A method of purifying an electronic medium

Abstract

The application provides a purification method for electronic medium on a biosensor, the electronic medium is provided with an amino group, and the purification method comprises the following steps: weighing low-purity electronic medium; reacting the electronic medium with an amino group protection reagent to obtain amino group protected electronic medium, dissolving the amino group protected electronic medium in an organic solvent, and obtaining high-purity amino group protected electronic medium through extraction; and reacting the high-purity amino group protected electronic medium with a deprotection agent to remove the amino group protection, and obtaining high-purity electronic medium. By using the purification method, the processing capacity of a single treatment is very large, the purification efficiency of the electronic medium is effectively improved, and the preparation cost is reduced. As one of the reagent components on the biosensor, the high-purity electronic medium obtained by using the purification method can be used for the preparation of the biosensor for blood glucose detection.

Description

Technical Field

[0001] This invention relates to a method for purifying electronic mediators, and particularly to a method for purifying electronic mediators used in diagnostic reagents such as blood glucose testing. Background Technology

[0002] Diabetes is a disease caused by a deficiency in insulin secretion or a reduced biological effect of insulin. Currently, there is no medical cure for diabetes. Early screening and corresponding preventative treatment are effective means of controlling blood sugar levels. Daily blood glucose monitoring using an enzyme electrode method is the most common method currently used.

[0003] In enzyme-based blood glucose biosensors (also known as blood glucose test strips), the enzymes used are macromolecules whose redox active centers are encapsulated within the enzyme protein molecule. Direct electron transfer between the enzyme and the electrode surface is difficult, and even if it were possible, the rate would be slow. Therefore, electron mediators are added to enzyme-based test strips to create electron-mediator-based biosensors.

[0004] Azure C is a biological staining agent, as well as a redox indicator and electron mediator, widely used in biochemical and electrochemical research. For example, it is used as an electron mediator in blood glucose biosensors. Commercially available Azure C has relatively low purity, generally between 40-50%, and also contains structurally similar substances such as Azure A, Azure B, and thionine. The proportions of these components vary significantly between different manufacturers, and even within the same manufacturer, there are substantial batch-to-batch variations, leading to considerable uncertainty in the practical application of commercially available Azure C. Therefore, obtaining high-purity Azure C is urgently needed to achieve more precise dosage and improved efficacy.

[0005] Commercially available azure C exists in the form of hydrochloride, but its solubility in water and organic solvents is very poor. Furthermore, the polarity of other components is very similar to that of azure C, making its purification extremely difficult. Given the chemical properties of azure C, HPLC purification can be used to obtain high-purity azure C with a purity of ≥99%. However, due to the solubility problem of azure C and the low sample loading volume of HPLC (typically about 5 mg per load, yielding only 1-2 mg of azure C per purification run), the purification efficiency of HPLC is very low, and the cost of purification is very high. Summary of the Invention

[0006] To address the problems in the prior art, this invention protects the amino group on the electron mediator with an amino protecting agent to obtain an easily separable intermediate. By removing the protecting group on the amino group through deprotection, a high-purity electron mediator is obtained.

[0007] This invention provides a method for purifying an electron mediator containing an amino group, comprising the following steps: weighing an electron mediator with low purity; reacting an amino protecting reagent with the electron mediator to obtain an amino-protected electron mediator; dissolving the amino-protected electron mediator in an organic solvent and extracting it to obtain a high-purity amino-protected electron mediator; reacting the high-purity amino-protected electron mediator with a deprotecting agent to deprotect the amino group and obtain a higher-purity electron mediator.

[0008] Furthermore, the electronic medium is selected from azurite C.

[0009] Furthermore, the amino protecting agent is selected from fluorenemethyloxycarbonyl chloride, ditert-butyl dicarbonate, triphenylchloromethane, benzyl chloroformate, p-toluenesulfonyl chloride, and trifluoroacetic anhydride.

[0010] Furthermore, the structural formula of the amino-protected electron mediator is as follows:

[0011]

[0012] Where R = H or X, X = amino protecting group, and the number of amino protecting groups X varies from 1 to 3.

[0013] Furthermore, amino protecting groups such as Fmoc, Boc, Trt, Cbz, Tos, or TFA.

[0014] Furthermore, the deprotecting agent is selected from diethylamine and trifluoroacetic acid.

