Coumarin-based fluorescent probe based on aminobenzaldehyde for preparing a reagent for detecting mercaptosuccinic acid and a preparation method thereof

Abstract

The present application belongs to the field of chemical analysis test, and particularly relates to a coumarin fluorescent probe based on aminobenzaldehyde for preparing a reagent for detecting mercaptosuccinic acid and a preparation method. 2-Aminobenzaldehyde and 7-(diethylamino)coumarin-3-carboxylic acid are dissolved in dichloromethane, 1-ethyl-(3-dimethylaminoaldehyde) carbonyldiimidazole hydrochloride (EDCI) and 1-hydroxybenzotriazole (HBOT) are added as condensing agents, and the reaction is stirred at room temperature. The obtained crude product is separated and purified by thin layer chromatography to obtain the final yellow product, the coumarin fluorescent probe Y-SH based on aminobenzaldehyde. The coumarin fluorescent probe prepared by the present application can rapidly detect mercaptosuccinic acid in deionized water.

Description

Technical Field

[0001] This invention belongs to the field of chemical analysis and testing, and specifically relates to an aminobenzaldehyde-based coumarin fluorescent probe for preparing a reagent for detecting mercaptosuccinic acid and its preparation method. Background Technology

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

[0003] Traditional methods for detecting mercaptosuccinic acid, such as high-performance liquid chromatography (HPLC) and capillary electrophoresis, suffer from drawbacks including high cost, complex operation, and susceptibility to interference. Fluorescent detection methods based on fluorescent probes, however, offer advantages such as high sensitivity, strong specificity, and fast response, attracting increasing attention. Therefore, developing a fluorescent probe capable of selectively recognizing mercaptosuccinic acid is of great significance for studying its physiological effects and pathological mechanisms. Summary of the Invention

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

[0005]

[0006] This invention also provides a method for preparing a 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: 7-(diethylamino)coumarin-3-carboxylic acid and 2-aminobenzaldehyde are dissolved in dichloromethane at a molar equivalent ratio of 1:1.1-2.0. Then, 1.8-2.0 molar equivalents of EDCI and 1.8-2.0 molar equivalents of HOBT are added as condensing agents. The reaction solution is stirred at room temperature for 10-12 hours. After the reaction is completed, the solvent is removed, and finally, the product is purified by thin-layer chromatography using a 1:1 volume ratio of petroleum ether and ethyl acetate as the developing solvent to obtain the yellow solid product Y-SH.

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

[0010] The probe Y-SH prepared in this invention uses a coumarin compound as the fluorescent group and benzaldehyde as the target. It is obtained through an amide condensation reaction and exhibits excellent fluorescence properties. When it recognizes mercaptosuccinic acid, the aldehyde group in the fluorescent probe and the double bond of the benzene ring attached to the aldehyde group react with the thiol and hydroxyl groups of mercaptosuccinic acid to form a seven-membered ring. This enhances photoinduced electron transfer, leading to quenching of the fluorophore, thereby achieving the function of detecting mercaptosuccinic acid.

[0011] Beneficial effects

[0012] The raw materials used in this invention are readily available, the synthesis method is simple, the reaction conditions are easy to control, and the pure product can be obtained through simple post-processing. The probe Y-SH reported in this invention solves the problem of detecting mercaptosuccinic acid in deionized water. From the fluorescence phenomenon, the fluorescence intensity of probe Y-SH at 470 nm decreased significantly after the addition of mercaptosuccinic acid, thus demonstrating the detection effect of probe Y-SH on mercaptosuccinic acid in this system. Attached Figure Description

[0013] Figure 1 The probe prepared for Example 1 was prepared at a ratio of 1 × 10⁻⁶. -5 Fluorescence spectra of deionized water at a concentration of mol / L after interaction with different thiol compounds and ions.

[0014] Figure 2 The probe prepared for Example 1 was prepared at a ratio of 1 × 10⁻⁶. -5 Fluorescence spectra of mercaptosuccinic acid at concentrations of mol / L reacting with different concentrations in deionized water.

