CN113336714A

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Info

Publication number: CN113336714A
Application number: CN202110714162.1A
Authority: CN
Inventors: 刘秀美; 陈晨
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2021-06-25
Filing date: 2021-06-25
Publication date: 2021-09-03

Abstract

Translated fromChinese

本发明属于电化学传感器技术领域，涉及一种化合物及其制备方法和应用，所述化合物为1‑(2‑((叔丁氧基羰基)氨基)乙氧基)‑5‑乙基吩嗪‑5‑鎓，具体结构如式Ⅰ所示：

这种化合物作为新型电子媒介体，以乙基吩嗪作为氧化还原中心将酶氧化反应产生的电子传递到电极上，侧链上的氨基乙氧基作为连接修饰中心，可以连接酶或者电极表面，有利于提高导电性和降低氧化还原电位，并提高电化学酶传感器的稳定性、灵敏度和抗干扰性。

The invention belongs to the technical field of electrochemical sensors, and relates to a compound, a preparation method and application thereof, wherein the compound is 1-(2-((tert-butoxycarbonyl)amino)ethoxy)-5-ethylphenazine -5-Onium, the specific structure is shown in formula I:

As a new type of electron mediator, this compound uses ethylphenazine as the redox center to transfer the electrons generated by the enzyme oxidation reaction to the electrode, and the aminoethoxy group on the side chain acts as the connecting modification center, which can connect the enzyme or the surface of the electrode. It is beneficial to improve the conductivity and reduce the redox potential, and improve the stability, sensitivity and anti-interference of the electrochemical enzyme sensor.

Description

Compound and preparation method and application thereof

Technical Field

The invention belongs to the technical field of electrochemical sensors, and relates to a compound, and a preparation method and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Electrochemical enzyme sensors are the most widespread form of biosensor in detection research due to their simple structure and superior performance. The principle of the electrochemical enzyme sensor is that the substrate is oxidized by the redox center of the enzyme, an electrochemical signal is generated between the enzyme and the gain-loss electrons of the electrode, and the electrochemical signal is captured by an electrochemical workstation, so that the detection of the substrate is realized. Such as glucose oxidase, lactate oxidase, horseradish peroxidase, etc., are commonly used in electrochemical enzyme sensors.

Electrochemical enzyme sensors have undergone three generations of development. The first generation of electrochemical enzyme sensors utilized molecular oxygen as the primary electron acceptor to determine substrate concentration by detecting oxygen consumption or hydrogen peroxide production, a method that is susceptible to atmospheric oxygen. The second generation electrochemical enzyme sensor uses a free electron mediator to replace oxygen in the first generation for electron transport, and can reduce the redox operating potential of the sensor, but has the defect that the mediator is easy to lose. The third generation of electrochemical enzyme sensors uses enzymes that can transfer electrons directly, so that after the substrate is oxidized by the enzyme, the enzyme can transfer electrons directly to the electrode.

In the development process of electrochemical enzyme sensors, the research on electron mediators has also received much attention. People begin to research the electronic mediator which can be immobilized so as to realize the purpose of fixing the electronic mediator on an electrode or connecting the electronic mediator on enzyme to solve the problem that the electronic mediator is easy to lose in the second generation sensor. Including typical electron mediators commonly used in the improved second generation sensors, such as ferrocene and its derivatives, tetrathiafulvalene, quinone and its derivatives, organic dyes, etc.

The electron mediator is a molecular conductive substance which can transfer electrons generated in an enzyme reaction process from an enzyme reaction center to the surface of an electrode in an electrochemical enzyme sensor so as to generate corresponding current change on the surface of the electrode. The currently used electronic mediators can be classified into organic small molecular mediators and high molecular mediators according to molecular structures and molecular weights. Wherein the small molecular mediator comprises ferrocene and derivatives thereof, organic dye, quinone and derivatives thereof, tetrathiafulvalene, fullerene, conductive organic salt and the like; the polymer medium includes valence-variable transition metal ion chelating polymer medium, redox polymer medium, enveloping polymer medium, monomer polymerization polymer medium, etc.

However, the inventors have found that the conventional electron mediator has poor conductivity, high oxidation-reduction potential, and is easily disturbed, resulting in a decrease in accuracy.

Disclosure of Invention

In order to improve the conductivity of an electron mediator and reduce the oxidation-reduction potential so as to avoid interference caused by various factors, the invention provides a compound and a preparation method and application thereof.

