阅读说明：本技术 一种绿色高效制备l-硒甲基硒代半胱氨酸的方法 (Method for green and efficient preparation of L-selenium methyl selenocysteine ) 是由 王小松 卢会芹 王兴东 王阳 李霁宇 于 2019-11-04 设计创作，主要内容包括：本发明公开了一种绿色高效制备L-硒甲基硒代半胱氨酸的方法,首先以L-丝氨酸甲酯盐酸盐为起始原料,通过乙酰化、氯化、甲硒化一锅法合成反应底物,再将所得反应底物在酶催化作用下水解生成L-硒甲基硒代半胱氨酸。本发明采用一锅法制备反应底物,既克服了中间体的不稳定性,又减少了中间体分离、提纯等繁琐步骤；采用酶催化水解,避免了强酸水解造成的环境污染、设备腐蚀,克服了硒醚键断裂及产物消旋化等缺点。本发明具有原料易得、价廉,操作方便,反应条件温和、绿色环保,产品单一、易分离,产率高、适合工业化生产的特点。(The invention discloses a green and efficient method for preparing L-selenomethylselenocysteine, which comprises the steps of firstly taking L-serine methyl ester hydrochloride as an initial raw material, synthesizing a reaction substrate through acetylation, chlorination and methyl selenocysteine one-pot method, and hydrolyzing the obtained reaction substrate under the enzyme catalysis to generate the L-selenomethylselenocysteine. The invention adopts a one-pot method to prepare the reaction substrate, which not only overcomes the instability of the intermediate, but also reduces the complicated steps of intermediate separation, purification and the like; the enzyme catalysis hydrolysis is adopted, thereby avoiding the environmental pollution and equipment corrosion caused by strong acid hydrolysis and overcoming the defects of selenoether bond breakage, product racemization and the like. The method has the characteristics of easily obtained raw materials, low price, convenient operation, mild reaction conditions, environmental protection, single product, easy separation, high yield and suitability for industrial production.)

1. A method for preparing L-selenium methyl selenocysteine in a green and high-efficiency manner is characterized by comprising the following steps:

(1) taking L-serine methyl ester hydrochloride as an initial raw material, and synthesizing a reaction substrate by acetylation, chlorination and methyl selenization one-pot method;

(2) hydrolyzing the reaction substrate obtained in the step (1) under the enzyme catalysis to generate the L-selenium methyl selenocysteine.

2. The method for green and efficient preparation of L-selenomethylselenocysteine according to claim 1, which is characterized in that the step (1) is specifically as follows:

(a) adding L-serine methyl ester hydrochloride and auxiliary reagent into the reaction medium under the condition of stirring, and cooling to 0 ~ 5 ℃;

(b) adding acetylation reagent, stirring for 2 ~ 5 hr, adding chlorination reagent, stirring for 0.5 ~ 2 hr, heating to 5 ~ 50 deg.C, stirring for 2 ~ 5 hr, and cooling to-5 ~ 0 deg.C;

(c) adding a selenizing reagent, stirring for 2 ~ 5 hours, heating to room temperature, and continuously stirring for 10 ~ 15 hours to obtain a reaction mixed solution;

(d) and (c) adjusting the pH of the reaction mixed solution obtained in the step (c) to be less than 4.0, extracting with ethyl acetate for 2 ~ 5 times, combining the extract, washing with water, drying, filtering, and evaporating under reduced pressure to remove ethyl acetate to obtain a reaction substrate.

3. The method for green and efficient preparation of L-selenomethylselenocysteine as claimed in claim 2, wherein the molar ratio of the L-serine methyl ester hydrochloride, the acetylation reagent, the chlorination reagent and the methyl selenization reagent is 1.0 (1.0 ~ 1.2): (1.0 ~ 1.6): 1.0 ~ 1.5.5.

