A fluorescent probe based on isoschizol and a preparation method and application thereof

Abstract

The application belongs to the technical field of fluorescent probes, and particularly relates to a fluorescent probe based on isosteviol and a preparation method and application thereof. The isosteviol and diethylene glycol bis-p-toluenesulfonate are dissolved in a solvent, and under the action of potassium carbonate, the carboxyl in the isosteviol nucleophilically substitutes the sulfonate group in the diethylene glycol bis-p-toluenesulfonate to obtain an intermediate. The intermediate and hydroxyquinoline are dissolved in a solvent, and under alkaline conditions, the hydroxyl in the hydroxyquinoline substitutes the sulfonate group in the intermediate to obtain the fluorescent probe. The fluorescent probe can selectively recognize maleic acid, and the preparation process is simple and effective.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of maleic acid detection, and particularly relates to a fluorescent probe based on isosteviol and a preparation method and application thereof. BACKGROUND

[0002] Fumaric acid is a commonly used food additive, and its derivatives can be used for treating multiple sclerosis, rheumatism, psoriasis, etc.; maleic acid is an activity inhibitor of sucrose cytidine hydrogenase and Krebs cycle glutathione coenzyme, and is often used as a new acidifying agent in food and beverages. However, maleic acid has stronger toxicity than fumaric acid, and excessive accumulation of maleic acid in the human body can cause Fanconi syndrome, nervous system diseases, cardiovascular diseases and cancer, etc. Since maleic acid can improve the glossiness of starch and other flour products and increase the viscosity and elasticity, it is used by businessmen and added into edible starch in the form of maleic acid starch ester. However, due to the limitation of technology and other conditions, there is a large amount of residual maleic acid as a raw material, which causes great food safety hazards of edible starch.

[0003] At present, the main methods for detecting maleic acid include high performance liquid chromatography, gas chromatography-mass spectrometry, ion mobility spectrometry, etc. These methods depend on expensive instrument equipment, have high cost, and the operation process is complex, so they are difficult to be widely used. SUMMARY

[0004] In order to solve the above technical problems, the application provides a fluorescent probe based on isosteviol and a preparation method and application thereof.

[0005] A first object of the application is to provide a preparation method of a fluorescent probe based on isosteviol, comprising the following steps:

[0006] Step 1, dissolving isosteviol and diethylene glycol bis-p-toluenesulfonate in a solvent, and under the action of potassium carbonate, the carboxyl group in isosteviol substitutes the sulfonate group in diethylene glycol bis-p-toluenesulfonate to obtain an intermediate.

[0007] It should be noted that the natural sweetener steviol has the characteristics of low cost, easy availability and stability, and often has multiple chiral sites; isosteviol is obtained by rearrangement under acidic conditions, and has multiple chiral centers and special rigid skeleton and groove structure, so that the isosteviol unit molecule becomes an ideal skeleton for constructing a molecular recognition fluorescent probe.

[0008] The present application takes steviol glycol as a reaction raw material, dissolves the steviol glycol in 10% sulfuric acid solution, carries out hydrolysis reaction at 70-80°C, filters, and recrystallizes at low temperature to form isosteviol. Then, the isosteviol and diethylene glycol bis-p-toluenesulfonate are dissolved in a solvent, and under the action of potassium carbonate, the oxygen atom of the carboxyl group in the isosteviol attacks the carbon atom of diethylene glycol bis-p-toluenesulfonate as a nucleophile; the carbon atom is connected with the sulfonate group to form a transition state; in the transition state, the sulfonate group is removed as a good leaving group, a nucleophilic substitution reaction occurs to generate a compound containing a tosyl group, and a multifunctional ethoxyethyl intermediate is obtained. Finally, the potassium carbonate and other solid residues are removed by filtration, and the crude product is obtained by concentrating the solution, and then purified by column chromatography to obtain a pure intermediate.

[0009] Preferably, the molar ratio of the isosteviol and diethylene glycol bis-p-toluenesulfonate is 3:4-5.

[0010] It should be further pointed out that potassium carbonate is used as a weak basic reagent to deprotonate the hydroxyl group of isosteviol to generate a strong nucleophilic oxygen anion, thereby improving its nucleophilicity. At the same time, potassium carbonate can promote the hydroxyl group of steviol glycol to participate in the substitution reaction to generate an intermediate containing a tosyl group. The present application uses a large amount of potassium carbonate and a non-polar solvent to promote the reaction equilibrium to move in the direction of generating the product. Preferably, the molar ratio of the isosteviol and potassium carbonate is 3:4-5.

[0011] Preferably, the reaction temperature for preparing the intermediate is 60-70°C, and the reaction time is 12-18h.

[0012] Preferably, the solvent for preparing the intermediate is dichloromethane or acetonitrile.

