Synthesis and application of a quaternary ammonium salt rhodamine fluorescent probe for detecting superoxide anion radicals

Abstract

The present invention discloses a fluorescent probe for detecting O2 ·‑ , which is constructed with silicon rhodamine as the fluorescent group and diphenylphosphinyl as the recognition group. In the presence of O2 ·‑ , O2 ·‑ will perform a nucleophilic attack on the phosphate ester, followed by 1,6 elimination, resulting in the decomposition of the quaternary ammonium salt, releasing the silicon rhodamine luminophore and promoting its ring-opening. Before the reaction, the silicon rhodamine quaternary ammonium salt 8 with a spiro structure remains non-fluorescent and has a low background; after reacting with O2 ·‑ , the free and ring-opened silicon rhodamine dye can be excited to emit near-infrared fluorescence, thereby achieving the purpose of detecting O2 ·‑ , and it will have good application prospects in the field of bio-related sciences. The fluorescent probe for detecting O2 * - in the present invention has the structure shown in Formula 8: #imgabs0#

Description

Technical Field

[0001] The present invention relates to a fluorescent probe, a preparation method and an application thereof, and more specifically to a fluorescent probe for detecting O2 ·- Fluorescent probe, preparation method and application. Background Art

[0002] Superoxide anion free radical (O2 ·- ) is an important reactive oxygen species (ROS) in biological systems and has long been recognized as a crucial cell signaling molecule in numerous physiological and pathological processes. ·- Usually as other endogenous ROS such as H2O2, hydroxyl radicals (·OH), singlet oxygen ( 1 O2) and other precursors. At the same time, O2 ·- In biology, it can also serve as an important medium for oxidation chain reactions and plays a very important role in maintaining the normal physiological functions of cells. ·- When the concentration exceeds the body's inherent clearance capacity, it can lead to a variety of diseases, such as Alzheimer's disease, rheumatoid arthritis, diabetes, and even cancer. In addition, there is increasing evidence that O2 ·- It is also a metabolic byproduct of some drugs, such as doxorubicin, cisplatin, and morphine. Therefore, specific detection of O2 in living cells and tissues is necessary. ·- It is of great significance for elucidating its multiple roles in biological and pharmacological processes. Summary of the invention

[0003] The purpose of the present invention is to provide a method for detecting O2 ·- The quaternary ammonium salt ion-type fluorescent probe is constructed with silicon rhodamine quaternary ammonium salt as the parent and diphenylphosphinoyl as the recognition group. ·- In the presence of O2 ·- It will attack the phosphoryl ester with nucleophilicity, causing the quaternary ammonium salt to decompose, and then release the silicon rhodamine luminophore and promote its ring opening. Before the reaction, the silicon rhodamine quaternary ammonium salt 8 with a spirocyclic structure remains in a non-fluorescent state with low background; after the reaction, the free and ring-opened silicon rhodamine dye can be excited to emit near-infrared fluorescence, thereby realizing the detection of O2 ·- It has a good application prospect in the field of biological related sciences.

[0004] The response mechanism of the fluorescent probe of the present invention is as follows:

[0005]

[0006] The invention also provides a preparation method and application of the fluorescent probe.

[0007] The technical solution of the present invention to solve the technical problem is as follows:

[0008] The method for detecting O2 ·- A fluorescent probe having a structure as shown in Formula 8:

[0009]

[0010] The present invention also provides a method for preparing the above-mentioned probe molecule, the specific process is:

[0011] (1) Under nitrogen protection, compound 1, diphenylphosphinoyl chloride and triethylamine were added to a dry reaction bottle equipped with a magnetic rod and dissolved in dichloromethane, and stirred at 0°C for 12 hours. After the reaction was completed, the solvent was evaporated under reduced pressure, and compound 3 was obtained after separation and purification by silica gel column chromatography.

[0012] (2) Under nitrogen protection, compound 3, carbon tetrabromide and triphenylphosphine were added to a dry reaction bottle equipped with a magnetic rod and dissolved in dichloromethane. The mixture was stirred at 25° C. for 3 hours. After the reaction was completed, compound 4 was obtained after separation and purification by silica gel column chromatography.

[0013] (3) Under nitrogen protection, compound 5 and an organic solvent were added to a dry reaction bottle equipped with a magnetic rod, the solution was cooled to -78°C, a strong base was added to the reaction bottle and the mixture was stirred at -78°C for 2 hours. Dichlorodimethylsilane was added dropwise to the reaction bottle, and after the addition was completed, the reaction mixture was slowly raised to room temperature and then stirred for 12 hours. Compound 6 was obtained by separation and purification.

[0014] (4) Add compound 6, 2-carboxybenzaldehyde and copper bromide to a sealable pressure-resistant tube equipped with a magnet, and stir the pressure-resistant tube at 140° C. for 5 hours to obtain compound 7 by separation and purification;

[0015] (5) Add compound 4, compound 7 and solvent to a reaction bottle containing a magnetic particle, and stir the reaction at room temperature for 12 hours. ·- Fluorescent probe 8.