[0015] Furthermore, the method for extracting high-purity amino-protected electron mediators is column chromatography.

[0016] Taking the purification of azure C as an example, this invention purifies azure C using a simple chemical method. First, azure C is reacted with an amino-protecting reagent to obtain amino-protected azure C. The amino-protecting reagent is selected from fluorenemethyloxycarbonyl chloride (Fmoc-Cl), di-tert-butyl dicarbonate (Boc2O), triphenylchloromethane (Trt-Cl), benzyl chloroformate (Cbz-Cl), p-toluenesulfonyl chloride (Tos-Cl), or trifluoroacetic anhydride (TFAA), etc. Amino-protected azure C is readily soluble in various organic solvents and can be purified by methods such as column chromatography to obtain high-purity amino-protected azure C. Then, amino deprotection is performed to obtain azure C with higher purity.

[0017]

[0018] Where R = H or X, X = amino protecting group (e.g., Fmoc or Boc or Trt or Cbz or Tos or TFA), different amino protecting reagents are used, and the number of amino protecting groups X varies from 1 to 3.

[0019] in,

[0020] Azure C:

[0021] Fluorenemethyloxycarbonyl chloride (Fmoc-Cl):

[0022] (Fmoc)2-Azure C:

[0023] Di-tert-butyl dicarbonate (Boc2O):

[0024] (Boc)2-Azure C:

[0025] Triphenylchloromethane (Trt-Cl):

[0026] Trt-Azure C:

[0027] One objective of this invention is to provide a method for purifying an amino-containing electron mediator, comprising the following steps: weighing a low-purity electron mediator; reacting an amino protecting agent with the electron mediator to obtain an amino-protected electron mediator; dissolving the amino-protected electron mediator in an organic solvent and extracting it to obtain a high-purity amino-protected electron mediator; reacting the high-purity amino-protected electron mediator with a deprotecting agent to deprotect the amino group and obtain a high-purity electron mediator.

[0028] Furthermore, the electronic medium is selected from azurite C.

[0029] Furthermore, the amino protecting agent is selected from fluorenemethyloxycarbonyl chloride, ditert-butyl dicarbonate, triphenylchloromethane, benzyl chloroformate, p-toluenesulfonyl chloride, or trifluoroacetic anhydride.

[0030] Furthermore, the structural formula of the amino-protected electron mediator is as follows:

[0031]

[0032] Where R = H or X, X = amino protecting group, and the number of amino protecting groups X varies from 1 to 3.

[0033] Furthermore, the amino protecting group is selected from Fmoc, Boc, Trt, Cbz, Tos, or TFA.

[0034] Furthermore, the deprotecting agent is selected from diethylamine and trifluoroacetic acid.

[0035] Furthermore, the method for extracting high-purity amino-protected electron mediators is column chromatography.

[0036] One objective of this invention is to provide a purification method for azure C using Fmoc-Cl as an amino protecting agent, comprising the following steps: preparing a mixture of low-purity azure C with sodium carbonate and dioxane; adding fluorenemethyloxycarbonyl chloride to the mixture to react and obtain a viscous product; dissolving the viscous product in an organic solvent, filtering out insoluble impurities, and drying to obtain an intermediate of azure C, (Fmoc)2-Azure C; dissolving the intermediate (Fmoc)2-Azure C in DMF, adding diethylamine to react, adding diethyl ether to precipitate a black solid, and drying to obtain high-purity azure C.

[0037] Furthermore, fluorenemethyloxycarbonyl chloride is added to the mixture after it is cooled to 10°C.

[0038] Furthermore, fluorenemethyloxycarbonyl chloride is added, and the reaction is first carried out in an ice-water bath, and then transferred to room temperature for further reaction.

[0039] Furthermore, the viscous product is dissolved in dichloromethane.

[0040] Furthermore, the intermediate (Fmoc)2-Azure C was dissolved in DMF, and diethylamine was added under an ice-water bath, followed by a reaction under an ice-water bath.

