Thiophenyl chloroformate-based resorufin-based fluorescent probes for detecting biological amines

Abstract

The present application belongs to the field of chemical analysis test, and particularly relates to a phenyl thiochloroformate-based resorcinol fluorescent probe for detecting biological amine. The resorcinol and phenyl thiochloroformate are reacted for 4-5 hours under the catalysis of N,N-diisopropyl ethylamine to obtain the target fluorescent probe. The fluorescent probe has the characteristics that the easily-obtained raw materials are used, and the thioester group structure is integrated into the fluorescent probe molecule through one-step reaction. The synthesis method is simple, the reaction condition is easy to control, and the product is convenient to purify. The fluorescent probe has superior performance in the detection of biological amine, high sensitivity and strong selectivity, and can rapidly and accurately identify biological amine in various solvents such as deionized water, aqueous solution of tris(hydroxymethyl) aminomethane hydrochloride, aqueous solution of cetyltrimethylammonium bromide, aqueous solution of sodium dodecyl sulfate or dimethyl sulfoxide solution, which provides a good precondition for the practical application of the fluorescent probe in biological samples.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of chemical analysis test, and particularly relates to a phenyl chlorothioformate-based resorcinol fluorescent probe for detecting biological amine. BACKGROUND

[0002] Biological amine (BAs) is a kind of organic compounds widely existing in organisms and foods, which is mainly produced by the action of microorganisms on proteins and amino acids. In food science, the presence of biological amine is often used as an important indicator to judge the freshness and degree of bacterial spoilage of food. Common biological amine includes histamine, cadaverine, tyramine, phenethylamine, etc., which widely exist in dairy products, meat products, condiments, wine and other foods rich in proteins and amino acids. The influence of biological amine on human body is mainly related to its allergenicity and toxicity. For example, histamine is a strong allergen, and excessive intake may cause allergic reactions, and even cause shock in severe cases. And cadaverine and other biological amine also have significant toxicity, and long-term intake may cause chronic damage to the human body, including damage to the liver and nervous system. Therefore, it is of great significance to develop reliable biological amine detection technology for food safety and human health protection.

[0003] Traditional biological amine detection methods include thin layer chromatography (TLC), capillary electrophoresis (CE), gas chromatography-mass spectrometry (GC-MS) and high performance liquid chromatography (HPLC), etc. Although the results of these methods are accurate and reliable, their operation is complex, time-consuming and requires large instruments, which greatly limits their practical application.

[0004] At present, although the fluorescent probe analysis method has achieved certain results in biological amine detection, it is also limited by the structure and performance of the probe. The current probe mostly uses coumarin, naphthalene derivatives and other fluorescent groups, and the fluorescence band is concentrated in the 350-500nm visible light region, so it is limited in distinguishing different biological amine and other biological molecules. Moreover, the existing probe targeting is focused on specific aldehyde ketone, anhydride structure, and cannot fully adapt to the diversified structure response of biological amine. Therefore, it is urgent to develop more diversified small molecule fluorescent probes. SUMMARY

[0005] The present application provides a phenyl chlorothioformate-based resorcinol fluorescent probe, and the structural formula of the fluorescent probe is as follows:

[0006]

[0007] The present application also provides a preparation method of a phenyl chlorothioformate-based resorcinol fluorescent probe, and the reaction equation is as follows:

[0008]

[0009] The specific preparation method is as follows: the resorcinol and phenyl chlorothioformate are fully mixed in anhydrous dichloromethane solution, then the reaction is carried out in an ice water bath under the catalysis of N,N-diisopropyl ethylamine (DIPEA) for 4-5 hours to ensure the completion of the reaction. After the reaction is completed, the organic solvent in the solution is effectively removed by the vacuum distillation technology. Then, the obtained compound is separated and purified by thin layer chromatography in a specific developing agent, and the obtained product is a yellow solid SYL-1.

[0010] The molar equivalent ratio of the resorcinol, the phenyl chlorothioformate and the N,N-diisopropyl ethylamine is 1:2:1.