4. The method for green and efficient preparation of L-selenomethylselenocysteine according to claim 2, which is characterized by comprising the following steps: the reaction medium is one or a mixture of two of dichloromethane, trichloromethane, 1, 2-dichloroethane, tetrahydrofuran, diethyl ether, ethylene glycol dimethyl ether, methyl tert-butyl ether, dioxane, acetonitrile, dimethylformamide and dimethyl sulfoxide; the acetylation reagent is acetyl chloride or acetylOne of bromine, acetic anhydride, ethyl acetate and acetic acid; the chlorination reagent is one of thionyl chloride, carbon oxychloride, phosphorus trichloride, phosphorus pentachloride, a mixture of triphenylphosphine and N-chlorosuccinimide, a mixture of triphenylphosphine and carbon tetrachloride and trimethylchlorosilane; the chemical formula of the methyl selenide reagent is CH₃SeM, wherein M is selected from H, Na, K or Li.

5. The method for green and efficient preparation of L-selenomethylselenocysteine according to claim 4, which is characterized by comprising the following steps: the reaction medium is tetrahydrofuran, the acetylation reagent is acetyl chloride, the chlorination reagent is thionyl chloride, and the methyl selenylation reagent is sodium methyl selenol.

6. The method for green high-efficiency preparation of L-selenomethylselenocysteine according to any one of claims 2 to 5, characterized in that: adding an acid assisting agent before adding the acetylation reagent in the step (b) and before adding the methyl selenide reagent in the step (c).

7. The method for green and efficient preparation of L-selenomethylselenocysteine according to claim 6, which is characterized by comprising the following steps: the acid assisting agent is selected from one of triethylamine, pyridine, sodium methoxide, sodium ethoxide, potassium tert-butoxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide and potassium hydroxide.

8. The method for green high-efficiency preparation of L-selenomethylselenocysteine as claimed in claim 1, wherein the step (2) is specifically that the reaction substrate obtained in the step (1) is dissolved in cobalt chloride aqueous solution, the pH is adjusted to 7.0 ~ 8.0.0, amino acylase is added, after stirring reaction for 4 ~ 110 hours at 20 ~ 40 ℃, the pH is adjusted to 5.0 ~ 6.0.0, activated carbon is added, stirring is carried out for 0.3 ~ 1.5.5 hours at 80 ~ 85 ℃ and 85 ℃, solid-liquid separation is carried out, the liquid is obtained, and the L-selenomethylselenocysteine is obtained after decompression concentration, separation and purification by strong acid cation exchange resin and recrystallization by water-alcohol solution.

9. The method for efficiently preparing L-selenomethylselenocysteine in a green way according to claim 8 is characterized in that the concentration of the cobalt chloride solution is 0.3 ~ 1mmol/L, the adding amount of the reaction substrate in the cobalt chloride solution is 22.4 ~ 67.2.2 g/L, the adding amount of the aminoacylase is 1.9 percent of the mass of the reaction substrate, ~ 2.4.4 percent of the mass of the aminoacylase, and the adding amount of the activated carbon is 0.1 ~ 0.5.5 percent of the mass of the reaction substrate.

10. The method for efficiently preparing L-selenomethylselenocysteine in green color according to claim 8 or 9, wherein the strong acid cation exchange resin is sequentially eluted by water and 3 ~ 8% ammonia water, the ammonia water eluate is evaporated to dryness under reduced pressure, and then is recrystallized by water-ethanol.

Technical Field

The invention belongs to the technical field of food additives, and particularly relates to a method for preparing L-selenium methyl selenocysteine in an environment-friendly and efficient manner.

Background

Selenium is a trace element necessary for human bodies, is an important component of various enzymes of the human bodies, has multiple functions of resisting oxidation, preventing cancer, detoxifying, promoting growth, improving immunity and the like, and once an organism lacks selenium, various diseases such as cardiovascular and cerebrovascular diseases, hypertension, metabolic syndrome, gastrointestinal diseases, diabetes, asthma, Parkinson's disease, liver diseases, cancer and the like can be directly caused, so that the huge effect of the selenium on human health cannot be replaced by other substances.

