A stable isotope labeling reagent for terminal alkyne compounds and its synthesis and application

Abstract

The present invention belongs to the field of analytical technology, and specifically relates to a stable isotope labeling reagent for terminal alkyne compounds and its synthesis and application. d 0 / d 5-1-benzyl-3-(3-azidopropyl)-imidazolium bromide, wherein the benzene ring is used as an isotope group, the imidazole salt is used as a mass spectrometry signal enhancement label, and the azide group is used as a reactive group. d 0 / d 5-Benzyl bromide is synthesized via a simple three-step reaction, using 5-benzyl bromide as the starting material. This labeling reagent can highly selectively label terminal alkynes via a copper-catalyzed azide-alkynyl cycloaddition (CuAAC) reaction. It boasts a simple synthesis, ease of operation, high labeling yield, mild reaction conditions, and a strong mass spectrometric signal from the labeled product. Combined with liquid chromatography-mass spectrometry, it enables accurate and sensitive detection of trace amounts of terminal alkynes.

Description

Technical Field

[0001] The present invention relates to a stable isotope labeling reagent for terminal alkyne compounds, a synthesis method and application thereof, and belongs to the technical field of analysis. Background Art

[0002] High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) is becoming the leading technique for the analysis of terminal alkyne compounds due to its high selectivity, high sensitivity, and ability to simultaneously monitor multiple analytes. Selected reaction monitoring (SRM) or multiple reaction monitoring (MRM) modes can selectively monitor specific analytes in the sample, effectively improving detection sensitivity. However, many terminal alkynes are unstable, exhibiting poor ionization efficiencies in positive ionization mode and exhibiting low abundance in real-world samples, posing significant challenges to HPLC-MS / MS analysis (J. Nat. Prod. 2022, 85, 105–114; J. Agric. Food Chem., 2023, 71, 14814-14824). In addition, the presence of complex matrices in actual samples is another thorny issue when using HPLC-MS / MS for analysis. Matrix effects caused by the sample matrix and co-eluted impurities may lead to significant deviations in quantitative results, thereby affecting the accuracy of the measurement results (Anal. Chim. Acta, 2016, 905, 106-114).

[0003] In order to solve these problems, stable isotope labeling technology has received widespread attention in recent years. This method uses light / heavy stable isotope labeling reagents to carry out specific chemical labeling reactions. 12 C) introduces the target analyte into the actual sample, and adds a heavy isotope label (such as D or 13 C) Introducing a standard as an internal standard, then mixing the lightly and heavily labeled samples for HPLC-MS / MS analysis, can effectively mitigate the impact of matrix effects by using a heavy isotope label as an internal standard. However, currently developed chemical labeling reagents have certain drawbacks, such as slow reaction rates, low yields, harsh reaction conditions, and weak labeled product signals. Furthermore, many labeling reagents require complex synthesis methods and are difficult to prepare, significantly limiting their application. Summary of the Invention

[0004] The present invention aims to provide a stable isotope labeling reagent for terminal alkyne compounds. The labeling reagent has the characteristics of simple synthesis, mild reaction conditions, strong selectivity, high labeling reaction yield, and strong labeled product signal. Combined with HPLC-MS / MS technology, it can achieve accurate and highly sensitive detection of terminal alkyne compounds.

[0005] 1. Stable isotope labeling reagents for terminal alkyne compounds and their synthesis

[0006] The stable isotope labeling reagent provided by the present invention uses a benzene ring as an isotope group, an imidazole salt as a mass spectrometry signal enhancement label, and an azide group as a reactive group. Its chemical name is d 0 / d 5-1-Benzyl-3-(3-azidopropyl)-imidazolium bromide, the chemical structure is shown below:

[0007] ,

[0008] Among them: When R=H, it is called d 0-1-Benzyl-3-(3-azidopropyl)-imidazolium bromide, abbreviated as d 0-ABI; when R=D, it is called d 5-1-Benzyl-3-(3-azidopropyl)-imidazolium bromide, abbreviated as d 5-ABI.