[0011] The developing agent is a mixed solvent of dichloromethane and methanol with a volume ratio of 60:1.

[0012] The application also provides an application of the resorcinol fluorescent probe based on the phenyl chlorothioformate. The resorcinol fluorescent probe SYL-1 based on the phenyl chlorothioformate can quickly identify various biological amines in various solution environments.

[0013] The detection solution environment includes deionized water (H2O), a trimethylol aminomethyl methane hydrochloride aqueous solution (Tris-Hcl buffer solution), a cetyl trimethyl ammonium bromide aqueous solution (CTAB, 4M), a sodium dodecyl sulfate aqueous solution (SDS, 4M) or a dimethyl sulfoxide solution (DMSO).

[0014] The biological amine is an aliphatic amine (putrescine (Put), cadaverine (Cad), spermidine (Spd), spermine (Spm)), an aromatic amine (2-phenylethylamine (Phm), tyramine (Tyr)) and a heterocyclic amine (tryptamine (Try), histamine (His)).

[0015] The resorcinol is used as a fluorophore in the structure of the compound, and the benzene ring and the nitrogen-oxygen heterocyclic ring system in the resorcinol molecule are the core components of the structure. The benzene ring is a stable ring structure composed of six carbon atoms, which endows the resorcinol molecule with high stability. At the same time, the presence of the benzene ring also enables the resorcinol to absorb and emit light of a specific wavelength, which is the basis for its fluorescent properties. The introduction of the nitrogen-oxygen heterocyclic ring makes the resorcinol molecule have more reaction sites and more complex electronic structure, thereby endowing it with unique chemical properties. This heterocyclic structure can interact with various chemical substances, including nucleophilic substitution, oxidation and reduction reactions, so that the resorcinol can be used as a chemical sensor or indicator to detect certain specific chemical substances. In addition to the above structural characteristics, the resorcinol also has a certain solubility. Because its molecular structure contains both hydrophilic groups (such as oxygen atoms) and hydrophobic groups (such as benzene rings), the resorcinol can be partially dissolved in water, which facilitates its application in aqueous solutions.

[0016] The thioester moiety in the structure of the prepared compound is a target point that can interact with biological amines (BAs). In the reaction of the probe SYL-1 with biological amines, the thioester group is aminated, and the resorufin fluorophore is released, thereby producing strong fluorescence.

[0017] Advantages

[0018] The raw material of the present application is easy to obtain, the thioester structure is introduced into the fluorescent probe through one-step reaction, the synthesis method is simple, the reaction condition is easy to control, and after the reaction is completed, the pure product can be obtained through simple post-treatment; as a fluorescent probe for biological amine detection, the resorufin-based fluorescent probe SYL-1 based on phenyl thiochloroformate has high sensitivity, good selectivity, and can quickly identify biological amines in various solutions, thereby providing a good precondition for the application of biological samples. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The fluorescence intensity column chart of the resorufin-based fluorescent probe SYL-1 based on phenyl thiochloroformate prepared in Example 1 before and after the reaction with biological amines (taking Cad as an example) at 605 nm in different solution environments.

[0020] Figure 2 The fluorescence spectrum of the resorufin-based fluorescent probe SYL-1 based on phenyl thiochloroformate prepared in Example 1 before and after the reaction with different biological amines (including aliphatic amines (putrescine (Put), cadaverine (Cad), spermidine (Spd), spermine (Spm)), aromatic amines (2-phenylethylamine (Phm), tyramine (Tyr)) and heterocyclic amines (tryptamine (Try), histamine (His)) in DMSO solution.

[0021] Figure 3 The fluorescence spectrum of the resorufin-based fluorescent probe SYL-1 based on phenyl thiochloroformate prepared in Example 1 after the reaction with biological amines (taking Cad as an example) in DMSO solution.

[0022] Figure 4 The fluorescence intensity column chart of the resorufin-based fluorescent probe SYL-1 based on phenyl thiochloroformate prepared in Example 1 at 605 nm after the reaction with biological amines (taking Cad as an example) in DMSO solution after adding five times of interfering substances.

