3,6-bis(4-boronic acid phenyl) carbazole, a preparation method thereof, and application thereof in glucose detection
By reacting 3,6-bis(4-boronicophenyl)carbazole with glucose to form a boron ester structure, highly sensitive and stable glucose detection is achieved through intramolecular charge transfer. This solves the problems of poor stability of enzymatic methods and poor water solubility of non-enzymatic probes in existing detection methods, and is suitable for clinical, food and environmental detection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- MOUTAI INST
- Filing Date
- 2026-04-02
- Publication Date
- 2026-06-09
AI Technical Summary
Existing glucose detection methods include enzymatic methods, which suffer from poor stability, high cost, and weak anti-interference ability, while non-enzymatic fluorescent detection probes have poor water solubility, weak fluorescence signals, and complex synthesis, making it difficult to meet the practical detection needs of high sensitivity, high stability, and low cost in aqueous systems.
3,6-bis(4-boronicophenyl)carbazole was used as a non-enzymatic fluorescent probe. It reacted with glucose in a DMSO:H2O mixed solvent to form a boron ester structure. The fluorescence intensity was attenuated by intramolecular charge transfer (ICT) to achieve quantitative detection of glucose concentration.
It achieves highly sensitive and stable glucose detection, avoiding enzyme inactivation and high cost issues. It has good water solubility and anti-interference ability, and is suitable for clinical, food and environmental testing.
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Figure CN122167462A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of organic chemistry, and in particular to 3,6-bis(4-boronic acid phenyl)carbazole, its preparation method, and its application in glucose detection. Background Technology
[0002] Glucose is a core energy source for living organisms, and its accurate concentration detection has irreplaceable value in clinical diagnosis, the food industry, environmental monitoring, and life science research. Excessive sugar intake is highly correlated with metabolic diseases such as obesity, hypertension, arteriosclerosis, and diabetes. With the prevalence of diabetes in my country continuing to rise, rapid, accurate, and stable glucose detection methods are crucial for early screening, disease monitoring, and prognostic assessment.
[0003] Existing glucose detection methods are mainly divided into instrumental analysis methods and sensor methods: instrumental methods such as high performance liquid chromatography and mass spectrometry have high accuracy, but the equipment is expensive, the pretreatment is cumbersome, the detection cycle is long, and it is difficult to detect quickly on site; electrochemical and fluorescence methods have become the mainstream due to their portability and sensitivity.
[0004] Current commercial glucose detection methods largely rely on biological enzyme catalytic systems such as glucose oxidase and glucose dehydrogenase, utilizing the current or fluorescence signal generated by the enzymatic reaction to quantify glucose. However, enzymatic methods have inherent limitations: 1. Poor biological stability: Enzymes are sensitive to environmental factors such as temperature, pH, metal ions, and organic solvents, and are easily inactivated and denatured. They require harsh storage and use conditions and have poor batch-to-batch consistency. 2. High preparation and use costs: The extraction, purification and immobilization processes of biological enzymes are complex and expensive, which is not conducive to low-cost and widespread application; 3. Limited anti-interference ability: In actual samples, coexisting substances such as ascorbic acid, uric acid, and dopamine can easily interfere with enzyme-catalyzed reactions, affecting the accuracy of detection.
[0005] To address the inherent limitations of enzymatic methods, the industry is actively developing detection technologies that do not involve biological enzymes. However, current non-enzymatic fluorescence detection systems still face several key technical bottlenecks: poor probe water solubility, weak fluorescence signals, insufficient response selectivity, lengthy synthesis routes, difficult purification, and difficulty in simultaneously meeting the practical detection requirements of aqueous systems, high sensitivity, high stability, low cost, and easy scalability. These limitations severely restrict the promotion and application of non-enzymatic glucose fluorescence detection methods in rapid clinical and field testing scenarios.
[0006] Therefore, developing a novel non-enzymatic fluorescent probe that is easy to synthesize, has excellent water solubility, strong fluorescence signal, high chemical stability, and specific response to glucose, and establishing a matching aqueous glucose detection method, is a technical problem that urgently needs to be solved in this field. Summary of the Invention
[0007] The present invention aims to provide 3,6-bis(4-boronic acid phenyl)carbazole and its preparation method, as well as its application in glucose detection, in order to solve the technical problems of poor stability, high cost, and weak anti-interference ability of enzymatic methods in glucose detection, and poor water solubility, weak fluorescence signal, complex synthesis and difficulty in meeting the actual detection requirements of high sensitivity and high stability in aqueous systems.