[0013] Preferably, the diethylene glycol bis-p-toluenesulfonate is prepared by the following steps: dissolving diethylene glycol and p-toluenesulfonyl chloride in dichloromethane, adding a base under ice water bath conditions, and stirring the reaction for 2-4h to form diethylene glycol bis-p-toluenesulfonate.

[0014] Preferably, the isosteviol is prepared by the following steps: taking steviol as a raw material, dissolving the steviol in sulfuric acid solution, and carrying out hydrolysis reaction at 70-80°C to form isosteviol.

[0015] Step 2, dissolving the intermediate and hydroxyquinoline in a solvent under basic conditions to replace the sulfonate group in the intermediate with the hydroxyl group in the hydroxyquinoline to obtain a fluorescent probe.

[0016] It should be noted that the intermediate and the hydroxyquinoline are dissolved in a solvent, and under alkaline conditions, the hydroxyl group in the hydroxyquinoline is deprotonated to form a strong nucleophilic alcohol anion; the alcohol anion attacks the carbon atom positive center in the intermediate to occur nucleophilic substitution reaction, and the sulfonate group is removed to form an ether bond. After the reaction is completed, potassium hydroxide is removed by filtration, the solution is concentrated to obtain a crude product, and then the crude product is purified by column chromatography to obtain a pure final product. Preferably, the molar ratio of the intermediate and the hydroxyquinoline is 2-3:3.

[0017] Preferably, the hydroxyquinoline is 8-hydroxyquinoline or 5-hydroxyquinoline.

[0018] The present application adds a basic reagent, which is used to deprotonate the hydroxyl group of the hydroxyquinoline to form a strong nucleophilic alcohol anion, and promote the alcohol anion to participate in the substitution reaction to generate the target product. Preferably, the basic reagent is potassium hydroxide, and the molar ratio of the intermediate and the potassium hydroxide is 2-3:5.

[0019] Preferably, the reaction temperature for preparing the fluorescent probe is 70-80°C, and the reaction time is 24-36h.

[0020] Preferably, the solvent for preparing the fluorescent probe is dichloromethane or acetonitrile.

[0021] The third object of the present application is to provide the use of the above-mentioned fluorescent probe in the detection of maleic acid.

[0022] Preferably, the method for detecting maleic acid comprises the following steps:

[0023] The fluorescent probe is dissolved in a solvent to obtain a fluorescent probe solution;

[0024] The fluorescent probe solution is added to the to-be-detected solution, and the mixture is uniformly mixed to obtain a test solution;

[0025] The excitation wavelength is selected as 325nm to determine the change of the fluorescence intensity of the test solution at 350-450nm; wherein, the volume ratio of the fluorescent probe solution and the to-be-detected solution is 1:1; the concentration of the fluorescent probe solution is 1×10 -5 mol / L, and the concentration of the to-be-detected solution is 5×10 -4 mol / L.

[0026] Or under the ultraviolet lamp, the change of the fluorescence of the test solution is observed, and when the test solution emits fluorescence, it indicates that the test solution contains maleic acid.

[0027] Compared with the prior art, the present application has the following beneficial effects:

[0028] The application takes the carboxyl group in isosteviol as a nucleophile, and a nucleophilic substitution reaction occurs between the carboxyl group in isosteviol and the sulfonate group in diethylene glycol bis-p-toluenesulfonate under the catalysis of potassium carbonate; thus, the natural properties of isosteviol are introduced into the fluorescent probe, and the biological compatibility and sensitivity of the fluorescent probe are enhanced. Hydroxyquinoline is used as a fluorescent group, and the hydroxyl group in hydroxyquinoline attacks the sulfonate group in the intermediate under alkaline conditions, thereby generating the fluorescent probe. The hydroxyquinoline hydroxyl group in the fluorescent probe can effectively absorb excitation light of a specific wavelength, thereby generating fluorescent emission. In the presence of maleic acid, the C=O and quinoline-N in the fluorescent probe form a stronger hydrogen bond with the carboxyl group of maleic acid, and the carboxyl group of maleic acid is closer to the quinoline group. The electron-withdrawing effect of the carboxyl group leads to a decrease in the electron cloud density of the proton of the quinoline group, thereby causing the chemical shift to shift to a low field. The C=O in the fluorescent probe forms a hydrogen bond with the carboxyl group of maleic acid, which corresponds to fluorescence quenching caused by a photoelectron transfer process. With the increase of the concentration of maleic acid, the π-π accumulation between the fluorophores quinoline is enhanced, leading to an excimer effect, thereby appearing and continuously enhancing the emission peak of the excimer association, corresponding to the appearance and enhancement of a new fluorescent emission peak; thus, the fluorescent probe prepared in the application becomes a fluorescent indicator signal for detecting maleic acid.

