Synthesis and Application of a Fluorescent Probe for Hydrogen Peroxide (H 2 O 2 ) Detection

Abstract

The present invention discloses a fluorescent probe for detecting H2O2. The probe is constructed with silicon rhodamine as the parent body and phenylborate structure as the recognition group. In the presence of H2O2, H2O2 first coordinates with the boron atom, and then oxidizes the B-C bond to form borate, which can be rapidly hydrolyzed in water, simultaneously releasing the silicon rhodamine luminophore. The free silicon rhodamine dye emits red fluorescence through excitation, thereby achieving the purpose of detecting H2O2. The fluorescent probe for detecting H2O2 in the present invention has the structure shown in Formula I: #imgabs0#

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 H 2 O 2 , a preparation method and an application thereof. Background Art

[0002] Excessive hydrogen peroxide (H 2 O 2 ) in the environment will cause serious environmental pollution. In addition, as a kind of reactive oxygen species (ROS), H 2 O 2 is an essential oxygen metabolite in the living system. On the one hand, H 2 O 2 is related to the normal functions of many cells. For example, more and more evidence supports its role as an oxidative stress marker and a messenger for cell signal transduction. On the other hand, abnormal H 2 O 2 levels are often associated with diseases. For example, abnormal generation or accumulation of H 2 O 2 in the mitochondrial electron transport chain can lead to the accumulation of oxidative stress and subsequent decline in the function of organ systems, including serious diseases such as cancer, diabetes, neurodegenerative Alzheimer's disease, Parkinson's disease and Huntington's disease. Considering the important role of H 2 O 2 and the advantages of fluorescence detection such as sensitivity, intuitiveness, simplicity and low cost, it is of great significance to develop new fluorescent probes. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to make up for the deficiencies of the existing technology and provide a fluorescent probe for detecting H 2 O 2 . The probe constructs a fluorescent probe with silicon rhodamine as the parent body and phenylborate as the recognition group. In the presence of H 2 O 2 , H 2 O 2 will coordinate with boron atoms, oxidize the B-C bond to form borate, which can be rapidly hydrolyzed in water, and at the same time release the silicon rhodamine luminophore, emitting red light, that is, the purpose of detecting H 2 O 2 can be achieved, and it will have good application prospects in the field of biological science.

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

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

[0006] The fluorescent probe for detecting fluoride ions according to the present invention has a structure shown in Formula I:

[0007]

[0008] The present invention also provides a preparation method of the above probe molecule, and the specific process is as follows:

[0009] (1) Under nitrogen protection, 3-bromo-N,N-dimethylaniline and an organic solvent are added to a dry reaction flask equipped with a magnetic stirrer. The solution is cooled to -78 °C, a strong base is added to the reaction flask, and the mixture is stirred at -78 °C for 2 hours. Dichlorodimethylsilane is added dropwise to the reaction flask. After the addition is complete, the reaction mixture is slowly warmed to room temperature and then stirred for another 12 hours. Compound III is obtained by separation and purification;

[0010] (2) Compound III, 2-carboxybenzaldehyde and copper bromide are added to a sealable pressure-resistant tube equipped with a magnetic stirrer. The pressure-resistant tube is heated at 140 °C for 5 hours. Compound II is obtained by separation and purification;

[0011] (3) Compound II, 4-(bromomethyl)phenylboronic acid pinacol ester and a solvent are added to a reaction flask equipped with a magnetic stirrer. The mixture is stirred at room temperature for 12 hours. The fluoride ion fluorescent probe I is obtained by separation and purification.

[0012] Among them, the strong base in step (1) is n-butyllithium, and the organic solvent is tetrahydrofuran; the solvent in step (3) is acetonitrile.

