A highly selective hypobromous acid fluorescent probe, preparation method and application

Abstract

The present invention relates to a highly selective fluorescent probe for identifying hypobromous acid. Specifically, the probe of the present invention can be used to measure, detect or screen hypobromous acid, can realize selective recognition of hypobromous acid, can realize rapid and highly sensitive analysis of hypobromous acid, and can also realize detection of hypobromous acid in real water samples, and has the advantages of simple synthesis, stable properties, and is conducive to commercial application.

Description

Technical Field

[0001] The invention belongs to the field of fluorescent probes, and specifically relates to a fluorescent probe of a pyrene compound based on a benzothiazoline group and an application thereof in measuring, screening or detecting hypobromous acid; the invention also provides a method for preparing the fluorescent probe. Background Art

[0002] Saltwater intrusion in coastal areas can lead to an increase in the concentration of bromide ions in water sources. Bromide ions will be oxidized to hypobromous acid during the chlorination of tap water, and the resulting hypobromous acid will react with natural organic matter (NOM) to form brominated disinfection byproducts (Br-DBPs), which will harm aquatic organisms and human health. Therefore, detecting the concentration of hypobromous acid in the water environment plays an important role in controlling the generation and transformation of brominated disinfection byproducts. And hypobromous acid, as a reactive oxygen species (ROS) in the human body, plays a key role in mediating physiological and pathological processes. It is a highly efficient oxidant and antibacterial agent in the human body, and is the product of the peroxidation reaction of bromide ions and hydrogen peroxide catalyzed by eosinophilic peroxidase (EPO). However, excessive hypobromous acid can cause severe irreversible damage to a variety of biological molecules and is an important cause of diseases such as inflammation and cancer. Therefore, in situ detection of hypobromous acid in living systems plays a key role in understanding the physiological and pathological processes mediated by hypobromous acid.

[0003] As a classic non-invasive detection method, fluorescent probe analysis is widely used in the detection of biomolecules in life systems due to its high selectivity, good sensitivity, real-time monitoring and high spatiotemporal resolution. So far, the construction of fluorescent probes for the detection of hypobromous acid is relatively limited, because the physicochemical properties of hypobromous acid and hypochlorous acid are very similar and the concentration of the former is much lower than that of the latter under physiological conditions. Therefore, the development of simple and highly selective probes for the detection of hypobromous acid in water environments and organisms has become an urgent research topic. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a class of highly sensitive and highly selective hypobromous acid fluorescent probes, as well as their preparation methods and uses, which have the characteristics of simple synthesis, high sensitivity, high selectivity and rapid response, and can not only be used for the measurement, screening or detection of hypobromous acid in water environments, but also for fluorescent imaging of cells.

[0005] Specifically, the present invention provides a fluorescent probe for measuring, detecting or screening hypobromous acid, having a structure shown in formula (I):

[0006]

[0007] In formula (I), R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 , R11 , R 12 and R 13 is a free hydrogen atom, a straight-chain or branched alkyl group, a straight-chain or branched alkoxy group, a sulfonic acid group, an ester group, or a carboxyl group; R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 and R 13 Can be the same or different.

[0008] In some specific embodiments of the present invention, the fluorescent probes of the present invention are R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 and R 13 The compound of formula (II) wherein all of them are hydrogen atoms has the following structural formula:

[0009]

[0010] The present invention also provides a method for preparing a fluorescent probe of formula (I), comprising the following steps: reacting a compound of formula (III) with a compound of formula (IV) to prepare a fluorescent probe of formula (I), wherein the reaction formula is as follows:

[0011]

[0012] In formula (I), (III) and (IV): wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 and R 13 is a free hydrogen atom, a straight-chain or branched alkyl group, a straight-chain or branched alkoxy group, a sulfonic acid group, an ester group, or a carboxyl group; R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 and R 13 Can be the same or different.

[0013] In some specific embodiments of the present invention, the compound of formula (IV) is dissolved in anhydrous ethanol, and then a certain molar ratio of the compound of formula (III) is added, and the mixture is stirred at room temperature for reaction. After the reaction is completed, the crude product is obtained by vacuum filtration, and then separated and purified by column chromatography to obtain a pure product.

[0014] In some specific embodiments of the present invention, the molar ratio of the compound of formula (IV) to the compound of formula (III) is 1:1-1:2.

[0015] In some specific embodiments of the present invention, the room temperature reaction time is 3-6 hours.

