A Fluorescent Probe Based on Double-Bond Structure and Its Application

Abstract

The present invention belongs to the field of chemical analysis and testing, and particularly relates to a fluorescent probe based on a double-bond structure and its application. First, using 4-aminostyrene and 7-(diethylamino)coumarin-3-carboxylic acid as raw materials, dichloromethane as a solvent, and 1-hydroxybenzotriazole (HOBT) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) as condensing agents, the final product crude is obtained by stirring at room temperature. The obtained crude product is then separated by column chromatography using petroleum ether and ethyl acetate as eluents to obtain the final yellow pure product. The fluorescent probe obtained by the present invention has good optical properties and fluorescence selectivity for 6-mercaptopurine (6-MP), and can realize the fluorescence detection of 6-MP.

Description

Technical Field

[0001] The invention belongs to the field of chemical analysis and testing, and particularly relates to a fluorescent probe based on a double bond structure and an application thereof. Background Art

[0002] 6-Mercaptopurine (6-MP) is a chemotherapeutic anticancer drug with immunosuppressive properties. Under the catalytic action of inosine nucleotide pyrophosphatase in the body, it is metabolized to sulfo-inosine nucleoside. It inhibits purine synthesis and is widely used in the treatment of acute lymphoblastic leukemia. The plasma concentration of 6-MP varies greatly among individuals, and its activity also varies with changes in plasma concentration. However, as a cytotoxic antitumor drug, it has serious side effects. Inadequate dosages are ineffective in curing the disease, while excessive dosages may cause adverse effects such as bone marrow suppression and liver problems, which limits its application. Therefore, the development of a simple and effective method for the quantitative detection of 6-MP is of great significance.

[0003] Currently, the main methods for detecting 6-MP include high-performance liquid chromatography (HPLC), electrochemical methods, surface plasmon resonance, Raman spectroscopy (RS), capillary electrophoresis (CE), and mass spectrometry (MS). Among these methods, electrochemical determination is the most widely reported, and many modified electrodes have been used for 6-MP detection. However, they are known to have poor reproducibility and complex electrode modification processes, which limit their application in actual samples. Detecting 6-MP by high-performance liquid chromatography coupled with mass spectrometry requires expensive equipment and often involves complex sample pretreatment. CE and RS also suffer from the shortcomings of HPLC and MS. Therefore, there is an urgent need for reagents and methods for the rapid, simple, and sensitive detection of 6-MP.

[0004] Compared to these methods, the fluorescent probe method offers advantages such as ease of use, high sensitivity, and good selectivity, making it an alternative technology for detecting 6-MP in complex samples. Traditional fluorescent probe methods rely on the complexation of 6-MP with copper ions to switch the probe's fluorescence. However, these methods require the presence of copper ions and are therefore unable to directly detect 6-MP. Summary of the Invention

[0005] The present invention provides a fluorescent probe based on a double bond structure, the structural formula of the fluorescent probe is:

[0006]

[0007] The present invention also provides a specific preparation method of a fluorescent probe based on a double bond structure: the chemical reaction formula for preparing the fluorescent probe is:

[0008]

[0009] The specific steps are as follows:

[0010] Dissolve 7-(diethylamino)coumarin-3-carboxylic acid, 1-hydroxybenzotriazole (HOBT), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) in dichloromethane at a molar ratio of 1:1.5:1.5, then add 1 - 2 equivalents of 4-aminostyrene, and stir at room temperature for 1 - 3 h. After the reaction is completed, evaporate dichloromethane, and finally purify by column chromatography using a mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 3:1 as the eluent to obtain the final pure yellow solid.

[0011] The present invention also provides the application of the above fluorescent probe: The prepared coumarin fluorescent probe based on a double bond structure is applied to the detection of 6-MP, directly detecting 6-MP in the aqueous phase, which is rapid, simple, highly selective, with a detection limit as low as 1 μg / mL, and has strong anti-interference ability against other thiol structures and anions.

