Amino-bridged tetraphenyl vinyl phenylboronic acid ester compound, synthesis method and application thereof, and glucose fluorescence detection method

Abstract

The invention relates to the technical field of biological small molecule fluorescence detection, and discloses an amino-bridged tetraphenylethylene phenylboronic acid ester compound, a synthesis method and application thereof and a glucose fluorescence detection method. Tetraphenylethylene is used as an aggregation-induced emission mother nucleus of the compound, a quaternized amino-bridged biphenylboronic acid recognition group is used, and the compound can be used for detecting glucose. The probe can be specifically combined with glucose to generate an aggregation-induced emission effect; the invention also provides a two-step synthesis method of the compound, the synthesis route is simple, the conditions are mild, and the product purity and yield are stable; the glucose detection method constructed based on the compound avoids inherent defects of traditional enzyme method detection, improves water solubility of sensing molecules and glucose binding dynamics performance, has the advantages of being high in detection sensitivity, wide in linear range, easy and convenient to operate and high in anti-interference capacity, and is suitable for rapid quantitative detection of glucose in a water phase system.

Description

Technical Field

[0001] This invention relates to the field of biomolecule fluorescence detection technology, specifically to an amino-bridged tetraphenylethylene phenylboronic acid ester compound, its synthesis method, application, and glucose fluorescence detection method. Background Technology

[0002] Glucose is a core energy source essential for maintaining normal physiological functions in living organisms, and its concentration is closely related to the body's metabolic state. In recent years, with the improvement of living standards and the diversification of dietary structures, daily sugar intake has increased significantly. Long-term excessive sugar intake can lead to various metabolic diseases such as obesity and diabetes, and the incidence of these diseases is increasing year by year and affecting younger people. Therefore, developing a simple, rapid, and highly sensitive glucose detection method is of great practical significance for the early screening, daily monitoring, and clinical diagnosis of metabolic diseases.

[0003] Among existing glucose detection technologies, fluorescence detection has been widely studied and applied due to its advantages such as fast response speed, high detection sensitivity, portability, and the ability to perform in-situ detection. Currently, most mainstream fluorescence glucose detection methods are based on the catalytic system of glucose oxidase. These methods rely on the catalytic activity of biological enzymes, but biological enzymes themselves suffer from poor stability, complex preparation processes, and stringent storage and usage conditions. They are easily deactivated by environmental factors such as temperature and pH, making it difficult to meet the needs of rapid on-site detection and long-term stable use, thus limiting the expansion of their application scenarios.

[0004] To address the inherent limitations of enzymatic detection methods, non-enzymatic fluorescent glucose detection technologies have become a research hotspot in recent years. Most reported non-enzymatic fluorescent detection technologies are based on photoinduced electron transfer mechanisms; however, the validity of this mechanism remains controversial within the industry. Some researchers have constructed phenylboronic acid sensors based on anthracene fluorophores, demonstrating that their fluorescence response to glucose originates from the depolymerization process of fluorescent molecule aggregates, rather than the previously believed photoinduced electron transfer effect. Other researchers have constructed aggregation-induced emission glucose sensors based on tetraphenylethylene phenylboronic acid. These sensors enhance fluorescence intensity by binding glucose to the sensing molecule to form dimers and inhibiting benzene ring rotation, thus achieving glucose detection. However, these sensing molecules have poor water solubility, resulting in insufficient binding kinetics for glucose in aqueous systems. Detection effectiveness is limited by the number of molecules bound, and there is a lack of simple, efficient, and scalable synthetic routes, hindering the realization of highly sensitive, wide-range quantitative glucose detection and industrial applications. Summary of the Invention

[0005] This invention aims to provide an amino-bridged tetraphenylethylene phenylboronic acid ester compound, its synthesis method, application, and glucose fluorescence detection method, in order to solve the inherent defects of existing glucose fluorescence detection technologies, such as poor stability, complex preparation, and harsh usage conditions of enzyme-dependent biological enzymes, and the poor water solubility, insufficient glucose binding kinetics, and limited detection effect due to the number of molecules bound, as well as the lack of simple and scalable synthetic routes for non-enzymatic aggregation-induced emission sensing molecules.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: an amino-bridged tetraphenylethylene phenylboronic acid ester compound, the structural formula of which is as follows: .

