Application of pomalidomide and various derivatives thereof as electrochemical luminescence probes

Abstract

The invention provides application of pomalidomide and derivatives thereof as electrochemical luminescence probes. Pomalidomide and a derivative probe thereof are respectively added into a potassium persulfate co-reactant solution, and an electrochemical luminescence reaction is carried out to generate an electrochemical luminescence signal. By modifying the molecular structure of the pomalidomide, the butyne derivative in the pomalidomide derivative has higher ECL efficiency (125%) and longer emission wavelength (696 nm) than classical ruthenium dipyridyl, can be prevented from being interfered by high-potential side reaction, and can effectively avoid ultraviolet region interference of a complex matrix through a near-infrared electrochemical luminescence signal; therefore, the method has a wider application prospect in the field of medical examination.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of electrochemistry, and particularly relates to the application of pomalidomide and derivatives thereof as electrochemiluminescence probes. BACKGROUND

[0002] Electrochemiluminescence refers to the process that a luminescent substance undergoes oxidation or reduction on the surface of an electrode, high-energy electron transfer occurs, an excited state is formed, and finally a photon is released to return to the ground state. Electrochemiluminescence has the advantages of high sensitivity, high selectivity, simple operation, small background signal, and low detection limit, and has wide application prospects in the fields of biology, medicine, environmental monitoring, etc., thus attracting great attention from researchers.

[0003] In an electrochemiluminescence system, there are usually two key components, namely a luminophore and a co-reactant. The design and development of a high-performance electrochemiluminescence luminophore is an important prerequisite for achieving high-sensitivity analysis. Among various electrochemiluminescence probes, small-molecule luminescent probes have the advantages of stable and repeatable production, small batch-to-batch difference, good biocompatibility, clear molecular structure, and easy modification, making small-molecule luminescent probes extremely attractive in the field of electrochemiluminescence detection. Currently, small-molecule probes used for electrochemiluminescence detection mainly include ruthenium bipyridine and derivatives, silane derivatives, thiophene derivatives, fluoroboropyrrole derivatives, coumarin derivatives, 9,10-diaromatic anthracene (DPA) derivatives, 1,1,2,2-tetraphenylethylene derivatives, fluorene derivatives, and porphyrin derivatives. Among them, drug small molecules are a good candidate for electrochemiluminescence luminophores due to their commercialization and easy availability. However, the drug small molecules reported in the literature are only used as co-reactants for electrochemiluminescence. So far, there has only been one report of using the drug norfloxacin as an electrochemiluminescence luminophore. The research on drug molecules as electrochemiluminescence probes is still in its initial stage. Therefore, it is urgent to develop new drug molecules as electrochemiluminescence probes to provide new possibilities for constructing high-performance electrochemiluminescence detection platforms for medical testing and studying drug-target interactions. SUMMARY

[0004] The application aims to provide the application of pomalidomide and its various derivatives as electrochemiluminescence probes. The derivatives include acetylene derivatives, nitration derivatives, hydroxylation derivatives, amino derivatives at position 5, fluorination derivatives, bromination derivatives, and iodination derivatives. A new electrochemiluminescence system of pomalidomide and its various derivatives / potassium persulfate is developed by taking potassium persulfate as a co-reactant, and the system has good luminescence effect. It is worth mentioning that the ECL phenomena of pomalidomide and its various derivatives mentioned above have not been reported. Compared with the high luminescence potential of-1.8 V of the classic ruthenium bipyridine small molecule probe in the potassium persulfate system, pomalidomide and its derivatives have lower cathode potentials (-1.2-1.5 V). In addition, by modifying the molecular structure of pomalidomide, the acetylene derivative of the new pomalidomide derivative has higher ECL efficiency (125%) and longer emission wavelength (696 nm) than the classic ruthenium bipyridine, can be immune to the interference of high potential side reactions, and can effectively avoid the interference of complex matrix in the ultraviolet region through near-infrared electrochemiluminescence signals, thus having a broader application prospect in the field of medical testing.