[0008] The 3,6-bis(4-boronic acid phenyl)carbazole in this scheme has the following structural formula:
[0009] The preparation of 3,6-bis(4-boronic acid phenyl)carbazole is carried out via the following synthetic route: .
[0010] Further, the specific steps are as follows: Step 1: Compound S1 (1.0 equivalent) was dissolved in a 1,4-dioxane / water mixed solvent (1.0 M / 0.25 M), and tetrakis(triphenylphosphine)palladium (0.1 equivalent), potassium carbonate (4 equivalent), and 1-bromo-4-iodobenzene (4 equivalent) were added sequentially. The mixture was stirred at 120°C for 12 hours under a nitrogen atmosphere, and then cooled to room temperature. The reaction solution was filtered through diatomaceous earth, and the filter cake was washed and extracted with dichloromethane. The organic phases were combined and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 4 / 1) to obtain compound S2. Step 2: At 0°C, sodium hydride (1.5 equivalents) was slowly added to a tetrahydrofuran (0.5 M) solution of S2 (1.0 equivalents). After stirring the mixture at room temperature for 0.5 hours, it was cooled to 0°C again, and 1-bromo-3-methoxypropane (2 equivalents) was added dropwise at this temperature. The mixture was then stirred at room temperature overnight. The reaction solution was quenched with saturated ammonium chloride aqueous solution, extracted with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 4 / 1) to give compound S3. Step 3: Add [1,1'-bis(diphenylphosphine)ferrocene]palladium(II) dichloride (0.1 equivalent), potassium acetate (5 equivalent), and bis-pinacolborate (4 equivalent) to a 1,4-dioxane (0.2 M) solution of S3 (1.0 equivalent). Stir the mixture at 120°C for 12 hours under a nitrogen atmosphere. After cooling to room temperature, filter through diatomaceous earth. Wash and extract the filter cake with dichloromethane. Combine the organic phases and concentrate under reduced pressure. Purify the crude product by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 4 / 1) to obtain compound S4. Step 4: Add methylboronic acid (10 equivalents) and trifluoroacetic acid (4 equivalents) to a solution of S4 (1.0 equivalents) in dichloromethane (0.5 M), stir overnight at room temperature, collect the solid by filtration, wash with dichloromethane and dry to obtain 3,6-bis(4-boronic acid phenyl)carbazole.
[0011] 3,6-Bis(4-Boratephenyl)carbazole is used for the detection of glucose concentration.
[0012] Further, the following steps are included: S1: Preparation of mother liquor: Weigh 4.93 mg of CA-2BA, add 10 mL of DMSO to dissolve, and prepare a 1 mM 3,6-bis(4-boronic acid phenyl)carbazole mother liquor; S2: Preparation of detection solution: Dissolve 2.8 g of anhydrous potassium carbonate and 0.475 g of potassium bicarbonate in 200 mL of deionized water, add 2.5 mL of 1 mM stock solution and 2.5 mL of DMSO, and bring the volume to 250 mL with deionized water to obtain a pH 10.5, 0.01 mM detection solution. The solvent ratio is DMSO:H2O = 1:20-1:60. S3: Preparation of glucose standard solution: Prepare a 10 mM stock solution of glucose and dilute it stepwise to obtain a series of standard solutions of 0.2 μM–8 mM. S4: Sample preparation and fluorescence detection: The detection solution and glucose solution are mixed in equal volumes and left to stand overnight; the emission spectrum is collected in the range of 310–700 nm with an excitation wavelength of 300 nm, a slit width of 2.5 nm / 2.5 nm, and a scanning speed of 1200 nm / min; the blank group without glucose is used as a reference, and the fluorescence intensity is negatively correlated with the glucose concentration to achieve quantitative detection.
