阅读说明：本技术 一种丙二醛乙醛加合物（maa）的合成方法 (Method for synthesizing Malonaldehyde Acetaldehyde Adduct (MAA) ) 是由 兰萍 胡治勇 习月 冉凯凯 于 2019-08-21 设计创作，主要内容包括：本发明公开了一种丙二醛乙醛加合物(MAA)的合成方法,包括以下步骤：步骤一、密闭容器中加入5克Malonildialdehyde-bis-dimethylacetal(Sigma)、5mL的去离子水和25mL的HCl,在室温下低速振摇反应1hr。本发明中,在步骤一中加入6NNaOH中和,再加入加冰丙酮100mL,提取沉淀物并溶于1.5去离子水中,加入与步骤四中去离子水等体积的冰丙酮,密封密闭容器并在在零下20摄氏度环境中放置1.5hr,抽滤反应物,收集沉淀,将产物真空干燥2-6hr或一整夜,将1.4 mmol的丙二醛、0.7 mmol的乙醛、30 mg的人血白蛋白和30 mL的PBS液体混匀,使用氮气密封,并置于37摄氏度的水浴中孵浴3天,然后取出并在4摄氏度的环境下过夜,除去未反应的丙二醛和乙醛,生成丙二醛乙醛(MAA)人血白蛋白加合物溶液。(The invention discloses a method for synthesizing Malonaldehyde Acetaldehyde Adduct (MAA), which comprises the following steps: step one, 5 g of Malonildehyde-bis-dimethyl acetal (Sigma), 5mL of deionized water and 25mL of HCl are added into a closed container, and the mixture is shaken at low speed at room temperature for reaction for 1 hr. In the invention, 6N NaOH is added in the first step for neutralization, 100mL of glacial acetone is added, the precipitate is extracted and dissolved in 1.5 deionized water, the glacial acetone with the same volume as that of the deionized water in the fourth step is added, the sealed container is sealed and placed in an environment at minus 20 ℃ for 1.5hr, the reactant is filtered by suction, the precipitate is collected, the product is dried in vacuum for 2-6hr or overnight, 1.4 mmol of malonaldehyde, 0.7 mmol of acetaldehyde, 30 mg of human albumin and 30 mL of PBS liquid are mixed uniformly, nitrogen is used for sealing, the mixture is placed in a water bath at 37 ℃ for 3 days, and then the mixture is taken out and placed overnight in an environment at 4 ℃ to remove unreacted malonaldehyde and acetaldehyde, so that Malonaldehyde (MAA) human albumin adduct solution is generated.)

1. A method for synthesizing a Malonaldehyde Acetaldehyde Adduct (MAA), comprising the steps of:

step one, adding 5 g of Malonildehyde-bis-dimethyl-ethyl (Sigma), 5mL of deionized water and 25mL of HCl into a closed container, and reacting for 1hr at low speed at room temperature;

step two, placing the reaction product on ice for cooling for 15min, and dropwise adding 6NNaOH for neutralization to neutrality;

step three, dropwise adding 100mL of glacial acetone, and placing on ice for reaction for 1 hr;

step four, suction filtering the reaction product in the step three, collecting the precipitate, and dissolving the precipitate in 1-1.5ml of deionized water;

step five, dropwise adding glacial acetone with the volume equal to that of the deionized water in the step four again;

step six, sealing the closed container and placing the container in an environment at the temperature of minus 20 ℃ for 1.5 hours;

step seven, carrying out suction filtration on the reactant again, collecting the precipitate, and carrying out vacuum drying on the product for 2-6hr or overnight;

step eight, transferring the mixture into a room-temperature sealed glass container, and storing the mixture in a dark place;

step nine, uniformly mixing 1.2-1.6 mmol of malondialdehyde, 0.4-0.8 mmol of acetaldehyde, 30 mg of human serum albumin and 30 mL of PBS liquid;

step ten, sealing by using nitrogen, and placing the sealed glass tube into a water bath at 37 ℃ for incubation bath for 3 days;

step eleven, taking out and standing overnight at the temperature of 4 ℃, and then removing unreacted malondialdehyde and acetaldehyde by using a dialysis bag;

and step twelve, performing SDS-PAGE verification and mass spectrum MAIDI-TOF-MS verification on the MAA adduct extracted in the step eleven.