[0015] Figure 3 The probe prepared for Example 1 was prepared at a ratio of 1 × 10⁻⁶. -5 Fluorescence intensity at 470 nm of mercaptosuccinic acid in deionized water at a concentration of mol / L after the addition of five times the interference of different mercapto compounds and ions.

[0016] Figure 4 The proton spectrum of the probe prepared in Example 1. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to specific embodiments.

[0018] Example 1

[0019] 2-Aminobenzaldehyde (90.9 mg, 0.75 mmol) and 7-(diethylamino)coumarin-3-carboxylic acid (130.7 mg, 0.5 mmol) were dissolved in 1.5 mL of dichloromethane, followed by the addition of EDCI (182.2 mg, 0.95 mmol) and HBOT (128.4 mg, 0.95 mmol). The reaction mixture was stirred at room temperature for 10 hours. After the reaction was complete, the solvent was removed under reduced pressure. The crude product was purified by thin-layer chromatography using a 1:1 (v / v) mixture of petroleum ether and ethyl acetate as the developing solvent, yielding a yellow solid product of 33.9 mg, 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-) to a 96-well plate. - SO4 2- PO4 - HSO3 - F - ,Br - The concentration is 1×10 -4 196 μL of deionized water and 2 μL of probe Y-SH (concentration of 1×10⁻⁶ mol / L) were added. -5 The fluorescence intensity of the solution in each well was measured using a microplate reader. The solution contained mercaptosuccinic acid (MtSA) and a control solution containing the probe Y-SH without any other compounds (blank) were used. The solutions in each well were thoroughly mixed, and the fluorescence intensity of each solution was measured using a microplate reader. The results showed that the fluorescence intensity of the probe at 470 nm decreased significantly after the addition of mercaptosuccinic acid; and the fluorescent probe showed no significant change in fluorescence intensity for other interfering substances, thus demonstrating the selective recognition of mercaptosuccinic acid by the fluorescent probe in deionized water.

[0021] Figure 1 The coumarin-based fluorescent probe based on aminobenzaldehyde prepared in Example 1 was used at 1×10 -5 A concentration of mol / L was observed in deionized water with different thiol compounds and ions (concentration of 1×10⁻⁶). -4 (mol / L)(6-mercaptopurine, L-cysteine, DL-homocysteine, DL-dithiothreitol, 2-aminoethanethiol, mercaptobenzimidazole, mercaptosuccinic acid, Cl) - SO4 2- PO4 - HSO3 - F - ,Br -Fluorescence spectra after blank treatment. The figure shows the changes in fluorescence intensity of the probe after adding different thiol compounds and ion solutions. When mercaptosuccinic acid solution was added, the fluorescence intensity of the fluorescent probe at 470 nm decreased significantly (curve indicated by the arrow); and the fluorescent probe did not show significant changes in fluorescence intensity for other interfering substances in deionized water, thus demonstrating the selective recognition of mercaptosuccinic acid by the fluorescent probe in deionized water.

[0022] Figure 2 The fluorescent probe prepared for Example 1 was used at 1×10 -5 Fluorescence spectra of Y-SH at a concentration of mol / L reacting with different concentrations of mercaptosuccinic acid in deionized water. The figures show that the fluorescence intensity of the probe solution gradually decreases with increasing mercaptosuccinic acid concentration. At a concentration of 20 μM, the fluorescence intensity of Y-SH tends to stabilize. At its lowest concentration of 1 μM, the fluorescence peak at 470 nm is still distinguishable from the background fluorescence curve without mercaptosuccinic acid, thus establishing the detection limit of this probe for mercaptosuccinic acid.