[0009] The present invention provides a method for synthesizing a stable isotope labeling reagent for a terminal alkyne compound, comprising the following steps:

[0010] (1) The first step of substitution reaction, intermediate Ⅰ ( d 0 / d Synthesis of 5-1-benzylimidazole

[0011] Will d 0 / d 5- was dissolved in tetrahydrofuran, imidazole and sodium hydroxide were dissolved in ultrapure water, and the resulting aqueous solution was added dropwise to the tetrahydrofuran solution under inert gas protection, and heated to react. After the reaction was completed, the mixture was cooled to room temperature and the solvent was removed by distillation under reduced pressure. The crude product was extracted three times with dichloromethane:water (3:1, v / v). The organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the solvent was removed by distillation under reduced pressure. Purification was performed by silica gel column chromatography to obtain intermediate I ( d 0 / d 5-1-benzylimidazole).

[0012] In step (1), d 0 / d The molar ratio of 5-benzyl bromide to imidazole is 1:1 to 1:3 (preferably 1:2 to 1:3). d 0 / dThe molar ratio of 5-benzyl bromide to sodium hydroxide is 1:1-1:2.5 (preferably 1:1.5-1:2.5), the volume ratio of the reaction solvent water to tetrahydrofuran is 1:1-1:8 (preferably 1:5-1:8), the reaction heating temperature is 65-85°C (preferably 70-80°C), and the reaction time is 2-8 h (preferably 4-8 h).

[0013] (2) The second step of salt formation reaction, intermediate II ( d 0 / d Synthesis of 5-1-benzyl-3-(3-bromopropyl)-imidazolium bromide

[0014] Dissolve intermediate I and 1,3-dibromopropane in acetonitrile and heat to react. After the reaction is completed, cool to room temperature, remove the solvent by distillation under reduced pressure, and obtain intermediate II ( d 0 / d 5-1-benzyl-3-(3-bromopropyl)-imidazolium bromide).

[0015] In step (2), the molar ratio of intermediate I to 1,3-dibromopropane is 1:1-1:20 (preferably 1:4-1:10), the reaction heating temperature is 80-100°C (preferably 85-95°C), and the reaction time is 4-12 h (preferably 6-12 h).

[0016] (3) The third step is substitution reaction, target product d 0 / d Synthesis of 5-1-Benzyl-3-(3-azidopropyl)-imidazolium Bromide

[0017] Dissolve intermediate II and sodium azide in N,N-dimethylformamide solvent and heat to react. After the reaction is completed, cool to room temperature, add appropriate amount of water and extract with n-butanol, wash with water and saturated brine, combine the organic phases, dry over anhydrous sodium sulfate, and remove the solvent under reduced pressure to obtain the target product. d 0 / d 5-1-Benzyl-3-(3-azidopropyl)-imidazolium bromide.

[0018] In step (3), the molar ratio of intermediate II to sodium azide is 1:1-1:6 (preferably 1:3-1:5), the reaction heating temperature is 40-65°C (preferably 50-65°C), and the reaction time is 4-24 h (preferably 8-24 h).

[0019] The synthesis mechanism is as follows:

[0020]

[0021] The first step is to use d 0 / dThe nucleophilic substitution reaction between 5-benzyl bromide and imidazole produces intermediate I. Under the action of sodium hydroxide, the nitrogen atom on the imidazole acts as a nucleophile to attack d 0 / d The central carbon atom in the 5-benzyl bromide molecule carries a partial positive charge, and the bromide ion leaves as a leaving group to obtain intermediate Ⅰ ( d 0 / d 5-1-benzylimidazole); in the second step, intermediate I can react with halogenated alkane 1,3-dibromopropane to form a quaternary ammonium salt to obtain intermediate II ( d 0 / d 5-1-benzyl-3-(3-bromopropyl)-imidazolium bromide); in the third step, the nitrogen atom of sodium azide acts as a nucleophile to attack the bromine atom in intermediate II, thereby replacing the bromine atom to generate the target product d 0 / d 5-1-Benzyl-3-(3-azidopropyl)-imidazolium bromide.

[0022] 2. Application of the stable isotope labeling reagent for terminal alkyne compounds provided by the present invention in food analysis, environmental analysis, life analysis, drug analysis, and natural product analysis, for detecting the concentration of terminal alkyne compounds in a sample, comprising the following steps:

[0023] (1) Prepare a 100 ng / mL methanol solution of terminal alkyne standard; d A methanol solution of 0-1-benzyl-3-(3-azidopropyl)-imidazolium bromide was prepared as a light labeling reagent solution. d A methanol solution of 5-1-benzyl-3-(3-azidopropyl)-imidazolium bromide was used as a heavy labeling reagent solution; and an aqueous solution of sodium ascorbate and copper sulfate pentahydrate was prepared.