[0005] Specifically, the application adopts the technical solution of: The application of pomalidomide and its derivatives as electrochemiluminescence probes: pomalidomide and its various derivative probes are added into a potassium persulfate co-reactant solution, electrochemiluminescence reactions are carried out, and electrochemiluminescence signals are generated.

[0006] The electrochemiluminescence signal of the application is collected by the following method: the glassy carbon electrode is polished to a smooth mirror surface with Al2O3 powder, and then sequentially ultrasonically cleaned in an aqueous HNO3 solution, anhydrous ethanol, and deionized water, and dried with N2; a three-electrode system is used for testing, a bare electrode is used as a working electrode, a platinum wire electrode is used as a counter electrode, Ag / AgCl is used as a reference electrode, a buffer solution is phosphate buffer or Tris-HCl buffer solution, and KCl or KNO3 is used as an electrolyte; the above three electrodes are inserted into a buffer solution containing pomalidomide and its derivatives and potassium persulfate co-reactant, a certain voltage is applied, and electrochemiluminescence radiation is generated on the surface of the working electrode.

[0007] In the application, the concentration of potassium persulfate is preferably 0.01-0.2 M ( Figure 16 ), the concentration of pomalidomide and its acetylene derivative is preferably 0.025-0.5 mg / mL ( Figure 17 ), and the pH of the buffer solution is preferably 5-9 ( Figure 18 ).

[0008] The structure of the pomalidomide derivative is: The application has the following advantages: The obtained pomalidomide derivative has the advantages that the pomalidomide acetylene derivative probe ECL has large light emission intensity, the electrochemiluminescence quantum yield is the highest, the electrochemiluminescence efficiency of the relative bipyridyl ruthenium is 125%, and the pomalidomide acetylene derivative is green, non-toxic, low in price, good in biocompatibility, and has good application prospect in the fields of electrochemiluminescence sensor devices and biomedicine. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 The pomalidomide acetylene derivative is 1 H NMR and structural formula; Figure 2 The pomalidomide acetylene derivative is 1 H NMR and structural formula; Figure 3 The pomalidomide acetylene derivative is 1 H NMR and structural formula; Figure 4 The pomalidomide acetylene derivative is 1 H NMR and structural formula; Figure 5 The ECL-potential diagram of the pomalidomide is Figure 6 The ECL-potential diagram of the pomalidomide acetylene derivative is Figure 7 The ECL-potential diagram of the pomalidomide acetylene derivative is Figure 8 The ECL-potential diagram of the pomalidomide acetylene derivative is Figure 9 The ECL-potential diagram of the pomalidomide acetylene derivative is Figure 10 The ECL-potential diagram of the pomalidomide acetylene derivative is Figure 11 The ECL-potential diagram of the pomalidomide acetylene derivative is Figure 12 The ECL-potential diagram of the pomalidomide acetylene derivative is Figure 13 The fluorescence excitation spectrum and emission spectrum diagram of the pomalidomide (left) and the pomalidomide acetylene derivative (right) are shown in the following figure: Figure 14 The electrochemiluminescence spectrum diagram of the pomalidomide (left) and the pomalidomide acetylene derivative (right) is shown in the following figure: Figure 15 The electrochemiluminescence intensity diagram of the pomalidomide (left) and the pomalidomide acetylene derivative (right) probe obtained by continuous scanning for 15 segments is shown in the following figure: Figure 16 The influence of different concentrations of potassium persulfate used for the pomalidomide (left) and the pomalidomide acetylene derivative (right) probe on the electrochemiluminescence intensity is shown in the following figure: Figure 17 Effect of different concentrations of the probe for pomalidomide (left) and its acetylene derivative (right) on electrochemiluminescence intensity; Figure 18 Effect of different pH buffers used for the probe for pomalidomide (left) and its acetylene derivative (right) on electrochemiluminescence intensity. DETAILED DESCRIPTION

[0010] The present application is further described below in conjunction with the accompanying drawings and specific examples, but the present application is not limited thereto.

