Pyrenyl fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a pyrenyl fluorescent probe and a preparation method and application thereof.The preparation method comprises the steps that 1-hydroxypyrene and triethylamine are added into a round-bottom flask with dichloromethane as a solvent to be cooled to 0 DEG C, then acryloyl chloride is added to react for 5 minutes, then the temperature is increased to the room temperature to react, after the reaction is finished, a reaction product is obtained, and the pyrenyl fluorescent probe is obtained. Carrying out water liquid separation, dichloromethane extraction, anhydrous sodium sulfate drying and rotary evaporation on the reaction mixture to remove a solvent, and purifying through column chromatography to obtain a white solid; when no to-be-detected substance is added, the probe has a weak fluorescence signal, and the fluorescence intensity is obviously enhanced after the cysteine is identified and combined, so that the probe can be used for monitoring the change of the concentration of the cysteine; the probe shows good selectivity, stability and light stability on cysteine, and detection on conditions such as pH and temperature proves that the probe has potential biological application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of functional materials and relates to fluorescent probe materials, in particular to a pyrene-based fluorescent probe and a preparation method and application thereof. Background Art

[0002] Cysteine ​​(Cys) is a sulfur-containing amino acid that is widely present in a variety of organisms. Although cysteine ​​plays an important role in protein synthesis, anti-oxidation and metabolism, its potential hazards cannot be ignored. Excessive cysteine ​​can have a negative impact on health, especially at high concentrations, which may lead to toxic reactions. Studies have shown that increased cysteine ​​levels are associated with the occurrence of a variety of diseases (such as cardiovascular disease, Alzheimer's disease and diabetes), which may be related to its ability to promote oxidative stress and inflammatory responses. In addition, cysteine ​​metabolites such as thiol compounds may also be cytotoxic, leading to cell damage and tissue inflammation. Long-term exposure to high concentrations of cysteine ​​may affect the nervous system, leading to neuronal damage and dysfunction. Therefore, although cysteine ​​plays an important physiological function in organisms, its hazards in an excessive state need to be paid attention to so that appropriate measures can be taken to manage its levels and reduce health risks.

[0003] Common methods for detecting cysteine ​​include spectroscopy, chromatography, fluorescent probe method, electrochemical method and mass spectrometry. Among them, the fluorescent probe method has become a popular means for detecting cysteine ​​due to its high sensitivity and selectivity. Fluorescent probes react specifically with cysteine ​​to produce fluorescent signals, which can achieve trace analysis at low concentrations. In addition, fluorescent probes also have the advantage of real-time dynamic monitoring, and are particularly suitable for cysteine ​​detection in complex biological samples. They are widely used in fields such as biomedicine and environmental science. Most of the existing fluorescent probes for detecting cysteine ​​are based on a single change in fluorescence intensity, which will lead to changes in probe concentration, fluctuations in radiated light, and systematic errors caused by different action positions and environments in cells. Secondly, it is challenging to distinguish between cysteine ​​(Hcy), homocysteine ​​(Hcy) and glutathione (GSH) in this field. Therefore, there is an urgent need for the emergence of fluorescent probes with stable properties and specificity for detecting cysteine. Summary of the invention

[0004] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a pyrene-based fluorescent probe and a preparation method and application thereof. The fluorescent probe has good selectivity, anti-interference ability and stability, and can specifically detect cysteine ​​in living cells.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A pyrene-based fluorescent probe, the structural formula is as follows:

[0007]

[0008] A method for preparing a pyrene-based fluorescent probe as claimed in claim 1, wherein the reaction formula is:

[0009]

[0010] The method for preparing the pyrene-based fluorescent probe comprises the following steps:

[0011] Step 1: Add 1-hydroxypyrene, excess acryloyl chloride and excess triethylamine into a round-bottom flask, use dichloromethane as a solvent, and stir at room temperature to allow 1-hydroxypyrene and acryloyl chloride to undergo a substitution reaction;

[0012] Step 2: After the reaction is completed, the organic phase is separated into water and extracted with dichloromethane and dried. The solvent is removed by rotary evaporation and then purified to obtain a white solid, which is the pyrene fluorescent probe.

