Preparation and application of a fluorescent probe for detecting hypochlorous acid based on rhodamine derivative

Abstract

The present application relates to a kind of new fluorescent probe RH based on rhodamine derivative, is specially used to quickly, sensitively detect hypochlorous acid (HOCl) in aqueous solution.The fluorescent probe RH of the present application can effectively exclude the interference of common active small molecules, ensure to realize accurate detection in complex physiological environment.The experimental results show that probe RH can realize the significant fluorescence enhancement of HOCl at the fluorescence emission wavelength of 552 nm, the detection limit is as low as 18.6 nM, has the fast response time 2 minutes, and keep good response characteristics when coexisting with other interfering substances.The present application provides a new, efficient detection technology for real-time monitoring of HOCl, has wide application prospect, especially in biological imaging, environmental monitoring and public health and other fields, can provide more reliable tool for related research and practical application.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of organic small molecule fluorescent probe, and particularly relates to a fluorescent probe based on rhodamine derivative for detecting hypochlorous acid and a preparation method and application thereof. BACKGROUND

[0002] Hypochlorous acid (HOCl) is an important oxidant, widely used in physiological environment, food safety and environmental monitoring. In physiological environment, HOCl is a key component of the immune system of the body, and white blood cells produce HOCl through enzymatic reaction, effectively killing bacteria and viruses, and enhancing immune defense. However, excessive HOCl can cause oxidative stress and damage cell health. In terms of food safety, HOCl is used as a disinfectant to remove microorganisms in food and prolong shelf life, but its residues can also pose a threat to human health. In environmental monitoring, HOCl is used as a disinfectant in water treatment to effectively remove pathogenic microorganisms in water, but its residues have potential toxicity to aquatic organisms and affect the ecological balance. Therefore, it is of great significance to develop efficient and sensitive detection methods to realize real-time monitoring of HOCl.

[0003] Traditional methods for detecting HOCl mainly rely on chemical analysis techniques such as colorimetric method, spectroscopic method and electrochemical method. These methods usually require complex experimental procedures, professional equipment, and have certain limitations in field application. Therefore, it is particularly important to find a rapid, sensitive and easy-to-operate detection technology. Fluorescent probes have become an ideal tool for detecting HOCl due to their high sensitivity, selectivity and real-time monitoring capability. In recent years, detection techniques based on fluorescent probes have made significant progress in the fields of biological imaging, environmental monitoring and disease diagnosis. Rhodamine, as a widely used fluorescent dye, has good light stability and strong fluorescence characteristics, making it an optimal material for designing fluorescent probes.

[0004] Although some fluorescent probes based on rhodamine have been developed for detecting different biomolecules and small molecules, there are still few specific probes for HOCl. The existing probes still have room for improvement in response speed, selectivity and sensitivity. Therefore, developing a new type of fluorescent probe based on rhodamine derivative, which can efficiently and selectively detect HOCl, will provide a new solution for related research and practical application. By optimizing the structure and function of the probe, its response capability to HOCl will be greatly improved, which will greatly promote the development of HOCl monitoring technology and provide more reliable tools for environmental monitoring and public health protection. SUMMARY

[0005] The application provides a fluorescent probe based on a rhodamine derivative for detecting HOCl, a preparation method and application thereof, and the fluorescent probe can be referred to as RH.

[0006] In a first aspect, the application provides a fluorescent probe based on a rhodamine derivative, which has a molecular formula of C 37 H 35 NO8S, and has the following structural formula:

[0007] .

[0008] The synthesis route of the fluorescent probe is as follows:

[0009]

[0010] In a second aspect, the application provides a preparation method of the fluorescent probe based on the rhodamine derivative, comprising the following steps:

[0011] Step 1: mixing compound R1, triethylamine and dichloromethane, adding triphosgene dropwise under the protection of nitrogen and ice bath, stirring at room temperature for 6 hours after dropwise addition, and removing the solvent to obtain compound R2;

[0012] Step 2: dissolving compound R3 and triethylamine in dichloromethane, adding compound R2 after stirring for 10 minutes, stirring at room temperature for 12 hours, removing the solvent, and purifying the crude product by column chromatography to obtain white solid compound, which is the fluorescent probe RH.