2. The method of claim 1, wherein in step four, deionized water is selected to be 1.5 mm.

3. The method of claim 2, wherein in each of the fourth and seventh steps, the reaction mixture is filtered using a buchner funnel plus # 1 filter paper.

4. The method of claim 3, wherein in step seven the product is vacuum dried overnight.

5. The method of claim 1 for the synthesis of a Malondialdehyde Acetaldehyde Adduct (MAA), wherein 1.4 mmol malondialdehyde and 0.7 mmol acetaldehyde are selected.

6. The method of claim 5, wherein 30 mg of human serum albumin, commercially available from Wuhan dean Biotechnology Limited, is selected as the Malondialdehyde Acetaldehyde Adduct (MAA).

Technical Field

The invention relates to the technical field of synthesis of malonaldehyde acetaldehyde adducts, in particular to a synthesis method of Malonaldehyde Acetaldehyde Adducts (MAA).

Background

Malondialdehyde, known by the english name Malondialdehyde or malonic dialdehyde, MDA for short. In chemical industry, the product can be obtained by condensation of acetaldehyde and ethyl formate under the action of alkali, and is mainly used as a raw material of a medical intermediate and a photosensitive pigment. In organisms, MDA is one of the most important products of membrane lipid peroxidation, the generation of the MDA can also aggravate membrane damage, and the degree of membrane lipid peroxidation can be known through the MDA so as to indirectly determine the damage degree of a membrane system and the stress resistance of plants. In vivo, free radicals act on lipid to generate peroxidation, and the oxidation end product is malondialdehyde, which can cause cross-linking polymerization of life macromolecules such as protein and nucleic acid, and has cytotoxicity. In 2017, 10 and 27, the world health organization international cancer research institution publishes a carcinogen list for preliminary reference arrangement, and malondialdehyde is in a category 3 carcinogen list.

Acetaldehyde, also known as acetaldehyde, is called AA for short. In chemical industry, the product can be prepared by oxidizing ethylene, ethanol and the like, is colorless and easy to flow liquid and has pungent smell. Acetaldehyde is widely present in organisms. In the human body, acetaldehyde is produced by metabolism mainly after ethanol (alcohol) is taken in. In 2017, 10 and 27, acetaldehyde related to the intake of alcoholic beverages is in a category-one carcinogen list, and acetaldehyde is in a category-2 carcinogen list, wherein the list of carcinogens published by international cancer research institute of world health organization is preliminarily collated for reference.

Acetaldehyde and malonaldehyde are relatively active chemical substances, and after being generated in vivo, the acetaldehyde and malonaldehyde have complex actions with proteins to form a relatively stable malonaldehyde-acetaldehyde adduct (MAA).

There are two major routes of in vivo MAA adduct origin:

ethanol metabolism produces acetaldehyde. Long-term alcohol intake can cause liver mitochondria to be dysfunctional, the capacity of oxidizing acetaldehyde is greatly reduced, the content of acetaldehyde in the liver is increased, and malondialdehyde is further generated. They undergo complex reactions with proteins to produce new adducts, malondialdehyde-acetaldehyde (MAA) protein adducts.

The peroxidation of polyunsaturated fatty acids produces malondialdehyde, which can be degraded in vivo to acetaldehyde, and further undergo complex reactions with proteins to form malondialdehyde-acetaldehyde (MAA) protein adducts.

Malondialdehyde-acetaldehyde (MAA) protein adduct produced in vivo, a heterologous substance to the immune system, produces corresponding antibodies in the body, and studies have shown that MAA antibody levels are directly proportional to the severity of coronary heart disease progression.

Accordingly, the present invention provides a novel process for the preparation of MAA adducts.