[0024] (2) Add 600 μL of ultrapure water, 100 μL of sample, 100 μL of light labeling reagent solution, 100 μL of ascorbic acid solution, and 100 μL of copper sulfate solution to a 1.5 mL centrifuge tube in sequence, and heat in a water bath to react to obtain reaction solution A;

[0025] (3) Add 600 μL of ultrapure water, 100 μL of terminal alkyne standard solution, 100 μL of heavy labeling reagent solution, 100 μL of ascorbic acid solution, and 100 μL of copper sulfate solution to a 1.5 mL centrifuge tube in sequence, and heat in a water bath to react to obtain reaction solution B;

[0026] (4) The reaction solution A and the reaction solution B are mixed in a volume ratio of 1:1, and the mixed solution is analyzed by HPLC-MS / MS; the peak area of ​​the light labeling product obtained after the light labeling reagent reaction is A1, and the concentration of the terminal alkyne compound in the sample is C1; the peak area of ​​the heavy labeling product obtained after the heavy labeling reagent reaction is A2, and the concentration of the terminal alkyne compound is C2. According to the formula A1 / A2 = C1 / C2, the concentration of the terminal alkyne compound in the sample can be calculated.

[0027] In step (1), d 0 / d The concentration of 5-1-benzyl-3-(3-azidopropyl)-imidazolium bromide methanol solution is 1.5~310×10 -5 mol / L (preferably 250×10 -5 mol / L), the concentration of sodium ascorbate aqueous solution is 0.5~10×10 -3 mol / L (preferably 3×10 -3 mol / L), the concentration of copper sulfate aqueous solution is 4~70×10 -6 mol / L (preferably 40×10 -6 mol / L).

[0028] In step (2) and step (3), the water bath heating reaction temperature is 20-70°C (preferably 60°C), and the reaction time is 1-90 min (preferably 60 min).

[0029] The detection mechanism is as follows:

[0030]

[0031] Sodium ascorbate is used to in situ reduce copper sulfate in solution to produce monovalent copper ions. The azide group and the terminal alkyne compound undergo a 1,3-dipolar cycloaddition reaction catalyzed by the monovalent copper ions to yield a 1,4-disubstituted 1,2,3-triazole-labeled product. Through the copper-catalyzed azide-alkynyl cycloaddition (CuAAC) reaction, a positively charged imidazole salt can be introduced into the labeled product as a mass spectrometry signal enhancement tag, thereby enhancing its ionization efficiency and improving detection sensitivity. Furthermore, the heavily labeled product obtained by introducing an isotopic group in this reaction can be used as an internal standard to mitigate matrix effects and improve detection accuracy.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The stable isotope labeling reagent for terminal alkyne compounds disclosed herein uses a benzene ring as an isotope labeling group, an imidazole salt as a mass spectrometry signal enhancing group, and an azide group as a reactive group. A CuAAC reaction is achieved between the azide group and the terminal alkyne compound under the catalysis of monovalent copper ions generated by sodium ascorbate and copper sulfate. Highly selective and efficient labeling of the terminal alkyne compound is achieved by adjusting the concentration of the labeling reagent, the concentrations of sodium ascorbate and copper sulfate, the reaction temperature, and the reaction time. Furthermore, the labeling reagent has the characteristics of simple synthesis, mild reaction conditions, high labeling reaction yield, and strong labeled product signal. Combined with HPLC-MS / MS technology, accurate and highly sensitive detection of the terminal alkyne compound can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The nuclear magnetic resonance of the light labeling reagent prepared in Example 1 of the present invention 1 H NMR spectrum.

[0035] Figure 2 This is a high-resolution mass spectrum of the light labeling reagent prepared in Example 1 of the present invention.

[0036] Figure 3 The nuclear magnetic resonance of the heavy labeling reagent prepared in Example 2 of the present invention 1 H NMR spectrum.

[0037] Figure 4 This is a high-resolution mass spectrum of the heavy labeling reagent prepared in Example 2 of the present invention.

