Aminobenzaldehyde-based coumarin fluorescent probe for preparing mercaptosuccinic acid detection reagent and preparation method thereof

Abstract

The invention belongs to the field of chemical analysis and testing, and particularly relates to an aminobenzaldehyde-based coumarin fluorescent probe for preparing a reagent for detecting mercaptosuccinic acid and a preparation method of the aminobenzaldehyde-based coumarin fluorescent probe. The preparation method comprises the following steps: dissolving 2-aminobenzaldehyde and 7-(diethylamino) coumarin-3-formic acid in dichloromethane, adding 1-ethyl-(3-dimethylamino aldehyde) carbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HBOT) as condensing agents, carrying out a stirring reaction at room temperature, carrying out separation and purification on the obtained crude product through a thin-layer chromatography, and carrying out recrystallization to obtain the coumarin derivative. The final yellow product, namely the aminobenzaldehyde-based coumarin fluorescent probe Y-SH, is obtained. The coumarin fluorescent probe prepared by the preparation method disclosed by the invention can be used for rapidly detecting mercaptosuccinic acid in deionized water.

Description

Technical Field

[0001] The present invention belongs to the field of chemical analysis and testing, and particularly relates to a coumarin-based fluorescent probe based on aminobenzaldehyde for preparing a reagent for detecting mercaptosuccinic acid and a preparation method thereof. Background Art

[0002] Mercaptosuccinic acid, as a sulfur-containing nucleophile, is an important bioactive substance with various physiological functions in vivo. Abnormal changes in its concentration are closely related to the occurrence and development of various diseases.

[0003] Traditional methods for detecting mercaptosuccinic acid include high performance liquid chromatography and capillary electrophoresis, which have disadvantages such as high cost, complex operation, and susceptibility to interference. Fluorescence detection methods based on fluorescent probes have advantages such as high sensitivity, strong specificity, and fast response speed, and have received increasing attention. Therefore, developing a fluorescent probe that can selectively recognize mercaptosuccinic acid is of great significance for studying its physiological functions and pathological mechanisms. Summary of the Invention

[0004] The present invention provides a coumarin-based fluorescent probe Y-SH based on aminobenzaldehyde, and the structural formula of the fluorescent probe is as follows:

[0005]

[0006] The present invention also provides a preparation method of the coumarin-based fluorescent probe based on aminobenzaldehyde. The chemical reaction formula for preparing the fluorescent probe is as follows:

[0007]

[0008] The specific preparation method is as follows: Dissolve 7-(diethylamino)coumarin-3-carboxylic acid and 2-aminobenzaldehyde in dichloromethane according to a molar equivalent of 1:1.1 - 2.0, then add 1.8 - 2.0 molar equivalents of EDCI and 1.8 - 2.0 molar equivalents of HOBT as condensing agents, and stir the reaction solution at room temperature for 10 - 12 hours. After the reaction is completed, remove the solvent, and finally purify by thin layer chromatography using a mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 1:1 to obtain a yellow solid product Y-SH.

[0009] The present invention also provides the application of the above fluorescent probe: The prepared coumarin-based fluorescent probe Y-SH based on aminobenzaldehyde can rapidly detect mercaptosuccinic acid in deionized water.

[0010] The probe Y-SH prepared by the present invention uses a coumarin compound as a fluorophore and benzaldehyde as a target, and is obtained through an amide condensation reaction, having good fluorescence properties. When it recognizes mercaptosuccinic acid, the aldehyde group in the fluorescent probe and the double bond of the benzene ring connected to the aldehyde group react with the mercapto group and hydroxyl group of mercaptosuccinic acid to form a seven-membered ring, strengthening the photoinduced electron transfer and resulting in the quenching of the fluorophore, thereby achieving the function of detecting mercaptosuccinic acid.

[0011] Beneficial effects

[0012] The raw materials of the present invention are easily obtained, the synthesis method is simple, the reaction conditions are easy to control, and a pure product can be obtained through simple post-treatment. The probe Y-SH reported in the present invention solves the problem of detecting mercaptosuccinic acid in deionized water. From the fluorescence phenomenon, after adding mercaptosuccinic acid, the fluorescence intensity of the probe Y-SH at 470 nm decreased significantly, thereby showing the detection effect of the probe Y-SH on mercaptosuccinic acid in this system. Description of the drawings

[0013] Figure 1 The fluorescence spectra of the probe prepared in Example 1 at a concentration of 1×10 -5 mol / L in deionized water after reacting with different mercapto compounds and ions.