[0013] The 1,3-hydroxyl group of glucose molecule undergoes condensation with the boric acid group of the probe to form a boron ester, which triggers intramolecular charge transfer (ICT), resulting in a decrease in the intensity of the fluorescence emission peak. The degree of decrease is positively correlated with the glucose concentration, thus achieving highly specific non-enzymatic detection.
[0014] Preferably, in S2, DMSO:H2O = 1:50.
[0015] The working principle and beneficial effects of this scheme are as follows: 3,6-Di(4-boronic acid phenyl)carbazole exhibits strong fluorescence emission properties and high fluorescence intensity in a DMSO:H2O mixed solvent with a solvent ratio of 1:50. When mixed with glucose molecules and left for more than 12 hours, the fluorescence intensity of the original emission peak of the fluorophore decreases, and the degree of fluorescence attenuation increases with increasing glucose concentration; the fluorescence intensity is inversely proportional to the glucose concentration. When glucose appears in the detection solution, the two hydroxyl groups at positions 1 and 3 of the glucose molecule undergo a condensation reaction with the borate hydroxyl group of the 3,6-di(4-boronic acid phenyl)carbazole molecule to form a borate ester structure. At this time, internal conversion (ICT) occurs in the molecule, which weakens the fluorescence.
[0016] 1. Probe advantages: With carbazole as the fluorescent core and bisphenylboronic acid as the recognition group, it has high fluorescence intensity, good water solubility, and strong chemical stability, and can be used stably in aqueous systems; 2. Synthetic advantages: The synthetic route is simple, the reaction conditions are mild, the purification operation is simple, and the reproducibility is good, making it suitable for laboratory preparation and large-scale scale-up; 3. Detection advantages: Completely non-enzymatic system, avoiding enzyme inactivation, interference, and high cost; strong specificity for glucose response, wide linear range, and low detection limit; 4. Application advantages: It can be used in clinical blood glucose monitoring, food sugar content analysis, environmental water sample testing and other scenarios. It is highly practical and has broad application prospects. Attached Figure Description
[0017] Figure 1 The fluorescence emission spectra of the fluorescent probe of this invention at different glucose concentrations are shown. Detailed Implementation
[0018] The following detailed explanation illustrates the specific implementation methods: Example 1: Preparation of 3,6-bis(4-boronic acid phenyl)carbazole Step 1: Compound S1 (1.0 equivalent) was dissolved in a 1,4-dioxane / water mixed solvent (1.0 M / 0.25 M), and tetrakis(triphenylphosphine)palladium (0.1 equivalent), potassium carbonate (4 equivalent), and 1-bromo-4-iodobenzene (4 equivalent) were added sequentially. The mixture was stirred at 120 °C for 12 hours under a nitrogen atmosphere, and then cooled to room temperature. The reaction solution was filtered through diatomaceous earth, and the filter cake was washed and extracted with dichloromethane. The organic phases were combined and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 4 / 1) to obtain compound S2. Step 2: At 0°C, sodium hydride (1.5 equivalents) was slowly added to a tetrahydrofuran (0.5 M) solution of compound S2 (1.0 equivalents). After stirring the mixture at room temperature for 0.5 hours, it was cooled to 0°C again, and 1-bromo-3-methoxypropane (2 equivalents) was added dropwise at this temperature. The mixture was then stirred at room temperature overnight. The reaction solution was quenched with saturated ammonium chloride aqueous solution, extracted with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 4 / 1) to give compound S3. Step 3: Add [1,1'-bis(diphenylphosphine)ferrocene]palladium(II) dichloride (0.1 equivalent), potassium acetate (5 equivalent), and bis-pinacolborate (4 equivalent) to a 1,4-dioxane (0.2 M) solution of compound S3 (1.0 equivalent). Stir the mixture at 120 °C for 12 hours under a nitrogen atmosphere. After cooling to room temperature, filter through diatomaceous earth. Wash and extract the filter cake with dichloromethane. Combine the organic phases and concentrate under reduced pressure. Purify the crude product by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 4 / 1) to obtain compound S4. Step 4: Add methylboronic acid (10 equivalents) and trifluoroacetic acid (4 equivalents) to a solution of compound S4 (1.0 equivalents) in dichloromethane (0.5 M), stir overnight at room temperature, collect the solid by filtration, wash with dichloromethane and dry to give 3,6-bis(4-boronic acid phenyl)carbazole.