Specifically, the invention is realized by the following technical scheme:

in a first aspect of the invention, a compound is 1- (2- ((tert-butoxycarbonyl) amino) ethoxy) -5-ethylphenazin-5-ium having the following specific structure:

in a second aspect of the invention, a method of preparing a compound, comprising:

step (1): dissolving N- (tert-butyloxycarbonyl) ethanolamine by dichloromethane, adding triethylamine and methylsulfonyl chloride for reaction, extracting and drying the crude product after the reaction is stopped, and performing rotary evaporation to obtain a sample 1;

step (2): dissolving 1-hydroxyphenyloxazine in N, N-dimethylformamide, adding potassium carbonate and the sample 1, heating for reaction, extracting after the reaction is stopped, drying, and performing rotary evaporation to obtain a sample 2;

and (3): dissolving the sample 2 in acetonitrile, adding iodoethane, heating for reaction, washing after the reaction is stopped, separating, and performing rotary evaporation to obtain the 1- (2- ((tert-butoxycarbonyl) amino) ethoxy) -5-ethylphenazine-5-onium.

In a third aspect of the present invention, an electron mediator is the compound described above and/or the compound obtained by the above production method.

In a fourth aspect of the present invention, an electrochemical enzyme sensor comprising said compound or a product obtained by a method for producing said compound.

In a fifth aspect of the invention, the use of said compound or a method for the preparation of said compound for the manufacture of an electrochemical enzyme sensor.

In the fifth aspect of the invention, the electrochemical enzyme sensor is applied to the fields of food safety, environmental detection and medicine development.

One or more embodiments of the present invention have the following advantageous effects:

(1) the novel compound 1- (2- ((tert-butoxycarbonyl) amino) ethoxy) -5-ethylphenazine-5-onium is used as a novel electron mediator of an electrochemical sensor, electrons generated by enzymatic oxidation are transferred to an electrode by taking ethylphenazine as a redox center, and aminoethoxy on a side chain is used as a connecting modification center and can be connected with the surface of the enzyme or the electrode, so that the rapid transfer of the electrons and the stability of the electrode structure are realized.

(2) The novel electronic mediator has good conductivity and lower oxidation-reduction potential, is beneficial to avoiding the interference of other factors, and can be used for manufacturing electrochemical enzyme sensors.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is a synthetic route to the preparation described in example 1;

FIG. 2 is a cyclic voltammogram of the electron mediator in example 1.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

At present, the electron mediator used for the electrochemical enzyme sensor in the prior art has the defects of higher oxidation-reduction potential, easy deletion and the like, and the invention provides a compound and a preparation method and application thereof in order to solve the problems.

In one or more embodiments of the invention, a compound is 1- (2- ((tert-butoxycarbonyl) amino) ethoxy) -5-ethylphenazin-5-ium having the following structure:

the 1- (2- ((tert-butoxycarbonyl) amino) ethoxy) -5-ethylphenazine-5-onium structure contains ethylphenazine and can be used as a redox center to realize rapid electron transfer, and meanwhile, the aminoethoxy on a side chain is used as a connecting modification center to stably connect enzyme or an electrode surface, so that the structure is favorable for reducing redox potential and avoiding loss of a mediator.

In one or more embodiments of the invention, a method of making a compound comprises:

In the preparation process, the adding sequence of the samples is controlled, and meanwhile, the adding types of the samples at each stage are controlled, so that certain influence is exerted on improving the purity of the product.

In one or more embodiments of the present invention, in step (1), the molar ratio of N- (tert-butoxycarbonyl) ethanolamine, triethylamine, and methylsulfonyl chloride is 8-12:10-15:10-15, preferably 10:13: 12. Under the addition proportion, the purity of the sample 1 can be improved, the generation of excessive impurities is avoided, the purification difficulty is reduced, and the purification efficiency is improved.

Or, in the step (1), the adding mode of the methylsulfonyl chloride is dropwise adding through a 0 ℃ constant pressure dropping funnel, and in the adding process, the temperature is too high, so that the reaction is not facilitated to be carried out, and therefore, the reaction can be effectively promoted to be carried out and the purity of the sample 1 can be improved through the dropwise adding mode through the 0 ℃ constant pressure dropping funnel.

Or, in the step (1), the reaction time is 8-15 min; preferably 10min, the reaction time is short, and the high-efficiency reaction is realized.

Or, in the step (1), the extraction method is to wash the sample 1 by using saturated sodium bicarbonate, saturated sodium chloride, a mixed solution of saturated sodium chloride and water and saturated sodium chloride in sequence, and the extraction method is favorable for quickly and efficiently purifying the sample 1 and avoids adverse effects of impurities on subsequent reactions.

Or, in the step (1), the rotary evaporation temperature is 30-50 ℃, preferably 40 ℃.