Disclosure of Invention

The invention aims to: in order to solve the problems that malonaldehyde acetaldehyde adduct used for predicting the severity of coronary heart disease process is generally generated in human body and is difficult to extract, the synthesis method of Malonaldehyde Acetaldehyde Adduct (MAA) is provided.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method of synthesizing a Malonaldehyde Acetaldehyde Adduct (MAA) comprising the steps of:

step one, adding 5 g of Malonildehyde-bis-dimethyl-ethyl (Sigma), 5mL of deionized water and 25mL of HCl into a closed container, and reacting for 1hr at low speed at room temperature;

step two, placing the reaction product on ice for cooling for 15min, and dropwise adding 6NNaOH for neutralization to neutrality;

step three, dropwise adding 100mL of glacial acetone, and placing on ice for reaction for 1 hr;

step four, suction filtering the reaction product in the step three, collecting the precipitate, and dissolving the precipitate in 1-1.5ml of deionized water;

step five, dropwise adding glacial acetone with the volume equal to that of the deionized water in the step four again;

step six, sealing the closed container and placing the container in an environment at the temperature of minus 20 ℃ for 1.5 hours;

step seven, carrying out suction filtration on the reactant again, collecting the precipitate, and carrying out vacuum drying on the product for 2-6hr or overnight;

step eight, transferring the mixture into a room-temperature sealed glass container, and storing the mixture in a dark place;

step nine, uniformly mixing 1.2-1.6 mmol of malondialdehyde, 0.4-0.8 mmol of acetaldehyde, 30 mg of human serum albumin and 30 mL of PBS liquid;

step ten, sealing by using nitrogen, and placing the sealed glass tube into a water bath at 37 ℃ for incubation bath for 3 days;

step eleven, taking out and standing overnight at the temperature of 4 ℃, and then removing unreacted malondialdehyde and acetaldehyde by using a dialysis bag;

and step twelve, performing SDS-PAGE verification and mass spectrum MAIDI-TOF-MS verification on the MAA adduct extracted in the step eleven.

As a further description of the above technical solution:

in the fourth step, deionized water is selected to be 1.5 mm.

As a further description of the above technical solution:

and in the fourth step and the seventh step, a Buchner funnel and No. 1 filter paper are selected to carry out suction filtration on the reactant.

As a further description of the above technical solution:

in step seven, the product was dried in vacuo overnight.

As a further description of the above technical solution:

1.4 mmol of malondialdehyde and 0.7 mmol of acetaldehyde were chosen.

As a further description of the above technical solution:

30 mg of human serum albumin, which is prepared by Wuhan Tanshan Biotechnology Limited, was selected.

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

1. in the invention, Malonildehyde-bis-dimethyllactol (Sigma), 5mL of deionized water and 25mL of HCl are added into a closed container, then 6N NaOH is added for neutralization, 100mL of glacial acetone is added, precipitate is extracted and dissolved in 1.5 deionized water, the glacial acetone with the same volume as the deionized water in step four is added dropwise again, the closed container is sealed and placed in an environment with the temperature of minus 20 ℃ for 1.5hr, the reactant is filtered again, the precipitate is collected, the product is dried in vacuum for 2-6hr or overnight to generate 100mg of malondialdehyde, 1.2-1.6 mmol of malondialdehyde, 0.4-0.8 mmol of acetaldehyde are mixed uniformly, 30 mg of human albumin and 30 mL of PBS liquid are sealed by using nitrogen and placed in a water bath with the temperature of 37 ℃ for 3 days, then the product is taken out and placed in an environment with the temperature of 4 ℃ for incubation, and unreacted malondialdehyde and acetaldehyde are removed by a dialysis bag, at this time, a 1 mg/ml solution of malonaldehyde acetaldehyde (MAA) human serum albumin adduct dissolved in 0.1M phosphate buffer was formed, and this synthesis was simpler than the conditions required for extraction in human body.

Drawings

FIG. 1 is a schematic illustration of SDS-PAGE validation of a method of synthesis of Malondialdehyde Acetaldehyde Adduct (MAA) in accordance with the present invention;

FIG. 2 is a schematic illustration of mass spectrometric verification of an adduct of a process for the synthesis of a Malondialdehyde Acetaldehyde Adduct (MAA) proposed by the present invention;

FIG. 3 is a schematic diagram of glyoxalated, malonaldehyde, and malonaldehyde-glyoxalated proteins according to the method of the invention for the synthesis of Malonaldehyde Acetaldehyde Adducts (MAA).