[0029] The fluorescent probe prepared in the application can realize the detection of maleic acid by the change of fluorescence under an ultraviolet lamp. When the fluorescent probe is added to a test solution, if the test solution emits fluorescence, it indicates that the test solution contains maleic acid. Meanwhile, the fluorescent probe prepared in the application has two emission wavelengths. After maleic acid is added, the fluorescence in the wavelength range of 350nm-400nm decreases, and the fluorescence intensity in the wavelength range of 400nm-450nm increases. The application realizes the detection of the concentration of maleic acid with higher sensitivity through the change of fluorescence intensity and the change of the fluorescent color of the solution as a double signal. The fluorescent probe prepared in the application can detect maleic acid in starch-rich food. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The fluorescence spectrum change of the fluorescent probe 1 prepared in Example 1 of the application under different concentrations of maleic acid.

[0031] Figure 2 The fluorescence spectrum change of the fluorescent probe 1 prepared in Example 1 of the application under different concentrations of fumaric acid.

[0032] Figure 3 The fluorescence spectrum change of the fluorescent probe 2 prepared in Example 2 of the application under different concentrations of maleic acid.

[0033] Figure 4 The fluorescence spectrum change of the fluorescent probe 2 prepared in Example 2 of the application under different concentrations of fumaric acid.

[0034] Figure 5 Photos of the fluorescent probe 1 prepared in Example 1 of the present application and the change in fluorescent color under ultraviolet light after maleic acid and fumaric acid are added to the fluorescent probe 1; wherein a is the fluorescent probe 1, b is after maleic acid is added, and c is after fumaric acid is added.

[0035] Figure 6 Photos of the fluorescent probe 2 prepared in Example 2 of the present application and the change in fluorescent color under ultraviolet light after maleic acid and fumaric acid are added to the fluorescent probe 2; wherein a is the fluorescent probe 2, b is after maleic acid is added, and c is after fumaric acid is added.

[0036] Figure 7 NMR hydrogen spectrum of the fluorescent probe 1 prepared in Example 1 of the present application, the fluorescent probe 1-maleic acid complex, and the fluorescent probe 1-fumaric acid complex; wherein a is the fluorescent probe 1, b is the fluorescent probe 1-maleic acid complex, and c is the fluorescent probe 1-fumaric acid complex.

[0037] Figure 8 NMR hydrogen spectrum of the fluorescent probe 2 prepared in Example 2 of the present application, the fluorescent probe 2-maleic acid complex, and the fluorescent probe 2-fumaric acid complex; wherein a is the fluorescent probe 2, b is the fluorescent probe 2-maleic acid complex, and c is the fluorescent probe 2-fumaric acid complex.

[0038] Figure 9 Three-dimensional structure diagram of the fluorescent probe 1 prepared in Example 1 of the present application, the fluorescent probe 1-maleic acid complex, and the fluorescent probe 1-fumaric acid complex calculated by B3LYP / 6-31G(d,p) of the present application; wherein a is the fluorescent probe 1, b is the fluorescent probe 1-maleic acid complex, and c is the fluorescent probe 1-fumaric acid complex.

[0039] Figure 10 Three-dimensional structure diagram of the fluorescent probe 2 prepared in Example 2 of the present application, the fluorescent probe 2-maleic acid complex, and the fluorescent probe 2-fumaric acid complex calculated by B3LYP / 6-31G(d,p) of the present application; wherein a is the fluorescent probe 2, b is the fluorescent probe 2-maleic acid complex, and c is the fluorescent probe 2-fumaric acid complex.

[0040] Figure 11 Determination of maleic acid in starch food by the fluorescent probe 1 prepared in Example 1 of the present application; wherein a is potato, b is sweet potato, and c is corn.

[0041] Figure 12 Determination of maleic acid in starch food by the fluorescent probe 2 prepared in Example 2 of the present application; wherein a is potato, b is sweet potato, and c is corn. DETAILED DESCRIPTION

[0042] In order to enable the technical personnel in the art to better understand the technical solutions of the present application and to implement them, the present application will be further described below in conjunction with specific examples and drawings.

[0043] In the description of the present application, unless specifically stated, the reagents used are commercially available, and the methods used are conventional techniques in the art.

[0044] Example 1

[0045] This embodiment provides a preparation method of a fluorescent probe based on isosteviol.