[0023] Figure 3 The fluorescent probe prepared for Example 1 was used at 1×10 -5 A solution of mercaptosuccinic acid at a concentration of mol / L was prepared in deionized water with five times the amount of other mercapto compounds and ionic solutions (concentration 1×10⁻⁶). -4 The fluorescence intensity at 470 nm after treatment with mercaptosuccinic acid (mol / L) is shown in the figure. The figure shows that when only mercaptosuccinic acid solution is added, the fluorescence intensity of the probe solution at 470 nm is significantly lower than that of the blank. However, the probe is less reactive to other interfering substances (L-cysteine, DL-homocysteine, DL-dithiothreitol, 2-aminoethanethiol, mercaptobenzimidazole, mercaptosuccinic acid, Cl...). - SO4 2- PO4 - HSO3 - F - ,Br - The blank sample showed no significant change in fluorescence. Subsequently, the fluorescence intensity of the probe at 470 nm was observed after adding 5 times the amount of other analog solutions to the detection system. This showed that the addition of other interfering substances did not lead to a decrease in the detection result of mercaptosuccinic acid by the probe, indicating that the fluorescent probe has strong anti-interference ability during the detection process.

[0024] Figure 4 The proton NMR spectrum of the coumarin-based fluorescent probe based on aminobenzaldehyde prepared in Example 1. 1H NMR (400MHz, CDCl3) δ10.92(s,1H),8.72(s,1H),7.68–7.62(m,2H),7.44–7.38(m,3H),6.61 (dd,J=8.8,2.5Hz,1H),6.47(d,J=8.4Hz,1H),3.41(q,J=8.8Hz,4H),1.19(t,J=8.0Hz,6H).

[0025] Example 2

[0026] 2-Aminobenzaldehyde (39.9 mg, 0.33 mmol) and 7-(diethylamino)coumarin-3-carboxylic acid (78.4 mg, 0.3 mmol) were dissolved in 1.0 mL of dichloromethane, followed by the addition of EDCI (103.6 mg, 0.54 mmol) and HBOT (72.9 mg, 0.54 mmol). The reaction mixture was stirred at room temperature for 10 hours. After the reaction was complete, the solvent was removed under reduced pressure. The crude product was purified by thin-layer chromatography using a 1:1 (v / v) mixture of petroleum ether and ethyl acetate as the developing solvent, yielding 22.3 mg of a yellow solid product in 20.4% yield.

[0027] Example 3

[0028] 2-Aminobenzaldehyde (242.3 mg, 2.0 mmol) and 7-(diethylamino)coumarin-3-carboxylic acid (261.3 mg, 1.0 mmol) were dissolved in 3.0 mL of dichloromethane, followed by the addition of EDCI (383.4 mg, 2.0 mmol) and HBOT (270.3 mg, 2.0 mmol). The reaction mixture was stirred at room temperature for 12 hours. After the reaction was complete, the solvent was removed under reduced pressure. The crude product was purified by thin-layer chromatography using a 1:1 (v / v) mixture of petroleum ether and ethyl acetate as the developing solvent, yielding 76.9 mg of a yellow solid product, 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 below: 。 2. A method for preparing a coumarin-based fluorescent probe based on aminobenzaldehyde as described in claim 1, characterized in that: The preparation method is as follows: 2-aminobenzaldehyde and 7-(diethylamino)coumarin-3-carboxylic acid are dissolved in dichloromethane, and then EDCI and HOBT are added as condensing agents. The reaction solution is stirred at room temperature. After the reaction is completed, the solvent is removed, and the crude product is separated and purified by thin-layer chromatography in the developing solvent to obtain the final product fluorescent probe.

3. The method for preparing a coumarin-based fluorescent probe based on aminobenzaldehyde as described in claim 2, characterized in that: The molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid, 2-aminobenzaldehyde, EDCI and HOBT is 1:1.1~2.0:1.8~2.0:1.8~2.

0.

4. The method for preparing a coumarin-based fluorescent probe based on aminobenzaldehyde as described in claim 2, characterized in that: The reaction time is 10-12 hours; the developing solvent is a mixture of petroleum ether and ethyl acetate in a volume ratio of 1:

1.

5. The application of the coumarin-based fluorescent probe based on aminobenzaldehyde as described in claim 1, characterized in that: The fluorescent probe is used to prepare reagents for detecting mercaptosuccinic acid.

6. The application of the coumarin-based fluorescent probe based on aminobenzaldehyde as described in claim 5, characterized in that: The reagent prepared by the fluorescent probe is used to detect mercaptosuccinic acid in deionized water by fluorescence quenching.

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