[0013] In some embodiments, in step 1, the molar ratio of compound R1 to triethylamine is 1:1.

[0014] In some embodiments, in step 1, the molar ratio of compound R1 to triphosgene is 3:1.

[0015] In some embodiments, in step 1, the solvent is removed by rotary evaporation to obtain compound R2 which is directly used in step 2 without treatment.

[0016] In some embodiments, in step 2, the molar ratio of compound R3 to compound R2 is 1:1.

[0017] In some embodiments, in step 2, the molar ratio of compound R3 to triethylamine is 1:1.

[0018] In some embodiments, in step 2, the solvent is removed by rotary evaporation.

[0019] In some embodiments, in step 2, petroleum ether: ethyl acetate (volume ratio 3:1) is used as an elution solvent for column chromatography.

[0020] In some embodiments, the method for preparing the fluorescent probe comprises:

[0021] Step 1: Add compound R1 (1.0 mmol), triethylamine (1.0 mmol), and dichloromethane (10 ml) to a 50 ml round-bottom flask. Under nitrogen and an ice bath, add triphosgene (0.33 mmol) dropwise. After the addition is complete, stir at room temperature for 6 hours. After the reaction is complete, remove the solvent by rotary evaporation to obtain compound R2.

[0022] Step 2: Dissolve compound R3 (1 mmol) and triethylamine (1.0 mmol) in 10 ml of dichloromethane. Stir for 10 minutes, then add compound R2 (1 mmol). Stir the mixed solution at room temperature for 12 hours, remove the solvent by rotary evaporation, and purify the crude product by column chromatography to obtain a white solid compound, which is the fluorescent probe RH.

[0023] In a third aspect, the present invention provides the use of the fluorescent probe based on rhodamine derivatives in detecting HOCl in a physiological environment.

[0024] In some embodiments, the suitable pH range for the application is 5-10.

[0025] In some embodiments, the minimum detection limit of the rhodamine derivative-based fluorescent probe for HOCl is 18.6 nM.

[0026] In some embodiments, the response time of the rhodamine derivative-based fluorescent probe to HOCl is less than 2 minutes.

[0027] The fluorescent probe of the present invention is used for identifying HOCl for the purpose of specific non-disease diagnosis.

[0028] In a fourth aspect, the present invention provides the use of the fluorescent probe based on rhodamine derivatives in the fluorescence imaging and detection of HOCl in plant cells.

[0029] The present invention has the following beneficial effects:

[0030] (1) The probe RH of the present invention has a unique structural design, a simple synthesis process, and is easy to separate and purify, effectively eliminating the interference of common active small molecules on the detection results. The probe shows high selectivity and sensitivity to HOCl and can achieve accurate identification in complex physiological environments.

[0031] (2) The mechanism of the fluorescent probe RH of the present invention in recognizing HOCl is as follows: Figure 1 shown.

[0032] The recognition mechanism of the probe of the application is that the strong oxidizing property of HOCl to sulfur acetal is utilized, after the probe reacts with HOCl, the response site is removed, the fluorophore is released, and the fluorescence at 550 nm is significantly enhanced. The response site is first proposed in the application and has no related report in other HOCl probes, and the application has important significance for the development of HOCl probes.