[0046] Step 1, preparation of intermediate:

[0047] 1.1) Dissolve 5 g of steviol in 10%, 250 mL of sulfuric acid solution, react at 75°C for 7 h to form an off-white solid, and then recrystallize in low-temperature ethanol after suction filtration to obtain light yellow isosteviol crystals with a yield of 67%. The reaction route is shown in Reaction Formula 1:

[0048]

[0049] The nuclear magnetic hydrogen spectrum data of isosteviol are as follows: 1 HNMR (500 MHz, Chloroform-d) δ 2.64 (dd, J = 18.6, 3.8 Hz, 1H), 2.17 (dtd, J = 13.5, 3.4, 1.5 Hz, 1H), 1.93-1.33 (m, 13H), 1.25 (s, 3H, 1.23-1.13 (m, 3H), 1.03 (td, J = 13.6, 4.2 Hz, 1H), 0.98 (s, 3H), 0.92 (td, J = 13.2, 4.4 Hz, 1H), 0.79 (s, 3H), indicating that isosteviol is successfully synthesized.

[0050] 1.2) Dissolve 2.653 g of diethylene glycol and 9.533 g of p-toluenesulfonyl chloride in 25 mL of dichloromethane, and then add 11.22 g of potassium hydroxide in portions under ice water bath conditions, and then magnetically stir for 3 h, remove the insoluble substances by suction filtration, and then concentrate the obtained solution and recrystallize with methanol to obtain colorless diethylene glycol bis-p-toluenesulfonate crystals with a yield of 73%. The reaction route is shown in Reaction Formula 2:

[0051]

[0052] The nuclear magnetic hydrogen spectrum data of diethylene glycol bis-p-toluenesulfonate are as follows: 1HNMR (400 MHz, chloroform-d) δ 7.79 (d, J = 8.4 Hz, 4H), 7.37 (d, J = 8.1 Hz, 4H), 4.23 - 4.02 (m, 4H), 3.73 - 3.58 (m, 4H), 2.46 (s, 6H). This indicates that diethylene glycol bis-p-toluenesulfonate salt was successfully synthesized.

[0053] 1.3) Dissolve 0.955 g of iso-steviol and 1.865 g of diethylene glycol bis-p- toluenesulfonate salt in 50 mL of acetonitrile; add 0.622 g of potassium carbonate, and react at 70 °C for 12 h; then filter the mixture, and concentrate the resulting solution to obtain a crude product, which is purified by column chromatography with a volume ratio of petroleum ether to ethyl acetate of 3:1 to obtain 2-(2-(phenylsulfonyloxy)ethoxy)ethyl (4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecahydro-6a,9-methanocyclo[a]naphthalene-4-carboxylate, denoted as Intermediate, in a yield of 89%, and its reaction scheme is shown in Reaction Formula 3:

[0054]

[0055] The nuclear magnetic hydrogen spectrum data of 2-(2-(phenylsulfonyloxy)ethoxy)ethyl (4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecahydro-6a,9-methanocyclo[a]naphthalene-4-carboxylate are as follows: 1HNMR (400 MHz, chloroform-d) δ 7.91-7.72 (m, 2H), 7.36 (d, J = 8.0 Hz, 2H), 4.28-4.04 (m, 4H), 3.81-3.55 (m, 4H), 2.60 (dd, J = 18.6, 3.7 Hz, 1H), 2.46 (s, 3H), 2.30-2.08 (m, 1H), 1.94-1.84 (m, 1H), 1.82 (s, 1H), 1.81-1.72 (m, 2H), 1.69 (h, J = 4.0, 3.2 Hz, 3H), 1.65 (t, J = 3.4 Hz, 1H), 1.62-1.45 (m, 3H), 1.45-1.32 (m, 3H), 1.32-1.22 (m, 2H), 1.20 (s, 3H), 1.14 (dd, J = 12.1, 2.3 z, 1H), 1.04 (dd, J = 13.5, 4.2 Hz, 1H), 0.99 (s, 2H), 0.91 (td, J = 13.2, 4.2 Hz, 1H), 0.69 (s, 3H), indicating that the application successfully synthesized 2-(2-(phenylsulfonyloxy)ethoxy)ethyl (4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecahydro-6a,9-methenocyclo[a]naphthalene-4-carboxylate.

[0056] Step 2, preparation of fluorescent probe:

[0057] Dissolve 1.4 g of intermediate and 0.435 g of 8-hydroxyquinoline in 30 mL of acetonitrile, then add 0.28 g of potassium hydroxide to obtain a mixture; heat the mixture at 80°C for 24 h, after the reaction is completed, filter out the precipitate to obtain a brown black oily liquid, and concentrate and purify by column chromatography, with a solvent system of a mixture of petroleum ether and ethyl acetate at a volume ratio of 2:1, to obtain 2-(2-(quinolin-8-yloxy)ethoxy)ethyl (4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecahydro-6a,9-methenocyclo[a]naphthalene-4-carboxylate, a fluorescent probe 1, with a yield of 55%, and its reaction route is shown in Reaction Formula 4:

[0058]