[0012] The specific application method is as follows: Add 2 μL of the test object solution (the test objects in each well are 6-MP, Cys (cysteine), HCY (homocysteine), NCY (N-acetylcysteine), DTT (dithiothreitol), PO4 2- 、S 2- 、HSO3 - 、F - 、Br - , with a concentration of 10 mg / mL), 196 μL of pure water, and 2 μL of the coumarin fluorescent probe based on a double bond structure (with a concentration of 1×10 -3 mol / L) into a 96-well plate. At the same time, use a solution containing the coumarin fluorescent probe without other thiol structures and ions (Blank) 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 the fluorescence intensity of the solution containing 6-MP at 550 nm is twice that of the solution without 6-MP; and there is no particularly obvious change in the fluorescence intensity of this fluorescent probe for other test objects, thus showing the selective recognition effect of this fluorescent probe for 6-MP in pure water.

[0013] The present invention solves the problem of the selective recognition of 6-MP by the fluorescent probe in different solvent systems (pure water, PBS, HEPES, Tris-HCl). From the reaction phenomenon, after adding 6-MP, the fluorescence intensity of the fluorescent probe in these several solvents increases significantly. At the same time, compared with the traditional method for detecting 6-MP, this method can directly detect 6-MP under aqueous phase conditions.

[0014] The beneficial effects of the present invention are as follows: The raw materials of the present invention are easily obtained. The double bond structure is introduced into the fluorescent probe through a one-step reaction. The synthesis method is simple, the reaction conditions are easy to control, and after the reaction, a pure product can be obtained through simple post-treatment; As a chemical probe for 6-MP detection, the coumarin fluorescent probe based on the double bond structure has high sensitivity, good selectivity, and can directly identify 6-MP in different solvents. For example, 6-MP can be identified in pure water, and the detection limit is 1 μg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The fluorescence spectra of the fluorescent probe prepared in Example 1 after reacting with different detection objects in pure water at a concentration of 1×10 -3 mol / L.

[0016] Figure 2 The fluorescence spectra of the fluorescent probe prepared in Example 1 after reacting with different concentrations of 6-MP in pure water at a concentration of 1×10 -3 mol / L.

[0017] Figure 3 The bar chart of the fluorescence intensity of the fluorescent probe prepared in Example 1 at 550 nm before and after reacting with 6-MP in different buffers.

[0018] Figure 4 The fluorescence intensity diagram of the fluorescent probe prepared in Example 1 at 550 nm after adding five-fold interfering objects when reacting with 6-MP in pure water at a concentration of 1×10 -3 mol / L.

[0019] Figure 5 The 1H NMR spectrum of the fluorescent probe prepared in Example 1. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] The present invention will be further described in detail below with reference to the embodiments:

[0021] Example 1:

[0022] Dissolve 7-(diethylamino)coumarin-3-carboxylic acid (0.261 g, 1 mmol), HOBT (0.201 g, 1.5 mmol), and EDCI (0.286 g, 1.5 mmol) in dichloromethane, then add 4-aminostyrene (0.119 g, 1 mmol), and stir the reaction at room temperature for 1 h. After the reaction is completed, evaporate dichloromethane, and finally use a mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 3:1 as the eluent, and purify by column chromatography to obtain 0.0432 g of the final pure product.

[0023] Figure 1 For the probe prepared in Example 1 at 1×10 -3Fluorescence spectra of the probe prepared in Example 1 at a concentration of 1×10

[0024] Figure 2 mol / L in 196 μL of pure water after reacting with different detection targets. As shown in the figure, after adding 2 μL of a solution of different detection targets with a concentration of 10 mg / mL, the change in the fluorescence intensity of the fluorescent probe is shown. When a 6-MP solution with a concentration of 10 mg / mL is added, the fluorescence intensity of the probe at 550 nm increases significantly (the curve indicated by the arrow); while in pure water, the probe shows no particularly obvious fluorescence change for other ions and thiol structures, thus demonstrating the selective recognition of 6-MP by the probe in this system. -3 Fluorescence spectra of the probe prepared in Example 1 at a concentration of 1×10