Most areas in the world are selenium-deficient, and 72 percent of areas in China belong to selenium-deficient areas and need to be supplemented with selenium. However, people have limited selenium obtained in daily diet, and the requirement of the human body on selenium cannot be met, so that the selenium supplement is urgently needed. The common selenium nutrition enhancer is L-selenium methyl selenocysteine, which is a selenium methylated derivative of the 21 st essential amino acid of human body, namely L-selenium selenocysteine, and the selenium methylation enhancer is widely existed in plants such as astragalus, garlic, onion, broccoli and the like and selenium-enriched yeast, has the advantages of definite chemical structure, small toxicity, high bioavailability, good selenium supplementing effect and the like, has the prevention effect on various tumors (such as breast cancer, prostate cancer, liver cancer and the like), has the auxiliary effect on the treatment of the cancers, and has wide application prospect. In 2002, L-selenomethylselenocysteine is recognized by the FDA in the United states as a latest generation selenium source dietary supplement; in 2009, L-selenomethylselenocysteine was approved by the ministry of health in our country as a novel nutritional supplement (new food additive variety No. 11 in 2009).

At present, the synthesis method of L-selenium methyl selenocysteine mainly comprises the following steps:

(Mono) chloroacetaldehyde Process

The method is a simulated hydantoin method, which comprises the steps of reacting methyl selenol salt with chloroacetaldehyde to prepare methyl selenyl acetaldehyde, cyclizing with cyanide to generate methyl selenyl methyl hydantoin, and then carrying out alkaline hydrolysis ring opening and acidification to obtain DL-selenium-methyl selenocysteine. The method has long reaction steps, uses hypertoxic cyanide, has huge risks in the aspects of safety, environmental protection and occupational health, and simultaneously obtains the product which is raceme and can obtain the L-selenium methyl selenocysteine with single configuration only by splitting.

(Jiangxi Chuanqi pharmaceutical industry Co., Ltd. a method for synthesizing selenium methyl selenocysteine by methyl seleno acetaldehyde, Chinese patent: ZL 200610124942.6).

Process for the preparation of (di) alpha-aminoacrylic acid derivatives

Firstly, methyl selenol and salt solution thereof and alpha-amino acrylic acid derivative are subjected to addition reaction to generate beta-methyl seleno-alpha-amino propionic acid derivative, then sodium bicarbonate, sodium hydroxide or potassium hydroxide are used for hydrolyzing and saponifying ester group in the beta-methyl seleno-alpha-amino propionic acid derivative, and hydrochloric acid or sulfuric acid is used for acidification to obtain carboxylic acid compound; and then hydrochloric acid or sulfuric acid is used for heating and hydrolyzing to remove amino protecting group-acetyl in the beta-methylseleno-alpha-aminopropionic acid derivative to obtain beta-methylseleno-alpha-aminopropionic acid hydrochloride or sulfate, and finally ammonia gas or triethylamine is used for neutralizing to obtain DL-selenium methylselenocysteine. The raw material alpha-acetaminoacrylic acid and the derivative of the alpha-acetaminoacrylic acid are difficult to source, expensive and complex in preparation process, and no manufacturer exists in China; the methyl selenol has low boiling point, is easy to volatilize, has high toxicity, is difficult to prepare and is not commercialized, and the salt of the methyl selenol is unstable, is difficult to purify and is not commercialized; the obtained final product is racemate DL-selenium methyl selenocysteine, and the single-configuration L-selenium methyl selenocysteine needs to be obtained by splitting, so that the process is long, the yield is low, the production cost is high, and the industrial production is not facilitated.

(Jiangxi Chuanqi pharmaceutical industry Co., Ltd. a method for preparing selenium methyl selenocysteine by using alpha-amino acrylic acid derivatives, Chinese patent: ZL 200710051362.3).

(tri) chloropropionine derivative process

(1) Sodium diselenide method: the method comprises the steps of firstly reacting chloropropionic acid with sodium diselenide to generate selenocysteine, then reducing and cracking Se-Se bonds by using sodium metal/liquid ammonia (-70 ℃), and then methylating to obtain the selenomethylselenocysteine.

