A nopinyl fluorescent probe for detecting methyl parathion based on enzyme inhibition mechanism, and its preparation method and application

Abstract

The present invention discloses a pinanyl fluorescent probe for detecting the organophosphorus pesticide methyl parathion based on an enzyme inhibition mechanism, as well as its preparation method and application. The fluorescent probe is: 4-(2-(4-(cyclopropylformyloxy-4′-(6,6-dimethyl-2-(4-nitrophenyl)-4,5,6,7-tetrahydro-2H-5,7-methyleneindazole-3-yl)-[1,1′-biphenyl]-3-yl)vinyl)-1-methylpyridinium iodide (abbreviated as: THIP-OCP). The compound can undergo enzymatic hydrolysis reaction with butyrylcholinesterase and, under irradiation with ultraviolet light at a wavelength of 365nm, The solution's fluorescence color changes from colorless to orange-yellow. Since methyl parathion effectively inhibits butyrylcholinesterase activity, when methyl parathion is added to the solution, the solution's fluorescence gradually changes from orange-yellow to colorless under ultraviolet irradiation at a wavelength of 365 nm. Therefore, this compound can be used as a fluorescent probe for detecting methyl parathion. This compound has many advantages, including a low detection limit (0.79 μg / mL) and a wide pH range (5-10), and has great application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of fine organic synthesis, and relates to a nopinyl fluorescent probe for detecting methyl parathion based on an enzyme inhibition mechanism, and a preparation method and application thereof. Background Art

[0002] Pesticides are an indispensable component of modern agricultural development, not only increasing crop yields but also eliminating pests and weeds. Methyl parathion is a highly toxic, highly effective, and broad-spectrum organophosphorus insecticide. Excessive use and improper handling of methyl parathion lead to residues in soil, water, and food, which then enter the human body through the food chain, posing a serious threat to the ecological environment and human safety. Therefore, developing an analytical method for the detection of methyl parathion is of great significance.

[0003] Currently, the main methods for detecting methyl parathion include electrochemical methods, enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), and surface-enhanced Raman spectroscopy (SERS). These traditional methods suffer from cumbersome pretreatment processes, complex operations, bulky detection instruments, and high costs, making them incapable of rapid detection of methyl parathion. Compared to these traditional detection methods, fluorescent probes have gained popularity due to their simple synthesis, good selectivity, high sensitivity, and ease of operation. Combining the principles of enzyme inhibition with fluorescent probe technology offers a new approach to achieving rapid, high-throughput, low-detection-line, and highly sensitive detection of pesticides. Therefore, developing an analytical method for detecting methyl parathion based on the principles of enzyme inhibition and fluorescent probe technology is of great significance. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the technical problem to be solved by the present invention is to provide a pinanyl fluorescent probe for detecting methyl parathion based on an enzyme inhibition mechanism, which can meet the usage requirements.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] A pinanyl fluorescent probe for detecting methyl parathion based on an enzyme inhibition mechanism, the structural formula of which is:

[0007]

[0008] The fluorescent probe has the molecular formula: C 40 H 37 N4IO4, named as: 4-(2-(4-(cyclopropylcarbonyloxy-4′-(6,6-dimethyl-2-(4-nitrophenyl)-4,5,6,7-tetrahydro-2H-5,7-endomethylindazol-3-yl)-[1,1′-biphenyl]-3-yl)vinyl)-1-methylpyridinium iodide, referred to as: THIP-OCP.

[0009] The method for preparing a nopinyl fluorescent probe for detecting methyl parathion based on an enzyme inhibition mechanism comprises the following steps:

[0010] 4-(2-(4′-(6,6-dimethyl-2-(4-nitrophenyl)-4,5,6,7-tetrahydro-2H-5,7-methanoindazol-3-yl)-4-hydroxy-[1,1′-biphenyl]-3-yl)vinyl)-1-methylpyridinium iodide (abbreviated as THIP-OH) is used as a raw material and subjected to an esterification reaction with cyclopropylcarbonyl chloride to obtain 4-(2-(4-(cyclopropylcarbonyloxy-4′-(6,6-dimethyl-2-(4-nitrophenyl)-4,5,6,7-tetrahydro-2H-5,7-methanoindazol-3-yl)-[1,1′-biphenyl]-3-yl)vinyl)-1-methylpyridinium iodide (abbreviated as THIP-OCP).

