A Coumarin Fluorescent Probe Based on Trifluorotoluene Derivatives and Its Application

Abstract

The present invention belongs to the field of chemical analysis and testing, and particularly relates to a coumarin-based fluorescent probe derived from trifluorotoluene and its application. 7-Hydroxycoumarin-4-acetic acid and p-bromotrifluorotoluene are dissolved in dimethyl sulfoxide solution, and the reaction solution is heated under reflux at 70-90 °C for 3-4 hours under the alkaline condition of potassium carbonate. The obtained crude product is separated and purified by thin layer chromatography to obtain the coumarin-based fluorescent probe SYL-3 derived from trifluorotoluene. The coumarin-based fluorescent probe obtained in the present invention can rapidly identify cadaverine (Cad) in an aqueous solution of cetyltrimethylammonium bromide (CTAB) surfactant.

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 derived from trifluorotoluene and its application. Background Art

[0002] Cadaverine (Cad) is a compound with a putrid odor produced by the hydrolysis of proteins when animal body tissues decay. It is a paste-like liquid at room temperature, can solidify and crystallize under deep freezing, emits fumes in the air, and can form a dihydrate. The structural formula of cadaverine is NH 2 (CH 2 ) 5 NH 2 , which is formed by the decarboxylation of lysine under the action of decarboxylase during protein spoilage. Higher levels of cadaverine can be found in the urine of patients with lysine metabolic defects. In addition, cadaverine is also a marker of food spoilage. Inhalation of excessive cadaverine may cause inflammation, spasm, edema, pulmonary edema or chemical pneumonia in the throat and bronchus, and even lead to death. Therefore, the development of simple and rapid detection techniques for Cad is of great significance for human life and health and food safety maintenance.

[0003] In recent years, optical imaging based on fluorescent probes has been widely used in various fields due to its advantages of high sensitivity, good selectivity, simple operation, low cost, and the ability to detect in real time in biological microenvironments.

[0004] Currently, a variety of chemical probes for Cad detection have been developed based on fluorescence methods, mainly using various supramolecular methods, including micelles embedded with dyes, sol-gel films, conjugated polymers, and other supramolecular methods. They amplify the sensing response through the self-assembly of the analyte-induced target. Most of these supramolecular structure probes have low selectivity and are susceptible to external stimuli (such as light, heat). Summary of the Invention

[0005] The present invention provides a coumarin-based fluorescent probe derived from trifluorotoluene, and the structural formula of the fluorescent probe is:

[0006]

[0007] The present invention also provides a preparation method of a coumarin-based fluorescent probe derived from trifluorotoluene, and its reaction equation is:

[0008]

[0009] The specific preparation method is as follows: Dissolve 7-hydroxy-coumarin-4-acetic acid and p-bromobenzotrifluoride in dimethyl sulfoxide solution. Under alkaline conditions (potassium carbonate), heat and react at 70-90 °C for 3-4 hours. After the reaction is complete, cool the reaction system to room temperature, remove the organic solvent by vacuum distillation, and separate and purify the obtained compound by thin-layer chromatography in the developing agent to obtain a pale yellow solid product SYL-3.

[0010] Among them, the molar equivalent ratio of 7-hydroxycoumarin-4-acetic acid, p-bromobenzotrifluoride and potassium carbonate is 1:1.1:1.1;

[0011] The developing agent is a mixed solvent of dichloromethane and methanol with a volume ratio of 20:1.

[0012] The present invention also provides an application of a coumarin-based fluorescent probe based on a benzotrifluoride derivative: The coumarin-based fluorescent probe SYL-3 based on a benzotrifluoride derivative can rapidly recognize cadaverine (Cad) in an aqueous solution of cetyltrimethylammonium bromide (CTAB) surfactant.

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

[0014] In the compound structure of the present invention, coumarin is used as a fluorescent group, an electron-donating group (hydroxyl group) is substituted at the 7th position of coumarin, and an electron-withdrawing group (carboxyl group) is substituted at the 4th position, which not only forms an electron push-pull of coumarin but also enhances the water solubility of the coumarin derivative. The ligand structure is mainly a benzotrifluoride derivative, and substitution at multiple sites leads to different physical and chemical properties.