[0041] Further, sodium carbonate was weighed and dissolved in water by stirring. Dioxane and low-purity Azure C were then added. The mixture was cooled to 10°C in an ice-water bath. Fluorene methyloxycarbonyl chloride was added in batches, and the reaction was carried out in an ice-water bath. The reaction was then transferred to room temperature to produce a viscous product. The supernatant was discarded, and the viscous product was dissolved in dichloromethane. Insoluble impurities were filtered off, and the filtrate was washed with water. Anhydrous sodium sulfate was dried, filtered, and concentrated to obtain the intermediate (Fmoc)2-Azure C. (Fmoc)2-Azure C was dissolved in DMF, and diethylamine was slowly added dropwise in an ice-water bath. The reaction was carried out in an ice-water bath, and diethyl ether was added. A black solid precipitated, which was filtered, washed with diethyl ether, and dried under vacuum at room temperature to obtain high-purity Azure C.

[0042] Furthermore, fluorenemethyloxycarbonyl chloride was added in batches, and the reaction was carried out in an ice-water bath for 1 hour, followed by a reaction at room temperature for 2 hours to produce a viscous product.

[0043] Furthermore, (Fmoc)2-Azure C was dissolved in DMF, and diethylamine was slowly added dropwise under an ice-water bath. The reaction was carried out under an ice-water bath for 1 hour.

[0044] One objective of this invention is to provide a method for purifying azure C using Boc2O as an amino protecting agent, comprising the following steps: weighing low-purity azure C, dissolving it in DMF, adding triethylamine and di-tert-butyl dicarbonate, reacting to obtain a viscous product, dissolving the viscous product in an organic solvent, and purifying it to obtain intermediate (Boc)2-Azure C; dissolving intermediate (Fmoc)2-Azure C in an organic solvent, adding trifluoroacetic acid, reacting and removing the solvent, adding acetonitrile to dissolve it, adding diethyl ether solution of hydrogen chloride, precipitating a black solid, filtering, washing and drying to obtain high-purity azure C.

[0045] Furthermore, triethylamine and di-tert-butyl dicarbonate were added under an ice-water bath.

[0046] Furthermore, the organic solvent is dichloromethane.

[0047] Furthermore, the viscous product dissolved in dichloromethane was dried, concentrated, and purified by column chromatography to obtain the intermediate product (Boc)2-Azure C.

[0048] Furthermore, the mobile phase for the column chromatography is ethyl acetate / petroleum ether = 1 / 10.

[0049] Further, weigh out Azure C with lower purity, dissolve it in DMF, add triethylamine and di-tert-butyl dicarbonate under an ice-water bath, react under an ice-water bath, then react at room temperature, filter, pour the filtrate into ice water and stir to precipitate a viscous product, discard the supernatant, dissolve the viscous substance in dichloromethane, dry with anhydrous sodium sulfate, concentrate, and precipitate by column chromatography at ethyl acetate / petroleum ether = 1 / 10 to obtain intermediate (Boc)2-Azure C; (Boc)2-Azure C is dissolved in dichloromethane, trifluoroacetic acid is added, react at room temperature, concentrate and evaporate the solvent, add acetonitrile and stir to dissolve, add diethyl ether solution of hydrogen chloride dropwise, precipitate a black solid, filter, wash with acetonitrile, and dry under vacuum at room temperature to obtain Azure C with higher purity.

[0050] One objective of this invention is to provide a purification method for azure C using Trt as an amino protecting agent, comprising the following steps: weighing low-purity azure C and triphenylchloromethane, dissolving them in DMF, adding triethylamine, reacting, filtering, diluting the filtrate with dichloromethane, washing with water, separating the layers, drying, filtering, concentrating, and obtaining the purified intermediate Trt-Azure C; dissolving the intermediate Trt-Azure C in an organic solvent, adding trifluoroacetic acid, removing the solvent, adding acetonitrile to dissolve, adding diethyl ether hydrochloride solution, precipitating a black solid, filtering, washing, and drying to obtain high-purity azure C.

[0051] Furthermore, the intermediate Trt-Azure C is soluble in dichloromethane, an organic solvent.

[0052] Furthermore, trifluoroacetic acid was added, and the reaction was carried out at room temperature.

[0053] Furthermore, the purification is performed by column chromatography.

[0054] Furthermore, the mobile phase for column chromatography was ethyl acetate / methanol = 20 / 1.