In one or more embodiments of the present invention, the specific operation in step (1) is: adding N- (tert-butyloxycarbonyl) ethanolamine into a round-bottom flask, dissolving the ethanolamine by using dichloromethane, adding triethylamine, dropwise adding methylsulfonyl chloride into a dropping funnel with a constant pressure of 0 ℃, stirring the mixture at room temperature for reaction, washing a crude product twice by using saturated sodium bicarbonate after the reaction is stopped, washing once by using saturated sodium chloride, washing once by using a mixed solution of the saturated sodium chloride and water, washing once by using the saturated sodium chloride, drying by using anhydrous sodium sulfate, and performing rotary evaporation at 40 ℃ by using a rotary evaporator.

In one or more embodiments of the invention, in step (2), the molar ratio of 1-hydroxyphenyloxazine to potassium carbonate to sample 1 is 0.5-1.2:1.5-2.5:0.8-1.5, preferably 1:2:1.2, and too high an amount of 1-hydroxyphenyloxazine added may result in more impurities, thereby increasing the difficulty of purification. The addition ratio of different reactants is well controlled, which is beneficial to obtaining a high-purity sample 2.

Or, in the step (2), the adding time of the sample 1 is 50 ℃ when the temperature is raised to 40-60 ℃; at this time, sample 1 had the best reaction efficiency with 1-hydroxyphenyloxazine.

Or, in the step (2), the reaction temperature is 70-90 ℃, preferably 80 ℃;

or, in the step (2), the reaction time is 1-4h, preferably 3 h;

or, in the step (2), the extraction method comprises washing with a mixed solution of water and ethyl acetate, washing the ethyl acetate layer with water, and then washing with saturated sodium chloride, and for the sample 2, it is necessary to purify with a mixed solution of water and ethyl acetate, which is beneficial to avoiding the generation of impurities.

Or, in the step (2), the rotary evaporation temperature is 30-50 ℃, preferably 40 ℃.

In one or more embodiments of the present invention, the specific operation of step (2) is: adding 1-hydroxyphenyloxazine into a round bottom flask, dissolving by using N, N-dimethylformamide, adding anhydrous potassium carbonate, adding the sample 1 when the temperature is raised to 40-60 ℃, heating and stirring for reaction, washing a crude product once by using a mixed solution of water and ethyl acetate after the reaction is stopped, washing an ethyl acetate layer twice by using water, washing once by using saturated sodium chloride, and carrying out rotary evaporation at the temperature of 30-50 ℃ by using a rotary evaporator.

In one or more embodiments of the present invention, in step (3), the molar ratio of sample 2 to iodoethane is 0.5-1.5:40-60, preferably 1:50, and an excessive amount of iodoethane generates impurities, and a too low amount of iodoethane does not achieve complete reaction with sample 2, resulting in low utilization.

Or, in the step (3), the heating reaction device is a sealed tube for heating, and the reaction temperature is 130-150 ℃, preferably 140 ℃; the tube sealing heating is beneficial to promoting the reaction, provides enough pressure and temperature for the reaction and reduces the generation of impurities.

Or, in the step (3), the reaction time is 8-12h, preferably 10 h;

or, in the step (3), the separation adopts a silica gel column separation mode; further, the volume ratio of the silica gel column gradient elution is ethyl acetate, dichloromethane: methanol 40:1, dichloromethane: methanol 30:1, dichloromethane: methanol 20:1, dichloromethane: methanol 10: 1; through silica gel column gradient elution, not only can the product purity be improved, but also the product yield can be improved.

Or, in the step (3), the rotary evaporation temperature is 30-50 ℃, preferably 40 ℃.

In one or more embodiments of the present invention, an electron mediator is a compound obtained by the above compound and/or the above compound preparation method.

The electron mediator, i.e. the molecular conductor, can effectively transfer electrons existing in the enzyme reaction center to the surface of the electrode, and then the current change is generated. The 1- (2- ((tert-butoxycarbonyl) amino) ethoxy) -5-ethylphenazine-5-onium is used as an electron mediator, and the compound contains conjugated pi electrons and can be overlapped with empty orbitals to be adsorbed by a substrate electrode. And secondly, the compound structure also has an electronic active group, so that the concentration of the group on the surface of the electrode can be increased, the sensing effect is good, the stability, the sensitivity and the reaction speed of the electrochemical enzyme sensor can be improved, and the immobilized enzyme cannot be poisoned. In addition, the compound 1- (2- ((tert-butoxycarbonyl) amino) ethoxy) -5-ethylphenazine-5-onium is in a low potential state in the redox reaction, so that the compound has stronger adsorption force on an electrode, and the service life and the anti-interference capability of an electrochemical enzyme sensor can be improved.