[0014] Figure 2 The fluorescence spectra of the probe prepared in Example 1 at a concentration of 1×10 -5 mol / L in deionized water after reacting with different concentrations of mercaptosuccinic acid.

[0015] Figure 3 The fluorescence intensity graph of the probe prepared in Example 1 at a concentration of 1×10 -5 mol / L in deionized water at 470 nm after reacting with mercaptosuccinic acid in the presence of five-fold interfering different mercapto compounds and ions.

[0016] Figure 4 The hydrogen spectrum of the probe prepared in Example 1. Detailed implementation manners

[0017] The following further details the present invention in combination with specific implementation manners.

[0018] Example 1

[0019] Dissolve 2-aminobenzaldehyde (90.9 mg, 0.75 mmol) and 7-(diethylamino)coumarin-3-carboxylic acid (130.7 mg, 0.5 mmol) in 1.5 mL of dichloromethane, then add EDCI (182.2 mg, 0.95 mmol) and HBOT (128.4 mg, 0.95 mmol), and stir the reaction mixture at room temperature for 10 hours. After the reaction is completed, remove the solvent under reduced pressure, and purify the crude product by thin-layer chromatography using a mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 1:1. Finally, 33.9 mg of yellow solid product is obtained with a yield of 18.6%.

[0020] The specific application method is as follows: Add 2 μL of different thiol compounds and ions (6-mercaptopurine, L-cysteine, DL-homocysteine, DL-dithiothreitol, 2-aminoethanethiol, mercaptobenzimidazole, mercaptosuccinic acid, Cl - , SO4 2- , PO4 - , HSO3 - , F - , Br - , with a concentration of 1×10 -4 mol / L), 196 μL of deionized water and 2 μL of probe Y-SH (with a concentration of 1×10 -5 mol / L) into a 96-well plate. At the same time, use a solution without other compounds (Blank) but containing probe Y-SH as a control. Mix the solution in each well evenly, and detect the fluorescence intensity of the solution in each well using a microplate reader. The results show that after adding mercaptosuccinic acid, the fluorescence intensity of the probe at 470 nm decreases significantly; and there is no obvious change in the fluorescence intensity of this fluorescent probe for other interfering substances, thus showing the selective recognition effect of this fluorescent probe for mercaptosuccinic acid in deionized water.

[0021] Figure 1 The coumarin-based fluorescent probe prepared in Example 1 was used at a concentration of 1×10 -5 mol / L in deionized water with different thiol compounds and ions (with a concentration of 1×10 -4 mol / L) (6-mercaptopurine, L-cysteine, DL-homocysteine, DL-dithiothreitol, 2-aminoethanethiol, mercaptobenzimidazole, mercaptosuccinic acid, Cl - , SO4 2- , PO4 - , HSO3 - , F - , Br -The fluorescence spectra after the action of blank. As shown in the figure, the changes in the fluorescence intensity of the probe after adding different mercapto compounds and ionic solutions are shown. When adding mercaptosuccinic acid solution, the fluorescence intensity of the fluorescent probe at 470 nm decreases significantly (the curve indicated by the arrow); and there is no obvious change in the fluorescence intensity of this fluorescent probe for other interfering substances in deionized water, thus showing the selective recognition effect of this fluorescent probe for mercaptosuccinic acid in deionized water.

[0022] Figure 2 The fluorescence spectra of the fluorescent probe prepared in Example 1 at a concentration of 1×10 -5 mol / L in deionized water after reacting with different concentrations of mercaptosuccinic acid. As shown in the figure, as the concentration of mercaptosuccinic acid increases, the fluorescence intensity of the probe solution gradually weakens. When its concentration is 20 μM, the fluorescence intensity of Y-SH tends to be stable. When its lowest concentration is 1 μM, the fluorescence peak at 470 nm can still be distinguished from the background fluorescence curve without adding mercaptosuccinic acid, thus obtaining the detection limit of this probe for mercaptosuccinic acid.