[0038] Figure 5 This is the MRM ion chromatogram of the terminal alkyne compound EE2 labeled with the light / heavy labeling reagent prepared in Examples 1 and 2 of the present invention. DETAILED DESCRIPTION

[0039] The present invention will be further explained below with reference to specific embodiments.

[0040] Example 1

[0041] Light labeling reagent synthesis

[0042] (1) Intermediate Ⅰ ( d Synthesis of 0-1-benzylimidazole

[0043] To a 100 mL three-necked flask, 2.5 g of benzyl bromide and 26 mL of tetrahydrofuran were added. To a 10 mL vial, 2.0 g of imidazole and 0.88 g of sodium hydroxide were added and dissolved in 4 mL of ultrapure water. Under argon protection, the resulting aqueous solution was added dropwise to the tetrahydrofuran solution. The reaction was heated at 75°C for 4 h, and the reaction progress was monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature and the solvent was removed by distillation under reduced pressure. The crude product was extracted three times with dichloromethane:water (3:1, v / v). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by distillation under reduced pressure. Purification was performed by silica gel column chromatography (dichloromethane:ethyl acetate = 10:1, v / v) to obtain 1.7 g of a white solid, namely, Intermediate I ( d 0-1-benzylimidazole), yield 74%.

[0044] (2) Intermediate II ( d Synthesis of 1-benzyl-3-(3-bromopropyl)-imidazolium bromide

[0045] 0.5 g of intermediate I and 3.2 g of 1,3-dibromopropane were dissolved in 10 mL of acetonitrile and refluxed at 90°C for 6 h. The reaction progress was monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature and the solvent was removed by distillation under reduced pressure. 0.89 g of colorless oily liquid, namely intermediate II ( d 0-1-benzyl-3-(3-bromopropyl)-imidazolium bromide), the yield was 78%.

[0046] (3) Target product d Synthesis of 0-1-Benzyl-3-(3-azidopropyl)-imidazolium Bromide

[0047] To a 100 mL three-necked flask, add 0.87 g of intermediate II and 0.63 g of sodium azide, dissolve in 50 mL of N,N-dimethylformamide, and heat at 55°C for 8 h. After the reaction is complete, cool to room temperature, add 150 mL of water, and extract with n-butanol. Wash with water and saturated brine. Combine the organic phases, dry over anhydrous sodium sulfate, and remove the solvent under reduced pressure to obtain 0.42 g of a light yellow oily liquid, the target product. d 0-1-Benzyl-3-(3-azidopropyl)-imidazolium bromide ( d 0-ABI), the yield was 54%.

[0048] Lightly labeled probes d 0-ABI product characterization:

[0049] H NMR 1 H NMR: (400 MHz, DMSO- d6) δ 9.59 (s, 1H), 7.91 – 7.86 (s, 2H), 7.48 – 7.35 (m, 5H), 5.47 (s, 2H), 4.27 (t, J = 7.0 Hz, 2H), 3.44 (t, J =6.6 Hz, 2H), 2.07 (p, J = 6.8 Hz, 2H).( Figure 1 );

[0050] High-resolution mass spectrometry: calcd. for C 13 H 16 N5 + 242.1400 [M –Br] + , found 242.1401.( Figure 2 );

[0051] From the above characterization, it can be seen that this example successfully synthesized a light-labeled probe. d 0-1-Benzyl-3-(3-azidopropyl)-imidazolium bromide ( d 0-ABI).

[0052] Example 2

[0053] Heavy labeling reagent synthesis

[0054] Compared with Example 1, the heavy labeling reagent is d 5-Benzyl bromide (R=D) is used as the starting material, and the remaining synthetic steps are the same as those for the light labeling reagent.

[0055] Heavy-duty labeled probes d 5-ABI product characterization:

[0056] H NMR 1 H NMR: (400 MHz, DMSO- d 6) δ 9.53 (s, 1H), 7.92 – 7.84 (s, 2H), 5.46 (s, 2H), 4.26 (t, J = 7.1 Hz, 2H), 3.44 (t, J = 6.5 Hz, 2H), 2.08 (p, J =6.8 Hz, 2H).( Figure 3 );

[0057] High-resolution mass spectrometry: calcd. for C 13 H 11 D5N5247.1714 [M –Br]+ ; found 247.1716.( Figure 4 );

[0058] From the above characterization, it can be seen that this example successfully synthesized the heavy labeling probe d 5-1-Benzyl-3-(3-azidopropyl)-imidazolium bromide ( d 5-ABI).