[0059] The nuclear magnetic hydrogen spectrum data of 2-(2-(quinolin-8-yloxy)ethoxy)ethyl (4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecahydro-6a,9-methenocyclo[a]naphthalene-4-carboxylate are as follows: 1H NMR (400 MHz, Chloroform-d) δ 8.93 (dd, J = 4.2, 1.8 Hz, 1H), 8.12 (dd, J = 8.3, 1.8 Hz, 1H), 7.51 - 7.37 (m, 3H), 7.13 (dd, J = 7.6, 1.3 z, 1H), 4.42 (t, J = 5.3 Hz, 2H), 4.30 - 4.14 (m, 2H), 4.05 (dd, J = 5.8, 4.7 Hz, 2H), 3.83 (ddd, J = 5.7, 4.0, 1.7 Hz, 2H), 2.58 (dd, J = 18.6, 3.7 Hz, 1H), 2.23 - 2.10 (m, 1H), 1.92 - 1.30 (m, 13H), 1.26 - 1.05 (m, 6H), 1.04 - 0.92 (m, 4H), 0.85 (td, J = 13.3, 4.4 Hz, 1H), 0.67 (s, 3H).

[0060] NMR carbon spectrum data are as follows: 13 C NMR (101 MHz, CDCl3) δ 222.20, 177.20, 154.67, 149.29, 140.42, 135.87, 129.55, 126.68, 121.57, 120.01, 109.36, 77.34, 77.03, 76.71, 69.40, 69.29, 68.38, 63.14, 57.17, 54.73, 54.31, 48.65, 48.40, 43.83, 41.49, 39.79, 39.42, 38.00, 37.91, 37.32, 28.89, 21.61, 20.29, 19.85, 18.91, 13.25, indicating that the 2-(2-(quinolin-8-yloxy)ethoxy)ethyl intermediate is successfully synthesized.

[0061] Example 2

[0062] The present embodiment provides a preparation method of a fluorescent probe based on isosweet alcohol.

[0063] Dissolve 1.4 g of intermediate and 0.435 g of 5-hydroxyquinoline in 30 mL of acetonitrile, then add 0.691 g of potassium carbonate to obtain a mixture; heat the mixture at 80°C for 24 h under reflux, after the reaction is completed, filter out the precipitate to obtain a brown black oily liquid, and concentrate and purify by column chromatography, the solvent system is a mixture of petroleum ether and ethyl acetate, the volume ratio of petroleum ether to ethyl acetate is 2:1, to obtain 2-(2-(isoquinolin-5-yloxy)ethoxy)ethyl (4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecacyclo-6a,9-methylenecyclo[a]naphthalene-4-carboxylate, the yield is 47%, which is fluorescent probe 2.

[0064] The nuclear magnetic hydrogen spectrum data of 2-(2-(isoquinolin-5-yloxy)ethoxy)ethyl (4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecacyclo-6a,9-methylenecyclo[a]naphthalene-4-carboxylate are as follows: 1 H NMR (400 MHz, Chloroform-d) δ 9.21 (d, J = 1.0 Hz, 1H), 8.53 (d, J = 5.8 Hz, 1H), 8.15-7.95 (m, 1H), 7.64-7.40 (m, 2H), 7.03 (dd, J = 7.4, 1.2 Hz, 1H), 4.39-4.16 (m, 4H), 4.05-3.95 (m, 2H), 3.84 (ddd, J = 5.5, 4.0, 1.3 Hz, 2H), 2.59 (dd, J = 18.5, 3.7 Hz, 1H), 2.24-2.14 (m, 1H), 1.90-1.30 (m, 13H), 1.19 (s, 3H), 1.17-1.06 (m, 3H), 1.06-0.94 (m, 4H), 0.86 (td, J = 13.3, 4.4 Hz, 1H), 0.70 (s, 3H).

[0065] The nuclear magnetic carbon spectrum data are as follows: 13C NMR (101 MHz, CDC13) δ 222.22, 178.02, 153.60, 151.81, 144.25, 130.29, 128.53, 127.38, 118.73, 114.22, 108.73, 77.34, 77.02, 76.70, 69.47, 69.41, 68.12, 63.08, 57.14, 54.67, 54.27, 48.66, 48.40, 43.86, 41.47, 39.75, 39.41, 38.01, 37.89, 37.30, 28.90, 21.61, 20.28, 19.83, 18.91, 13.27. It indicated that the 2-(2-(isoquinoline-5-yloxy)ethoxy)ethyl (4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecahydro-6a,9-methanocyclo[a]naphthalene-4-carboxylate was successfully synthesized.