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Referring to fig. 1-3, a method for synthesizing malonaldehyde-acetaldehyde adduct (MAA) includes the following steps:

step one, adding 5 g of Malonildehyde-bis-dimethyl-ethyl (Sigma), 5mL of deionized water and 25mL of HCl into a closed container, and reacting for 1hr at low speed at room temperature;

step two, placing the reaction product on ice for cooling for 15min, and dropwise adding 6NNaOH for neutralization to neutrality;

step three, dropwise adding 100mL of glacial acetone, and placing on ice for reaction for 1 hr;

step four, suction filtering the reaction product in the step three, collecting the precipitate, and dissolving the precipitate in 1-1.5ml of deionized water;

step five, dropwise adding glacial acetone with the volume equal to that of the deionized water in the step four again, wherein the solution becomes turbid;

step six, sealing the closed container and placing the container in an environment at the temperature of minus 20 ℃ for 1.5 hours, and observing the precipitation of needle-shaped white crystals;

seventhly, performing suction filtration on the reactant again, collecting the precipitate, and performing vacuum drying on the product for 2-6hr or overnight to obtain NaMDA;

step eight, transferring the product into a room-temperature sealed glass container, storing in a dark place, and generating malondialdehyde when obvious white needle-shaped crystals are separated out;

step nine, uniformly mixing 1.2-1.6 mmol of malondialdehyde, 0.4-0.8 mmol of acetaldehyde, 30 mg of human serum albumin and 30 mL of PBS liquid;

step ten, sealing by using nitrogen, and placing the sealed glass tube into a water bath at 37 ℃ for incubation bath for 3 days;

step eleven, taking out and standing overnight at 4 ℃, and then removing unreacted malonaldehyde and acetaldehyde by using a dialysis bag to obtain a product, namely a malonaldehyde acetaldehyde (MAA) human serum albumin adduct solution of 1 mg/ml dissolved in 0.1M phosphate buffer solution;

and twelfth, verifying the MAA adduct extracted in the eleventh step by SDS-PAGE and mass spectrum MAIDI-TOF-MS, verifying that the HAS molecular weight is increased from 67kDa to about 100kDa by SDS-PAGE after the addition reaction, and verifying that the adduct is successfully prepared, wherein the mass spectrum MAIDI-TOF-MS verifies that the mass-to-charge ratio is 36600, the charge amount is 3 and the actual molecular weight is 102.8 kDa. Much higher than the normal 65kda molecular weight of BSA, indicating successful adduct synthesis.

Example 2

Referring to fig. 1-3, a method for synthesizing malonaldehyde-acetaldehyde adduct (MAA) includes the following steps:

step one, adding 5 g of Malonildehyde-bis-dimethyl-ethyl (Sigma), 5mL of deionized water and 25mL of HCl into a closed container, and reacting for 1hr at low speed at room temperature;

step two, placing the reaction product on ice for cooling for 15min, and dropwise adding 6NNaOH for neutralization to neutrality;

step three, dropwise adding 100mL of glacial acetone, and placing on ice for reaction for 1 hr;

step four, adopting a Buchner funnel and No. 1 filter paper to filter and filter the reaction product in the step three, collecting the precipitate, dissolving the precipitate in 1.5ml of deionized water, adding the No. 1 filter paper into the Buchner funnel to facilitate extraction and crystallization, adding the No. 1 filter paper into the Buchner funnel to facilitate washing and precipitation with glacial acetone;

step five, dropwise adding glacial acetone with the volume equal to that of the deionized water in the step four again, wherein the solution becomes turbid;

step six, sealing the closed container and placing the container in an environment at the temperature of minus 20 ℃ for 1.5 hours, and observing the precipitation of needle-shaped white crystals;

step seven, adding No. 1 filter paper into a Buchner funnel to pump and filter the reactant, collecting the precipitate, and vacuum-drying the product for 2-6hr or overnight to obtain NaMDA, wherein the Buchner funnel is added with No. 1 filter paper to facilitate extraction and crystallization;

step eight, transferring the product into a room-temperature sealed glass container, storing in a dark place, and generating malondialdehyde when obvious white needle-shaped crystals are separated out;

step nine, uniformly mixing 1.2-1.6 mmol of malondialdehyde, 0.4-0.8 mmol of acetaldehyde, 30 mg of human serum albumin and 30 mL of PBS liquid;