[0015] In the structure of the prepared compound, the ether bond is the action target and can interact with cadaverine (Cad). In the structure of the probe SYL-3, the benzotrifluoride part is an electron donor unit, and the coumarin ring is in a low-energy level orbital as an electron acceptor unit. When the -O-benzotrifluoride part has a specific response to Cad, the ether bond breaks, which will significantly inhibit the intramolecular charge transfer (ICT) process, resulting in fluorescence quenching, thereby achieving the function of recognizing Cad.

[0016] Beneficial effects

[0017] The present invention solves the problem of specific recognition of Cad by the probe SYL-3 in the CTAB solvent system. From the reaction phenomenon, when the probe SYL-3 is added to the CTAB surfactant aqueous solution and the fluorescence intensity is measured, when Cad is added, the fluorescence intensity of the probe SYL-3 at 450 nm decreases significantly, thereby showing the specific recognition effect of the probe SYL-3 on Cad in this system. Description of the drawings

[0018] Figure 1 Fluorescence spectra of the coumarin-based fluorescent probe SYL-3 derived from trifluorotoluene derivative prepared in Example 1 after reacting with different biogenic amines (putrescine (Put), tyramine (Tyr), histamine (His), tryptamine (Try), spermidine (Spd), spermine (Spm), dopamine (DA), cadaverine (Cad)) in CTAB surfactant solution.

[0019] Figure 2 Comparison chart of fluorescence intensity at 450 nm of the coumarin-based fluorescent probe SYL-3 derived from trifluorotoluene derivative prepared in Example 1 after reacting with Cad in 0.2 - 1 mM CTAB surfactant solution.

[0020] Figure 3 Fluorescence spectra of the coumarin-based fluorescent probe SYL-3 derived from trifluorotoluene derivative prepared in Example 1 after reacting with different concentrations of Cad in 0.4 mM CTAB surfactant aqueous solution.

[0021] Figure 4 1H NMR spectrum of the coumarin-based fluorescent probe SYL-3 derived from trifluorotoluene derivative prepared in Example 1.

[0022] Figure 5 Absorption spectra of the fluorescent probe SYL-5 prepared in Comparative Example 1 after reacting with different biogenic amines in CTAB surfactant aqueous solution.

[0023] Figure 6 Fluorescence spectra of the fluorescent probe SYL-5 prepared in Comparative Example 1 after reacting with different biogenic amines in CTAB surfactant aqueous solution. Detailed implementation mode

[0024] The present invention will be described in detail below in conjunction with specific implementation modes.

[0025] Example 1

[0026] Dissolve 7-hydroxycoumarin-4-acetic acid (150 mg, 0.681 mmol) and p-bromotrifluorotoluene (169 mg, 0.749 mmol) in 4 mL of dimethyl sulfoxide solution. In the presence of potassium carbonate (104 mg, 0.749 mmol), heat and react at 85 °C for 3.5 hours. After the reaction is completed, remove the solvent by reduced pressure distillation to obtain a yellow solid. The obtained crude product is separated and purified by thin layer chromatography (developing agent volume ratio: dichloromethane:methanol = 20:1) to obtain 135.6 mg of a pale yellow solid SYL-3, with a yield of 55%.

[0027] The specific application method is as follows: Add 2 μL of 10 mM biogenic amine solution (the BAs in each well are putrescine (Put), tyramine (Tyr), histamine (His), tryptamine (Try), spermidine (Spd), spermine (Spm), dopamine (DA), cadaverine (Cad)), 196 μL of 0.4 mM CTAB surfactant solution, and 2 μL of 2 mM coumarin-based fluorescent probe SYL-3 based on trifluorotoluene into the 96-well plate. At the same time, use the solution containing the fluorescent probe SYL-3 without adding biogenic amine as a control. Mix the solution in each well evenly and measure the fluorescence intensity of the solution in each well. The results show that Cad has an obvious quenching effect on the probe SYL-3, thus showing the specific recognition effect of the probe SYL-3 on Cad in the CTAB solvent system.

[0028] Figure 1 It is the fluorescence spectrogram of the coumarin-based fluorescent probe based on trifluorotoluene derivative prepared in Example 1 after reacting with different biogenic amines (putrescine (Put), tyramine (Tyr), histamine (His), tryptamine (Try), spermidine (Spd), spermine (Spm), dopamine (DA), cadaverine (Cad)) in 0.4 mM CTAB surfactant solution. The figure shows the change in the fluorescence intensity of the probe SYL-3 after adding different biogenic amine solutions. When Cad is added, the fluorescence intensity of the probe SYL-3 at 450 nm decreases significantly, thus showing the specific recognition effect of the compound on Cad in this system.

