Synthesis and application of a fluorescent probe for biothiol detection based on silicon rhodamine

Abstract

The present invention discloses a fluorescent probe for detecting biothiol, wherein the probe uses a p-nitrobenzoyl structure as a recognition group and silicon rhodamine as a matrix to construct the fluorescent probe. Under the condition of the presence of biothiol, the probe reacts with the biothiol to cleave the ester group of the p-nitrobenzoyl group, and then the silicon rhodamine luminophore is freed through 1,6 elimination. The free silicon rhodamine dye can be excited to emit red fluorescence, thereby achieving the purpose of detecting biothiol. The fluorescent probe for detecting biothiol in the present invention has a structure as shown in Formula I:

Description

Technical Field

[0001] The present invention relates to a fluorescent probe, a preparation method and an application, and more specifically to a fluorescent probe for detecting biological thiol, a preparation method and an application. Background Art

[0002] Biothiols, including glutathione (GSH), cysteine ​​(Cys), homocysteine ​​(Hcy) and hydrogen sulfide (H2S), are a class of important small molecule thiol amino acids and their derivatives that exist in cells. On the one hand, biothiols have the functions of maintaining the redox balance of various physiological processes, regulating the homeostatic structure of cells, and participating in intracellular signal conversion, redox reaction, cell proliferation and apoptosis. On the other hand, diseases such as neurodegeneration, cancer, cardiovascular disease, and skin damage are closely related to abnormal levels of biothiols. The main reason for the generation of reactive oxygen species (ROS) is mitochondrial damage, and biothiols are antioxidants that protect cells and even tissues from free radical oxidation and endogenous reactive oxygen species. Not only that, the level of biothiols in cancer cells will also increase significantly. Therefore, by monitoring the level of biothiols, normal and diseased cell tissues, even tumor cell tissues, can be distinguished. Considering the important role of biothiols and the advantages of fluorescence detection such as sensitivity, intuition, simplicity and low cost, the development of new fluorescent probes will be of great significance. Summary of the invention

[0003] The technical problem to be solved by the present invention is to make up for the shortcomings of the prior art and provide a fluorescent probe for detecting biothiols. The probe is constructed by using silicon rhodamine as a parent and p-nitrobenzoyl as a recognition group. Under the condition of the presence of biothiols, the probe reacts with the biothiols to cleave the ester group of the p-nitrobenzoyl group, and then the silicon rhodamine luminophore is freed through 1,6 elimination. The free silicon rhodamine dye is excited to emit red fluorescence, and the purpose of detecting biothiols can be achieved. The present invention has a good application prospect in the field of biological sciences.

[0004] The invention also provides a preparation method and application of the fluorescent probe.

[0005] The technical solution of the present invention to solve the technical problem is as follows:

[0006] The fluorescent probe for detecting biothiols of the present invention has a structure as shown in Formula I:

[0007]

[0008] The present invention also provides a method for preparing the above-mentioned probe molecule, the specific process is:

[0009] (1) Add 4-hydroxybenzyl alcohol to a dry reaction bottle equipped with a magnetic bar, and operate the reaction bottle in anhydrous and oxygen-free conditions. Add a solvent and place it at 0°C, add triethylamine, and then add 4-nitrobenzoyl chloride dissolved in the solvent and stir at 0°C for 4 hours. Separate and purify to obtain compound V;

[0010] (2) Add compound V and solvent to a reaction bottle containing a magnetic particle, and then cool the reaction solution to 0°C. Add phosphorus tribromide dropwise to the reaction solution. After the addition is complete, slowly warm the reaction mixture to room temperature and continue stirring for 2 hours. Compound IV is isolated;

[0011] (3) Under nitrogen protection, add 3-bromo-N, N-dimethylaniline and an organic solvent to a dry reaction bottle equipped with a magnetic bar, cool the solution to -78°C, add a strong base to the reaction bottle and stir the mixture at -78°C for 2 hours. Add dichlorodimethylsilane dropwise to the reaction bottle, slowly warm the reaction mixture to room temperature after the addition is complete, and then continue stirring for 12 hours. Compound III is obtained by separation and purification;

[0012] (4) Add compound III, 2-carboxybenzaldehyde and copper bromide to a sealable pressure-resistant tube equipped with a magnet, and heat the pressure-resistant tube at 140° C. for 5 hours. Separate and purify to obtain compound II;

[0013] (5) Adding compound II, compound IV (4-(bromomethyl)phenyl-4-nitrobenzoate) and a solvent to a reaction bottle equipped with a magnetic particle, stirring at 40° C. for 12 hours, and separating and purifying to obtain the biothiol fluorescent probe I.

