture was extracted with DCM and H2O. The organic phase was collected, dried over anhydrous Na2SO4, and the crude product was purified by column chromatography to obtain compound 2.

[0052] (3) Compound 2, (5-bromo-[2]thienyl)-(4-hydroxyphenyl) ketone, tetra(triphenylphosphine)palladium, and K2CO3 were dissolved in THF / H2O (v / v, 9 / 1) at a molar ratio of 1:1.2:0.016:4. The mixture was heated under N2 protection and stirred under reflux at 70°C for 5 hours. Then, the solution was removed under reduced pressure, and the crude product was purified by column chromatography to obtain compound 3.

[0053] (4) Compound 3 and malononitrile were dissolved in anhydrous dichloromethane at a molar ratio of 1:3 and stirred at 0°C under nitrogen protection. 6.5 equivalents of titanium tetrachloride (equivalent to compound 3) were slowly added and stirred at 0°C for half an hour. Then, 8 equivalents of pyridine (equivalent to compound 3) were slowly added and stirred at 0°C for half an hour. Finally, the mixture was heated at 40°C for one hour. After cooling to room temperature, the mixture was extracted with DCM and H2O, and the organic phase was collected and dried over anhydrous Na2SO4. The crude product was purified by column chromatography to obtain compound 4.

[0054] (5) Compound 4 and triphenylphosphine were dissolved in THF at a molar ratio of 1:2.75 in an appropriate amount of water. The mixture was stirred at 40°C for 5 hours under nitrogen protection. Then, the solution was removed under reduced pressure, and the crude product was dissolved in 0.5M HCl and washed three times with dichloromethane. The crude amine product was obtained by evaporation of the aqueous solution and used directly without further purification. 2.4 equivalents of 3,5-dioxanecarboxylic acid, 2.4 equivalents of EDC, and 2.4 equivalents of NHS were dissolved in DMF and stirred at 0°C for 2 hours. The crude amine product and 2.4 equivalents of DIPEA were added to the above reaction mixture, and the mixture was stirred at room temperature for six hours. The solution was removed under reduced pressure, and the crude product was purified by column chromatography to obtain compound 5.

[0055] (6) Compounds 5, 1,3-propanesulfonyl lactone, and Cs₂CO₃ were dissolved in DMF at a molar ratio of 1:10:5. The mixture was stirred at 30°C for 48 hours, and the reaction was removed under reduced pressure. The reactants were dissolved in water, and the crude product was purified by high performance liquid chromatography to obtain CHD-TPEAI. The mass spectrum of CHD-TPEAI is shown below. Figure 1 As shown, the NMR spectrum is as follows Figure 2 As shown, the successful synthesis of CHD-TPEAI is demonstrated. Figure 3 As shown, CHD-TPEAI has a wide UV absorption range, and the molecular probe has a strong absorption peak at 465 nm.

[0056] Example 2

[0057] In vitro experiments using CHD-TPEAI for imaging of thiol protein oxidation

[0058] β-lactoglobulin was incubated with DTT in Tris-HCl buffer overnight at 4°C. Excess DTT was removed by centrifugation using an ultrafiltration tube to obtain the thiol protein. The thiol protein was then incubated with CHD-TPEAI and H2O2 at 37°C for 1 hour. The fluorescence of the sample was measured using an FLS-980 Edinburgh fluorescence spectrometer (Ex = 465 nm, Em = 550-750 nm). Figure 4 The results show that the molecular probe only emits fluorescence when thiol protein and hydrogen peroxide are present simultaneously, indicating that the probe responds to hyposulfonic acid protein.

[0059] To further verify the specificity of CHD-TPEAI for hyposulfonic acid proteins, H2O2, ATP, Cys, Gly, GSH, GSSH, TCEP, insulin, BSA, and β-lactoglobulin were added to CHD-TPEAI and incubated in Tris-HCl buffer at 37°C for 1 hour. Fluorescence of different samples was measured using an FLS-980 Edinburgh fluorescence spectrometer. Figure 5 The results show that CHD-TPEAI reacts only with hyposulfonate proteins (BLG-SOH or BSA-SOH), while other small molecules (H2O2, ATP, Cys, Gly, GSH, GSSH, TCEP) and biomacromolecules (insulin, BSA, BLG) have little effect on the fluorescence of CHD-TPEAI, indicating that the probe has high specificity for hyposulfonate proteins.

[0060] Example 3

[0061] CHD-TPEAI for biological experiments involving thiol protein oxidation imaging

[0062] Cytotoxicity assay

[0063] The cytotoxicity of the CHD-TPEAI molecular probe to cells was measured using 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) assays in HeLa (human cervical cancer cells). HeLa cells were incubated with different concentrations of CHD-TPEAI (0, 5, 10, 20, and 40 μM), followed by incubation for different durations (0, 2, 4, 8, 12, and 24 hours). The absorbance was then measured after MTT treatment. Figure 6 The results show that as the probe concentration and incubation time increase, the cells still maintain a relatively high survival rate, indicating that the probe has good biocompatibility and is basically non-toxic to cells.

[0064] Fluorescence imaging of hyposulfonic acid protein in live cells

[0065] HeLa cells were cultured in confocal dishes for 24 h. HeLa cells were incubated with different concentrations of H₂O₂ (0, 100, 500, 1000 μM) for 30 min, then co-incubated with CHD-TPEAI for 2 h. Finally, the cells were incubated with a nuclear tracer dye (Hoechst) for 10 min to obtain confocal images. Figure 7 The results show that as the concentration of hydrogen peroxide increases, more intracellular proteins are oxidized into hyposulfonate proteins, and the red fluorescence of the probe in the cell becomes stronger, indicating that the molecular probe of this application can image hyposulfonate proteins in living cells.