[0033] (3) The fluorescent probe RH of the application has high selectivity and sensitivity for HOCl detection, and can realize specific recognition of HOCl in a complex physiological environment. Experimental results show that the probe can still maintain obvious fluorescence enhancement when coexisting with other competitive analytes, and the response time to different concentrations of HOCl is rapid, and the detection limit is as low as 18.6 nM. In addition, the probe RH shows a relatively stable fluorescence signal in the pH range of 5 to 10, which improves its reliability in practical application. Through imaging experiments in plant cells, the fluorescent probe RH can effectively detect and image HOCl in plants, showing its wide application potential in the fields of biomedicine and plant science. BRIEF DESCRIPTION OF DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0035] Figure 1 The mechanism of the fluorescent probe RH of the application for recognizing HOCl is shown;

[0036] Figure 2 The nuclear magnetic resonance hydrogen spectrum of the fluorescent probe RH of the application (solvent is CDCl3) is shown;

[0037] Figure 3 The nuclear magnetic resonance carbon spectrum of the fluorescent probe RH of the application (solvent is CDCl3) is shown;

[0038] Figure 4 The fluorescence selectivity diagram of the fluorescent probe RH of the application for recognizing HOCl is shown, and the excitation wavelength is 520 nm;

[0039] Figure 5 The ultraviolet absorption selectivity diagram of the fluorescent probe RH of the application for recognizing HOCl is shown, and the excitation wavelength is 520 nm;

[0040] Figure 6 The competitive test diagram of the fluorescent probe RH of the application is shown, the excitation wavelength is 530 nm, and the emission wavelength is 550 nm;

[0041] Figure 7 The fluorescence titration graph of the fluorescent probe RH of the present application recognizing HOCl, the excitation wavelength is 530 nm;

[0042] Figure 8 The fluorescence titration graph of the fluorescent probe RH of the present application recognizing HOCl, the excitation wavelength is 530 nm;

[0043] Figure 9 The minimum detection limit graph of the fluorescent probe RH of the present application recognizing HOCl, the excitation wavelength is 530 nm, and the emission wavelength is 550 nm;

[0044] Figure 10 The pH application range test graph of the fluorescent probe RH of the present application recognizing HOCl, the excitation wavelength is 530 nm, and the emission wavelength is 550 nm;

[0045] Figure 11 The relationship curve between the fluorescence intensity of the fluorescent probe RH of the present application at the maximum emission wavelength 550 nm and the pH value (ranging from 1 to 14);

[0046] Figure 12 The fluorescence imaging graph of the fluorescent probe RH of the present application recognizing HOCl in onion epidermal cells.

[0047] Embodiment Mode

[0048] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0049] Example 1

[0050] The preparation method of the fluorescent probe molecule for HOCl detection in the present embodiment is as follows:

[0051] (1) Preparation of compound R2

[0052] Compound R1 (240.0 mg, 1.0 mmol), triethylamine (101.2 mg, 1.0 mmol) and dichloromethane (10 ml) were added to a 50 ml round-bottom flask, and a solution of triphosgene (296.7 mg, 0.33 mmol) in dichloromethane was added dropwise under nitrogen and ice bath protection. After the dropwise addition was completed, the reaction was stirred at room temperature for 6 hours. After the reaction was completed, the excess solvent was removed by rotary evaporation to obtain compound R2, which was directly subjected to the next reaction.

[0053] (1) Preparation of compound RH

[0054] Compound R3 (387.4 mg, 1 mmol) and triethylamine (101.2 mg, 1.0 mmol) were dissolved in 10 ml of dichloromethane and stirred for 10 minutes. Compound R2 (302.8 mg, 1 mmol) was added. The mixed solution was stirred at room temperature for 12 hours, and the excess solvent was removed by rotary evaporation. The crude product was purified by column chromatography to obtain a white solid compound, which is a fluorescent probe for HOCl detection.

[0055] H NMR spectroscopy: 1 H NMR (400 MHz, CDCl3) 8.04 (d, J = 7.4 Hz, 1H), 7.73 – 7.59 (m, 2H), 7.46 – 7.34 (m, 2H), 7.22 (d, J = 7.5 Hz, 1H), 7.12 (d, J= 2.2 Hz, 1H), 6.92 – 6.86 (m, 2H), 6.86 – 6.76 (m, 2H), 6.61 (s, 1H), 6.48 (d, J = 2.5 Hz, 1H), 6.39 (dd, J = 8.9, 2.6 Hz, 1H), 4.48 – 4.23 (m, 3H),4.04 – 3.90(m, 1H), 3.82 (s, 4H), 3.49 – 3.31 (m, 4H), 3.28 – 3.13 (m, 1H), 3.14 – 3.02 (m, 1H), 2.74 – 2.46(m, 2H), 1.20 (t, J = 7.0 Hz, 6H).