[0014] Wherein, the solvent in step (1) is tetrahydrofuran; the solvent in step (2) is dichloromethane; the strong base in step (3) is n-butyl lithium, and the organic solvent is tetrahydrofuran; and the solvent in step (5) is acetonitrile.

[0015] The preparation process of the preparation method can refer to the following equation:

[0016]

[0017] The present invention also provides an application of the fluorescent probe, which can be used for the detection of biothiols in an aqueous phase. Compared with the prior art, the present invention has the advantages that the silicon rhodamine fluorescent probe obtained by the present invention can achieve efficient detection of biothiols, and the synthesis method is simple and feasible with high yield, so it is an ideal biothiols detection tool. In addition, the probe has excellent biocompatibility, so it has broad application prospects in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1is the hydrogen nuclear magnetic resonance spectrum of the reaction product V of Example 1;

[0019] Figure 2 is the hydrogen nuclear magnetic resonance spectrum of the reaction product IV of Example 2;

[0020] Figure 3 is the hydrogen nuclear magnetic resonance spectrum of the reaction product III of Example 3;

[0021] Figure 4 is the hydrogen nuclear magnetic resonance spectrum of the reaction product II of Example 4;

[0022] Figure 5 is the hydrogen nuclear magnetic resonance spectrum of the reaction product I of Example 5;

[0023] Figure 6 is a graph showing the change in fluorescence intensity of the biothiol detection fluorescent probe I in Example 6 in response to Cys over time;

[0024] Figure 7 This is a graph showing the variation of the fluorescence intensity of the fluorescent probe I for detecting biothiol with the Cys concentration in Example 6;

[0025] Figure 8 is the linear fit of Cys concentration and fluorescence intensity in Example 6;

[0026] Fig. 9 This is a fluorescence intensity diagram of the selectivity experiment of the biothiol detection fluorescent probe I in Example 6. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. It is impossible to list all implementation methods here. All technical solutions obtained by equivalent replacement or equivalent transformation methods fall within the protection scope of the present invention.

[0028] Example 1

[0029] 4-Hydroxybenzyl alcohol (600 mg, 4.8 mmol) was placed in a dry 100 mL reaction bottle, filled with nitrogen for protection, and anhydrous tetrahydrofuran (6 mL) was added to the reaction bottle. After the reaction solution was lowered to 0°C. Triethylamine (489 mg, 4.8 mmol) was added to the reaction bottle, and the reaction solution was stirred at 0°C for 10 minutes. 4-Nitrobenzoyl chloride (986 mg, 5.3 mmol) dissolved in anhydrous tetrahydrofuran was slowly added dropwise to the reaction solution, and the mixture was stirred at 0°C for 4 hours. After the reaction was completed, triethylammonium chloride was filtered to remove triethylammonium chloride, the filtrate was collected and the solvent was removed under reduced pressure. The reaction mixture was diluted with 30 mL of dichloromethane, washed with saturated sodium bicarbonate aqueous solution (20 mL × 2), and then washed once with saturated sodium chloride aqueous solution (20 mL), the organic phase was collected, and the combined organic phase was dried over anhydrous sodium sulfate. After the residual solvent was removed under reduced pressure, the product was separated and purified using silica gel column chromatography (dichloromethane:methanol=40:1) to obtain 911 mg of compound V (yellow solid) with a yield of 69%.

[0030] Compound V 1 H NMR (400MHz, Chloroform-d) δ: 8.37 (d, J=2.8Hz, 4H), 7.45 (d, J=8.6Hz, 2H), 7.22 (d, J=8.6Hz, 2H), 4.72 (s, 2H), 2.08 (s, 1H).