[0066] Experimental test

[0067] 1. Fluorescence test

[0068] The fluorescent probe 1 prepared in Example 1 and the fluorescent probe 2 prepared in Example 2 were respectively dissolved in ethanol to prepare a fluorescent probe 1 solution and a fluorescent probe 2 solution with a concentration of 1 x 10 -5 mol / L; and 1.5 mL of the fluorescent probe 1 solution and 1.5 mL of the fluorescent probe 2 solution were taken in two portions; then a gradient concentration of fumaric acid and maleic acid solution was added to the fluorescent probe 1 solution and the fluorescent probe 2 solution respectively for titration; wherein the gradient concentration was 0.01 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.5 mM, 0.8 mM, 1 mM, 2 mM, 3 mM, 4 mM and 5 mM, and the corresponding fluorescence intensity was tested each time. The fluorescence during titration was measured in a standard rectangular quartz cuvette using a Horiba Fluoro Max-4 fluorescence spectrophotometer, and the corresponding emission value during the entire titration process was recorded.

[0069] Figure 1 The fluorescence spectrum change of the fluorescent probe 1 prepared in Example 1 under different concentrations of maleic acid is shown in FIG. 1. As shown in FIG. 1, the fluorescent probe 1 showed fluorescence emission at a wavelength of 400 nm when excited at 325 nm. With the increase of the concentration of the maleic acid solution from 0.01 mM to 5 mM, the fluorescence intensity at 400 nm gradually decreased, and a new emission peak appeared at a wavelength of 488 nm, and the fluorescence intensity gradually increased, forming a typical ratio fluorescence signal. Figure 1

[0070] Figure 2 ​Fluorescence spectrum changes of the fluorescent probe 1 prepared in Example 1 under different concentrations of fumaric acid. As shown in Figure 2 , when fumaric acid was added to the fluorescent probe 1 solution, no significant fluorescence change was observed with the increase of the concentration of the fumaric acid solution, and the Stokes shift of the fluorescent probe 1 was 88 nm.

[0071] Figure 3 Fluorescence spectrum changes of the fluorescent probe 2 prepared in Example 2 under different concentrations of maleic acid. As shown in Figure 3 , the fluorescent probe 2 also showed a similar maleic acid response as the fluorescent probe 1. When excited at 325 nm, the fluorescent probe 2 showed fluorescence emission at a wavelength of 364 nm; with the increase of the concentration of the maleic acid solution from 0.01 mM to 5 mM, the fluorescence intensity at 364 nm gradually decreased, while a new emission peak appeared at a wavelength of 442 nm, and the fluorescence intensity gradually increased, forming a typical ratiometric fluorescence signal.

[0072] Figure 4 Fluorescence spectrum changes of the fluorescent probe 2 prepared in Example 2 under different concentrations of fumaric acid. As shown in Figure 4 , after adding fumaric acid to the fluorescent probe 2 solution, there was no significant fluorescence change. The Stokes shift of the fluorescent probe 2 was 78 nm.

[0073] Figure 5 Photos of the fluorescent probe 1 prepared in Example 1 and after adding maleic acid and fumaric acid to the fluorescent probe 1 under ultraviolet light; wherein a is the fluorescent probe 1, b is the addition of maleic acid, and c is the addition of fumaric acid. As can be seen from Figure 5 , the fluorescent probe 1 and the fluorescent probe added with fumaric acid both had no fluorescence change, and only the fluorescent probe 1 added with maleic acid showed green fluorescence under ultraviolet light.

[0074] Figure 6 Photos of the fluorescent probe 2 prepared in Example 2 and after adding maleic acid and fumaric acid to the fluorescent probe 2 under ultraviolet light; wherein a is the fluorescent probe 2, b is the addition of maleic acid, and c is the addition of fumaric acid. As can be seen from Figure 6 , the fluorescent probe 2 and the fluorescent probe added with fumaric acid both had no fluorescence change, and only the fluorescent probe 2 added with maleic acid showed light blue fluorescence under ultraviolet light.

[0075] It is thus shown that the fluorescent probe prepared in the present application has fluorescence effect and selective recognition effect for maleic acid.

[0076] 2. Nuclear magnetic resonance hydrogen spectrum test

[0077] To further explore the exact mechanism of the fluorescence spectrum change of the fluorescent probe 1 prepared in Example 1 and the fluorescent probe 2 prepared in Example 2 in the maleic acid solution, the present application respectively dissolves the fluorescent probe 1 and the fluorescent probe 2 prepared in Example 2 with deuterated DMSO as a blank solution; after mixing with maleic acid and fumaric acid according to a molar ratio of 1:1, the present application respectively dissolves them with deuterated DMSO to obtain four kinds of to-be-tested solutions, which are recorded as the fluorescent probe 1-maleic acid complex, the fluorescent probe 1-fumaric acid complex, the fluorescent probe 2-maleic acid complex and the fluorescent probe 2-fumaric acid complex, and carries out nuclear magnetic resonance testing, and the results are shown in Figure 7 and Figure 8 .