[0013] The preparation process of the preparation method can be referred to the following chemical equation:

[0014]

[0015] The present invention also provides the application of the fluorescent probe. This fluorescent probe can be used for the detection of H 2 O 2 in the aqueous phase. Compared with the prior art, the advantages of the present invention are as follows: The silicon rhodamine-based fluorescent probe obtained by the present invention can achieve efficient detection of H 2 O 2 , and the synthesis method is simple and feasible with a high yield. Therefore, it is an ideal detection tool for H 2 O 2 . In addition, this probe has excellent biocompatibility and thus has broad application prospects in the biomedical field. Description of the Drawings

[0016] Figure 1 is the nuclear magnetic resonance hydrogen spectrum of the reaction product III in Example 1;

[0017] Figure 2is the 1H NMR spectrum of the reaction product II in Example 2;

[0018] Figure 3 is the 1H NMR spectrum of the reaction product I in Example 3;

[0019] Figure 4 is Example 4H 2 O 2 is the graph showing the change of fluorescence intensity of the fluorescent probe I over time;

[0020] Figure 5 is Example 4H 2 O 2 is the graph showing the change of fluorescence intensity of the fluorescent probe I with the concentration of H 2 O 2 ;

[0021] Figure 6 is Example 4 for H 2 O 2 is the linear fitting of the concentration and fluorescence intensity;

[0022] Figure 7 is Example 4H 2 O 2 is the fluorescence intensity graph of the selectivity experiment of the fluorescent probe I. Detailed implementation mode

[0023] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to 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 used to limit the present invention. It is impossible to enumerate all the implementation manners here. All technical solutions obtained by using equivalent replacements or equivalent transformations fall within the protection scope of the present invention.

[0024] Example 1

[0025] Under nitrogen protection, 3-bromo-N,N-dimethylaniline (3.0 g, 14.99 mmol) and tetrahydrofuran (40 mL) were added to a 250 mL dry flask equipped with a magnetic stirrer. The solution was cooled to -78 °C, and n-butyllithium (2.5 M in n-hexane, 6.3 mL, 15.74 mmol) was added dropwise to the reaction flask, and stirring was continued at -78 °C for 2 hours. Dichlorodimethylsilane (0.871 mL, 9 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 (30 mL) was added to quench the reaction, the solvent was removed under reduced pressure, the organic phase was extracted with ethyl acetate (50 mL × 3), the combined organic phases were washed with saturated brine, and dried over anhydrous sodium sulfate. After removing the remaining solvent under reduced pressure, silica gel column chromatography (petroleum ether ∶Ethyl acetate = 20:1) to obtain 1.8 g of Compound III (yellow oily liquid) by separation and purification, with a yield of 80%.

[0026] Of Compound III 1 H NMR (400 MHz, Chloroform-d) δ 7.28 - 7.25 (m, 2H), 6.96 (d, J = 2.7 Hz, 2H), 6.94 (d, J = 7.1 Hz, 2H), 6.80 - 6.77 (m, 2H), 2.95 (s, 12H), 0.56 (s, 6H).

[0027] Example 2

[0028] Add Compound III (1.0 g, 3.35 mmol), o-carboxybenzaldehyde (2.5 g, 16.75 mmol) and copper bromide (74.8 mg, 0.335 mmol) to a 25 mL pressure-resistant tube equipped with a magnetic stirrer. Heat the pressure-resistant tube at 140 °C for 5 hours. After the reaction is completed, cool it to room temperature, dissolve the reaction mixture in 5 mL of dichloromethane, and obtain 445.3 mg of Compound II (white solid) by silica gel column chromatography (petroleum ether:ethyl acetate:triethylamine = 20:1:1), with a yield of 31%.

[0029] Of Compound II 1 NMR (400 MHz, Chloroform-d) δ 7.97 (d, J = 7.6 Hz, 1H), 7.64 (td, J = 7.5, 1.2 Hz, 1H), 7.54 (td, J = 7.5, 0.9 Hz, 1H), 7.31 (d, J = 7.6 Hz, 1H), 6.98 (d, J = 2.9 Hz, 2H), 6.79 (d, J = 8.9 Hz, 2H), 6.55 (dd, J = 8.5, 2.9 Hz, 2H), 2.97 (s, 12H), 0.65 (s, 3H), 0.62 (s, 3H).