[0023] Figure 5 The hydrogen spectrum of the resorufin-based fluorescent probe SYL-1 based on phenyl thiochloroformate prepared in Example 1. DETAILED DESCRIPTION

[0024] The present application will be described in detail below in combination with specific implementation manners.

[0025] Example 1

[0026] Halogen (100 mg, 1.0 mmol) and phenyl thiochloroformate (161.2 mg, 2 mmol) were dissolved in 10 mL of anhydrous dichloromethane. The reaction was carried out in an ice-water bath for 4 h in the presence of a basic catalyst, DIPEA (0.2 mL). After the reaction was completed, the solvent was removed by vacuum distillation to obtain a yellow solid. The crude product was purified by thin-layer chromatography (developing solvent volume ratio: dichloromethane:methanol = 60:1). The final product, SYL-1, was a yellow solid (103.56 mg, yield 63%).

[0027] The specific application method is as follows:

[0028] Add 2 μL of 10 mM cadaverine solution, 196 μL of detection solution (deionized water, phosphate-buffered saline (PBS), Tris-HCl buffer, HEPES buffer, 4 mM cationic surfactant CTAB aqueous solution, 4 mM anionic surfactant SDS aqueous solution, or DMSO solution), and 2 μL of 5 mM cinnamyl chloride-based halogenated fluorescent probe SYL-2 to each well of a 96-well plate. A control solution containing SYL-2 without cadaverine was used. Mix the solutions in each well thoroughly and measure the fluorescence intensity of each solution.

[0029] Figure 1 The bar chart shows the fluorescence intensity at 605 nm of the phenyl thiocarbamate-based fluorescent probe SYL-1, prepared in Example 1, before and after interaction with biogenic amines (using Cad as an example) in different solution environments. The chart shows that before the addition of Cad, the fluorescence intensity of the probe solution was relatively weak in different solutions. After the addition of Cad, the fluorescence intensity of probe SYL-1 generally increased under different solution conditions. Good detection performance was observed in deionized water (H2O), Tris-HCl buffer, CTAB aqueous solution, SDS aqueous solution, and DMSO solution.

[0030] A certain mass of the fluorescent probe SYL-1 was weighed and dissolved in dimethyl sulfoxide to prepare a probe stock solution with a concentration of 5M. Then, a series of 10M biogenic amine stock solutions were prepared using deionized water, including aliphatic amines (putrescine, cadaverine, spermidine, spermine), aromatic amines (2-phenylethylamine, tyramine), and heterocyclic amines (tryptophan, histamine). At room temperature, 2 μL of each biogenic amine stock solution was added to a 96-well plate using a pipette. Then, 196 μL of screening solution was added to each well sequentially to ensure a consistent total volume of the reaction system. Finally, 2 μL of the probe stock solution was accurately measured and added to the well plate, and thoroughly mixed to ensure that the concentration of probe SYL-1 in each well was 50 μM and the concentration of biogenic amine was 100 μM. The solutions in each well were mixed thoroughly, and the fluorescence intensity of the solution in each well was measured. The results showed that biogenic amines significantly enhanced the fluorescence of probe SYL-1, demonstrating the rapid recognition of biogenic amines by probe SYL-1 in various solution environments.

[0031] Figure 2 The fluorescence spectra of SYL-1, a halogenated fluorescent probe based on phenyl thiochloroformate prepared in Example 1, before and after interaction with different biogenic amines (including aliphatic amines such as putrescine (Put), cadaverine (Cad), spermidine (Spd), and spermine (Spm)), aromatic amines such as 2-phenylethylamine (Phm) and tyramine (Tyr)), and heterocyclic amines such as tryptamine (Try) and histamine (His)) in DMSO solution are shown in the figure. The changes in fluorescence intensity of probe SYL-1 after adding different biogenic amine solutions are illustrated. When different types of biogenic amine solutions were added, the fluorescence intensity of probe SYL-1 at 605 nm was significantly enhanced, demonstrating the recognition effect of compound SYL-1 on various biogenic amines in this system.