[0056] Carbon NMR spectroscopy determination: 13 C NMR (101 MHz, CDCl3) δ 169.5, 159.0, 153.0,152.7, 149.7, 136.9, 134.9, 129.6, 129.1, 128.9, 127.1, 126.6, 124.9,124.1,117.3, 116.3, 113.7, 109.7, 108.6, 104.8, 97.6, 96.4, 83.6, 70.7, 65.8, 55.3,44.5, 42.5, 34.3.

[0057] H NMR spectrum Figure 2 As shown, the carbon NMR spectrum is as follows Figure 3 shown.

[0058] Example 2

[0059] The probe RH prepared in Example 1 was accurately weighed and dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mM solution for use. The HOCl (10 mM) solution was prepared by weighing NaOCl (13.7) and dissolving in 10 ml of deionized water, and then transferring to a 10 mL volumetric flask and diluting to volume, and storing at -20 °C (freshly prepared). The fluorescence and UV absorption of the PBS solution containing probe RH (10 μM) were tested using a fluorescence spectrometer and a UV spectrophotometer.

[0060] As shown in Figure 4 and Figure 5 , in the PBS buffer solution, the probe alone has no fluorescence emission at 550 nm and no UV absorption peak at 520 nm. When other analytes are added, no changes in the emission peak and absorption peak of the probe are induced. Only after the addition of HOCl (100 μM), a significant fluorescence enhancement at 550 nm occurs, and the absorption peak at 520 nm is significantly increased. This indicates that the probe RH has a high selectivity for HOCl response.

[0061] Example 3

[0062] The concentration of probe RH in Example 1 was kept at 10 μM, and the effect of other competitive analytes on the response of the probe to HOCl was investigated.

[0063] As shown in Figure 6 , in the PBS solution containing probe RH (10 μM), after the addition of HOCl and other competitive analytes, the fluorescence of the probe at 550 nm was significantly increased, and the fluorescence was not induced to decrease by the addition of analytes. This indicates that the probe can achieve specific recognition of HOCl under complex physiological conditions.

[0064] Example 4

[0065] The concentration of probe RH in Example 1 was kept at 10 μM, and the response time of the probe to HOCl was investigated.

[0066] HOCl (100 μM) was added to the PBS solution containing probe RH (10 μM), and the fluorescence change of the probe at 550 nm was recorded. As shown in Figure 7 , without the addition of HOCl, the fluorescence of probe RH at 550 nm had almost no change within 0 to 30 minutes. However, after the addition of HOCl, its fluorescence signal was significantly enhanced within 2 minutes. This experimental result shows that the detection of the probe for HOCl has a relatively fast response time.

[0067] Example 5

[0068] The concentration of probe RH in Example 1 was kept at 10 μM, and the UV absorption and fluorescence emission changes of the probe to different concentrations of HOCl were investigated.

[0069] As shown in Figure 8 and Figure 9 , the single probe has almost no absorption peak and fluorescence emission at 520 nm and 550 nm. However, after adding different concentrations of HOCl (0-100 μM) solution, the absorption peak at 520 nm and the fluorescence emission at 550 nm are significantly enhanced. As shown in Figure 10 , by linear fitting the fluorescence intensity of probe RH at 550 nm to the concentration of HOCl added, the linear equation Y=68.76X+24.58 is obtained, and it shows a high linear correlation. According to the minimum detection limit equation LOD=3σ / k, the detection limit is calculated to be 18.6 nM. This shows that the probe RH has high sensitivity for the detection of HOCl.