[0016] Wherein, the solvent in steps (1) and (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.

[0017] The preparation process of the preparation method can refer to the following equation:

[0018]

[0019] The present invention also provides an application of the fluorescent probe, which can be used for O2 ·-Compared with the prior art, the advantages of the present invention are: the silicon rhodamine fluorescent probe obtained by the present invention can realize efficient detection of O2 ·- , and the synthesis method is simple and feasible with high yield, so it is an ideal O2 ·- In addition, the probe has excellent biocompatibility and thus has broad application prospects in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of the reaction product 3 in Example 1;

[0021] Figure 2 is the hydrogen nuclear magnetic resonance spectrum of the reaction product 4 in Example 2;

[0022] Figure 3 is the hydrogen nuclear magnetic resonance spectrum of the reaction product 6 in Example 3;

[0023] Figure 4 is the hydrogen nuclear magnetic resonance spectrum of the reaction product 7 in Example 4;

[0024] Figure 5 is the hydrogen nuclear magnetic resonance spectrum of the reaction product 8 in Example 5;

[0025] Figure 6 O2 in Example 6 ·- Detection of the fluorescence intensity of fluorescent probe 8 over time;

[0026] Figure 7 O2 in Example 6 ·- Detection of fluorescence intensity of fluorescent probe 8 with O2 ·- Concentration variation graph;

[0027] Figure 8 is the O2 in Example 6 ·- Linear fitting of concentration and fluorescence intensity;

[0028] Fig. 9 O2 in Example 6 ·- Fluorescence intensity diagram of the selectivity experiment for detecting fluorescent probe 8. DETAILED DESCRIPTION

[0029] 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.

[0030] Example 1

[0031] Under nitrogen protection, compound 1 (1.24 g, 10 mmol), diphenylphosphinoyl chloride (2.36 g, 10 mmol), triethylamine (2.0 g, 20 mmol) and ultra-dry dichloromethane (20 mL) were added to a 100 mL dry flask equipped with a magnet; the flask was placed at 0 ° C, stirred for 1 h, slowly returned to room temperature and stirred for 5 hours. After the reaction, the organic layer was washed with water and saturated sodium chloride aqueous solution, respectively, and dried over anhydrous sodium sulfate. After the residual solvent was removed under reduced pressure, silica gel column chromatography (petroleum ether: ethyl acetate = 2: 1) was used for separation and purification to obtain 1.60 g of compound 3 (white solid), with a yield of 51%.

[0032] Compound 3 1 H NMR(400MHz, Chloroform-d)δ 7.90-7.85 (m, 4H), 7.54 (td, J=7.2, 1.6Hz, 2H), 7.46 (td, J=8.0, 4.0Hz, 4H), 7.22 (d, J=8.8Hz, 1H), 7.17-7.15 (m, 2H), 4.59 (s, 2H).

[0033] Example 2

[0034] Under nitrogen protection, compound 3 (324 mg, 1.0 mmol), carbon tetrabromide (663 mg, 2.0 mmol), triphenylphosphine (656 mg, 2.5 mmol) and ultra-dry dichloromethane (10 mL) were added to a 100 mL dry flask equipped with a magnetic pole; the mixture was stirred at 25°C for 3 hours. After the reaction was completed, the remaining solvent was removed under reduced pressure, and the remaining reaction mixture was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 3: 1, v: v) to obtain 302 mg of compound 4 (white solid), with a yield of 78%.

[0035] Compound 4 1 H NMR (400MHz, Chloroform-d) δ7.91-7.86 (m, 4H), 7.54 (td, J=7.6, 1.6Hz, 2H), 7.49-7.45 (m, 4H), 7.27-7.25 (m, 2H), 7.19-7.16 (m, 2H), 4.41 (s, 2H).

[0036] Example 3

[0037] Under nitrogen protection, compound 5 (3.0 g, 15 mmol) and tetrahydrofuran (40 mL) were added to a 250 mL dry flask equipped with a magnetic device. The solution was cooled to -78 ° C, n-butyl lithium (2.5 M in n-hexane, 6.3 mL, 15.7 mmol) was added dropwise to the reaction flask, and stirring was continued at -78 ° C for 2 hours. Dichlorodimethylsilane (0.87 mL, 9.0 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 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 over anhydrous sodium sulfate. After the residual solvent was removed under reduced pressure, silica gel column chromatography (petroleum ether: ethyl acetate = 20: 1) was used to separate and purify to obtain 1.80 g of compound 6 (yellow oily liquid), with a yield of 80%.

[0038] Compound 6 1 H NMR (400MHz, Chloroform-d) δ 7.28-7.25 (m, 2H), 6.96 (d, J=2.8Hz, 2H), 6.94 (d, J=7.2Hz, 2H), 6.80-6.77 (m, 2H), 2.95 (s, 12H), 0.56 (s, 6H).