[0011] The specific preparation method of the compound THIP-OCP includes:

[0012] (1) 0.113-0.170 g (0.2-0.3 mmol) of THIP-OH, 0.031-0.052 g (0.3-0.5 mmol) of cyclopropylcarbonyl chloride, and 2000-2500 mL of anhydrous dichloromethane were added sequentially into a three-necked flask equipped with a stirrer and reacted at 0-10°C for 10-12 h under nitrogen protection;

[0013] (2) The reaction solution is distilled to recover the solvent, thereby obtaining a crude product of the compound THIP-OCP;

[0014] (3) The crude product of compound THIP-OCP was purified by silica gel column chromatography (dichloromethane:methanol=10:1) to obtain compound THIP-OCP as a brown-red powder.

[0015] The compound THIP-OCP can specifically undergo an enzymatic hydrolysis reaction with butyrylcholinesterase. Under irradiation with ultraviolet light at a wavelength of 365nm, the fluorescent color of the solution changes from colorless to orange-yellow. Methyl parathion can inhibit the activity of butyrylcholinesterase. Under irradiation with a 365nm ultraviolet lamp, the fluorescent color of the probe solution changes from orange-yellow to colorless.

[0016] Beneficial Effects: Compared with existing technologies, the compound THIP-OCP of the present invention can specifically detect methyl parathion and sensitively measure its content in solution. The detection range for methyl parathion is 0.8 to 450 μg / mL, with a detection limit as low as 0.79 μg / mL. As a fluorescent probe for the detection of methyl parathion, it has many advantages, including easy synthesis, good selectivity, and high sensitivity, and has promising application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the fluorescence spectrum of the compound THIP-OCP interacting with different pesticides;

[0018] Figure 2 This is the fluorescence spectrum of the compound THIP-OCP reacting with different concentrations of methyl parathion. DETAILED DESCRIPTION

[0019] The present invention will be further described below with reference to specific embodiments.

[0020] Example 1

[0021] Preparation of compound THIP-OCP

[0022] The reaction formula is as follows:

[0023]

[0024] The specific steps are as follows:

[0025] 0.113-0.170 g (0.2-0.3 mmol) of compound THIP-OH, 0.031-0.052 g (0.3-0.5 mmol) of cyclopropylcarbonyl chloride, and 20 mL of anhydrous acetonitrile were added sequentially to a three-necked flask equipped with a stirrer. The mixture was reacted at 10°C under nitrogen protection for 5 hours. The solvent was recovered by distillation from the reaction solution to obtain a crude product of compound THIP-OCP. The crude product was then chromatographed on a silica gel column (dichloromethane:methanol = 10:1) to obtain a brownish-red powder compound THIP-OCP with a yield of 56%. 1 H NMR (600MHz, DMSO-d6) δ8.96 (d, J=6.2Hz, 2H), 8.37 (dd, J=8.9, 3.8Hz, 2H), 8.28 (dd, J=20.0, 4.2Hz, 3H), 8.00 (d, J=8. 1Hz, 2H), 7.92-7.86 (m, 3H), 7.85-7.81 (m, 3H), 7.76 (d, J=16.3Hz, 1H), 7.34 (dd, J=8.4, 4.9Hz, 1H), 4.31 (s, 3H), 3.26 (t, J=5.3Hz, 1H), 3.11 (dd, J=15.8, 3.0Hz, 1H), 3.00-2.93 (m, 1H), 2.78 (dt, J=9.6, 5.8Hz, 1H), 2.40 (tq, J=8.5, 5.1, 4 .0Hz, 1H), 2.10 (tq, J=10.5, 5.9, 5.3Hz, 1H), 1.46 (d, J=10.2Hz, 4H), 1.12 (ddt, J=29.2, 7.8, 3.7Hz, 4H), 0.77 (s, 3H).13 C NMR (150MHz, DMSO-d6) δ: 173.25, 152.57, 150.79, 149.05, 148.89, 145.86, 145.77, 144.41, 139.42, 137.29, 132.83, 131.94, 129.56, 128.68, 127.66, 126.80, 126.26, 125.29, 124.42, 123.58, 113.78, 41.22, 31.61, 30.29, 29. 48, 29.16, 26.73, 26.23, 22.55, 21.73, 14.41, 12.50, 10.51.HRMS (m / z): [M] + calcd for C 40 H 37 N4O4 + , 637.2809; found, 637.2818.