[0078] Figure 7 FIG. 1 is a nuclear magnetic hydrogen spectrum of the fluorescent probe 1, the fluorescent probe 1-maleic acid complex and the fluorescent probe 1-fumaric acid complex prepared in Example 1; wherein a is the fluorescent probe 1, b is the fluorescent probe 1-maleic acid complex, and c is the fluorescent probe 1-fumaric acid complex. As shown in Figure 7 , compared with the nuclear magnetic hydrogen spectrum of the fluorescent probe 1, the protons of the quinoline group in the fluorescent probe 1 after being combined with maleic acid show downfield shifts due to the deshielding effect thereof, and the specific downfield shifts are ΔδH1=0.1ppm, ΔδH2=0.25ppm, ΔδH3=0.17ppm, ΔδH4=0.12ppm, ΔδH5=0.12ppm, ΔδH6=0.15ppm. In the nuclear magnetic hydrogen spectrum of the fluorescent probe 1-fumaric acid complex, the chemical shifts are almost unchanged.

[0079] Figure 8 FIG. 2 is a nuclear magnetic hydrogen spectrum of the fluorescent probe 2, the fluorescent probe 2-maleic acid complex and the fluorescent probe 2-fumaric acid complex prepared in Example 2; wherein a is the fluorescent probe 2, b is the fluorescent probe 2-maleic acid complex, and c is the fluorescent probe 2-fumaric acid complex. As shown in Figure 8 , after the isochinoline protons of the fluorescent probe 2 are mixed with maleic acid, downfield shifts occur, and the specific downfield shifts are ΔδH1=0.21ppm, ΔδH2=0.06ppm, ΔδH3=0.19ppm, ΔδH4=0.14ppm, ΔδH5=0.13ppm, ΔδH6=0.15ppm; and the fluorescent probe 2-fumaric acid complex does not have significant changes.

[0080] Therefore, the change of the downfield shifts may be due to the fact that the fluorescent probe 1 and the fluorescent probe 2 form complexes with maleic acid through intermolecular hydrogen bonds, which affects the chemical environment of the fluorophore protons and shifts their signals to the low field. It is shown that the fluorescent probes prepared in the present application can form complexes with maleic acid, thereby affecting the chemical environment of the fluorophore protons, having a fluorescence effect on maleic acid and a selective recognition effect.

[0081] 3. Density functional theory research

[0082] The present application uses Gaussian16 software to calculate the three-dimensional structure diagram of the fluorescent probe 1-maleic acid complex, the fluorescent probe 1-fumaric acid complex, the fluorescent probe 2-maleic acid complex and the fluorescent probe 2-fumaric acid complex at the B3LYP / 6-31G(d,p) level by using the IEFPCM solvent model and the GD3BJ dispersion correction.

[0083] Figure 9 The three-dimensional structure diagram of the fluorescent probe 1, the fluorescent probe 1-maleic acid complex and the fluorescent probe 1-fumaric acid complex calculated by B3LYP / 6-31G(d,p); wherein a is the fluorescent probe 1, b is the fluorescent probe 1-maleic acid complex, and c is the fluorescent probe 1-fumaric acid complex. As shown in Figure 9 The fluorescent probe 1 forms intermolecular hydrogen bonds in the maleic acid solution, as shown by the red arrow. Compared with the three-dimensional structure diagram of the fluorescent probe 1 in the fumaric acid solution, the C=O and quinoline-N in the fluorescent probe 1 form stronger hydrogen bonds with the carboxyl group of maleic acid, and the carboxyl group of maleic acid is closer to the quinoline group. The electron-withdrawing effect of the carboxyl group causes the electron cloud density of the proton of the quinoline group to decrease, thereby causing its chemical shift to shift to the low field. The hydrogen bond between the C=O of the fluorescent probe 1 and the carboxyl group of maleic acid corresponds to the fluorescence quenching caused by the photoelectron transfer process. With the increase of the concentration of maleic acid, the π-π accumulation between the fluorophores quinoline is enhanced, leading to the excimer effect, thereby appearing and continuously enhancing the emission peak of the excimer association, corresponding to the appearance and enhancement of the new fluorescence emission peak. This shows that the fluorescent probe prepared by the present application has fluorescence effect and strong selective recognition effect on maleic acid.

[0084] Figure 10 The three-dimensional structure diagram of the fluorescent probe 2, the fluorescent probe 2-maleic acid complex and the fluorescent probe 2-fumaric acid complex calculated by B3LYP / 6-31G(d,p); wherein a is the fluorescent probe 2, b is the fluorescent probe 2-maleic acid complex, and c is the fluorescent probe 2-fumaric acid complex. As shown in Figure 10 The formation of intermolecular hydrogen bonds in the fluorescent probe 2-maleic acid complex, as shown by the red arrow. Similarly, the complexation of the fluorescent probe 2 with maleic acid causes the proton of the isoquinoline group to shift to the low field, and the fluorescence intensity changes proportionally.