[0031] Example 2

[0032] Compound V (300 mg, 1.1 mmol) was added to a 100 mL reaction bottle equipped with a magnetic son, and dichloromethane (10 mL) was added to reduce the reaction solution to 0 ° C. Phosphorus tribromide (0.1 mL, 1.1 mmol) was slowly added dropwise to the reaction solution. After the addition was completed, the reaction solution was slowly heated to room temperature and stirred for 2 hours. After the reaction was completed, the reaction was quenched with saturated sodium bicarbonate aqueous solution (20 mL). The organic phase was extracted with dichloromethane (20 mL × 3), the combined organic phases were dried over anhydrous sodium sulfate, and the residual solvent was removed under reduced pressure. Silica gel column chromatography (petroleum ether: ethyl acetate = 5: 1) was used to separate and purify to obtain compound IV (light yellow solid) 265 mg, with a yield of 72%.

[0033] Compound IV 1 H NMR (400MHz, Chloroform-d) δ: 8.37 (s, 4H), 7.49 (d, J=8.7Hz, 2H), 7.22 (d, J=8.6Hz, 2H), 4.53 (s, 2H).

[0034] Example 3

[0035] Under nitrogen protection, 3-bromo-N, N-dimethylaniline (2.0 g, 10 mmol) and anhydrous tetrahydrofuran (26 mL) were added to a 250 mL dry flask equipped with a magnetic. The solution was cooled to -78 ° C, n-butyl lithium (2.5 M in n-hexane, 4.3 mL, 10.7 mmol) was added dropwise to the reaction flask, and stirring was continued at -78 ° C for 2 hours. Dichlorodimethylsilane (0.49 mL, 5.1 mmol) was added dropwise, and the reaction mixture was slowly warmed to room temperature and stirred for 12 hours. After the reaction was completed, water (40 mL) was added to quench, the solvent was removed under reduced pressure, and the organic phase was extracted with ethyl acetate (50 mL × 3), and the combined organic phase was washed with saturated brine and dried with anhydrous sodium sulfate. After the residual solvent was removed under reduced pressure, silica gel column chromatography (petroleum ether: ethyl acetate = 30: 1) was used to separate and purify to obtain 1.13 g of compound III (yellow oily liquid), with a yield of 74.3%.

[0036] Compound III 1 H NMR (400MHz, Chloroform-d) δ: 7.29 (dd, J=7.0, 7.0Hz, 2H), 6.99 (d, J=2.6Hz, 2H ), 6.97 (d, J=7.1Hz, 2H), 6.81 (dd, J=8.4, 2.8Hz, 2H), 2.97 (s, 12H), 0.58 (s, 6H).

[0037] Example 4

[0038] Compound III (484 mg, 1.7 mmol), o-carboxybenzaldehyde (1.3 g, 8.4 mmol) and copper bromide (37 mg, 0.17 mmol) were added to a 25 mL pressure tube equipped with a magnetic device. The pressure tube was heated at 140 ° C for 5 hours. After the reaction was completed, it was cooled to room temperature, and the reaction mixture was dissolved in 5 mL of dichloromethane and subjected to silica gel column chromatography (petroleum ether: ethyl acetate: triethylamine = 20: 1: 1) to obtain 343 mg of compound II (white solid), with a yield of 48%.

[0039] Compound II 1 H NMR (400MHz, Chloroform-d) δ: 7.97 (d, J=7.4Hz, 1H), 7.65 (td, J=7.4, 1.3Hz, 1H), 7.55 (td, J=7.6, 1.0Hz, 1H), 7.32 (d, J=7. 7Hz, 1H), 6.98 (d, J=2.9Hz, 2H), 6.79 (d, J=8.8Hz, 2H), 6.55 (dd, J=9.0, 3.0Hz, 2H), 2.97 (s, 12H), 0.65 (s, 3H), 0.62 (s, 3H).