[0055] Further, weigh out azure C with lower purity and triphenylchloromethane, dissolve them in DMF, add triethylamine dropwise, react at room temperature, filter, dilute the filtrate with dichloromethane, wash with water, separate the layers, dry with anhydrous sodium sulfate, filter, concentrate, and precipitate by column chromatography in an ethyl acetate / methanol = 20 / 1 system to obtain intermediate Trt-Azure C; Trt-Azure C is dissolved in dichloromethane, trifluoroacetic acid is added, react at room temperature, concentrate and evaporate the solvent, add acetonitrile and stir to dissolve, add diethyl ether solution of hydrogen chloride dropwise, precipitate a black solid, filter, wash with acetonitrile, and dry under vacuum at room temperature to obtain azure C with higher purity.

[0056] The beneficial effects of this invention are as follows: This invention utilizes an amino protecting agent to form commercially available azure C (i.e., low purity) into amino-protected azure C (i.e., an intermediate in the purification process) that is easily soluble in various organic solvents. This intermediate is then purified, and after deprotection, high-purity azure C is finally obtained. Using the purification reagents and methods described in this invention, the throughput of a single purification is very large, effectively improving the purification efficiency of commercially available azure C and reducing preparation and purification costs. Furthermore, it allows for more precise dosage of azure C in biosensors, improving its performance. The purification method described in this invention has a large throughput per cycle and yields a large amount of high-purity product, effectively meeting the preparation needs of biosensors such as those used for blood glucose detection and improving production efficiency. Attached Figure Description

[0057] Figure 1 This is the LC-MS spectrum of commercially available Azure C (Azure C with low purity before purification).

[0058] Figure 2 This is the LC-MS mass spectra of a commercially available Azure C device, specifically the mass spectrum corresponding to the peak position at T=1.55 min.

[0059] Figure 3 This is the LC-MS spectrum of Azure C purified by using Fmoc as a protecting group in Example 1.

[0060] Figure 4 This is the LC-MS spectrum of Azure C purified by using Boc as a protecting group in Example 2.

[0061] Figure 5 This is the LC-MS spectrum of Azure C purified by using Trt as a protecting group in Example 3. Detailed Implementation

[0062] The present invention will now be described in detail with reference to specific embodiments. These specific embodiments are merely limited enumerations without departing from the spirit of the present invention, and do not preclude other specific implementation schemes arising by combining prior art with the present invention by those skilled in the art.

[0063] The present invention provides a method for purifying commercially available azurite C, comprising:

[0064] Step 1: The amino protecting reagent reacts with low-purity azure C to obtain amino-protected azure C.

[0065] Step 2: The amino-protected azurite C obtained in Step 1 is readily soluble in various organic solvents. High-purity amino-protected azurite C (intermediate) is obtained by conventional separation methods (e.g., column chromatography).

[0066] Step 3: Deprotect the intermediate obtained in Step 2 by amino grouping to obtain azurite C with high purity.

[0067] Commonly used amino protecting reagents are selected from, but not limited to, fluorenemethyloxycarbonyl chloride (Fmoc-Cl), ditert-butyl dicarbonate (Boc2O), triphenylchloromethane (Trt-Cl), benzyl chloroformate (Cbz-Cl), p-toluenesulfonyl chloride (Tos-Cl), trifluoroacetic anhydride (TFAA), etc.

[0068] The LC-MS (Liquid Chromatography-Mass Spectrometry) analysis method for Azure C is as follows:

[0069] Instrument: Waters Acquity UPLC H-Class+SQD2 LC-MS / MS, Column: Acquity BEH C181.7μm, 21×50mm Column.

[0070] Mobile phase: acetonitrile / water (0.1% formic acid), acetonitrile % = 10-90% (3 min).

[0071] Flow rate: 0.4 ml / min.

[0072] Detection wavelength: 200-800nm ​​full scan.

[0073] Column temperature: 40℃.

[0074] Based on the above LC-MS analysis, the content of C in commercially available azure is 40%. Figure 1 As shown. Analysis of the C mass spectrum [M-Cl] of azure. + =242.35, as Figure 2 As shown.

[0075] Example 1: Purification of Azure C (Azure C) using Fmoc-Cl as an amino protecting agent

[0076] A method for purifying azurite C using fluorenemethyloxycarbonyl chloride (Fmoc-Cl) as an amino protecting agent.