[0025] Figure 3 mol / L in 196 μL of pure water after reacting with different concentrations of 6-MP. As shown in the figure, as the concentration of 6-MP decreases, the fluorescence intensity of the probe gradually weakens. When its final concentration is 1 μg / mL, the fluorescence peak at 550 nm can still be distinguished from the background fluorescence curve without 6-MP, thus indicating that the detection limit of the probe for 6-MP is low. -3 Bar chart of the fluorescence intensity of the fluorescent probe prepared in Example 1 at 550 nm before and after reacting with 6-MP in different buffers. As shown in the figure, before adding 6-MP, the fluorescence intensity of the probe solution with a concentration of 1×10

[0026] Figure 4 mol / L is weak in the aqueous phase and in three buffers: PBS, HEPES, and Tris-HCl; after adding 2 μL of 6-MP with a concentration of 10 mg / mL, the fluorescence of the fluorescent probe at 550 nm is significantly enhanced in several solvent conditions, and the fluorescence intensity is increased by 2 times, thus demonstrating that the probe can selectively recognize 6-MP in different solvents. -3 Fluorescence intensity diagram of the fluorescent probe prepared in Example 1 at a concentration of 1×10 2- mol / L in pure water at 550 nm after adding 2 μL of 6-MP with a concentration of 10 mg / mL and five-fold interfering protein. As shown in the figure, when only the 6-MP solution is added, the fluorescence intensity of the probe solution at 550 nm increases significantly relative to the blank, while the probe shows no significant fluorescence change for other detection targets (Cys, HCY, NCY, DTT, PO4 2- 、S 3- 、HSO - 、F -) There was no particularly obvious fluorescence change; subsequently, after adding 5 times other protein solutions to the detection system, the fluorescence intensity change of the probe at 550nm showed that the addition of other interfering objects did not lead to a decrease in the 6-MP detection results of the probe, which shows that the fluorescent probe has a strong anti-interference ability during the detection process.

[0027] Figure 5 This is the hydrogen spectrum of the coumarin fluorescent probe based on double bond structure prepared in Example 1. 1 H(400MHz, DMSO-d6): δ10.79(s,1H),8.77(s,1H),7.72(dd,J=16.8,8.8Hz,3H),7.48(d,J=8.6Hz,2H),6.86(dd,J=9.1,2.5Hz,1H),6. 70-6.66(m,2H),5.80(d,J=1.0Hz,1H),5.77-5.75(m,1H),5.20(dd,J=11.2,1.0Hz,1H),3.51(q,J=7.2Hz,4H),1.16(t,J=7.2Hz,6H).

[0028] Example 2

[0029] 7-(Diethylamino)coumarin-3-carboxylic acid (0.261 g, 1 mmol), HOBT (0.201 g, 1.5 mmol), and EDCI (0.286 g, 1.5 mmol) were dissolved in dichloromethane, and 4-aminostyrene (0.119 g, 1 mmol) was added. The mixture was stirred at room temperature for 3 h. After the reaction, the dichloromethane was evaporated, and the product was purified by column chromatography using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 3:1 as the developing solvent to obtain 0.0454 g of the final pure product.

[0030] Example 3

[0031] 7-(Diethylamino)coumarin-3-carboxylic acid (0.261 g, 1 mmol), HOBT (0.201 g, 1.5 mmol), and EDCI (0.286 g, 1.5 mmol) were dissolved in dichloromethane, and 4-aminostyrene (0.238 g, 2 mmol) was added. The mixture was stirred at room temperature for 1 h. After the reaction, the dichloromethane was evaporated, and the product was purified by column chromatography using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 3:1 to obtain 0.0653 g of the final pure product.

Claims

1. An application of a fluorescent probe based on a double bond structure, characterized in that: The fluorescent probe is used to prepare a reagent for detecting 6-MP in pure water, PBS, HEPES or Tris-HCl aqueous phase conditions. The structural formula of the fluorescent probe is: 。

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