Coumarin fluorescent probe based on benzoxazole derivatives and application thereof

Abstract

The application belongs to the field of chemical analysis test, and particularly relates to a coumarin fluorescent probe based on benzoxazole derivative and application thereof. 7-hydroxy-4-methyl coumarin-3-acetic acid and 6-bromobenzo[d]oxazole are dissolved in dimethyl sulfoxide solution, and the reaction solution is heated to reflux under the alkaline condition of potassium carbonate at 70-90 DEG C for 3-4 hours. The obtained crude product is separated and purified by thin layer chromatography to obtain the coumarin fluorescent probe SYL-4 based on benzoxazole derivative. The coumarin fluorescent probe obtained in the application can quickly identify biological amines (BAs) in a cetyltrimethylammonium bromide (CTAB) surfactant aqueous solution.

Description

Technical Field

[0001] The present invention belongs to the field of chemical analysis and testing, and in particular relates to a coumarin fluorescent probe based on benzoxazole derivatives and applications thereof. Background Art

[0002] Biogenic amines (BAs) are nitrogen-containing compounds synthesized by enzymes from various organisms through the decarboxylation of amino acids. Based on the number of amino groups in their structure, BAs can be divided into monoamines (compounds containing one amine group) and polyamines (compounds containing multiple amine groups). BAs can be produced endogenously by plants and animals and exogenously by microorganisms during food processing and improper storage. Excessive BAs can be cytotoxic and lead to serious consequences. For example, histamine and tyramine can induce inflammatory symptoms, while putrescine and cadaverine interact with amine oxidase and increase the toxicity of histamine. Furthermore, putrescine, cadaverine, spermidine, and spermine can react with nitrite in meat products containing nitrite and nitrate curing agents to form carcinogenic N-nitrosamines. Therefore, the development of simple and rapid detection technologies for BAs is of great significance for the early diagnosis of diseases and maintaining food safety.

[0003] Traditional methods for detecting BAs include thin-layer chromatography, capillary electrophoresis, gas chromatography-mass spectrometry (GC-MS), and high-performance liquid chromatography (HPLC). However, these methods are often characterized by cumbersome sample preparation, complex operations, lengthy processing times, and the need for large instrumentation, significantly limiting their practical application. In recent years, fluorescent probe-based optical imaging has been widely used in various fields due to its high sensitivity, excellent selectivity, ease of operation, low cost, and ability to detect BAs in real-time within biological microenvironments.

[0004] A variety of chemical probes for BAs detection have been developed based on fluorescence methods, primarily employing various supramolecular approaches, including dye-embedded micelles, sol-gel films, conjugated polymers, and other supramolecular methods. These amplify the sensing response through analyte-induced target self-assembly. However, most of these supramolecular probes have low selectivity and are easily affected by external stimuli (e.g., light and heat). Summary of the Invention

[0005] The present invention provides a coumarin-based fluorescent probe based on benzoxazole derivatives, the structural formula of the fluorescent probe is:

[0006]

[0007] The present invention also provides a method for preparing a coumarin-based fluorescent probe based on a benzoxazole derivative, the reaction equation of which is:

[0008]

[0009] The specific preparation method is as follows: 7-hydroxy-4-methylcoumarin-3-acetic acid and 6-bromobenzo[d]oxazole are dissolved in dimethyl sulfoxide solution. Under alkaline conditions (potassium carbonate), the reaction is heated at 70-90°C for 3-4 hours. After the reaction is complete, the reaction system is cooled to room temperature, and the organic solvent is removed by vacuum distillation. The resulting compound is separated and purified by thin-layer chromatography in a developing solvent to obtain the white solid product SYL-4.

[0010] The molar equivalent ratio of 7-hydroxy-4-methylcoumarin-3-acetic acid, 6-bromobenzo[d]oxazole and potassium carbonate is 1:1.1:1.1; and the developing solvent is a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 3:1.

[0011] The present invention also provides an application of a coumarin fluorescent probe based on a benzoxazole derivative: the coumarin fluorescent probe SYL-4 based on a benzoxazole derivative can quickly identify BAs in a CTAB surfactant aqueous solution.

[0012] The biogenic amines are tyramine, putrescine, tryptamine, spermidine, spermine, benzylamine, dopamine, and histamine.

[0013] The concentration of the CTAB surfactant aqueous solution was 0.2-1 mM.

[0014] The compound structure of this invention utilizes coumarin as a fluorescent group. An electron-donating group (hydroxyl) is substituted at position 7, and an electron-withdrawing group (carboxyl) is substituted at position 3. This allows the coumarin to generate electron-pushing and electron-pulling reactions, further enhancing the water solubility of the coumarin derivative. The ligand structure is primarily a benzoxazole derivative, and substitution at multiple sites results in diverse physical and chemical properties.