[0013] Preferably, the molar ratio of 1-hydroxypyrene, acryloyl chloride and triethylamine is 1:1.5:1.5.

[0014] Preferably, in the step 1, 1-hydroxypyrene, triethylamine and dichloromethane are added sequentially in proportion to a round-bottom flask, cooled to 0° C., acryloyl chloride is added and reacted for 5 minutes, and then the temperature is raised to room temperature for reaction.

[0015] Preferably, the reaction process of step 1 is monitored by thin layer chromatography.

[0016] Furthermore, the developing solvent of the thin layer chromatography method is PE:EA=10:1, R f =0.2~0.4.

[0017] Preferably, the drying in step 2 is performed by drying with anhydrous Na2SO4.

[0018] Preferably, the purification in step 2 is performed by column chromatography, and the eluent is PE:EA=10:1.

[0019] The present invention also protects the use of the pyrene-based fluorescent probe described above in the process of detecting cysteine.

[0020] Compared with the prior art, the present invention has the following technical effects:

[0021] The invention adopts 1-hydroxypyrene and acryloyl chloride as raw materials, and synthesizes a fluorescent probe for specifically detecting cysteine ​​through a one-step reaction. In the case where no test substance is added, the probe itself has a weak fluorescent signal. After combining with cysteine, the thiol group of Cys and the double bond of the reaction site of the probe first undergo Michael addition reaction, the thiol group attacks the α, β unsaturated double bond of the probe, and then the amino group attacks the carbonyl carbon to undergo an intramolecular cyclization reaction, and finally forms a seven-membered ring structure and a raw material product, the conjugated system of the probe is destroyed, and under the excitation of a wavelength of 349nm, the fluorescent signal at 389nm is activated, and the fluorescence intensity is significantly enhanced, thereby realizing the monitoring of the change in cysteine ​​concentration. The probe exhibits strong selectivity, stability and photostability in cysteine ​​detection, and through the detection of conditions such as pH and temperature, it is proved that the probe has potential biological application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the 1H NMR spectrum of the fluorescent probe of the present invention;

[0023] Figure 2 is the 13C NMR spectrum of the fluorescent probe of the present invention;

[0024] Figure 3 It is a linear relationship diagram between the fluorescence intensity of the fluorescent probe of the present invention and GSH, Hcy, and Cys at different excitation wavelengths;

[0025] Figure 4 is a graph showing the effect of different pH values ​​on the fluorescence intensity of the fluorescent probe of the present invention;

[0026] Figure 5 is a time response graph of the fluorescent probe of the present invention to Cys at room temperature;

[0027] Figure 6 is a time response graph of the fluorescent probe of the present invention to Cys at 36°C;

[0028] Figure 7 It is a fluorescence response diagram of the fluorescence intensity of the fluorescent probe of the present invention as the Cys concentration increases;

[0029] Figure 8 It is a linear relationship diagram of the fluorescence intensity of the fluorescent probe of the present invention in the range of 0-40 μM Cys concentration and the change of Cys concentration;

[0030] Fig. 9 It is the fluorescence response diagram of Cys, other amino acids and ions in cells at 389nm after adding fluorescent probe;

[0031] Fig.10It is a fluorescence response diagram of the fluorescent probe of the present invention and other high-concentration amino acids or high-concentration ions at 389 nm. DETAILED DESCRIPTION

[0032] The specific contents of the present invention are further explained in detail below in conjunction with embodiments.

[0033] In the following examples, triethylamine was used as a catalyst and dichloromethane (DCM) was used as a solvent;

[0034] The developing solvent for thin layer chromatography (TLC) and the eluent for column chromatography used for purification were both petroleum ether (PE) and ethyl acetate (EA), and PE:EA=10:1.