In one or more embodiments of the invention, the electrochemical enzyme sensor prepared by the novel compound has higher accuracy and sensitivity and higher stability.

In one or more embodiments of the invention, the use of the compound or a method of making the compound in the manufacture of an electrochemical enzyme sensor; further, the enzyme in the electrochemical enzyme sensor is selected from glucose oxidase, lactate oxidase, and horseradish peroxidase.

In one or more embodiments of the invention, the electrochemical enzyme sensor is applied to the fields of food safety, environmental detection and medicine development.

The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the invention and not limiting.

Example 1

Preparation of 2- ((tert-butoxycarbonyl) amino) ethyl methanesulfonate:

adding N- (tert-butoxycarbonyl) ethanolamine (20mmol) into a round-bottom flask, dissolving the ethanolamine with 50mL of dichloromethane, adding triethylamine (26mmol), dropwise adding methylsulfonyl chloride (24mmol) into a dropping funnel at a constant pressure of 0 ℃, stirring the mixture at room temperature for reaction, washing a crude product after the reaction is stopped twice with 20mL of saturated sodium bicarbonate, once with 20mL of saturated sodium chloride, once with 10mL of mixed solution of saturated sodium chloride and 5mL of ultrapure water, once with 10mL of saturated sodium chloride, drying the product with anhydrous sodium sulfate, and performing rotary evaporation at 40 ℃ by using a rotary evaporator to obtain 2- ((tert-butoxycarbonyl) amino) ethyl methanesulfonate with the yield of 85%.

¹HNMR(400MHz,CDCl₃-d₁)δ4.21(t,2H),3.41(t,2H)，2.97(s,3H),1.38(s,9H).

Preparation of 1- ((tert-butoxycarbonyl) amino) ethoxyphenazine:

adding 1-hydroxyphenyloxazine (2.55mmol) into a round-bottom flask, dissolving the 1-hydroxyphenyloxazine with 50mL of N, N-dimethylformamide, adding anhydrous potassium carbonate (5.1mmol), adding a sample 1(3.06mmol) when the temperature is increased to 50 ℃, heating the mixture to 80 ℃, stirring the mixture for reaction, washing a crude product once with a mixed solution of 100mL of ultrapure water and 50mL of ethyl acetate after the reaction is stopped, taking an ethyl acetate layer, washing the ethyl acetate layer twice with 50mL of ultrapure water, washing with 20mL of saturated sodium chloride once, and performing rotary evaporation at 40 ℃ by using a rotary evaporator to obtain 1- (tert-butoxy) -N- (2- (phenazine-1-yloxy) ethyl) ethylamine with the yield of 74%.

¹HNMR(400MHz,CDCl₃-d₁)δ8.40(d,1H),8.24(d,1H)，7.86(t,1H),7.86(t,1H),7.86(t,1H),7.75(d,1H),7.18(d,1H),4.39(t,2H),3.77(q,2H),1.47(s,9H).

Preparation of 1- (2- ((tert-butoxycarbonyl) amino) ethoxy) -5-ethylphenazin-5-ium:

adding sample 2(0.15mmol) into a round-bottom flask, dissolving the sample with about 1mL of acetonitrile, adding iodoethane (7.5mmol), stirring the mixture in a sealed tube at 140 ℃, stopping the reaction after reacting for 10 hours, washing the mixture with diethyl ether, separating the crude product by a silica gel column, and performing gradient elution on the silica gel column by respectively using ethyl acetate, dichloromethane, methanol 40:1, dichloromethane, methanol 30:1, dichloromethane, methanol 20:1, dichloromethane, methanol 10:1, and finally performing rotary evaporation on the target component by using a rotary evaporator at 40 ℃ to obtain 1- (2- (((tert-butoxycarbonyl) amino) ethoxy) -5-ethylphenazine-5-onium.

¹HNMR(400MHz,DMSO-d₆)δ8.28(d,1H),8.27(d,1H)，7.91(t,1H),7.88(t,1H),7.86(t,1H),7.86(d,1H),7.18(d,1H),4.20(m,2H),3.85(m,2H),3.64(t,2H),1.59(dd,3H)。

Example 2:

measurement of oxidation-reduction potential of the electron mediator:

the electron mediator 1- (2- ((tert-butoxycarbonyl) amino) ethoxy) -5-ethylphenazine-5-ium from example 1 was dissolved in PBS buffer and measured using an electrochemical workstation in which a glassy carbon electrode was the working electrode, Ag/AgCl was the reference electrode, a platinum wire was the counter electrode, and cyclic voltammetric scans were performed at a voltage of-1V to 1V.