[0016] In some specific embodiments of the present invention, 1-pyrenecarboxaldehyde and 2-aminothiophenol in a molar ratio of 1:2 are dissolved in ethanol and stirred at room temperature for 6 hours. After the reaction is completed, vacuum filtration is performed to obtain a crude product, which is separated and purified by column chromatography to obtain a pure fluorescent probe of formula (II).

[0017] The present invention also provides a fluorescent probe composition for measuring, screening or detecting hypobromous acid, which comprises the fluorescent probe of formula (I) of the present invention.

[0018] In some specific embodiments of the present invention, the fluorescent probe of formula (I) has the following structure:

[0019]

[0020] In some specific embodiments of the present invention, the fluorescent probe composition further comprises a solvent, an acid, a base, a buffer solution or a combination thereof.

[0021] The present invention also provides the use of the fluorescent probe of formula (I) in the preparation, measurement, screening or detection of hypobromous acid reagents.

[0022] The present invention also provides a method for detecting the presence of hypobromous acid in a sample or determining the content of hypobromous acid in a sample, comprising:

[0023] a) contacting the fluorescent probe of formula (I) or (II) with a sample to form a fluorescent compound;

[0024] b) determining the fluorescence property of the fluorescent compound.

[0025] In some specific embodiments of the invention, the sample is a chemical sample.

[0026] In some embodiments of the invention, the sample is a biological sample.

[0027] In some specific embodiments of the invention, the sample is an aqueous environment sample.

[0028] The present invention also provides a kit for detecting the presence of hypobromous acid in a sample or determining the content of hypobromous acid in a sample, which comprises the fluorescent probe of formula (I) or formula (II).

[0029] The present invention also provides the use of the fluorescent probe of formula (I) or (II) in cell fluorescence imaging.

[0030] Compared with the prior art, the present invention has the following significant advantages and effects:

[0031] 1. High selectivity and strong anti-interference ability. The hypobromous acid fluorescent probe of the present invention reacts with potassium ion, calcium ion, sodium ion, magnesium ion, zinc ion, ferrous ion, iron ion, copper ion, aluminum ion, tin ion, nitrate ion, chloride ion, carbonate ion, sulfate ion, sulfide ion, cysteine, glutathione, tert-butyl peroxide, hydroxyl radical, hydrogen peroxide, superoxide anion, nitric oxide, singlet oxygen, peroxynitrite, hypochlorous acid, tert-butyl peroxide radical, bromide ion and hypobromous acid, thereby realizing selective recognition of hypobromous acid. The fluorescent probe of the present invention shows high selectivity and strong anti-interference ability.

[0032] 2. The hypobromous acid fluorescent probe of the present invention reacts sensitively and rapidly with hypobromous acid, thus being beneficial to the detection of hypobromous acid.

[0033] 3. The hypobromous acid fluorescent probe of the present invention has low cytotoxicity and is conducive to application under physiological conditions.

[0034] 4. The hypobromous acid fluorescent probe of the present invention has good stability and can be stored and used for a long time.

[0035] 5. The hypobromous acid fluorescent probe of the present invention is simple to synthesize, which is conducive to commercial promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0037] Figure 1 is the change of fluorescence spectrum of the probe (5 μM) before and after adding hypobromous acid (0-30 μM);

[0038] Figure 2 is the working curve of the probe (5 µM) for quantitative analysis of different concentrations of hypobromous acid (0-30 µM);

[0039] Figure 3 is the response time of the probe (5 µM) after the addition of hypobromous acid (30 µM);

[0040] Figure 4It is a test of the ability of the probe (5 µM) to selectively recognize hypobromous acid. The numbers 1-29 are: blank, potassium ion, calcium ion, sodium ion, magnesium ion, zinc ion, ferrous ion, iron ion, copper ion, aluminum ion, tin ion, nitrate ion, chloride ion, carbonate ion, sulfate ion, sulfur ion, cysteine ​​(500 µM), glutathione (500 µM), tert-butyl peroxide, hydroxyl radical, hydrogen peroxide, superoxide anion, nitric oxide, singlet oxygen, peroxynitrite, hypochlorous acid (20 µM), tert-butyl peroxide (unless otherwise specified, the concentration of other analytes is 100 µM). The bar graph represents the fluorescence intensity value of the probe at 470 nm in the presence of different analytes;

[0041] Figure 5 Fluorescent probe cytotoxicity test diagram;

[0042] Figure 6 Fluorescent probe imaging of HeLa cells;

[0043] Figure 7 Image of zebrafish using fluorescent probes;

[0044] Figure 8 Fluorescence intensity graph of hypobromous acid during tap water disinfection. DETAILED DESCRIPTION

[0045] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention and should not be used to limit the protection scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the protection scope of the present invention.