[0011] The synthesis steps of the pomalidomide butyl acetylene derivative are as follows: 5.0 g of 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione, 50 mL of N,N-dimethylformamide, 1.2 g of 1-amino-3-butynyl and 7.0 g of N,N-diisopropylethylamine are mixed, heated to 95°C and reacted for 16 h; TLC is used to monitor the completion of the reaction, 100 mL of water is added to quench the reaction, 100 mL of ethyl acetate is used to extract twice, the combined ethyl acetate is washed with 100 mL of water once, the organic solvent is removed under reduced pressure, and then the product is purified by column chromatography. 1 H NMR (400 MHz, CDCl3-d) δ 8.06 (s, 1H), 7.53-7.49 (m, 1H), 7.14-7.12 (m, 1H), 6.93-6.91 (m, 1H), 6.49 (t, J = 5.5 Hz, 1H), 4.96-4.89 (m, 1H), 3.49 (q, J = 6.7 Hz, 2H), 2.91 – 2.68 (m, 3H), 2.56-2.52 (m, 2H), 2.18-2.05 (m, 2H), 1.30 – 1.16 (m, 2H). Figure 1 ) The synthesis steps of the 4-nitro-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione are as follows: the raw material 4-nitrophthalimide (1.0 g, 1.0 eq) is weighed into a reaction bottle, 10 mL of acetic acid is added, stirring is started, the raw material 3-amino-2,6-piperidinedione hydrochloride (0.8 g, 1.0 eq) and anhydrous sodium acetate (0.5 g, 1.2 eq) are added to the reaction bottle, and heated to 115 o C for 4 h. TLC is used to detect the completion of the reaction, and the temperature is lowered to 25 oC, add water 20 mL to the reaction solution, extract with 30 mL of isopropyl alcohol three times, combine the organic phase and concentrate to get the crude product, purify by column chromatography (DCM:MeOH 20:1) to get 150 mg of product 4-nitro-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione. 1 H NMR (400 MHz, DMSO-d6) δ 11.19 (s, 1H), 8.39-8.33 (m, 1H), 8.27-8.22 (m, 1H), 8.16-8.11 (m, 1H), 5.24-5.19 (m, 1H), 2.95-2.86 (m, 5.4 Hz, 1H), 2.65-2.61 (m, 1H), 2.58-2.47 (m, 1H), 2.13-2.06 (m, 1H). Figure 2 ) The synthesis of the 5-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione is as follows: weigh the starting material 5-nitrophthalimide (1.0 g, 1.0 eq) into a reaction bottle, add acetic acid 10 mL, start stirring, add the starting material 3-amino-2,6-piperidinedione hydrochloride (0.8 g, 1.1 eq) and anhydrous sodium acetate (0.5 g, 1.2 eq) to the reaction bottle, heat to 115 o C for 4 hours. TLC detects that the reaction is complete, cool to 25 o C, add water 20 mL to the reaction solution, extract with 30 mL of isopropyl alcohol three times, combine the organic phase and concentrate to get the crude product, purify by column chromatography (DCM:MeOH 20:1) to get 150 mg of product 4-nitro-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione. o C for 4 hours. TLC detects that the reaction is complete, cool to 25 o C, filter out the Pd / C, concentrate the filtrate to get 150 mg of product 5-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione. 1H NMR(400 MHz, DMSO-d6) δ 11.08 (s, 1H), 7.54-7.52 (m, 1H), 6.97 -6.96(m, 1H), 6.86-6.83(m, 1H), 6.55 (s, 2H), 5.06-5.01 (m, 1H), 2.93-2.84 (m, 1H), 2.64-2.48 (m, 2H), 2.06-1.98 (m, 1H). ( Figure 3 ) The synthesis steps of the 4-hydroxy-2-(2,6-dioxopiperidin-3-yl)isodihydroindole-1,3-diketone sodium salt are as follows: Weigh 1.0 g (1.0 eq) of 4-hydroxyphthalimide into a reaction flask, add 10 mL of acetic acid, start stirring, add 1.1 g (1.1 eq) of 3-amino-2,6-piperidinediketone hydrochloride and 0.6 g (1.2 eq) of anhydrous sodium acetate into the reaction flask, and heat to 115 °C. o The reaction was carried out at C for 4 hours. TLC analysis confirmed the reaction was complete, and the temperature was then lowered to 25°C. o C. Add 20 mL of water to the reaction solution, extract three times with 30 mL of acetonitrile, combine the organic phases and concentrate to obtain the crude product. Purify by column chromatography (DCM:MeOH 10:1) to obtain 200 mg of the product 4-hydroxy-2-(2,6-dioxadipin-3-yl)isoindoline-1,3-dione. Weigh 150 mg (1.0 eq) of the starting material 4-hydroxy-2-(2,6-dioxadipin-3-yl)isoindoline-1,3-dione into a reaction flask, add 5 mL of ethanol and 5 mL of water, start stirring, add sodium hydroxide (29 mg, 1.0 eq), and stir at room temperature for 2 hours until clear. Lyophilize the reaction solution to obtain 170 mg of the product 4-hydroxy-2-(2,6-dioxadipin-3-yl)isodihydroindoline-1,3-dione sodium salt. 1 H NMR (400MHz, D2O-d2) δ 7.37-7.32 (m, 1H), 6.91-6.87 (m, 1H), 6.83-6.79 (m, 1H), 2.88-2.42 (m, 1H), 2.30-2.05 (m, 3H). ( Figure 4 ) The remaining molecules include pomalidomide (CAS No.: 19171-19-8, molecular formula: C). 13 H 11 N3O4), pomalidomide fluorinated derivatives (CAS No.: 2244520-92-9, molecular formula: C 13H9FN2O4), pomalidomide bromide derivative (CAS No: 2093536-12-8, molecular formula: C 13 H9BrN2O4), pomalidomide iodide derivative (CAS No: 959150-64-2, molecular formula: C 13 H9IN2O4), all purchased from Aladdin Biochem Technology Co., Ltd.