[0077]

[0078] Weigh 15g (141.5mmol) of sodium carbonate, add 150ml of water and stir to dissolve. Then add 150ml of dioxane and 10g (36mmol) of commercially available Azure C (40% purity). Cool to 10°C in an ice-water bath. Add 23.28g (90mmol) of fluorenemethyloxycarbonyl chloride in portions, react in an ice-water bath for 1 hour, then at room temperature for 2 hours to produce a viscous product. Discard the supernatant, dissolve the viscous product in dichloromethane, filter to remove insoluble impurities, and wash the filtrate with water. Dry with anhydrous sodium sulfate, filter, and concentrate to obtain 3.4g of intermediate (Fmoc)2-Azure C. (Fmoc)2-Azure C is amino-protected Azure C.

[0079] The prepared (Fmoc)2-Azure C was dissolved in 75 ml of DMF, and 20 ml of diethylamine was slowly added dropwise under an ice-water bath. The reaction was carried out for 1 hour under an ice-water bath. 500 ml of diethyl ether was added, and a black solid precipitated. The solid was filtered and washed with diethyl ether. The solution was dried under vacuum at room temperature to obtain 2.3 g of Azure C (purity: 84.6%). Figure 3 As shown.

[0080] Example 2: Purification of Azure C using Boc2O as an amino protecting agent

[0081] A method for purifying azurite C using di-tert-butyl dicarbonate (Boc2O) as an amino protecting agent.

[0082]

[0083] Weigh 5 g (18 mmol) of commercially available 40% Azure C and dissolve it in 100 ml of DMF. Add 15 ml (108 mmol) of triethylamine and 16.5 ml (72 mmol) of di-tert-butyl dicarbonate under an ice-water bath. React for 1 hour under an ice-water bath, then transfer to room temperature and react for another 3 hours. Filter the solution, pour the filtrate into ice water, stir to precipitate a viscous product, discard the supernatant, dissolve the viscous substance in dichloromethane, dry with anhydrous sodium sulfate, and concentrate. Column chromatography (e.g., using a SiO2 silica gel column) at an ethyl acetate / petroleum ether ratio of 1 / 10 yields 1.6 g of intermediate (Boc)2-Azure C. (Boc)2-Azure C is amino-protected Azure C.

[0084] The prepared intermediate (Boc)2-Azure C was dissolved in 15 ml of dichloromethane, and 3 ml of trifluoroacetic acid was added. The reaction was carried out at room temperature for 4 hours. The solvent was concentrated and evaporated to dryness, and 30 ml of acetonitrile was added and stirred to dissolve. 3 ml of diethyl ether hydrochloride solution was added dropwise, and a black solid precipitated. The solid was filtered, washed with acetonitrile, and dried under vacuum at room temperature to obtain 1 g of Azure C (purity: 95%). Figure 4 As shown.

[0085] Example 3: Purification of Azure C using Trt as an amino protecting agent

[0086] A method for purifying azurite C using triphenylchloromethane (Trt-Cl) as an amino protecting agent.

[0087]

[0088] Weigh 5 g (18 mmol) of 40% Azure C and 15 g (54 mmol) of triphenylchloromethane, dissolve them in 100 mL of DMF, and then add 12.5 mL (90 mmol) of triethylamine dropwise. React at room temperature for 1 hour. Filter, dilute the filtrate with 400 mL of dichloromethane, wash with water, separate the layers, dry with anhydrous sodium sulfate, filter, and concentrate. Column chromatography (e.g., using a SiO2 silica gel column) in an ethyl acetate / methanol = 20 / 1 system yields 2.2 g of the intermediate Trt-Azure C. Trt-Azure C is amino-protected Azure C.

[0089] The prepared intermediate Trt-Azure C was dissolved in 30 mL of dichloromethane, and 3 mL of trifluoroacetic acid was added. The reaction was carried out at room temperature for 1 hour. The solvent was concentrated and evaporated to dryness, and 30 mL of acetonitrile was added and stirred to dissolve. 3 mL of diethyl ether hydrochloride solution was added dropwise, and a black solid precipitated. The solid was filtered, washed with acetonitrile, and dried under vacuum at room temperature to obtain 1.24 g of Azure C (purity: 97%). Figure 5 As shown.

[0090] Example 4: Direct preparation and purification of commercially available Azure C using HPLC (Comparative Example)

[0091] As a comparative example of the present invention, the method of direct purification of commercially available Azure C by HPLC is as follows.