[0015] The prepared compound's structure features an ether bond as a target site for interaction with biogenic amines (BAs). In the probe SYL-4, the benzoxazole moiety serves as an electron donor, while the coumarin ring, located in a low-energy orbital, serves as an electron acceptor. When the -O-benzoxazole moiety specifically interacts with BAs, the ether bond breaks, significantly inhibiting the intramolecular charge transfer (ICT) process and leading to fluorescence quenching, thereby achieving BA recognition.

[0016] Beneficial effects

[0017] The present invention solves the problem of rapid identification of BAs by the probe SYL-4 in a CTAB solvent system. The fluorescence intensity of the probe SYL-4 at 460 nm was measured when the probe SYL-4 was added to an aqueous CTAB surfactant solution. The fluorescence intensity of the probe SYL-4 at 460 nm decreased significantly after the addition of different BAs, demonstrating that the probe SYL-4 can recognize a variety of BAs in this system. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the fluorescence spectrum of the benzoxazole derivative-based coumarin fluorescent probe SYL-4 prepared in Example 1 after it reacted with different BAs (tyramine (Tyr), putrescine (Put), tryptamine (Try), spermidine (Spd), spermine (Spm), benzylamine (Benzylamine), dopamine (DA), and histamine (His)) in a CTAB surfactant solution.

[0019] Figure 2 This is a comparison chart of the fluorescence intensity at 460 nm of the benzoxazole derivative-based coumarin fluorescent probe SYL-4 prepared in Example 1 after reacting with BAs (taking Spm as an example) in a 0.2-1 mM CTAB surfactant solution.

[0020] Figure 3 This is a fluorescence spectrum of the benzoxazole derivative-based coumarin fluorescent probe SYL-4 prepared in Example 1 after it acts with different concentrations of BAs (taking Spm as an example) in a 0.4 mM CTAB surfactant aqueous solution.

[0021] Figure 4 This is the hydrogen spectrum of the coumarin-based fluorescent probe SYL-4 based on benzoxazole derivatives prepared in Example 1. DETAILED DESCRIPTION

[0022] The present invention is described in detail below with reference to specific embodiments.

[0023] Example 1

[0024] 7-Hydroxy-4-methylcoumarin-3-acetic acid (100 mg, 0.426 mmol) and 6-bromobenzo[d]oxazole (93 mg, 0.469 mmol) were dissolved in 4 mL of dimethyl sulfoxide solution. The reaction was heated at 85°C in the presence of potassium carbonate (65 mg, 0.469 mmol) for 3.5 hours. After the reaction was completed, the solvent was removed by distillation under reduced pressure to obtain a yellow solid. The crude product was separated and purified by thin-layer chromatography (developing solvent volume ratio: petroleum ether: ethyl acetate = 3:1) to obtain 85.6 mg of SYL-4 as a white solid, with a yield of 57%.

[0025] The specific application method is as follows: 2uL of 10mM BAs solution (the BAs in each well are (tyramine (Try), tryptamine (Try), dopamine (DA), spermidine (Spd), benzylamine (Benzylamine), histamine (His), putrescine (Put), spermine (Spm)), 196uL of 4mM CTAB surfactant solution and 2uL of 5mM benzoxazole-based coumarin fluorescent probe SYL-4) are added to a 96-well plate, and a solution containing the fluorescent probe SYL-4 without BAs is used as a comparison. The solution in each well is mixed evenly, and the fluorescence intensity of the solution in each well is measured. The results show that BAs has a significant quenching effect on the probe SYL-4, thereby demonstrating the rapid recognition effect of the probe SYL-4 on BAs in the CTAB solvent system.

[0026] Figure 1 Fluorescence spectra of the benzoxazole derivative-based coumarin fluorescent probe prepared in Example 1 after interaction with different BAs (tyramine (Tyr), tryptamine (Try), dopamine (DA), spermidine (Spd), benzylamine (Benzylamine), histamine (His), putrescine (Put), and spermine (Spm)) in a 4 mM CTAB surfactant solution. The figure shows the change in fluorescence intensity of the probe SYL-4 after the addition of different BA solutions. The fluorescence intensity of the probe SYL-4 at 460 nm decreased significantly with the addition of different BAs, demonstrating the compound's recognition of multiple BAs in this system.

[0027] Figure 2 This is a comparison of the fluorescence intensity at 460 nm of the coumarin-based fluorescent probe based on benzoxazole derivatives prepared in Example 1 after reacting with BAs (taking Spm as an example) in a 0.2-1 mM CTAB surfactant aqueous solution. The figure shows that as the concentration of the CTAB surfactant aqueous solution increases, the fluorescence intensity of the probe SYL-4 solution and the Spm+SYL-4 solution system both show a downward trend. Among them, in the CTAB surfactant aqueous solution of different concentrations, compared with the probe solution without Spm, the fluorescence intensity decreased by about 2 times after the addition of Spm, and the best detection effect was achieved when the CTAB surfactant solution concentration was 0.4 mM. This shows that the probe can identify BAs in a CTAB surfactant aqueous solution with a concentration range of 0.2-1 mM.