[0007] A method for synthesizing the above-described amino-bridged tetraphenylethylene phenylboronic acid ester compound includes the following steps: (1) Synthesis of p-dimethylamino-substituted tetraphenylethylene: p-dimethylamino-substituted tetraphenylethylene was prepared by Suzuki coupling reaction with 1-bromo-1,2,2-triphenylethylene and 4-(N,N-dimethylamino)phenylboronic acid as raw materials under palladium catalyst. (2) Synthesis of the target compound: Under inert gas protection, 4-bromomethylphenylboronic acid ester and the p-dimethylamino-substituted tetraphenylethylene obtained in step (1) were dissolved in anhydrous acetonitrile. After stirring at room temperature, solvent removal by vacuum distillation, and purification by column chromatography, the target amino-bridged tetraphenylethylene phenylboronic acid ester compound was obtained.

[0008] Preferably, as an improvement, in step (1), the palladium catalyst is tetra(triphenylphosphine)palladium, the reaction solvent is a mixture of tetrahydrofuran and potassium carbonate aqueous solution, the reaction is carried out by reflux overnight, the product is purified by silica gel column chromatography, and the eluent is a mixture of dichloromethane and petroleum ether in a volume ratio of 1:6.

[0009] Preferably, as an improvement, the inert gas in step (2) is argon, the reaction time is 12-24 hours, and the eluent for column chromatography purification is a mixture of ethyl acetate and petroleum ether at a volume ratio of 1:10.

[0010] Application of an amino-bridged tetraphenylethylene phenylboronic acid ester compound in glucose fluorescence detection.

[0011] A glucose fluorescence detection method, using the above-mentioned amino-bridged tetraphenylethylene phenylboronic acid ester compound as a fluorescence sensing molecule, includes the following steps: (1) Prepare a detection solution for the fluorescent sensing molecule, wherein the solvent of the detection solution is a mixture of dimethyl sulfoxide and water; (2) Mix the glucose sample solution to be tested with the detection solution in equal volumes, and let it stand to obtain the sample to be tested; (3) The fluorescence spectrum of the sample to be tested is collected by a fluorescence spectrophotometer, and the glucose is quantitatively detected based on the fluorescence intensity of the characteristic emission peak.

[0012] Preferably, as an improvement, the volume ratio of dimethyl sulfoxide to water in the mixed solvent is 1:10 to 1:30.

[0013] Preferably, as an improvement, the detection solution uses a potassium carbonate-potassium bicarbonate buffer system to adjust the pH to 10.0 to 11.0, and the concentration of the fluorescent sensing molecule in the detection solution is 0.005 mM to 0.02 mM.

[0014] Preferably, as an improvement, the settling time in step (2) is not less than 12 hours; the excitation wavelength of fluorescence spectroscopy in step (3) is 310 nm, the emission spectrum collection range is 320 nm to 700 nm, and the excitation slit width and the emission slit width are both 5 nm.

[0015] Preferably, as an improvement, in step (3), the fluorescence emission peak intensity at 500 nm is used as the quantitative basis, a linear standard curve of fluorescence intensity and glucose concentration is established in advance, and the glucose concentration is calculated by the fluorescence intensity of the sample to be tested.

[0016] Advantages of this solution: 1. The amino-bridged tetraphenylethylene phenylboronic acid ester compound designed in this invention achieves a significant improvement in water solubility and an enhancement in glucose binding kinetics through an amino quaternization bridging structure, making it fully suitable for the detection of glucose in aqueous liquid phases.