[0029] Figure 2 It is the comparison chart of the fluorescence intensity at 450 nm of the coumarin-based fluorescent probe based on trifluorotoluene derivative prepared in Example 1 after reacting with Cad in 0.2 - 1 mM CTAB surfactant aqueous solution. The figure shows that as the concentration of the CTAB surfactant aqueous solution increases, the fluorescence intensities of both the probe SYL-3 solution and the Cad + SYL-3 solution system show a downward trend. Among them, compared with the probe solution without adding Cad in CTAB surfactant aqueous solutions of different concentrations, after adding Cad, the fluorescence intensity decreases by about 4 times, and the best detection effect is achieved when the concentration of the CTAB surfactant solution is 0.4 mM. Thus, it shows that this probe can recognize Cad in the CTAB surfactant aqueous solution with a concentration range of 0.2 - 1 mM.

[0030] Figure 3Fluorescence spectra of the coumarin-based fluorescent probe SYL-3 prepared in Example 1 based on trifluorotoluene derivatives after reacting with different concentrations of Cad in an aqueous solution of 0.4 mM CTAB surfactant. As shown in the figure, as the concentration of Cad increases, the fluorescence intensity of the SYL-3 probe solution gradually weakens. When the Cad concentration is in the range of 0 - 500 μM, the higher the concentration, the more obvious the fluorescence quenching phenomenon of the probe. When its final concentration is 10 μM, the fluorescence peak of the probe SYL-3 at 450 nm can still be distinguished from the background fluorescence curve without adding Cad, thus indicating that the detection limit of this probe for Cad is low and the sensitivity is high.

[0031] Figure 4 1H NMR spectrum of the coumarin fluorescent probe SYL-3 prepared in Example 1 based on trifluorotoluene derivatives. 1 HNMR(400MHz,DMSO)δ10.65(s,1H),7.59(d,J=8.8Hz,2H),6.83(m,2H),6.74(d,J=2.4Hz,2H),6.12(s,2H),2.37(s,2H).

[0032] Example 2

[0033] Dissolve 7-hydroxycoumarin-4-acetic acid (300 mg, 1.362 mmol) and p-bromotrifluorotoluene (338 mg, 1.498 mmol) in 8 mL of dimethyl sulfoxide solution. In the presence of potassium carbonate (208 mg, 1.498 mmol), heat the reaction at 85 °C for 3.5 hours. After the reaction is completed, remove the solvent by distillation under reduced pressure to obtain a yellow solid. After separation and purification by thin-layer chromatography (developing agent volume ratio: dichloromethane: methanol = 20:1), 278.6 mg of the final product SYL-3 is obtained, and the yield is 57%.

[0034] Example 3

[0035] Dissolve 7-hydroxycoumarin-4-acetic acid (300 mg, 1.362 mmol) and p-bromotrifluorotoluene (338 mg, 1.498 mmol) in 8 mL of dimethyl sulfoxide solution. In the presence of potassium carbonate (208 mg, 1.498 mmol), heat the reaction at 90 °C for 4 hours. After the reaction is completed, remove the solvent by distillation under reduced pressure to obtain a yellow solid. After separation and purification by thin-layer chromatography (developing agent volume ratio: dichloromethane: methanol = 20:1), 278.1 mg of the final product SYL-3 is obtained, and the yield is 56%.

[0036] Comparative Example 1

[0037] 7-Hydroxycoumarin-4-acetic acid (100 mg, 1.0 mmol) and 2-chlorobenzoxazole (76.8 mg, 1.1 mmol) were dissolved in 8 mL of dimethyl sulfoxide solution. In the presence of potassium carbonate (69 mg, 1.1 mmol), the reaction was heated at 80 °C for 3 hours. After the reaction was completed, the solvent was removed by distillation under reduced pressure to obtain a yellow solid. After separation and purification by thin-layer chromatography (developing agent volume ratio: dichloromethane: methanol = 40:1), 49.1 mg of the final product SYL-5 was obtained, with a yield of 32%. The reaction equation is as follows:

[0038]