[0032] Figure 3 The fluorescence spectra of the SYL-1 halogenated fluorescent probe, prepared in Example 1 and reacted with different concentrations of biogenic amines (Cad, for example) in DMSO solution are shown. The figures show that the fluorescence intensity of the SYL-1 probe solution increases significantly with increasing Cad concentration. When the Cad concentration varies from 1 to 300 μM, the higher the Cad concentration, the more significant the fluorescence enhancement. Even at a final Cad concentration of 1 μM, the fluorescence peak of the SYL-1 probe at 605 nm is still effectively distinguishable from the background fluorescence curve without Cad. This demonstrates that the SYL-1 probe exhibits a low detection limit and high sensitivity for biogenic amines (Cad, for example) in DMSO solution.

[0033] Figure 4The fluorescence intensity at 605 nm of the phenyl thiocarbamate-based fluorescent probe SYL-1, prepared in Example 1, was obtained by reacting it with a biogenic amine (Cad, for example) in DMSO solution after adding five times the amount of interfering substances (glycine (GLY), alanine (ALA), 2-aminopyridine (APR), aniline (Ani), o-phenylenediamine (1,2-PD), thiourea (TU), cysteine ​​(Cys), and proline (Pro)). The figure shows that the fluorescence intensity of the probe solution containing Cad is significantly enhanced compared to the fluorescence intensity of the probe itself, while the fluorescence intensity of the probe solution containing other interfering substances is lower. Furthermore, adding five times the equivalent amount of other competing substances to the mixture of probe SYL-1 and Cad did not affect the fluorescence intensity of the probe. This demonstrates that the fluorescent probe exhibits strong anti-interference ability during detection.

[0034] Figure 5 The proton spectrum of SYL-1, a halogenated fluorescent probe based on phenyl thiochloroformate prepared in Example 1. 1 HNMR(400MHz,DMSO)δ7.98(d,J=8.8Hz,1H),7.68(d,J=2.4Hz,1H),7.60-7.52(m,3H),7 .49–7.46(m,1H),7.39-7.36(m,3H),6.86(dd,J=9.8,2.0Hz,1H),6.34(d,J=2.0Hz,1H).

[0035] Example 2

[0036] Halogen (200 mg, 2.0 mmol) and phenyl thiochloroformate (322.4 mg, 4.0 mmol) were dissolved in 20 mL of anhydrous dichloromethane. The reaction was carried out in an ice-water bath for 4 h in the presence of an alkaline catalyst, DIPEA (0.4 mL). After the reaction was completed, the solvent was removed by vacuum distillation to obtain a yellow solid. The crude product was purified by thin-layer chromatography (developing solvent volume ratio: dichloromethane:methanol = 60:1), and the final product SYL-1 was a yellow solid (217.39 mg, yield 66%).

[0037] Example 3

[0038] Halogen (50.0 mg, 0.5 mmol) and phenyl thiochloroformate (80.6 mg, 1.0 mmol) were dissolved in 5 mL of anhydrous dichloromethane. The reaction was carried out in an ice-water bath for 5 h in the presence of an alkaline catalyst, DIPEA (0.1 mL). After the reaction was completed, the solvent was removed by vacuum distillation to obtain a yellow solid. The crude product was purified by thin-layer chromatography (developing solvent volume ratio: dichloromethane:methanol = 60:1), and the final product SYL-1 was a yellow solid (48.72 mg, yield 59%).

Claims

1. An application of a halogenated fluorescent probe based on phenyl thiochloroformate, characterized in that: The phenyl thiochloroformate-based halogenated fluorescent probe is used to prepare reagents for recognizing biogenic amines in solution; The solution is: deionized water, tris(hydroxymethyl)aminomethane hydrochloride aqueous solution, hexadecyltrimethylammonium bromide aqueous solution, sodium dodecyl sulfate aqueous solution, or dimethyl sulfoxide solution; The structural formula of the fluorescent probe is: ； The biogenic amine is selected from one of putrescine, cadaverine, spermidine, spermine, 2-phenylethylamine, tyramine, tryptamine, and histamine.

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