[0026] Example 2

[0027] The compound THIP-OCP was prepared into 1×10 -5 M PBS buffer solution (pH = 7.4, DMSO / PBS = 2 / 8, 10mM), and at the same time, carbaryl, methomyl, thiacloprid, imidacloprid, methyl parathion, chlorpyrifos, dichlorvos, and dimethoate were dissolved in DMSO to prepare solutions with a concentration of 400μg / mL respectively. The fluorescence emission spectrum of the compound THIP-OCP in the presence of different pesticides was measured on a fluorescence spectrophotometer using fluorescence spectrometry titration method, as shown in FIG. Figure 1 The results show that the fluorescence emission intensity is lowest in the presence of methyl parathion. This indicates that the compound THIP-OCP can be used as a fluorescent probe to detect methyl parathion using the enzyme inhibition principle.

[0028] Example 3

[0029] The compound THIP-OCP was prepared into 1×10 -5 M PBS buffer solution (pH = 7.4, DMSO / PBS = 2 / 8, 10mM), and methyl parathion was dissolved in PBS buffer to prepare solutions with concentrations of 0, 50, 100, 150, 200, 250, 300, 350, 400 and 450 μg / mL. The fluorescence emission spectrum of the compound THIP-OCP in the presence of different concentrations of methyl parathion was measured on a fluorescence spectrophotometer using fluorescence spectrometry. Figure 2The results showed that as the concentration of methyl parathion in the solution gradually increased from 0 μg / mL to 450 μg / mL, the fluorescence emission intensity of the compound THIP-OCP at 547 nm gradually decreased. This indicates that the compound can be used as a fluorescent probe for the sensitive detection of methyl parathion.

Claims

1. A pininyl fluorescent probe THIP-OCP for detecting methyl parathion based on an enzyme inhibition mechanism, characterized in that: Its structural formula is:

2. The method for preparing the nopinanyl fluorescent probe for detecting methyl parathion based on the enzyme inhibition mechanism according to claim 1, characterized in that: The steps include: 4-(2-(4′-(6,6-dimethyl-2-(4-nitrophenyl)-4,5,6,7-tetrahydro-2H-5,7-methanoindazol-3-yl)-4-hydroxy-[1,1′-biphenyl]-3-yl)vinyl)-1-methylpyridinium iodide (abbreviated as THIP-OH) was used as a raw material and subjected to an esterification reaction with cyclopropylcarbonyl chloride to obtain 4-(2-(4-(cyclopropylcarbonyloxy-4′-(6,6-dimethyl-2-(4-nitrophenyl)-4,5,6,7-tetrahydro-2H-5,7-methanoindazol-3-yl)-[1,1′-biphenyl]-3-yl)vinyl)-1-methylpyridinium iodide (abbreviated as THIP-OCP).

3. Use of the compound THIP-OCP according to claim 1 in detecting methyl parathion, wherein the purpose of the use is not to diagnose or treat a disease.

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