[0012] Example 1 A glassy carbon electrode with a diameter of 3 mm was polished to a smooth mirror surface with 1.0 μm, 0.3 μm and 0.05 μm Al2O3 powder in turn, and then ultrasonically cleaned in HNO3 solution (volume ratio of concentrated nitric acid to water 1:1), anhydrous ethanol and deionized water for 3 minutes, and dried with N2. The above electrode was inserted into a 0.1 mol / L pH 7.4 phosphate buffer solution containing 0.4 mg / mL pomalidomide and its derivatives, 0.1 mol / L potassium persulfate and 0.1 mol / L KCl. Using cyclic voltammetry, a linear scanning voltage of 0 V ~-1.8 V was applied, and the scanning speed was 0.1 V / s. The high voltage of the photomultiplier tube was set to 800 V, and the electrochemiluminescence signals generated on the surface of the working electrode corresponding to the eight probes were detected respectively, and strong electrochemiluminescence signals were obtained. The ECL emission potential of pomalidomide was-1.384 V, and the intensity was 16360 (see Figure 5 ). The ECL emission potential of pomalidomide butynyl derivative was-1.443 V, and the intensity was 12000 (see Figure 6 ). The ECL emission potential of nitro derivative was-1.457 V, and the intensity was 9832 (see Figure 7 ). The ECL emission potential of hydroxyl derivative was-1.323 V, and the intensity was 12490 (see Figure 8 ). The ECL emission potential of 5-amino substituted derivative was-1.431 V, and the intensity was 3359 (see Figure 9 ). The ECL emission potential of fluorinated derivative was-1.254 V, and the intensity was 112 (see Figure 10 ). The ECL emission potential of brominated derivative was-1.288 V, and the intensity was 224 (see Figure 11 ). The ECL emission potential of iodinated derivative was-1.225 V, and the intensity was 621 (see Figure 12 ).