The oxidation-reduction potential Em is-0.2V vs Ag/AgCl, and electrochemical detection can be carried out at a low working potential so as to avoid the influence of various interference factors.

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A compound is characterized in that the compound is 1- (2- ((tert-butoxycarbonyl) amino) ethoxy) -5-ethylphenazine-5-onium, and the specific structure is shown as a formula I:

2. a method for preparing a compound, comprising:

3. The method for preparing a compound according to claim 2, wherein in the step (1), the molar ratio of N- (tert-butoxycarbonyl) ethanolamine to triethylamine to methylsulfonyl chloride is 8-12:10-15:10-15, preferably 10:13: 12;

or, in the step (1), the addition mode of the methylsulfonyl chloride is dropwise adding by a dropping funnel with a constant pressure of 0 ℃;

or, in the step (1), the reaction time is 8-15 min; preferably, 10 min;

or, in the step (1), the extraction method comprises washing with saturated sodium bicarbonate, saturated sodium chloride, a mixed solution of saturated sodium chloride and water, and saturated sodium chloride in sequence;

4. The process for preparing a compound as claimed in claim 2, wherein the specific operation in step (1) is: adding N- (tert-butyloxycarbonyl) ethanolamine into a round-bottom flask, dissolving the ethanolamine by using dichloromethane, adding triethylamine, dropwise adding methylsulfonyl chloride into a dropping funnel with a constant pressure of 0 ℃, stirring the mixture at room temperature for reaction, washing a crude product twice by using saturated sodium bicarbonate after the reaction is stopped, washing once by using saturated sodium chloride, washing once by using a mixed solution of the saturated sodium chloride and water, washing once by using the saturated sodium chloride, drying by using anhydrous sodium sulfate, and performing rotary evaporation at 40 ℃ by using a rotary evaporator.

5. The process for the preparation of a compound as claimed in claim 2, wherein in step (2), the molar ratio of 1-hydroxyphenyloxazine to potassium carbonate to sample 1 is from 0.5 to 1.2:1.5 to 2.5:0.8 to 1.5, preferably 1:2: 1.2;

or, in the step (2), the adding time of the sample 1 is 50 ℃ when the temperature is raised to 40-60 ℃;

or, in the step (2), the reaction temperature is 70-90 ℃, preferably 80 ℃;

or, in the step (2), the reaction time is 1-4h, preferably 3 h;

or, in the step (2), the extraction method comprises washing with a mixed solution of water and ethyl acetate, washing the ethyl acetate layer with water, and then washing with saturated sodium chloride;

or, in the step (2), the rotary evaporation temperature is 30-50 ℃, preferably 40 ℃;

or, the specific operation of the step (2) is as follows: adding 1-hydroxyphenyloxazine into a round bottom flask, dissolving by using N, N-dimethylformamide, adding anhydrous potassium carbonate, adding the sample 1 when the temperature is raised to 40-60 ℃, heating and stirring for reaction, washing a crude product once by using a mixed solution of water and ethyl acetate after the reaction is stopped, washing an ethyl acetate layer twice by using water, washing once by using saturated sodium chloride, and carrying out rotary evaporation at the temperature of 30-50 ℃ by using a rotary evaporator.

6. The process for the preparation of a compound as claimed in claim 2, wherein in step (3), the molar ratio of sample 2 to iodoethane is from 0.5 to 1.5:40 to 60, preferably from 1: 50;

or, in the step (3), the heating reaction device is a sealed tube for heating, and the reaction temperature is 130-150 ℃, preferably 140 ℃;

or, in the step (3), the reaction time is 8-12h, preferably 10 h;

or, in the step (3), the separation adopts a silica gel column separation mode; further, the volume ratio of the silica gel column gradient elution is ethyl acetate, dichloromethane: methanol 40:1, dichloromethane: methanol 30:1, dichloromethane: methanol 20:1, dichloromethane: methanol 10: 1;

7. An electron mediator, which is the compound according to claim 1 and/or the compound obtained by the method for producing the compound according to any one of claims 2 to 6.

8. An electrochemical enzyme sensor comprising the compound according to claim 1 or the product obtained by the production method of the compound according to any one of claims 2 to 6.

9. Use of a compound according to claim 1 or a process for the preparation of a compound according to any one of claims 2 to 6 in the manufacture of an electrochemical enzyme sensor; further, the enzyme in the electrochemical enzyme sensor is selected from glucose oxidase, lactate oxidase, and horseradish peroxidase.

10. Use of the electrochemical enzyme sensor according to claim 8 in the fields of food safety, environmental testing, and medical research and development.