[0035] Example

[0036] This example provides a method for preparing a pyrene-based fluorescent probe, and the reaction formula is:

[0037]

[0038] The specific steps include:

[0039] 1-Hydroxypyrene (44 mg, 0.2 mmol), 3 mL DCM, and triethylamine (30.3 mg, 0.3 mmol) were added to a 25 mL round-bottom flask equipped with a stirrer. The temperature was cooled to 0°C, and acryloyl chloride (20.8 mg, 0.3 mmol) was added. After reacting for 5 minutes, the temperature was raised to room temperature for reaction. The reaction was monitored by TLC (PE: EA = 10: 1). After the reaction was completed for 8 to 10 hours, the reaction solution was poured into 10 mL of water and stirred thoroughly and separated. The organic phase was extracted three times with DCM, the organic phases were combined, dried and filtered with anhydrous Na2SO4, and the solvent was removed by rotary evaporator. Column chromatography was performed with PE: EA = 10: 1 to obtain 0.076 g of a white solid probe with a yield of 90%. The structural formula is:

[0040]

[0041] The pyrene-based fluorescent probe prepared in the example was tested as follows:

[0042] (1) Figure 1 and Figure 2 As shown, the basic data of pyrene-based fluorescent probe are as follows:

[0043] 1H NMR (600MHz, CDCl3) δ8.08–8.04(m,3H),7.97(s,2H),7.93(s,2H),7.89(t,J=7.6Hz,1H),7.7 3–7.71(m,1H),6.71(d,J=17.4Hz,1H),6.47(dd,J=17.4,10.5Hz,1H),6.05(d,J=10.5Hz,1H).

[0044] 13C NMR (151MHz, CDCl3) δ164.2,143.4,132.5,130.4,130.3,128.7,127.5,127.1,126.4,125.6,124.9,124.7,124.3,123.8,122.4,119.5,119.0.

[0045] (2) The selectivity experiment of pyrene-based fluorescent probe for three biothiols is as follows:

[0046] The acetonitrile-phosphate buffer solution (CH3CN: PBS (10mM, pH=7.4, v / v, 3 / 7)) was selected as the buffer system for the probe fluorescence titration experiment. In this system, the probe (10μM) and the main biothiol Cys / Hcy / GSH (30μM) responded respectively, and the changes in their fluorescence intensity were observed, such as Figure 3 As shown, the fluorescence intensity of the three biothiols has changed, among which Cys has the most obvious change. Under the excitation wavelength of 349nm, the probe produces an obvious fluorescence emission peak at 389nm. Hcy and GSH have relatively small changes, with similar degrees of change, and are difficult to distinguish. From the above phenomenon, it can be assumed that the probe has a detection effect on Cys.

[0047] (3) The pH range selectivity experiment of pyrene-based fluorescent probe for Cys is as follows:

[0048] In order to study the effect of pH on the performance of fluorescent probes, a series of phosphate buffer solutions (CH3CN / PBS, v / v, 3 / 7) with different pH values ​​were prepared for fluorescence spectrum analysis of Cys. Figure 4 As shown, the probe is relatively stable in the pH range of 1.0-10.0 and has good stability. However, during the reaction between the probe and Cys, the fluorescence response intensity is relatively low in the pH range of 1.0-5.0, and in the pH range of 6.0-10.0, the fluorescence response of the probe to Cys is strong and relatively stable. Because the pH value of most cells is 7.4, the probe can be applied to the cells later, so the environment of pH = 7.4 is preferred, and the probe has the possibility of entering the cell for detection.

[0049] (4) The temperature range selectivity experiment of pyrene-based fluorescent probe for Cys is as follows:

[0050] Fluorescence stability assessment of fluorescent probes, such as Figure 5 As shown in the figure, under room temperature, it was observed that the maximum absorption wavelength of the probe did not change significantly within 3 hours, which indicates that the probe has good photostability. At the same time, it was observed that it took 3 hours for the probe to reach equilibrium with Cys. Therefore, the reaction rate was increased by optimizing the temperature and raising the room temperature to 36°C. Figure 6 As shown in Figure 6, the probe still maintains good photostability. The fluorescence intensity of the probe and Cys reaches a certain value within 30 minutes, and the reaction of the probe and Cys reaches equilibrium. At the same time, it can be concluded from Figure 6 that the optimal reaction time of the probe and Cys is 30 minutes, at which time the reaction has reached equilibrium. The above experimental results once again show that the probe is suitable for further cell detection experiments.