[0023] Figure 3 The fluorescence intensity diagram of the fluorescent probe prepared in Example 1 at a concentration of 1×10 -5 mol / L in deionized water at 470 nm after reacting with mercaptosuccinic acid in the presence of five-fold interfering other mercapto compounds and ionic solutions (concentration of 1×10 -4 mol / L). As shown in the figure, when only adding mercaptosuccinic acid solution, the fluorescence intensity of the probe solution at 470 nm decreases significantly relative to the blank, while there is no particularly obvious fluorescence change of this probe for other interfering substances (L-cysteine, DL-homocysteine, DL-dithiothreitol, 2-aminoethanethiol, mercaptobenzimidazole, mercaptosuccinic acid, Cl - 、SO4 2- 、PO4 - 、HSO3 - 、F - 、Br - 、blank); then the change in the fluorescence intensity of the probe at 470 nm after adding 5-fold other analogue solutions to the detection system shows that the addition of other interfering substances does not lead to a decrease in the detection result of this probe for mercaptosuccinic acid, indicating that this fluorescent probe has strong anti-interference ability during the detection process.

[0024] Figure 4 The 1H NMR spectrum of the coumarin-based fluorescent probe prepared in Example 1. 11H NMR (400 MHz, CDCl3) δ 10.92 (s, 1H), 8.72 (s, 1H), 7.68–7.62 (m, 2H), 7.44–7.38 (m, 3H), 6.61 (dd, J = 8.8, 2.5 Hz, 1H), 6.47 (d, J = 8.4 Hz, 1H), 3.41 (q, J = 8.8 Hz, 4H), 1.19 (t, J = 8.0 Hz, 6H).

[0025] Example 2

[0026] Dissolve 2-aminobenzaldehyde (39.9 mg, 0.33 mmol) and 7-(diethylamino)coumarin-3-carboxylic acid (78.4 mg, 0.3 mmol) in 1.0 mL of dichloromethane, then add EDCI (103.6 mg, 0.54 mmol) and HBOT (72.9 mg, 0.54 mmol), and stir the reaction mixture at room temperature for 10 hours. After the reaction is completed, remove the solvent under reduced pressure, and purify the crude product by thin-layer chromatography using a mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 1:1 as the eluent. Finally, 22.3 mg of a yellow solid product is obtained, with a yield of 20.4%.

[0027] Example 3

[0028] Dissolve 2-aminobenzaldehyde (242.3 mg, 2.0 mmol) and 7-(diethylamino)coumarin-3-carboxylic acid (261.3 mg, 1.0 mmol) in 3.0 mL of dichloromethane, then add EDCI (383.4 mg, 2.0 mmol) and HBOT (270.3 mg, 2.0 mmol), and stir the reaction mixture at room temperature for 12 hours. After the reaction is completed, remove the solvent under reduced pressure, and purify the crude product by thin-layer chromatography using a mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 1:1 as the eluent. Finally, 76.9 mg of a yellow solid product is obtained, with a yield of 21.1%.

Claims

1. A coumarin-based fluorescent probe based on aminobenzaldehyde, characterized in that: The structural formula of the fluorescent probe is shown as follows:

2. A preparation method of the coumarin-based fluorescent probe based on aminobenzaldehyde as described in claim 1, characterized in that: The preparation method is as follows: Dissolve 2-aminobenzaldehyde and 7-(diethylamino)coumarin-3-carboxylic acid in dichloromethane, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole as condensing agents. The reaction solution is stirred at room temperature. After the reaction is completed, the solvent is removed. The obtained compound is separated and purified by thin-layer chromatography in a developing agent to obtain the final product probe Y-SH.

3. The preparation method of the coumarin-based fluorescent probe based on aminobenzaldehyde according to claim 2, characterized in that: The molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid, 2-aminobenzaldehyde, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole is: 1:1.1 - 2.0:1.8 - 2.0:1.8 - 2.

0.

4. The preparation method of the aminobenzaldehyde-based coumarin fluorescent probe according to claim 2, characterized in that: The reaction time is: 10 - 12 hours; the developing agent is: a mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 1:

1.

5. Use of a coumarin-based fluorescent probe based on aminobenzaldehyde as described in claim 1, characterized in that: The fluorescent probe is used for preparing a reagent for detecting mercaptosuccinic acid.

6. Use of the aminobenzaldehyde-based coumarin fluorescent probe according to claim 5, characterized in that: The reagent prepared from the fluorescent probe is used for detecting mercaptosuccinic acid by fluorescence quenching in deionized water.

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