[0059] Application Examples

[0060] Food testing applications

[0061] The light and heavy labeling reagents prepared in Examples 1 and 2 were used to detect terminal alkynes. The specific steps are as follows:

[0062] (1) Prepare a 100 ng / mL methanol solution of ethinyl estradiol (EE2) standard; prepare 250×10 - 5 mol / L d A methanol solution of 0-1-benzyl-3-(3-azidopropyl)-imidazolium bromide was used as a light labeling reagent solution to prepare 250×10 -5 mol / L d A methanol solution of 5-1-benzyl-3-(3-azidopropyl)-imidazolium bromide was used as a heavy labeling reagent solution; 3×10 -3 mol / L sodium ascorbate aqueous solution and 40×10 -6 mol / L copper sulfate aqueous solution.

[0063] (2) Add 600 μL of ultrapure water, 100 μL of the actual pork sample after liquid-liquid extraction, 100 μL of light labeling reagent solution, 100 μL of ascorbic acid solution, and 100 μL of copper sulfate solution to a 1.5 mL centrifuge tube, and heat in a 60°C water bath for 60 min to obtain reaction solution A;

[0064] (3) Add 600 μL of ultrapure water, 100 μL of EE2 solution, 100 μL of heavy labeling reagent solution, 100 μL of ascorbic acid solution, and 100 μL of copper sulfate solution to a 1.5 mL centrifuge tube, heat in a 60°C water bath for 60 min, and obtain reaction solution B;

[0065] (4) The above reaction solution A and reaction solution B were mixed in a volume ratio of 1:1, and the mixed solution was analyzed by HPLC-MS / MS; the peak area of ​​the light-labeled product obtained after the light-labeled reagent reaction was A1, and the concentration of EE2 in the sample was C1; the peak area of ​​the heavy-labeled product obtained after the heavy-labeled reagent reaction was A2, and the concentration of EE2 was C2. According to the formula A1 / A2 = C1 / C2, the concentration of EE2 in the sample can be calculated.

[0066] The labeling reagent reacts with the terminal alkyne to form a CuAAC reaction. The MRM ion chromatogram of the EE2 labeled product is shown in Figure 2. Figure 5 As shown, the isotope effect is less than 0.05 min, meeting the requirements for mass spectrometry quantification. Furthermore, after labeling with a light labeling reagent, the mass spectrometry response signal of the EE2-labeled product was enhanced 4532-fold, measured in peak area, compared to the pre-labeling period (Table 1).

[0067] Table 1 Enhancement of mass spectrometric response signals of alkyne compound EE2 before and after labeling with light probe

[0068] .

Claims

1. A stable isotope labeling reagent for terminal alkyne compounds, characterized in that: The chemical structure of the isotope labeling reagent is shown below: ， Among them: When R=H, it is called d 0-1-Benzyl-3-(3-azidopropyl)-imidazolium bromide, abbreviated as d 0-ABI; when R=D, it is called d 5-1-Benzyl-3-(3-azidopropyl)-imidazolium bromide, abbreviated as d 5-ABI.

2. The method for synthesizing a stable isotope labeling reagent for a terminal alkyne compound according to claim 1, wherein: The steps include: (1) The first step of substitution reaction: d 0 / d 5-Benzyl bromide is dissolved in tetrahydrofuran, imidazole and sodium hydroxide are co-dissolved in ultrapure water, and the resulting aqueous solution is added dropwise to the tetrahydrofuran solution under the protection of inert gas, and heated to 65-85°C for reaction for 2-8 hours. After the reaction is completed, the mixture is cooled to room temperature, and the solvent is removed by distillation under reduced pressure. The organic phases are extracted, combined, dried over anhydrous sodium sulfate, filtered, and the solvent is removed by distillation under reduced pressure. The mixture is purified by silica gel column chromatography to obtain intermediate I, which is designated as d 0 / d 5-1-Benzylimidazole; (2) Second step salt formation reaction: Dissolve intermediate I and 1,3-dibromopropane in acetonitrile, heat to 80-100°C and react for 4-12 hours. After the reaction, cool to room temperature, remove the solvent by vacuum distillation, and purify by silica gel column chromatography to obtain intermediate II, which is recorded as d 0 / d 5-1-Benzyl-3-(3-bromopropyl)-imidazolium bromide; (3) The third step of substitution reaction: dissolve intermediate II and sodium azide in N,N-dimethylformamide solvent, heat to 40~65℃ and react for 4~24h; after the reaction, cool to room temperature, extract, wash, combine the organic phases, dry over anhydrous sodium sulfate, and remove the solvent under reduced pressure to obtain the target product, which is recorded as d 0 / d 5-1-Benzyl-3-(3-azidopropyl)-imidazolium bromide.