[0046] Example 1: Synthesis of compound of formula (II)

[0047] The synthetic design route is as follows:

[0048]

[0049] Implementation Plan 1: 230 mg (1 mmol) of 1-pyrenecarboxaldehyde and 125 mg (1 mmol) of 2-aminobenzenethiol were dissolved in 15 mL of anhydrous ethanol, placed in a flask, reacted with nitrogen at room temperature for 3 hours, and then filtered to obtain a light yellow solid; the crude product was separated by column chromatography using a mixed system of dichloromethane and methanol (v / v, 60:1) to obtain a pure product, and 105 mg of a yellow pure compound of formula (II) was obtained, with a yield of 31%.

[0050] Embodiment 2: 230 mg (1 mmol) of 1-pyrenecarboxaldehyde and 187 mg (1.5 mmol) of 2-aminobenzenethiol were dissolved in 15 mL of anhydrous ethanol, placed in a flask, reacted with nitrogen at room temperature for 3 hours, and then filtered to obtain a light yellow solid; the crude product was separated by column chromatography using a mixed system of dichloromethane and methanol (v / v, 60:1) to obtain a pure product, and 115 mg of yellow pure compound of formula (II) was obtained, with a yield of 34%.

[0051] Embodiment 3: 230 mg (1 mmol) of 1-pyrenecarboxaldehyde and 250 mg (2 mmol) of 2-aminobenzenethiol were dissolved in 15 mL of anhydrous ethanol, placed in a flask, reacted with nitrogen at room temperature for 3 hours, and then filtered to obtain a light yellow solid; the crude product was separated by column chromatography using a mixed system of dichloromethane and methanol (v / v, 60:1) to obtain a pure product, and 130 mg of yellow pure compound of formula (II) was obtained, with a yield of 38%.

[0052] Embodiment 4: 230 mg (1 mmol) of 1-pyrenecarboxaldehyde and 250 mg (2 mmol) of 2-aminobenzenethiol were dissolved in 15 mL of anhydrous ethanol, placed in a flask, reacted with nitrogen at room temperature for 6 hours, and then filtered to obtain a light yellow solid; the crude product was separated by column chromatography using a mixed system of dichloromethane and methanol (v / v, 60:1) to obtain a pure product, and 150 mg of yellow pure compound of formula (II) was obtained, with a yield of 44%.

[0053] The H NMR spectrum, C NMR spectrum and mass spectrum data of the compound of formula (II) prepared in Embodiment 1-4 are as follows:

[0054] 1 H NMR (400 MHz, DMSO- d 6) δ (ppm): 8.41 (t, J = 10.0 Hz, 2H), 8.35 – 8.28(m, 4H), 8.20 – 8.15 (m, 2H), 8.10 (t, J = 8.0 Hz, 1H), 7.56 (s, 1H), 7.03 (d, J = 8.0 Hz, 1H), 6.97 (t, J = 8.0 Hz, 1H), 6.80 (d, J = 8.0 Hz, 1H), 6.65 (t, J = 8.0 Hz, 1H), 3.52 (s, 1H).

[0055] 13C NMR (100 MHz, DMSO- d 6) δ (ppm): 148.49, 136.38, 131.28, 131.01,130.64, 128.45, 127.91, 127.85, 127.13, 126.91,126.09, 125.92, 125.45,124.53, 124.37, 124.23, 122.89, 121.85, 119.31, 109.43, 66.64.

[0056] HRMS (ESI): Calcd for C 23 H 15 NS [M+H] + Found, 338.1002.

[0057] Example 2: Testing the concentration gradient of the fluorescent probe for hypobromous acid

[0058] Multiple parallel samples with a probe concentration of 5 μM were prepared in 10 mL colorimetric tubes, and then different concentrations of hypobromous acid were added to the test system. After shaking evenly, the system was left to stand for 20 minutes, and the fluorescence intensity change was tested using a fluorescence spectrometer. The above determination was carried out in a pure water (10 mM PBS, pH 7.4) system, and the probe used was the probe prepared in Example 1. All spectral tests were measured at 25 °C. The results are shown in Figure 2. Figure 1 shown.

[0059] from Figure 1 It can be clearly seen that with the increase of hypobromous acid concentration, the fluorescence intensity at 470 nm gradually increases. Figure 2 It can be seen that the fluorescence intensity shows a good linear relationship after the addition of the fluorescent probe (5 µM) to hypobromous acid (0-30 µM), which proves that hypobromous acid can be quantitatively analyzed with the help of this fluorescent probe.