[0085] 4. Detection of maleic acid in a starch sample

[0086] This invention extends this detection system to the detection of maleic acid in starch samples. In this invention, 10g of starch-rich food (potato, sweet potato, or corn) is chopped and placed in a 500mL beaker, and 200mL of ethanol is added to obtain a mixture. The mixture is stirred for 12 hours, then filtered and the filtrate is collected. 30mL of the filtrate is measured and divided into three portions: one as a blank control, and the other two portions are treated with 1.75mg of maleic acid and 1.75mg of fumaric acid, respectively, to obtain three test solutions. Fluorescent probe 1 prepared in Example 1 and fluorescent probe 2 prepared in Example 2 are dissolved in ethanol to prepare solutions with a concentration of 1×10⁻⁶. -5 mol / L fluorescent probe 1 solution and fluorescent probe 2 solution; then add 1.5 mL of fluorescent probe 1 solution and fluorescent probe 2 solution to each of the three test solutions to obtain test solutions, and perform fluorescence spectroscopy on these six test solutions.

[0087] Figure 11 The fluorescent probe 1 prepared in Example 1 was used to determine maleic acid in starchy foods; where a is potato, b is sweet potato, and c is corn. Figure 11 As shown, compared with the fluorescence spectrum of fluorescent probe 1 mixed with starch food, no significant fluorescence change was observed after fluorescent probe 1 was mixed with starch food with added fumaric acid; after fluorescent probe 1 was mixed with starch food with added maleic acid, the fluorescence intensity in the wavelength range of 400nm to 420nm was weakened.

[0088] Figure 12 The fluorescent probe 2 prepared in Example 2 was used to determine maleic acid in starchy foods; where a is potato, b is sweet potato, and c is corn. Figure 12 As shown, compared with the fluorescence spectrum of fluorescent probe 1 mixed with starch food, no significant fluorescence change was observed after fluorescent probe 2 was mixed with starch food with added fumaric acid; after fluorescent probe 2 was mixed with starch food with added maleic acid, the fluorescence intensity at 364 nm decreased, while a new emission peak appeared at 442 nm, and its fluorescence intensity increased.

[0089] This demonstrates that the fluorescent probe of the present invention can qualitatively detect maleic acid in starchy foods such as potatoes, sweet potatoes, and corn.

[0090] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range, as well as any value between the two endpoints, can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described in this invention to avoid redundancy. Although preferred embodiments of this invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments, and all such changes and modifications fall within the scope of this invention.

[0091] It will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the spirit or scope of the application. Such modifications and variations are considered to be within the scope of the application.

Claims

1. A method for preparing a fluorescent probe based on isosteviol, characterized in that, Includes the following steps: Isosteviol and diethylene glycol bis-toluenesulfonate were dissolved in a solvent, and under the action of potassium carbonate, the carboxyl group in isosteviol nucleophilically replaced the sulfonate group in diethylene glycol bis-toluenesulfonate to obtain an intermediate. The intermediate and hydroxyquinoline were dissolved in a solvent, and under alkaline conditions, the hydroxyl groups in the hydroxyquinoline replaced the sulfonate groups in the intermediate to obtain a fluorescent probe. The structural formula of the intermediate is as follows: 。 2. The method for preparing the fluorescent probe according to claim 1, characterized in that, The molar ratio of the intermediate to hydroxyquinoline is 2~3:

3.

3. The method for preparing the fluorescent probe according to claim 1, characterized in that, The hydroxyquinoline is 8-hydroxyquinoline or 5-hydroxyquinoline.

4. The method for preparing the fluorescent probe according to claim 1, characterized in that, The molar ratio of isosteviol to diethylene glycol bis(p-toluenesulfonate) is 3:4~5; The molar ratio of isosteviol to potassium carbonate is 3:4~5.

5. The method for preparing the fluorescent probe according to claim 1, characterized in that, The reaction temperature for preparing the fluorescent probe is 70℃~80℃, and the reaction time is 24h~36h.

6. The method for preparing the fluorescent probe according to claim 1, characterized in that, The reaction temperature for preparing the intermediate is 60℃~70℃, and the reaction time is 12h~18h.

7. The method for preparing the fluorescent probe according to claim 1, characterized in that, The alkaline conditions are achieved by adding an alkaline reagent, which is potassium hydroxide. The molar ratio of the intermediate to potassium hydroxide is 2~3:

5.

8. The method for preparing the fluorescent probe according to claim 1, characterized in that, The solvents used to prepare the fluorescent probe and the intermediate are both dichloromethane or acetonitrile.

9. A fluorescent probe prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the fluorescent probe of claim 9 in detecting maleic acid for non-disease detection and treatment purposes.

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