[0017] 2. The compound synthesis method provided by this invention can be completed in just two classical organic reactions. The reaction conditions are mild, the operation is simple, and the purity and yield of the product are stable. It does not require demanding reaction equipment and is suitable for industrial-scale production.

[0018] 3. This invention achieves detection based on the enhanced aggregation effect of glucose-induced compounds, which is not limited by the number of molecules bound. Compared with existing detection mechanisms, it has a wider linear detection range and higher detection sensitivity.

[0019] 4. The glucose detection method of the present invention is a non-enzymatic catalytic system, which avoids the inherent defects of poor stability, complex preparation and harsh storage conditions of biological enzymes. The detection operation is simple, the response is fast and the fluorescence signal is stable, making it suitable for rapid on-site detection scenarios.

[0020] 5. The detection system of the present invention has excellent specificity for glucose. It can still achieve accurate detection of glucose even in the presence of common interfering substances in biological systems. It has strong anti-interference ability and can be applied to the detection of complex biological samples. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the molecular synthesis of the amino-bridged tetraphenylethylene phenylboronic acid ester compound described in this invention. Figure 2 This is a fluorescence spectrum of the detection system as a function of glucose concentration in an embodiment of the present invention; Figure 3 This is a graph showing the linear relationship between fluorescence intensity and glucose concentration in the detection system of this invention. Detailed Implementation

[0022] The following detailed description, through specific embodiments, further illustrates the following: all reagents used in the following examples are analytical grade reagents; the experimental water is deionized water; the fluorescence spectrophotometer used is a Hitachi F7100 fluorescence spectrophotometer; and the target compound used is the amino-bridged tetraphenylethylene phenylboronic acid ester compound as described in claim 1, whose molecular structure is as follows: Figure 1 As shown in the attached figure, this figure is the chemical structural formula of the sensing molecule designed in this invention, which is used to clarify the structural composition and group connection mode of the core compound of this invention.

[0023] First, the target compound was synthesized. The synthesis of p-dimethylamino-substituted tetraphenylethylene was carried out first. 3.0 g of 1-bromo-1,2,2-triphenylethylene, 2.1 g of 4-(N,N-dimethylamino)phenylboronic acid, and 310 mg of tetra(triphenylphosphine)palladium were placed in a three-necked flask. After evacuation, nitrogen was introduced for 3 minutes to purge the flask. 70 mL of tetrahydrofuran and 9 mL of 3.0 mol / L potassium carbonate aqueous solution were degassed separately for 30 minutes and then added to the three-necked flask. The resulting mixture was refluxed overnight. After cooling to room temperature, the reaction solution was diluted with dichloromethane and washed successively with water and saturated sodium chloride solution. After drying with anhydrous sodium sulfate and filtration, the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography using a 1:6 volume ratio mixture of dichloromethane and petroleum ether as the eluent. Finally, 2.0 g of a white solid product, p-dimethylamino-substituted tetraphenylethylene, was obtained, with a yield of 72%.

[0024] Subsequently, the target amino-bridged tetraphenylethylene phenylboronic acid ester compound was synthesized. Under argon protection, 3 g of 4-bromomethylphenylboronic acid ester and 1.5 g of the previously prepared p-dimethylamino-substituted tetraphenylethylene were dissolved in 150 mL of anhydrous acetonitrile. The mixture was stirred overnight at room temperature. After the reaction was completed, the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography using a 1:10 volume ratio mixture of ethyl acetate and petroleum ether as the eluent. Finally, the target product, the amino-bridged tetraphenylethylene phenylboronic acid ester compound, was obtained with a yield of 58%, thus completing the preparation of the target compound.