[0060] Example 3: Testing the response time of the probe to hypobromous acid

[0061] The probe (5 µM) was placed in the test system, and then 30 µM hypobromous acid was added to the test system. After shaking evenly, the fluorescence intensity change was immediately tested using a fluorescence spectrometer. The above determination was carried out in a pure water (10 mM PBS, pH 7.4) system, the probe used was the probe prepared in Example 1, and all spectral tests were measured at 25 °C.

[0062] Depend on Figure 3It can be clearly seen that after hypobromous acid was added, the fluorescence intensity at 470 nm reached a maximum value and remained unchanged after about 10 minutes of detection, which shows that the probe reacts quickly with hypobromous acid and can provide a rapid analytical method for the determination of hypobromous acid.

[0063] Example 4: Testing the selectivity of fluorescent probes for hypobromous acid

[0064] Analytes No. 1-29 are: blank, potassium ion, calcium ion, sodium ion, magnesium ion, zinc ion, ferrous ion, iron ion, copper ion, aluminum ion, tin ion, nitrate ion, chloride ion, carbonate ion, sulfate ion, sulfide ion, cysteine ​​(500 μM), glutathione (500 μM), tert-butyl peroxide, hydroxyl radical, hydrogen peroxide, superoxide anion, nitric oxide, singlet oxygen, peroxynitrite, hypochlorous acid (20 μM), tert-butyl peroxide radical, bromide ion and hypobromous acid (20 μM) (unless otherwise specified, the concentrations of other analytes are 100 μM). The bar graph represents the fluorescence intensity value of the probe at 470 nm in the presence of different analytes. The above determination was carried out in a pure water system (10 mM PBS, pH 7.4), the probe used was the probe prepared in Example 1, and all spectral tests were measured at 25 °C. Specifically, multiple parallel samples with a probe concentration of 5 µM were prepared in 10 mL colorimetric tubes, and then a certain amount of analyte was added, shaken, and the fluorescence intensity value was measured after standing for 20 minutes. Figure 4 shown.

[0065] from Figure 4 It can be seen that the probe of the present invention has a high selectivity for hypobromous acid and can react specifically with hypobromous acid. The fluorescence spectrum changes significantly before and after the reaction, while the fluorescence intensity of other common analytes in the water environment does not change significantly after the probe reacts with the probe.

[0066] Example 5: Fluorescent probe cytotoxicity test

[0067] The cell counting kit (CCK-8) was used to detect the effect of the probe on the viability of HeLa cells; briefly, a cell count of 1×10 6 cells•mL -1 HeLa cells were seeded on a 96-well plate and cultured in a 5% CO2 / 95% air incubator at 37°C for 12 h. Then, probes of different concentrations (0, 5, 10, 20, 30 µM) were suspended in the culture medium and incubated for 12 h. Subsequently, CCK-8 solution was added to each well and incubated for 2 h, and the absorbance at 450 nm was measured.

[0068] from Figure 5It can be clearly seen that the probe has low cytotoxicity, which is conducive to its application under physiological conditions.

[0069] The probe used in the above test is the probe prepared in Example 1.

[0070] Example 6: Probe detection limit test and calculation

[0071] The detection limit was calculated by fluorescence titration. The detection limit calculation formula is as follows:

[0072] Detection limit = 3σ / k

[0073]

[0074] σ is the standard deviation of the fluorescence intensity of the blank probe, k is Figure 2 The slope of a linear relationship graph.

[0075] The detection limit of the probe of formula (II) for hypobromous acid was calculated to be 119 nM.

[0076] Example 7: Fluorescence microscopic imaging of hypobromous acid in living cells using fluorescent probes

[0077] First, image HeLa cells without probe ( Figure 6 Panel A), after incubation of the probe (10 µM) in cells for 20 minutes, the cells showed a faint blue light ( Figure 6 Group B); another group of cells was added with NAC (100 µM, which has the function of removing HOBr) for 20 minutes and then incubated with the probe (10 µM) for 20 minutes before imaging. The fluorescence of group B was weakened ( Figure 6 Group C in the middle); another group of cells was incubated with the probe (10 µM) for 20 minutes and then HOBr (20 µM) was added for 20 minutes before imaging. The fluorescence was significantly enhanced compared to that of group B ( Figure 6 Group D).

[0078] By calculating the fluorescence intensity of the blue channel, it can be seen that the probe can detect both endogenous hypobromous acid in cells and exogenous hypobromous acid in cells.

[0079] Example 8 Fluorescence imaging analysis of fluorescent probe (10 µM) in zebrafish.