[0028] Figure 3Fluorescence spectra of the coumarin fluorescent probe SYL-4 based on benzoxazole derivatives prepared in Example 1 after the action of BAs (taking Spm as an example) at different concentrations in a 0.4mM CTAB surfactant aqueous solution. The figure shows that the fluorescence intensity of the SYL-4 probe solution gradually weakens with the increase of the Spm concentration. When the Spm concentration is within the range of 1-500uM, the higher the Spm concentration, the more obvious the probe fluorescence quenching phenomenon. When its final concentration is 10uM, the fluorescence peak of the probe SYL-4 at 460nm can still be distinguished from the background fluorescence curve without Spm, thereby concluding that the probe has a low detection limit and high sensitivity for BAs.

[0029] Figure 4 This is the hydrogen spectrum of the benzothiazole derivative-based coumarin fluorescent probe SYL-4 prepared in Example 1. 1 HNMR (400MHz, DMSO) δ10.93(s,1H),8.38-7.97(m,1H),7.68(d,J=8.4Hz,2H),7.31–6.48(m,4H),3.57(s,2H),2.86(m,2H).

[0030] Example 2

[0031] 7-Hydroxy-4-methylcoumarin-3-acetic acid (200 mg, 0.852 mmol) and 6-bromobenzo[d]oxazole (186 mg, 0.938 mmol) were dissolved in 8 mL of dimethyl sulfoxide. The mixture was heated at 85°C in the presence of potassium carbonate (130 mg, 0.938 mmol) for 3.5 hours. After completion of the reaction, the solvent was removed by distillation under reduced pressure to yield a yellow solid. Thin-layer chromatography (developing solvent volume ratio: petroleum ether: ethyl acetate = 3:1) was used to separate and purify the product, yielding 157.3 mg of the final product, SYL-4, with a yield of 52%.

[0032] Example 3

[0033] 7-Hydroxy-4-methylcoumarin-3-acetic acid (200 mg, 0.852 mmol) and 6-bromobenzo[d]oxazole (186 mg, 0.938 mmol) were dissolved in 8 ml of dimethyl sulfoxide (DMSO). The mixture was heated at 90°C in the presence of potassium carbonate (130 mg, 0.938 mmol) for 4 hours. After completion of the reaction, the solvent was removed by distillation under reduced pressure to yield a yellow solid. Thin-layer chromatography (developing solvent, volume ratio: petroleum ether:ethyl acetate = 3:1) was used to separate and purify the product, SYL-4 (176.8 mg), with a yield of 59%.

Claims

1. A coumarin-based fluorescent probe based on a benzoxazole derivative, characterized in that: The structural formula of the fluorescent probe is: ； The preparation method of the coumarin-based fluorescent probe based on benzoxazole derivatives is as follows: 7-hydroxy-4-methylcoumarin-3-acetic acid and 6-bromobenzo[d]oxazole are dissolved in a dimethyl sulfoxide solution, the reaction solution is heated to reflux under alkaline conditions, and after the reaction is complete, the reaction system is cooled to room temperature, the organic solvent is removed by reduced pressure distillation, and the resulting compound is separated and purified by thin-layer chromatography in a developing solvent to obtain a white solid product, namely, the coumarin-based fluorescent probe based on benzoxazole derivatives.

2. The coumarin-based fluorescent probe based on benzoxazole derivatives according to claim 1, characterized in that: The base is potassium carbonate; the molar equivalent ratio of the 7-hydroxy-4-methylcoumarin-3-acetic acid, 6-bromobenzo[d]oxazole and potassium carbonate is 1:1.1:1.

1.

3. The coumarin-based fluorescent probe based on benzoxazole derivatives according to claim 1, characterized in that: The heating reflux reaction temperature is 70-90° C., and the reaction time is 3-4 hours.

4. The coumarin-based fluorescent probe based on benzoxazole derivatives according to claim 1, characterized in that: The developing solvent is a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 3:

1.

5. A use of the coumarin-based fluorescent probe based on benzoxazole derivatives as claimed in claim 1, characterized in that: The coumarin fluorescent probe based on benzoxazole derivatives is used to prepare a reagent for rapidly identifying biogenic amines BAs in a cetyltrimethylammonium bromide (CTAB) surfactant aqueous solution.

6. The use of a coumarin-based fluorescent probe based on a benzoxazole derivative as claimed in claim 5, characterized in that: The concentration of the CTAB surfactant aqueous solution is 0.2-1 mM.

7. The use of a coumarin-based fluorescent probe based on a benzoxazole derivative as claimed in claim 5, characterized in that: The biogenic amines are tyramine, putrescine, tryptamine, spermidine, spermine, benzylamine, dopamine and histamine.

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