[0025] Next, the solutions for glucose fluorescence detection were prepared. First, an amino-bridged tetraphenylethylene phenylboronic acid ester stock solution was prepared. The target compound obtained from the above synthesis was accurately weighed, dissolved in dimethyl sulfoxide, and shaken well to obtain a target compound stock solution with a molar concentration of 1 mM. Then, the fluorescence sensing molecular detection solution was prepared. Anhydrous potassium carbonate and potassium bicarbonate were accurately weighed and added to a 250 mL volumetric flask. 200 mL of deionized water was added, and after the solids were completely dissolved, 2.5 mL of the above-prepared target compound stock solution was added, followed by 7.5 mL of dimethyl sulfoxide. After thorough mixing, the volume was adjusted to 250 mL with deionized water to obtain a detection solution with a pH of 10.5 and a target compound concentration of 0.01 mM. The volume ratio of the mixed solvent dimethyl sulfoxide to water in this detection solution was 1:25. Simultaneously, a 0.01 mM dimethyl sulfoxide solution of the target compound was prepared. The above 1 mM mother liquor was taken into a 100 mL volumetric flask, 90 mL of dimethyl sulfoxide was added and shaken well. The volume was then adjusted to 100 mL with dimethyl sulfoxide to obtain a single-molecule sensing molecular solution for control testing.

[0026] Next, prepare glucose standard solutions. Accurately weigh glucose into a 25 mL volumetric flask, add 20 mL of deionized water, and after the solid is completely dissolved, dilute to 25 mL with deionized water to obtain a 10 mM glucose standard stock solution. Take 8 mL of the above 10 mM glucose standard stock solution, add 2 mL of deionized water to obtain an 8 mM glucose standard solution. Using the same stepwise dilution method, prepare a series of glucose standard solutions of different concentrations: 6 mM, 4 mM, 2 mM, 1.8 mM, 1.6 mM, 1.2 mM, 1 mM, 0.8 mM, 0.4 mM, 0.2 mM, 160 μM, and 120 μM.

[0027] Subsequently, the samples to be tested were prepared and fluorescence detected. 2 mL of the prepared fluorescence sensing molecular detection solution was placed in multiple sample vials, and 2 mL of glucose standard solution of different concentrations was added to each vial. After thorough mixing, the samples were allowed to stand overnight to obtain a series of samples containing different concentrations of glucose. Simultaneously, a blank control sample was prepared by placing 2 mL of the fluorescence sensing molecular detection solution in a sample vial, adding 2 mL of deionized water, and then thoroughly mixing and allowing it to stand overnight to obtain the blank control sample.

[0028] After the sample has settled, fluorescence spectra are collected. Take 2 mL of the prepared sample and place it in a 10 mm... In a 10mm cuvette, the cuvette was placed in a fluorescence spectrophotometer. The excitation wavelength was set to 310nm, the emission spectrum collection range to 320nm-700nm, the excitation slit width to 5nm, the emission slit width to 5nm, and the scan speed to 1200nm / min. The fluorescence spectra of each sample were acquired. Using the same instrument parameters, the fluorescence spectra of a blank control sample and a single-molecule sensing molecular solution were acquired for comparative analysis.

[0029] The fluorescence emission spectra of the samples to be tested at different glucose concentrations collected in this embodiment are as follows: Figure 2 As shown in the attached figure, the data comes from actual measurements taken with a Hitachi F7100 fluorescence spectrophotometer. It visually demonstrates the gradual increase in fluorescence intensity of the detection system as glucose concentration increases. The test results show that the dimethyl sulfoxide solution of the single-molecule target compound has no obvious fluorescence emission peak at 500 nm, while the fluorescence sensing molecular detection solution shows a characteristic fluorescence emission peak of the oligomer at 500 nm, but its fluorescence intensity is low. When glucose is added to the detection solution, the intensity of the fluorescence emission peak at 500 nm significantly increases, and the fluorescence intensity of this characteristic peak increases systematically with increasing glucose concentration.