[0051] (5) The fluorescence spectrum titration experiment of pyrene-based fluorescent probe and Cys is as follows:

[0052] The optimal environment for detection was CH3CN / PBS (10mM, pH=7.40, v / v, 3 / 7) as the buffer solvent. Under this condition, the fluorescence changes of the reaction system were observed after 30 minutes of reaction. To further explore the fluorescence response of the probe and Cys, a fluorescence titration experiment was conducted on the fluorescent probe and Cys. Under the optimal environment, the fluorescence response of the probe to Cys was as follows: Figure 7 As shown in the figure, under the excitation wavelength of 349 nm, the emission peak at 389 nm increases with the increase of the concentration of added Cys. And the probe shows an excellent linear relationship in the range of 0-40 μM Cys concentration, as shown in the figure. Figure 8 As shown, the correlation coefficient (R 2 ) was 0.9916, and the linear regression equation was F = 10.45 [Cys] + 125. The detection limit of this method for Cys was 0.049 μM (detection limit = 3SD / K).

[0053] (6) The selectivity experiment of pyrene-based fluorescent probe for Cys is as follows:

[0054] The detection probe is highly selective for Cys. Considering the complexity of the intracellular environment, a series of selectivity experiments were performed. Fig. 9 As shown, when other ions (30 μM) and amino acids (30 μM) were added, little change in fluorescence intensity was observed. Even when Hcy / GSH was added, the change in fluorescence intensity was relatively small. However, when Cys (30 μM) was added to the reaction system, a significant increase in fluorescence intensity occurred. This result demonstrates that the probe is specific, highly selective, and highly sensitive to Cys.

[0055] To evaluate whether the specific selectivity of the probe for Cys would be interfered by other high concentrations of amino acids or high concentrations of ions, further fluorescence experiments were performed, such as Fig.10 As shown in the figure, even in the environment of high concentrations of other ions (300 μM) and amino acids (300 μM), the fluorescence intensity of the probe still only changed slightly, which once again proved the special selectivity of the probe for Cys.

Claims

1. A pyrene-based fluorescent probe, characterized in that: The structural formula is as follows:

2. A method for preparing a pyrene-based fluorescent probe as claimed in claim 1, characterized in that: The reaction formula is:

3. The method for preparing a pyrene-based fluorescent probe according to claim 2, wherein: The following steps are involved: Step 1: Add 1-hydroxypyrene, excess acryloyl chloride and excess triethylamine into a round-bottom flask, use dichloromethane as a solvent, and stir at room temperature to allow 1-hydroxypyrene and acryloyl chloride to undergo a substitution reaction; Step 2: After the reaction is completed, the organic phase is separated into water and extracted with dichloromethane and dried. The solvent is removed by rotary evaporation and then purified to obtain a white solid, which is the pyrene fluorescent probe.

4. The method for preparing the pyrene-based fluorescent probe according to claim 3, wherein: The molar ratio of 1-hydroxypyrene, acryloyl chloride and triethylamine is 1:1.5:1.

5.

5. The method for preparing the pyrene-based fluorescent probe according to claim 3, characterized in that: In the step 1, 1-hydroxypyrene, triethylamine and dichloromethane are added in a round-bottom flask in proportion and then cooled to 0° C., acryloyl chloride is added and reacted for 5 minutes, and then the temperature is raised to room temperature for reaction.

6. The method for preparing a pyrene-based fluorescent probe according to claim 3, wherein: The reaction process of step 1 is monitored by thin layer chromatography.

7. The method for preparing a pyrene-based fluorescent probe according to claim 6, wherein: The developing solvent of the thin layer chromatography is PE:EA=10:1, R f =0.2~0.

4.

8. The method for preparing a pyrene-based fluorescent probe according to claim 3, wherein: The drying described in step 2 is performed by drying with anhydrous Na2SO4.

9. The method for preparing a pyrene-based fluorescent probe according to claim 3, wherein: The purification described in step 2 is carried out by column chromatography, and the eluent is PE:EA=10:

1.

10. Use of the pyrene-based fluorescent probe as claimed in claim 1 in the detection of cysteine.

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