[0013] Example 2 The pomalidomide powder was dissolved in ultrapure water for fluorescence spectrum analysis, and the maximum excitation wavelength and emission wavelength were 392 nm and 500 nm respectively (see Figure 13), the maximum excitation wavelength and the emission wavelength of the pomalidomide butynyl derivative powder dissolved in ultrapure water for fluorescence spectrum analysis were 422 nm and 528 nm, respectively (see Figure 13 ).

[0014] Example 3 A glassy carbon electrode with a diameter of 3 mm was polished to a smooth mirror surface with 1.0 μm, 0.3 μm and 0.05 μm Al2O3 powder, respectively, and then ultrasonically cleaned in HNO3 solution (volume ratio of concentrated nitric acid to water 1:1), anhydrous ethanol and deionized water for 3 minutes, and dried with N2. The above electrode was inserted into a 0.1 mol / L pH 7.4 phosphate buffer solution containing 0.4 mg / mL pomalidomide and pomalidomide butynyl derivative, 0.1 mol / L potassium persulfate and 0.1 mol / L KCl. The cyclic voltammetry was used, a linear scanning voltage of 0 V to-1.8 V was applied, the scanning speed was 0.1 V / s, and the electrochemiluminescence intensity in the range of 400-950 nm was collected. The high voltage of the photomultiplier tube was set to 800 V, and the electrochemiluminescence signals generated on the surface of the working electrode corresponding to the two probes were detected, respectively, to obtain the ECL maximum emission wavelength of pomalidomide as 500 nm and the ECL maximum emission wavelength of pomalidomide butynyl derivative as 696 nm (see Figure 14 ).

[0015] Example 4 The electrochemical test was performed using a three-electrode system, a bare electrode was used as the working electrode, a platinum wire electrode was used as the counter electrode, and Ag / AgCl was used as the reference electrode. The above working electrode was inserted into a 0.1 M pH 7.4 phosphate buffer solution containing 0.4 mg / mL pomalidomide and pomalidomide butynyl derivative, 0.1 mol / L potassium persulfate and 0.1 mol / L KCl. The cyclic voltammetry was used, a linear scanning voltage of 0 V to-1.8 V was applied, and the scanning speed was 0.1 V / s. The high voltage of the photomultiplier tube was set to 800 V, 15 continuous scans were performed, and the electrochemiluminescence signals of pomalidomide and its butynyl derivative were recorded, respectively. The electrochemiluminescence signals remained basically unchanged, the relative standard deviation of the ECL 15 signals of pomalidomide was 2%, and the relative standard deviation of the ECL 15 signals of pomalidomide butynyl derivative was 4.9% (see Figure 15 ).

[0016] Example 5 The three-electrode system was used for electrochemical test, the bare electrode was used as the working electrode, the platinum wire electrode was used as the counter electrode, and Ag / AgCl was used as the reference electrode. The above working electrode was inserted into 0.1 M pH 7.4 phosphate buffer solution containing 0.4 mg / mL pomalidomide and pomalidomide butynyl derivative, different concentrations of potassium persulfate and 0.1 mol / L KCl. The cyclic voltammetry method was used to measure the electrochemiluminescence under the condition of different concentrations of potassium persulfate, and the curves of the electrochemiluminescence intensity of pomalidomide and pomalidomide butynyl derivative respectively versus the concentration of potassium persulfate were drawn (see Figure 16 ).

[0017] Example 6 The three-electrode system was used for electrochemical test, the bare electrode was used as the working electrode, the platinum wire electrode was used as the counter electrode, and Ag / AgCl was used as the reference electrode. The above working electrode was inserted into 0.1 M pH 7.4 phosphate buffer solution containing different concentrations of pomalidomide and pomalidomide butynyl derivative, 0.1 mol / L potassium persulfate and 0.1 mol / L KCl. The cyclic voltammetry method was used to measure the electrochemiluminescence, and the curves of the electrochemiluminescence intensity versus the concentration of pomalidomide and its butynyl derivative were drawn (see Figure 17 ).