[0066] In vivo fluorescence imaging of hyposulfonate protein

[0067] A HeLa-bearing tumor-bearing nude mouse model was established for in vivo imaging using molecular probes. All animal experiments were conducted in accordance with the Laboratory Animal Care Principles of the People's Republic of China. Nude mice aged 6-8 weeks were injected with HeLa (human cervical cancer cells), and the tumors were allowed to grow to 100 mm. 3 Imaging was performed on the tumor. The molecular probe (CHD-TPEAI) from Example 1 was injected into the tumor of mice, and the same concentration of CHD-TPEAI was injected into normal tissue on the opposite side of the tumor as a control group. Subsequently, the mice were imaged at different time points (0, 1, 2, 4, 12, 24 h) after injection. The results are as follows: Figure 8 As shown. From Figure 8 As can be seen, compared with the control group, the fluorescence signal in the tumor tissue was significantly enhanced 4 hours after injection, indicating that the content of hyposulfonate protein increases during tumor development. Even 24 hours after injection, a strong fluorescence signal was still observed in the tumor tissue compared with the control group. This may be because the probe is immobilized on hyposulfonate protein, allowing it to remain in the tumor tissue for a long time. These results demonstrate that the prepared CHD-TPEAI can specifically image hyposulfonate protein in vivo for extended periods.

[0068] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An aggregation-induced emission molecular probe, characterized in that, It has the structure shown in equation (I); Equation (I).

2. A method for preparing the aggregation-induced emission molecular probe as described in claim 1, characterized in that, include: The compound with the structure shown in formula (II) was reacted with 1,3-propanesulfonyl lactone to obtain the aggregation-induced emission molecular probe with the structure shown in formula (I). Equation (II).

3. The preparation method according to claim 2, characterized in that, Cs2CO3 was added as a catalyst during the reaction. The molar ratio of the compound with the structure shown in formula (II), 1,3-propanesulfonyl lactone and Cs2CO3 was 1:9.9-10.1:4.9-5.

1. The reaction temperature was 25-35 °C and the reaction time was 40-48 h.

4. The preparation method according to claim 2, characterized in that, The preparation method of the compound with the structure shown in formula (II) includes: The compound with the structure shown in formula (III) was first reduced with triphenylphosphine, and the reaction product was reacted with 3,5-dioxanecarboxylic acid to obtain the compound with the structure shown in formula (II). Equation (III).

5. The preparation method according to claim 4, characterized in that, The molar ratio of the compound with the structure shown in formula (III) to triphenylphosphine is 1:2.7-2.

8. The reduction reaction is carried out under nitrogen protection at a temperature of 35-45 °C for 4-6 h. The molar ratio of the reaction product of the compound with the structure shown in formula (III) to 3,5-dioxanecarboxylic acid is 1:2.3-2.

5. The reaction time with 3,5-dioxanecarboxylic acid is 1-3 h.

6. The preparation method according to claim 4, characterized in that, The preparation method of the compound with the structure shown in formula (III) includes: The compound with the structure shown in formula (IV) is reacted with malononitrile to generate the compound with the structure shown in formula (III); Formula (IV).

7. The preparation method according to claim 6, characterized in that, The molar ratio of the compound with the structure shown in formula (IV) to malononitrile is 1:2.9-3.

1.

8. The preparation method according to claim 6, characterized in that, The method for preparing the compound with the structure shown in formula (IV) includes: The compound with the structure shown in formula (V) was reacted with (5-bromo-[2]thienyl)-(4-hydroxyphenyl)one to generate the compound with the structure shown in formula (IV); Formula (V).

9. The preparation method according to claim 8, characterized in that, The molar ratio of the compound with the structure shown in formula (V) to (5-bromo-[2]thiophene)-(4-hydroxyphenyl) ketone is 1:1.1-1.3, the reaction temperature is 60-80 °C, and the reaction time is 4-6 h.

10. The preparation method according to claim 8, characterized in that, The method for preparing the compound with the structure shown in formula (V) includes: The compound with the structure shown in formula (VI) is reacted with pinacol diboronic acid ester to generate the compound with the structure shown in formula (V); Formula (VI).

11. The preparation method according to claim 10, characterized in that, The molar ratio of the compound with the structure shown in formula (VI) to pinacol diboronic acid ester is 1:1.8-1.9, and the specific reaction conditions are heating and stirring under reflux at 80-85℃ overnight.

12. The preparation method according to claim 10, characterized in that, The method for preparing the compound with the structure shown in formula (VI) includes: The compound with the structure shown in formula (VI) is obtained by reacting 1,1-bis(4'-hydroxybenzene)-2-(4''-bromobenzene)-2-phenylethylene with 2-azidoethyl 4-toluenesulfonic acid.

13. The preparation method according to claim 12, characterized in that, The molar ratio of 1,1-bis(4'-hydroxybenzene)-2-(4''-bromobenzene)-2-phenylethylene to 2-azidoethyl 4-toluenesulfonic acid was 1:2.4-2.6, the reaction temperature was 75-85℃, and the reaction time was 7-9 h.

14. The application of the aggregation-induced emission molecular probe as described in claim 1 in the preparation of reagents for thiol protein oxidation imaging.

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