[0092] Instrument: Waters Prep150 preparative liquid chromatograph, column: C18 OBD TM 5μm 19×150mm Column.

[0093] Mobile phase: acetonitrile / water (0.05% trifluoroacetic acid), acetonitrile % = 15-55% (10 min).

[0094] Flow rate: 20 ml / min.

[0095] Detection wavelength: 610nm.

[0096] Column temperature: room temperature.

[0097] Sample concentration: 5 mg / ml in N,N-dimethylformamide, injection volume: 1 ml.

[0098] Collect the main product (Azure C) at T = 3.6–4.4 min.

[0099] Analysis showed that HPLC was used to directly purify azurite C, and 1-2 mg of azurite C could be collected each time. The purity of the purified azurite C was ≥99%.

[0100] Analyzing and comparing the experimental results of Examples 1, 2, and 3 of the present invention with that of Comparative Example 4, it can be seen that the purification method of the present invention has a very large single-stage purification capacity, with a processing capacity at least in grams. The amount of high-purity azure C obtained after purification is also very large, at least in grams. In contrast, the direct purification of azure C by HPLC (Example 4) has a very small single-stage purification capacity, only in milligrams, and the amount of high-purity azure C obtained is also very small, only in milligrams. Therefore, the present invention can effectively increase the single-stage purification capacity and improve the purification yield, resulting in high purification efficiency and low cost. Experimental verification shows that azure C purified by the method of the present invention can be used as an electronic mediator reagent for blood glucose detection in the preparation of blood glucose biosensors. The prepared biosensors can meet various detection requirements such as blood glucose detection. Therefore, azure C purified by the method of the present invention can be used as an electronic mediator for blood glucose and other biosensors.

Claims

1. A method for purifying an electronic medium having an amino group, characterized by, The process includes the following steps: weighing a low-purity electron mediator; reacting an amino-protecting reagent with the electron mediator to obtain an amino-protected electron mediator, wherein the structural formula of the amino-protected electron mediator is as follows: Wherein, R = H or X, X = amino protecting group, the number of amino protecting groups X varies from 1 to 3, the amino-protected electron mediator is dissolved in an organic solvent, and high-purity amino-protected electron mediator is obtained by extraction; the high-purity amino-protected electron mediator reacts with a deprotecting agent, the amino group is deprotected, and high-purity electron mediator is obtained, wherein the amino protecting group is selected from Fmoc or Boc or Trt or Cbz or Tos or TFA.

2. The purification method according to claim 1, characterized by, The electronic medium is selected from azure C.

3. The purification method according to claim 1, characterized by, The amino protecting agent is selected from fluorenemethyloxycarbonyl chloride, ditert-butyl dicarbonate, triphenylchloromethane, benzyl chloroformate, p-toluenesulfonyl chloride, or trifluoroacetic anhydride.

4. The purification method according to claim 1, characterized in that, The deprotecting agent is selected from diethylamine and trifluoroacetic acid.

5. The purification method according to claim 1, characterized by, The method for extracting high-purity amino-protected electron mediators is column chromatography.

6. The purification method according to claim 3, characterized by, The process includes the following steps: weighing low-purity Azure C, dissolving it in DMF, adding triethylamine and di-tert-butyl dicarbonate, reacting to obtain a viscous product, dissolving the viscous product in an organic solvent, purifying it to obtain intermediate (Boc)2-Azure C; dissolving intermediate (Boc)2-Azure C in an organic solvent, adding trifluoroacetic acid, reacting to remove the solvent, adding acetonitrile to dissolve it, adding diethyl ether solution of hydrogen chloride, precipitating a black solid, filtering, washing, and drying to obtain high-purity Azure C.

7. The purification method according to claim 3, characterized by, The process includes the following steps: Weigh low-purity Azure C and triphenylchloromethane, dissolve them in DMF, add triethylamine, react, filter, dilute the filtrate with dichloromethane, wash and separate the layers with water, dry, filter, concentrate, and purify to obtain intermediate Trt-Azure C; Dissolve intermediate Trt-Azure C in an organic solvent, add trifluoroacetic acid, remove the solvent, add acetonitrile to dissolve, add diethyl ether solution of hydrogen chloride, precipitate a black solid, filter, wash, and dry to obtain high-purity Azure C.

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