[0019] Example 2: Application of 3,6-bis(4-boronic acid phenyl)carbazole in the detection of glucose concentration Experimental methods S1: Preparation of 3,6-bis(4-boronic acid phenyl)carbazole (CA-2BA) stock solution: Weigh 4.93 mg of CA-2BA, add 10 mL of DMSO, shake the solution well to obtain a 1 mM 3,6-bis(4-boronic acid phenyl)carbazole stock solution.
[0020] Preparation of S2: 3,6-Di(4-boronic acid phenyl)carbazole detection solution: Weigh 2.8 g of anhydrous potassium carbonate and 0.475 g of potassium bicarbonate into a 250 mL volumetric flask, then add 200 mL of deionized water. After complete dissolution, add 2.5 mL of 3,6-di(4-boronic acid phenyl)carbazole stock solution and 2.5 mL of DMSO, shake well, and finally dilute to 250 mL with deionized water to obtain a detection solution with pH 10.5 and a 3,6-di(4-boronic acid phenyl)carbazole concentration of 0.01 mM (solvent ratio of DMSO:H2O = 1:50, which is optimal). Immediately after preparation, mix with an equal volume of glucose solution to prepare a glucose-containing detection sample.
[0021] S3: Preparation of glucose solution: (1) Preparation of 10 mM glucose solution: Weigh 45 mg of glucose into a 25 mL volumetric flask, add 20 mL of deionized water, and after it is completely dissolved, dilute to 25 mL with deionized water to obtain a glucose solution with a molar concentration of 10 mM.
[0022] (2) Preparation of glucose solutions of different concentrations: Take 8 mL of the prepared 10 mM glucose solution and add 2 mL of deionized water to obtain an 8 mM glucose solution. Using the same method, dilute stepwise to obtain glucose solutions with concentrations of 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.
[0023] S4: Preparation of glucose detection samples: Take 2 mL of the 3,6-bis(4-borate phenyl)carbazole detection solution from S2 into multiple sample bottles, then add 2 mL of glucose solution of different concentrations to each sample bottle, shake well, let stand overnight, and then collect fluorescence spectra.
[0024] S5: Preparation of blank sample: Take 2 mL of 3,6-bis(4-boratephenyl)carbazole detection solution (step (2)) into the sample bottle, then add 2 mL of deionized water, shake well, let stand overnight, and then collect fluorescence spectrum.
[0025] S6: Acquisition of fluorescence spectra (1) Acquisition of fluorescence spectrum of glucose-containing sample: Take 2 mL of the prepared glucose-containing sample from S4 into a cuvette (10 mm) (10 mm), then place the cuvette in a fluorescence spectrophotometer (Hitachi F7100) and collect the fluorescence spectrum. Instrument parameters: Excitation wavelength (ex): 300 nm; Emission spectrum collection range: 310 nm-700 nm; Slit width: 2.5 nm (ex), 2.5 nm (em); Scanning speed: 1200 nm / min.
[0026] (2) Fluorescence spectrum acquisition of blank sample: Take 2 mL of blank sample from S5 into a cuvette (10 mm) (10 mm), then place the cuvette in a fluorescence spectrophotometer (Hitachi F7100) and collect the fluorescence spectrum. Instrument parameters: Excitation wavelength (ex): 300 nm; Emission spectrum collection range: 310 nm-700 nm; Slit width: 2.5 nm (ex), 2.5 nm (em); Scanning speed: 1200 nm / min.
[0027] like Figure 1 As shown, the excitation wavelength is 300 nm. The arrow indicates that the glucose concentration gradually increases. The fluorescence intensity decreases significantly with increasing glucose concentration, indicating that the probe can be used for the quantitative detection of glucose.
[0028] Conclusion: 3,6-Di(4-boronic acid phenyl)carbazole exhibits strong fluorescence emission properties and high fluorescence intensity in a DMSO:H2O mixed solvent with a solvent ratio of 1:50. When mixed with glucose molecules and left for more than 12 hours, the fluorescence intensity of the original emission peak of the fluorophore decreases, and the degree of fluorescence attenuation increases with increasing glucose concentration. The fluorescence intensity is inversely proportional to the glucose concentration, enabling efficient detection of glucose concentration in the liquid phase within the range of 0.5 mM-10 mM.