[0018] Example 7 The three-electrode system was used for electrochemical test, the bare electrode was used as the working electrode, the platinum wire electrode was used as the counter electrode, and Ag / AgCl was used as the reference electrode. The above working electrode was inserted into 0.1 M different pH phosphate buffer solution containing 0.4 mg / mL pomalidomide and pomalidomide butynyl derivative, 0.1 mol / L potassium persulfate and 0.1 mol / L KCl. The cyclic voltammetry method was used to measure the electrochemiluminescence of pomalidomide and pomalidomide butynyl derivative respectively under the condition of different pH, and the curves of the electrochemiluminescence intensity of pomalidomide and pomalidomide butynyl derivative versus the pH of potassium persulfate solution were drawn (see Figure 18 ).

[0019] Example 8 The three-electrode system was used for electrochemical test, the bare electrode was used as the working electrode, the platinum wire electrode was used as the counter electrode, and Ag / AgCl was used as the reference electrode. The above electrode was inserted into 0.1 mol / L pH 7.4 phosphate buffer solution containing pomalidomide and pomalidomide butynyl derivative, 0.1 mol / L potassium persulfate and 0.1 mol / L KCl. The cyclic voltammetry method was used, and the linear scanning voltage of 0V ~ -1.8V was applied, the scanning speed was 0.1 V / s, the high voltage of the photomultiplier tube was set to 700 V, and the electrochemiluminescence signals generated by pomalidomide and pomalidomide butynyl derivative respectively on the surface of the working electrode were detected (see IThe corresponding amount of electricity generated is Q. f Additionally, a 3 mm diameter glassy carbon electrode was successively polished and ground with 1.0 μm, 0.3 μm, and 0.05 μm Al₂O₃ powders until a smooth mirror surface was achieved. It was then sequentially immersed in HNO₃ solution (concentrated nitric acid to water volume ratio 1:1), anhydrous ethanol, and deionized water for ultrasonic cleaning for 3 minutes, and dried with N₂. A three-electrode system was used, with the bare glassy carbon electrode as the working electrode, a platinum wire electrode as the counter electrode, and Ag / AgCl as the reference electrode. The bare glassy carbon electrode was inserted into a solution containing 1.0 mmol / L [Ru(bpy)₃]. 2+ In an acetonitrile solution containing 0.1 mol / L tetrabutylperchlorate, a linear scan voltage of -1.0 V to -1.8 V was applied at a scan rate of 0.2 V / s. The photomultiplier tube voltage was set to 700 V, and the electrochemiluminescence signal generated on the working electrode surface was detected. I ° The corresponding amount of electricity generated is Q. ° f From the formula Φ ECL = Φ ° ECL ( I Q ° f / I ° Q f The electrochemiluminescence efficiency of pomalidomide was calculated. Φ ECL The electrochemiluminescence efficiency of pomalidomide butyne derivatives was 50%. Φ ECL It is 125%.

[0020] The above description is merely a typical 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 within the protection scope of the present invention.

Claims

1. The application of pomalidomide and its derivatives as electrochemiluminescent probes, characterized in that, Pomalidomide and its derivative probes were added to a potassium persulfate co-reactant solution to carry out an electrochemiluminescence reaction and generate an electrochemiluminescence signal.

2. The application according to claim 1, characterized in that, Electrochemiluminescence signals were acquired using the following method: a three-electrode system was used for testing, with a glassy carbon electrode as the working electrode, a platinum wire electrode as the counter electrode, and Ag / AgCl as the reference electrode. The buffer solution was either phosphate buffer or Tris-HCl buffer solution, and the electrolyte was KCl or KNO3. The three electrodes were inserted into a buffer solution containing pomalidomide and its derivatives and potassium persulfate as co-reactants. A certain voltage was applied, and electrochemiluminescence radiation was generated on the surface of the working electrode.

3. The application according to claim 2, characterized in that, The concentration of potassium persulfate is 0.01~0.2 M.

4. The application according to claim 2, characterized in that, The concentration of pomalidomide and its derivatives is 0.025~0.5 mg / mL.

5. The application according to claim 2, characterized in that, The pH of the buffer solution is 5-9.

6. The application according to claim 1, characterized in that, The structure of pomalidomide derivatives is as follows: .

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