step ten, sealing by using nitrogen, and placing the sealed glass tube into a water bath at 37 ℃ for incubation bath for 3 days;

step eleven, taking out and standing overnight at 4 ℃, and then removing unreacted malonaldehyde and acetaldehyde by using a dialysis bag to obtain a product, namely a malonaldehyde acetaldehyde (MAA) human serum albumin adduct solution of 1 mg/ml dissolved in 0.1M phosphate buffer solution;

and twelfth, verifying the MAA adduct extracted in the eleventh step by SDS-PAGE and mass spectrum MAIDI-TOF-MS, verifying that the HAS molecular weight is increased from 67kDa to about 100kDa by SDS-PAGE after the addition reaction, and verifying that the adduct is successfully prepared, wherein the mass spectrum MAIDI-TOF-MS verifies that the mass-to-charge ratio is 36600, the charge amount is 3 and the actual molecular weight is 102.8 kDa. Much higher than the normal 65kda molecular weight of BSA, indicating successful adduct synthesis.

Example 3

Referring to fig. 1-3, a method for synthesizing malonaldehyde-acetaldehyde adduct (MAA) includes the following steps:

step one, adding 5 g of Malonildehyde-bis-dimethyl-ethyl (Sigma), 5mL of deionized water and 25mL of HCl into a closed container, and reacting for 1hr at low speed at room temperature;

step two, placing the reaction product on ice for cooling for 15min, and dropwise adding 6NNaOH for neutralization to neutrality;

step three, dropwise adding 100mL of glacial acetone, and placing on ice for reaction for 1 hr;

step four, adopting a Buchner funnel and No. 1 filter paper to filter and filter the reaction product in the step three, collecting the precipitate, dissolving the precipitate in 1.5ml of deionized water, adding the No. 1 filter paper into the Buchner funnel to facilitate extraction and crystallization, adding the No. 1 filter paper into the Buchner funnel to facilitate washing and precipitation with glacial acetone;

step five, dropwise adding glacial acetone with the volume equal to that of the deionized water in the step four again, wherein the solution becomes turbid;

step six, sealing the closed container and placing the container in an environment at the temperature of minus 20 ℃ for 1.5 hours, and observing the precipitation of needle-shaped white crystals;

step seven, adding No. 1 filter paper into a Buchner funnel to pump and filter the reactant, collecting the precipitate, and vacuum-drying the product for 2-6hr or overnight to obtain NaMDA, wherein the Buchner funnel is added with No. 1 filter paper to facilitate extraction and crystallization;

step eight, transferring the product into a room-temperature sealed glass container, storing in a dark place, and generating malondialdehyde when obvious white needle-shaped crystals are separated out;

step nine, uniformly mixing 1.4 mmol of malondialdehyde, 0.7 mmol of acetaldehyde, 30 mg of human serum albumin prepared by Wuhan worn-biology Limited company and 30 mL of PBS liquid, selecting 1.4 mmol of malondialdehyde and 0.7 mmol of acetaldehyde, and improving the actual molecular weight through mass spectrum MAIDI-TOF-MS verification during subsequent generated adduct inspection;

step ten, sealing by using nitrogen, and placing the sealed glass tube into a water bath at 37 ℃ for incubation bath for 3 days;

step eleven, taking out and standing overnight at 4 ℃, and then removing unreacted malonaldehyde and acetaldehyde by using a dialysis bag to obtain a product, namely a malonaldehyde acetaldehyde (MAA) human serum albumin adduct solution of 1 mg/ml dissolved in 0.1M phosphate buffer solution;

and twelfth, verifying the MAA adduct extracted in the eleventh step by SDS-PAGE and mass spectrum MAIDI-TOF-MS, verifying that the HAS molecular weight is increased from 67kDa to about 100kDa by SDS-PAGE after the addition reaction, and verifying that the adduct is successfully prepared, wherein the mass spectrum MAIDI-TOF-MS verifies that the mass-to-charge ratio is 36600, the charge amount is 3 and the actual molecular weight is 109.8 kDa. Much higher than the normal 65kda molecular weight of BSA, indicating successful adduct synthesis.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.