[0070] Example 6

[0071] The concentration of fluorescent probe RH in Example 1 was kept at 10 μM to investigate the pH application range of the probe for HOCl. As shown in Figure 11 , the relationship curve between the fluorescence intensity of probe RH at the maximum emission wavelength of 550 nm and the pH value (range 1 to 14) is shown. Between pH 5 and 10, the fluorescence intensity of probe RH is relatively stable; while after adding HOCl, the fluorescence intensity is significantly enhanced. Therefore, the pH value between 5 and 10 is determined as the working application range of probe RH for detecting HOCl.

[0072] Example 7

[0073] The fluorescent probe RH in Example 1 was applied to the detection and imaging of HOCl in onion epidermal cells. As shown in Figure 12 , 10 μM of probe RH solution was added to the culture solution of onion epidermal cells, and after 30 minutes of incubation at room temperature, imaging was performed, and the results showed Figure 12 (A2) has weak fluorescence, indicating that the single probe has almost no fluorescence emission. Subsequently, 10 μM of probe RH solution was added to the culture solution of onion epidermal cells under the same conditions, and after 30 minutes of incubation, HOCl (100 μM) was added, and after 60 minutes of further incubation, imaging was performed, and Figure 12 (B2) was obtained, in which the red fluorescence was significantly enhanced. The results of onion epidermal cell experiments showed that the new probe RH can effectively realize the fluorescence imaging and detection of HOCl in plant cells.

[0074] The above merely provides the preferred embodiment of the present application, and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A fluorescent probe based on a rhodamine derivative, the molecular formula of which is C 37 H 35 NO8S has the following structural formula: 。 2. The method for preparing a fluorescent probe based on a rhodamine derivative according to claim 1, comprising: ； Step 1: Compound R1, triethylamine, and dichloromethane were mixed, and triphosgene was added dropwise under nitrogen and ice bath protection. After the addition was complete, the mixture was stirred at room temperature for 6 hours. After the reaction was complete, the solvent was removed to obtain compound R2; Step 2: Compound R3 and triethylamine were dissolved in dichloromethane, stirred for 10 minutes, and then compound R2 was added. After stirring at room temperature for 12 hours, the solvent was removed and the crude product was purified by column chromatography to obtain a white solid compound, which was the fluorescent probe RH.

3. The preparation method according to claim 2, wherein In step 1, the molar ratio of compound R1 to triethylamine is 1:

1.

4. The preparation method according to claim 2, wherein In step 1, the solvent was removed by rotary evaporation to give compound R2 which was used directly in step 2 without further treatment.

5. The preparation method according to claim 2, wherein In step 2, the molar ratio of compound R3 to compound R2 is 1:

1.

6. The preparation method according to claim 2, wherein include: Step 1: Add 1.0 mmol of compound R1, 1.0 mmol of triethylamine, and 10 ml of dichloromethane to a 50 ml round-bottom flask. Under nitrogen and an ice bath, add 0.33 mmol of triphosgene dropwise. After the addition is complete, stir at room temperature for 6 hours. After the reaction is complete, remove the solvent by rotary evaporation to obtain compound R2. Step 2: Dissolve 1 mmol of compound R3 and 1.0 mmol of triethylamine in 10 ml of dichloromethane. After stirring for 10 minutes, add 1 mmol of compound R2. After stirring the mixed solution at room temperature for 12 hours, remove the solvent by rotary evaporation. The crude product is purified by column chromatography to obtain a white solid compound, which is the fluorescent probe RH.

7. Use of the fluorescent probe based on rhodamine derivatives according to claim 1 in the preparation of a reagent for detecting HOCl in a physiological environment, wherein: The suitable pH value range for the application is 5-10; and the minimum detection limit of the fluorescent probe based on the rhodamine derivative for HOCl is 18.6 nM.

8. Use of the rhodamine derivative-based fluorescent probe according to claim 1 in fluorescence imaging and detection of HOCl in plant cells.

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