[0080] First, image the zebrafish without the probe ( Figure 7 In Figure 7, Group A, the zebrafish were incubated with the probe (10 µM) for 20 minutes and the fluorescence was slightly enhanced, indicating that the probe entered the zebrafish (Figure 7, Group B). Another group of zebrafish were incubated with NaBr (100 µM) for 20 minutes and then with the probe (10 µM) for 20 minutes before imaging. NaBr was oxidized to HOBr, and the fluorescence was enhanced compared to Group B ( Figure 7The other three groups of cells were incubated with the probe (10 µM) for 20 minutes and then imaged with HOBr (20 µM, 50 µM, 100 µM) for 20 minutes. The fluorescence was significantly enhanced compared to that of group B, and continued to increase with the increase of HOBr concentration ( Figure 7 DF group).

[0081] By calculating the fluorescence intensity of the blue channel, it can be seen that the probe not only has a good fluorescence imaging effect on the exogenous hypobromous acid in the zebrafish, but also has a good fluorescence imaging effect on the endogenous hypobromous acid in the zebrafish.

[0082] Example 9 Detection of Hypobromous Acid during Tap Water Disinfection

[0083] Adjust the pH of sodium hypochlorite (4 mM) to 7.4, take 5 mL, and then add different concentrations of Br − (4, 12, 30 μM) for 1 hour. During this reaction, hypochlorous acid will convert Br − Oxidized to hypobromous acid. Add 20 μL of the reaction solution to 10 mL of PBS (10 mM, pH=7.4), add 5 μM of the probe to measure the fluorescence intensity (labeled 4-6). The concentration of hypobromous acid can be detected by the fluorescence intensity of the solution. In order to eliminate sodium hypochlorite and Br − The effects of sodium hypochlorite (8 μM) and Br on − The fluorescence response of the probe (30 μM) to the probe (labeled as 2 and 3 respectively). Labeled as 1 is the fluorescence intensity of the probe (5 μM) measured in 10 mL PBS (10 mM, pH=7.4) (labeled as 1).

[0084] Probe pair Br − and sodium hypochlorite have no obvious fluorescence response. However, when both are added at the same time, Br − It will be oxidized to HOBr by sodium hypochlorite, so the fluorescence intensity of the solution will increase with the increase of Br − The above results show that the probe can be used to detect hypobromous acid during tap water disinfection, which provides an effective method for quantifying the generation of brominated disinfection by-products.

[0085] Example 10 Application test of fluorescent probe in actual water samples

[0086] The practicality of the probe in detecting hypobromous acid in actual water samples was further explored. First, the actual water samples were filtered to remove some insoluble impurities in the water samples. Samples B and C were diluted 3 times with ultrapure water, and tap water was not diluted. Then two hypobromous acid concentrations within the online relationship range (0.5 and 1, respectively) were selected as the concentrations of exogenously added hypobromous acid, and the above method was used to detect the concentration of hypobromous acid in the water samples. As can be seen from Table 1, the probe has a good recovery rate for different concentrations of hypobromous acid in different water samples, indicating that the probe can successfully detect the concentration of hypobromous acid in complex water samples.

[0087] Table 1

[0088]

[0089] Although the present invention is described using the above embodiments, it should be understood that the present invention may be further modified and varied without departing from the spirit of the present invention, and these modifications and variations are all within the scope of protection of the present invention.

Claims

1. A fluorescent probe composition for measuring, screening or detecting hypobromous acid, comprising a fluorescent probe having a chemical structural formula as shown in formula (II), wherein formula (II) is 。 2. The fluorescent probe composition according to claim 1, characterized in that: The fluorescent probe composition further comprises a solvent, an acid, a base, a buffer solution or a combination thereof.

3. Use of a fluorescent probe having a chemical structure as shown in formula (II) in the preparation of a reagent for measuring, screening or detecting hypobromous acid, wherein formula (II) is 。 4. A method for detecting the presence of hypobromous acid in a sample or determining the content of hypobromous acid in a sample for non-disease diagnosis and treatment purposes, comprising: a) contacting a fluorescent probe having a chemical structure as shown in formula (II) with a sample to form a fluorescent compound, wherein the formula (II) is ； b) determining the fluorescence property of the fluorescent compound.

5. The method according to claim 4, wherein the sample is a chemical sample, a biological sample or an aqueous environment sample.

6. Use of a fluorescent probe having a chemical structure as shown in formula (II) in cell fluorescence imaging, wherein the application is for non-disease diagnosis and treatment purposes, wherein formula (II) is 。

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