[0029] When glucose appears in the detection solution, the two hydroxyl groups at positions 1 and 3 of the glucose molecule undergo a condensation reaction with the borate hydroxyl group on the 3,6-bis(4-boronic acid phenyl)carbazole molecule to form a borate ester structure. At this time, the molecule undergoes internal conversion (ICT), which weakens the fluorescence.
[0030] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific structures and characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure 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 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. 3,6-Di(4-boronic acid phenyl)carbazole, characterized in that: The structural formula is shown in S5 below: .
2. The method for preparing 3,6-bis(4-boronic acid phenyl)carbazole according to claim 1, characterized in that: It was prepared using the following synthetic route: 。 3. The method for preparing 3,6-bis(4-boronic acid phenyl)carbazole according to claim 2, characterized in that: The specific steps are as follows: Step 1: Compound S1 was dissolved in a 1,4-dioxane / water mixed solvent, and tetra(triphenylphosphine)palladium, potassium carbonate and 1-bromo-4-iodobenzene were added sequentially. The mixture was heated and stirred under a nitrogen atmosphere, and then cooled to room temperature. The reaction solution was filtered to obtain a filter cake, which was washed and extracted with dichloromethane. The organic phases were combined and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain compound S2. Step 2: At 0°C, sodium hydride was slowly added to a tetrahydrofuran solution of compound S2. After stirring at room temperature, the mixture was cooled to 0°C again, and 1-bromo-3-methoxypropane was added dropwise at this temperature. The mixture was then stirred at room temperature overnight. The reaction solution was quenched with a saturated ammonium chloride aqueous solution, extracted with ethyl acetate, and the organic phase was washed, dried, and concentrated. The residue was purified by silica gel column chromatography to obtain compound S3. Step 3: Add [1,1'-bis(diphenylphosphine)ferrocene]palladium(II) dichloride, potassium acetate and bispinacolborate to the 1,4-dioxane solution of compound S3. Heat and stir the mixture under a nitrogen atmosphere, cool to room temperature and filter. Wash the filter cake with dichloromethane and extract. Combine the organic phases and concentrate under reduced pressure. The crude product is purified by silica gel column chromatography to obtain compound S4. Step 4: Add methylboric acid and trifluoroacetic acid to a dichloromethane solution of compound S4, stir overnight at room temperature, filter and collect the solid, wash and dry to obtain 3,6-bis(4-boronic acid phenyl)carbazole.
4. The application of 3,6-bis(4-boronic acid phenyl)carbazole according to any one of claims 1 to 3 in the detection of glucose concentration.
5. The application of 3,6-bis(4-boronic acid phenyl)carbazole in glucose detection according to claim 4, characterized in that: Includes the following steps: S1: Preparation of mother liquor: Weigh CA-2BA, dissolve it in DMSO, and prepare 3,6-bis(4-boronic acid phenyl)carbazole mother liquor; S2: Preparation of detection solution: Dissolve anhydrous potassium carbonate and potassium bicarbonate in deionized water, add mother liquor and DMSO, and dilute with deionized water to obtain detection solution with pH 10.
5. The solvent ratio is DMSO:H2O = 1:20-1:
60. S3: Preparation of glucose standard solution: Prepare glucose into a stock solution and dilute it stepwise to obtain a series of standard solutions of 0.2 μM–8 mM; S4: Sample preparation and fluorescence detection: The detection solution and glucose solution are mixed in equal volumes and left to stand overnight; the emission spectrum is collected in the range of 310–700 nm with an excitation wavelength of 300 nm, a slit width of 2.5 nm / 2.5 nm, and a scanning speed of 1200 nm / min; the blank group without glucose is used as a reference, and the fluorescence intensity is negatively correlated with the glucose concentration to achieve quantitative detection.
6. The application of 3,6-bis(4-boronic acid phenyl)carbazole according to claim 5 in glucose detection, characterized in that: In S2, DMSO:H2O = 1:50.