3. The method for synthesizing a stable isotope labeling reagent for a terminal alkyne compound according to claim 2, wherein: In step (1), d 0 / d The molar ratio of 5-benzyl bromide to imidazole is 1:1~1:3, d 0 / d The molar ratio of 5-benzyl bromide to sodium hydroxide is 1:1~1:2.

5.

4. The method for synthesizing a stable isotope labeling reagent for a terminal alkyne compound according to claim 2, wherein: In step (2), the molar ratio of intermediate I to 1,3-dibromopropane is 1:1 to 1:

20.

5. The method for synthesizing a stable isotope labeling reagent for a terminal alkyne compound according to claim 2, wherein: In step (2), the molar ratio of intermediate II to sodium azide is 1:1 to 1:

6.

6. Use of the stable isotope labeling reagent for terminal alkyne compounds according to claim 1 in food analysis, environmental analysis, life analysis, drug analysis and natural product analysis, wherein the purpose of the use is not disease diagnosis or treatment.

7. The use of a stable isotope labeling reagent for terminal alkyne compounds according to claim 6, characterized in that: It is used to detect the concentration of terminal alkyne compounds in samples. The specific steps are as follows: (1) Prepare a 100 ng / mL methanol solution of terminal alkyne standard; the concentration is 1.5~310×10 -5 mol / L d 0-1-Benzyl-3-(3-azidopropyl)-imidazolium bromide methanol solution was used as a light labeling reagent solution with a concentration of 1.5~310×10 -5 mol / L d 5-1-Benzyl-3-(3-azidopropyl)-imidazolium bromide methanol solution was used as the heavy labeling reagent solution; the concentration was 0.5~10×10 -3 mol / L sodium ascorbate aqueous solution and concentration of 4~70×10 -6 mol / L copper sulfate aqueous solution; (2) Add 600 μL of ultrapure water, 100 μL of sample, 100 μL of light labeling reagent solution, 100 μL of ascorbic acid solution, and 100 μL of copper sulfate solution to a 1.5 mL centrifuge tube, heat in a water bath to 20-70°C and react for 1-90 minutes to obtain reaction solution A; (3) Add 600 μL of ultrapure water, 100 μL of terminal alkyne standard solution, 100 μL of heavy labeling reagent solution, 100 μL of ascorbic acid solution, and 100 μL of copper sulfate solution to a 1.5 mL centrifuge tube, heat in a water bath to 20-70°C and react for 1-90 min to obtain reaction solution B; (4) The reaction solution A and the reaction solution B are mixed in a volume ratio of 1:1, and the mixed solution is analyzed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS); the peak area of ​​the light labeling product obtained after the light labeling reagent reaction is A1, and the concentration of the terminal alkyne compound in the sample is C1; the peak area of ​​the heavy labeling product obtained after the heavy labeling reagent reaction is A2, and the concentration of the terminal alkyne compound is C2. According to the formula A1 / A2 = C1 / C2, the concentration of the terminal alkyne compound in the sample can be calculated.

8. The use of a stable isotope labeling reagent for terminal alkyne compounds according to claim 7, wherein: In step (1), d 0 / d The concentration of 5-1-benzyl-3-(3-azidopropyl)-imidazolium bromide methanol solution is 250×10 -5 mol / L, the concentration of sodium ascorbate aqueous solution is 3×10 -3 mol / L, the concentration of copper sulfate aqueous solution is 40×10 -6 mol / L.

Images