[0030] In this embodiment, the linear fitting results of the fluorescence emission peak intensity at 500 nm and glucose concentration of the detection system are as follows: Figure 3 As shown in the attached figure, this data is derived from the quantitative analysis and linear fitting of measured fluorescence spectral data, used to clarify the quantitative linear range and fitting relationship of the detection method of this invention. The results show a good linear relationship between fluorescence intensity and glucose concentration, which can be used to establish a standard curve for the quantitative detection of glucose samples with unknown concentrations.

[0031] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. An amino-bridged tetraphenylethylene phenylboronic acid ester compound, characterized in that: The structural formula of the compound is as follows: 。 2. A method for synthesizing the amino-bridged tetraphenylethylene phenylboronic acid ester compound according to claim 1, characterized in that: Includes the following steps: (1) Synthesis of p-dimethylamino-substituted tetraphenylethylene: p-dimethylamino-substituted tetraphenylethylene was prepared by Suzuki coupling reaction with 1-bromo-1,2,2-triphenylethylene and 4-(N,N-dimethylamino)phenylboronic acid as raw materials under palladium catalyst. (2) Synthesis of the target compound: Under inert gas protection, 4-bromomethylphenylboronic acid ester and the p-dimethylamino-substituted tetraphenylethylene obtained in step (1) were dissolved in anhydrous acetonitrile. After stirring at room temperature, solvent removal by vacuum distillation, and purification by column chromatography, the target amino-bridged tetraphenylethylene phenylboronic acid ester compound was obtained.

3. The synthesis method according to claim 2, characterized in that: In step (1), the palladium catalyst is tetra(triphenylphosphine)palladium, the reaction solvent is a mixture of tetrahydrofuran and potassium carbonate aqueous solution, the reaction is carried out by reflux overnight, the product is purified by silica gel column chromatography, and the eluent is a mixture of dichloromethane and petroleum ether in a volume ratio of 1:

6.

4. The synthesis method according to claim 2, characterized in that: In step (2), the inert gas is argon, the reaction time is 12-24 hours, and the eluent for column chromatography purification is a mixture of ethyl acetate and petroleum ether at a volume ratio of 1:

10.

5. The application of the amino-bridged tetraphenylethylene phenylboronic acid ester compound of claim 1 in glucose fluorescence detection.

6. A method for detecting glucose fluorescence, characterized in that: Using the amino-bridged tetraphenylethylene phenylboronic acid ester compound of claim 1 as a fluorescence sensing molecule includes the following steps: (1) Prepare a detection solution containing the fluorescent sensing molecule, wherein the solvent of the detection solution is a mixture of dimethyl sulfoxide and water; (2) Mix the glucose sample solution to be tested with the detection solution in equal volumes, and let it stand to obtain the sample to be tested; (3) The fluorescence spectrum of the sample to be tested is collected by a fluorescence spectrophotometer, and the glucose is quantitatively detected based on the fluorescence intensity of the characteristic emission peak.

7. The glucose fluorescence detection method according to claim 6, characterized in that: The volume ratio of dimethyl sulfoxide to water in the mixed solvent is 1:10 to 1:

30.

8. The glucose fluorescence detection method according to claim 6, characterized in that: The detection solution is prepared by adjusting the pH to 10.0 to 11.0 using a potassium carbonate-potassium bicarbonate buffer system, and the concentration of the fluorescent sensing molecule in the detection solution is 0.005 mM to 0.02 mM.

9. The glucose fluorescence detection method according to claim 6, characterized in that: The static treatment time in step (2) shall not be less than 12 hours; the excitation wavelength of fluorescence spectroscopy in step (3) shall be 310 nm, the emission spectrum collection range shall be 320 nm to 700 nm, and the excitation slit width and the emission slit width shall both be 5 nm.

10. The glucose fluorescence detection method according to claim 6, characterized in that: In step (3), the fluorescence emission peak intensity at 500 nm is used as the quantitative basis. A linear standard curve of fluorescence intensity and glucose concentration is established in advance, and the glucose concentration is calculated by the fluorescence intensity of the sample to be tested.

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