[0039] The specific application method is as follows: 2 μL of 10 mM biogenic amine solution (the biogenic amines in each well are putrescine (Put), tyramine (Tyr), tryptamine (Try), spermidine (Spd), spermine (Spm), benzylamine (Phm), cadaverine (Cad)), 196 μL of 0.4 mM CTAB surfactant solution and 2 μL of 5 mM benzoxazole-based coumarin fluorescent probe SYL-5 were added to a 96-well plate respectively. At the same time, a solution containing the fluorescent probe SYL-5 without biogenic amine was used as a control. The solutions in each well were mixed evenly, and the fluorescence intensity of the solution in each well was measured. The results showed that the above biogenic amines had an obvious quenching effect on the probe SYL-5, thus indicating that SYL-5 had a recognition effect on the above seven biogenic amines in the CTAB solvent system.

[0040] Compared with the coumarin fluorescent probe SYL-3 based on trifluorotoluene derivatives prepared in Example 1, different ligands were connected to the same coumarin dye for both of them, showing different screening effects. SYL-3 only had an obvious specific recognition effect on Cad; SYL-5 had a recognition effect on a variety of biogenic amines, with poor specificity.

[0041] Figure 5 To compare the absorption spectra of the coumarin fluorescent probe SYL-5 based on benzoxazole derivatives prepared in Example 1 after reacting with different biogenic amines (putrescine (Put), tyramine (Tyr), tryptamine (Try), spermidine (Spd), spermine (Spm), benzylamine (Phm), cadaverine (Cad)) in a 0.4 mM CTAB surfactant solution. As shown in the figure, the change in the absorption spectrum of the probe SYL-5 after adding different biogenic amine solutions is shown. As shown in the figure, when different biogenic amines were added, the absorption peak of the probe SYL-5 at 320 nm decreased significantly, thus indicating that the compound had a recognition effect on various biogenic amines in this system, but the selectivity was poor.

[0042] Figure 6Fluorescence spectra of the coumarin-based fluorescent probe SYL-5 prepared for Comparative Example 1 after reacting with different biogenic amines (putrescine (Put), tyramine (Tyr), tryptamine (Try), spermidine (Spd), spermine (Spm), benzylamine (Phm), cadaverine (Cad)) in a 0.4 mM CTAB surfactant solution. As shown in the figure, the changes in the fluorescence spectra of the probe SYL-5 after adding different biogenic amine solutions are shown. As shown in the figure, when different biogenic amines are added, the fluorescence intensity of the probe SYL-5 at 450 nm is significantly quenched, indicating that the compound has a recognition effect on various biogenic amines in this system, but the selectivity is poor.

Claims

1. A coumarin fluorescent probe based on trifluorotoluene derivatives, characterized in that: the structural formula of the fluorescent probe is: 。 2. A preparation method of the coumarin fluorescent probe based on trifluorotoluene derivatives as described in claim 1, characterized in that: the preparation method is: dissolving 7-hydroxycoumarin-4-acetic acid and p-bromotrifluorotoluene in dimethyl sulfoxide solution, heating the reaction solution under reflux at 70-90 °C for 3-4 hours under the alkaline condition of potassium carbonate, after the reaction is complete, cooling the reaction system to room temperature, distilling off the organic solvent under reduced pressure, and separating and purifying the obtained compound by thin layer chromatography in the developing agent to obtain a light yellow solid product, namely the coumarin fluorescent probe based on trifluorotoluene derivatives as described in claim 1.

3. The preparation method of the coumarin fluorescent probe based on trifluorotoluene derivatives as described in claim 2, characterized in that: the molar ratio of 7-hydroxycoumarin-4-acetic acid, p-bromotrifluorotoluene and potassium carbonate is 1:1.1:1.

1.

4. The preparation method of the coumarin fluorescent probe based on trifluorotoluene derivatives as described in claim 2, characterized in that: the developing agent is a mixed solvent of dichloromethane and methanol with a volume ratio of 20:

1.

5. An application of the coumarin fluorescent probe based on trifluorotoluene derivatives as described in claim 1, characterized in that: the application of the coumarin fluorescent probe based on trifluorotoluene derivatives in the preparation of a reagent for rapidly identifying cadaverine in an aqueous solution of cetyltrimethylammonium bromide surfactant.

6. The application of the coumarin fluorescent probe based on trifluorotoluene derivatives as described in claim 5, characterized in that: the concentration of the aqueous solution of cetyltrimethylammonium bromide surfactant is 0.2-1 mM.

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