[0030] Example 3

[0031] Add Compound III (42.8 mg, 0.1 mmol), acetonitrile (2 mL) and 4-(bromomethyl)phenylboronic acid pinacol ester (34.4 mg, 0.1 mmol) successively to a 10 mL reaction flask equipped with a magnetic stirrer, and react at room temperature for 12 hours. After the reaction is completed, remove the solvent under reduced pressure, and the remaining reaction mixture is separated and purified by silica gel column chromatography (dichloromethane:methanol = 10:1) to obtain 18.1 mg of Compound I (white solid) with a yield of 30%, as an H 2 O 2 Fluorescent probe.

[0032] Of Compound I 1NMR (400 MHz, Chloroform-d) δ 8.14 (d, J = 2.8 Hz, 1H), 7.95 (d, J = 7.6 Hz, 1H), 7.67 (m, 2H), 7.55 (t, J = 7.6 Hz, 1H), 7.50 (d, J = 7.6 Hz, 2H), 7.23 (s, 1H), 7.17 (d, J = 8.8 Hz, 1H), 7.00 (d, J = 8.0 Hz, 2H), 6.91 (d, J = 9.2 Hz, 2H), 6.60 (dd, J = 8.8, 2.8 Hz, 2H), 5.76 (s, 2H), 4.00 (d, J = 21.6 Hz, 6H), 2.96 (s, 6H), 1.27 (s, 12H), 0.70 (s, 3H), 0.65 (s, 3H).

[0033] Example 4

[0034] (1) Spectral properties of probe I in response to H 2 O 2 at different times

[0035] The PBS solution of probe I was treated with 200 μM of H 2 O 2 and its fluorescence emission spectrum was measured using a fluorometer with 643 nm as the excitation light.

[0036] The emission spectrum is as Figure 4 shown. The probe responds rapidly and the fluorescence intensity increases significantly within 1 min.

[0037] (2) Spectral properties of probe I in response to different concentrations of H 2 O 2 The PBS solution of probe I was treated with 20 μM, 40 μM, 60 μM, 80 μM, 100 μM, 120 μM, 140 μM, 160 μM, 180 μM, 200 μM of H

[0038] respectively, and its fluorescence emission spectrum was measured using a fluorometer with 643 nm as the excitation light. 2 O 2 The emission spectrum is as

[0039] shown. The change in the fluorescence intensity of the probe shows a positive correlation with the concentration of H Figure 5 O 2 O 2 .< Figure 6 For the linear fitting of the concentration of H 2 O 2 and the luminescence intensity.

[0040] (3) Selectivity test of probe I

[0041] Use 100 μM of NO respectively 2 - 、GSH, CYS, HCY, Na + 、K + 、Zn 2+ 、Fe 3+ 、Mg 2+ 、Ca 2+ 、Cu 2+ 、Ba 2+ 、H 2 O 2 to treat the PBS solution of probe I, and use a fluorescence photometer to measure its fluorescence emission spectrum with 643 nm as the excitation light.

[0042] Figure 7 It shows that probe I has a specific response to H 2 O 2 and has no response to other analytes.

[0043] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A fluorescent probe for detecting hydrogen peroxide, characterized in that: The probe has a structure as shown in Formula I:

2. A method for preparing a fluorescent probe for detecting hydrogen peroxide according to claim 1, characterized in that: The following steps are involved: Compound II is dissolved in ultra-dry acetonitrile, 4-bromomethylphenylboronic acid pinacol ester 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, and the mixture is separated and purified by silica gel column chromatography to obtain a fluorescent probe as shown in Formula I; 3. The preparation method according to claim 2, characterized in that: The amount of the compound II used is 1.0 times the mole of 4-bromomethylphenylboronic acid pinacol ester.

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 is 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 hydrogen peroxide.

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