[0040] Example 5

[0041] Compound II (50 mg, 0.12 mmol), acetonitrile (2 mL) and compound IV (4-(bromomethyl)phenyl-4-nitrobenzoate) (41 mg, 0.12 mmol) were added to a 10 mL reaction bottle equipped with a magnetic rod, and the mixture was reacted at room temperature for 12 hours. After the reaction, the solvent was removed under reduced pressure, and the remaining reaction mixture was separated and purified by silica gel column chromatography (dichloromethane: methanol = 10: 1) to obtain 34 mg of compound I (yellow solid) with a yield of 42%, which was used as a biothiol fluorescent probe.

[0042] Compound I 1 H NMR (400MHz, Chloroform-d) δ: 8.25 (q, J=9.0Hz, 4H), 8.15 (d, J=3.0Hz, 1H), 7.94 (d, J=7.6HZ, 1H) , 7.67 (td, J=7.4, 1.3HZ, 1H), 7.56 (t, J=6.5HZ, 2H), 7.28 (d, J=7.7HZ, 1H), 7.17 (d, J=9.0Hz, 1H), 7 .09 (d, J=8.7HZ, 2H), 7.00 (d, J=8.7Hz, 2H), 6.93 (d, J=2.8HZ, 1H), 6.87 (d, J=8.9HZ, 1H), 6.57 (dd , J=9.0, 2.9Hz, 1H), 5.31 (s, 2H), 3.75 (s, 3H), 3.73 (s, 3H), 2.96 (s, 6H), 0.70 (s, 3H), 0.61 (s, 3H).

[0043] Example 6

[0044] (1) Spectral properties of probe I's response to Cys at different times

[0045] A PBS solution of probe I was treated with 100 μM Cys, and its fluorescence emission spectrum was measured using a fluorescence spectrophotometer with 643 nm as the excitation light.

[0046] The emission spectrum is Figure 6 As shown, the probe responded quickly, with the fluorescence intensity increasing by about 14 times within 55 min.

[0047] (2) Spectral properties of probe I in response to different concentrations of Cys

[0048] The PBS solutions of probe I were treated with 5 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM, 90 μM and 100 μM Cys, respectively, and their fluorescence emission spectra were measured using a fluorescence spectrophotometer with 643 nm as the excitation light.

[0049] The emission spectrum is Figure 7 As shown in the figure, the change of probe fluorescence intensity showed a positive correlation with Cys concentration. Figure 8 is the linear fit of Cys concentration and luminescence intensity.

[0050] (3) Selectivity test of probe I

[0051] 100 μM Mg 2+ , Fe 3+ , Ca 2+ 、Zn 2+ , K + 、Na + , Cu 2+ 、NO2 - , ClO - 、S2O3 2- 、SO4 2- ,Leu,Gly,His,Ala,Ser,Val,HS - The PBS solution of probe I was treated with , Hcy, GSH, and Cys, and its fluorescence emission spectrum was measured using a fluorescence spectrophotometer with 643 nm as the excitation light.

[0052] Fig. 9 The results showed that probe I specifically responded to biothiols but had no response to other analytes.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A fluorescent probe for detecting biothiols, characterized in that: The probe has a structure as shown in Formula I: 。 2. A method for preparing a fluorescent probe for detecting biothiols according to claim 1, characterized in that: The following steps are involved: Compound II is dissolved in ultra-dry acetonitrile, compound IV is added, and the mixture is stirred at room temperature for 12 hours. After the reaction is completed, the solvent is evaporated under reduced pressure to remove the solvent, and the fluorescent probe for detecting biothiols as shown in formula I is obtained after separation and purification by silica gel column chromatography; 。 3. The preparation method according to claim 2, characterized in that: The amount of compound II used is 1.0 times the molar amount of compound IV.

4. The preparation method according to claim 2, characterized in that: During the separation and purification by silica gel column chromatography, methanol: dichloromethane with a volume ratio of 1:10 was used as the eluent.

5. A use of the fluorescent probe as claimed in claim 1, characterized in that: The probe is used for preparing a detection reagent for biothiol, and the biothiol is glutathione, cysteine ​​and homocysteine.

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