[0039] Example 4

[0040] Compound 6 (1.0 g, 3.4 mmol), o-carboxybenzaldehyde (2.5 g, 16.8 mmol) and copper bromide (74 mg, 0.34 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 445 mg of compound 7 (white solid), with a yield of 31%.

[0041] Compound 7 1 H NMR(400MHz, Chloroform-d)δ 7.97 (d, J=7.6Hz, 1H), 7.64 (td, J=7.6, 1.2Hz, 1H), 7.54 (td, J=7.6, 1.2Hz, 1H), 7.31 (d, J=7.6Hz, 1H), 6.98 ( d, J=2.8Hz, 2H), 6.79 (d, J=9.2Hz, 2H), 6.55 (dd, J=8.8, 2.8Hz, 2H), 2.97 (s, 12H), 0.65 (s, 3H), 0.62 (s, 3H).

[0042] Example 5

[0043] Compound 7 (42 mg, 0.10 mmol), ultra-dry acetonitrile (2 mL) and compound 4 (39 mg, 0.1 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 reaction mixture was separated and purified by silica gel column chromatography (dichloromethane: methanol = 10: 1) to obtain 25 mg of compound 8 (white solid) with a yield of 43%. As O2 ·- Fluorescent probes.

[0044] Compound 8 1 H NMR(400MHz, Chloroform-d)δ 8.23 (d, J=2.4Hz, 1H), 7.97 (d, J=7.6Hz, 1H), 7.81-7.75 (m, 4H), 7.70 (td, J=7.6, 0.8Hz , 1H), 7.58 (t, J=7.2Hz, 2H), 7.51 (t, J=7.2Hz, 1H), 7.44-7.40 (m, 4H), 7.29 (d, J=7.6Hz, 1H), 7.13 (d, J=9.2Hz, 1H), 7.01-6.94 (m, 5H), 6.90 (d, J=8.8Hz, 2H), 6.60 (dd, J=9.2, 2. 8Hz, 1H), 5.69(s, 2H), 3.88(s, 3H), 3.87(s, 3H), 2.98(s, 6H), 0.74(s, 3H), 0.66(s, 3H).

[0045] Example 6

[0046] (1) In different time gradients, probe 8 reacts with O2 ·- Spectral properties of the response

[0047] The probe 8 was dissolved in DMSO and the DMSO solution of KO2 was added (KO2 was used as O2 ·- Source), after incubation for a certain period of time, the fluorescence emission spectrum was measured in PBS buffer (1% DMSO) using a fluorescence spectrophotometer with 643 nm as the excitation light.

[0048] The emission spectrum is Figure 6 As shown, the probe responded quickly and the fluorescence intensity was significantly enhanced within 1 minute.

[0049] (2) Probe 8 for different concentration gradients of O2 ·- Spectral properties of the response

[0050] Probe 8 was dissolved in DMSO, and 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, and 50 μM DMSO solutions of KO2 were added respectively. After incubation for 1 min, its fluorescence emission spectrum was measured in PBS buffer (1% DMSO) using a fluorescence photometer with 643 nm as the excitation light.

[0051] The emission spectrum is Figure 7 As shown, the change of probe fluorescence intensity is related to O2 ·- The concentration showed a positive correlation trend. Figure 8 For O2 ·- Linear fitting of concentration and luminescence intensity. According to the detection limit calculation formula: 3σ / k, the detection limit of the probe was calculated to be 0.37μM.

[0052] (3) Selectivity test of probe 8

[0053] The probe 8 was dissolved in DMSO and 100 μM H2O2 and ClO were added. - , 1 O2, ·OH, t-BuOOH, NO2 - , GSH, CYS, HCY, Na + , K + 、Zn 2+ , Fe 3+ Mg 2+ , Ca 2+ , Cu 2+ , Ba 2+ 、O2 ·- The solution was incubated for 1 min, and then its fluorescence emission spectrum was measured in PBS buffer (1% DMSO) using a fluorescence spectrophotometer with 643 nm as the excitation light.

[0054] Fig. 9 Display, probe 8 for O2 ·- There is a specific response, but no obvious response to other test substances.

[0055] 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 superoxide anion free radicals, characterized in that: The probe has a structure as shown in Formula 8:

2. A method for preparing a fluorescent probe for detecting superoxide anion free radicals as claimed in claim 1, characterized in that: The following steps are involved: Compound 7 was dissolved in ultra-dry acetonitrile, and compound 4 was added, and the mixture was stirred at room temperature for 12 hours. After the reaction was completed, the solvent was evaporated under reduced pressure, and the fluorescent probe shown in Formula 8 was 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 4 used is 1.0 times the molar amount of compound 7.

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 superoxide anion free radicals.

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