（Ioanna A, Wiro M P B, Menge. Synthesis of novel se-substituted selenocysteine derivatives as potential kidney selective prodrugs ofbiologically active selenol compounds：evaluation of kinetics of β-eliminationreactions in rat renal cytosol. J. Med. Chem, 1996, 39: 2040-2046）。

(2) Sodium methylselenolate method: the method is not beneficial to large-scale production because the source of the raw material of the chloropropionic acid is difficult, the production cost is high, the yield is low.

（Muhammed M, Kalyanam N. Manufacturing processes for se-methyl-L-selenocysteine, US06794537B1, 2002）。

(tetra) N-t-butoxyacyl-L-serine-beta-lactone method

Reacting N-tert-butoxy acyl-L-serine with azodicarbonic diester in the presence of trialkyl (aryl) phosphorus or phosphite to generate beta-lactone, reacting with methyl selenol or its salt to generate tert-butoxy acyl protected selenium methyl selenocysteine, and removing protecting group to obtain selenium methyl selenocysteine. The preparation of the substrate N-tert-butoxy acyl-L-serine-beta-lactone is difficult, and the related reaction raw materials and protective agents have high price, long reaction time and low total yield.

（Julian, E Spallholz. A method of using synthetic L-Se-methylselenocysteine as a nutriceutical and a method of its synthesis,EP1205471A1, 2001）。

(penta) 2, 3-dihalopropionitrile process

Firstly, the methyl selenol salt selectively reacts with 2, 3-dihalo propionitrile to generate nucleophilic substitution reaction to generate 2-halogen-3-methyl seleno propionitrile, then acidolysis is carried out to obtain 2-halogen-3-methyl seleno propionic acid, and finally ammoniation is carried out to obtain DL-selenium methyl selenocysteine. In the method, the substrate dibromopropionitrile is prepared by addition of acrylonitrile and liquid bromine, and a large amount of volatile liquid bromine with high toxicity is used, so that the method has the advantages of high operation difficulty, high equipment requirement, serious pollution and high environmental protection cost; the nucleophilic substitution reaction of the selenium alkoxide and the substrate has poor selectivity and low yield; the final product is raceme, and an optically active substance with a single configuration needs to be obtained by resolution, so the process is complicated, long and low in yield, is only suitable for laboratory preparation and is not suitable for industrial production.

(Wangling, a method for synthesizing, racemizing and splitting selenomethylselenocysteine, Chinese patent: CN 201010107900.8).

In summary, these synthetic methods have disadvantages, such as: the raw materials are difficult to source and high in price; the synthesis process is complex and the yield is low; the reaction conditions are harsh, and the equipment requirement is high; strong acid and virulent raw materials are involved, and the environmental pollution is serious; and the target molecule is raceme (DL-configuration), needs to be resolved, and the like, so that the target molecule and the DL-configuration are difficult to realize large-scale production.

In recent years, with the diversification of selenium supplement products, the demand of the market for L-selenomethylselenocysteine is continuously increased, and a green and efficient synthesis method is urgently needed by production enterprises.

Disclosure of Invention

Based on the defects of the prior art, the invention aims to provide a method for preparing L-selenium methyl selenocysteine in a green and high-efficiency manner, which takes L-serine methyl ester hydrochloride as a raw material, prepares L-2-acetamido-3-methyl seleno propionic acid by a one-pot method, and prepares the L-selenium methyl selenocysteine by enzymatic hydrolysis.

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

a method for preparing L-selenium methyl selenocysteine in a green and high-efficiency manner comprises the following steps:

(1) taking L-serine methyl ester hydrochloride as an initial raw material, and synthesizing a reaction substrate (L-2-acetamido-3-methylselenopropionic acid) through acetylation, chlorination and methylselenide in one pot;

(2) hydrolyzing the reaction substrate obtained in the step (1) under the enzyme catalysis to generate the L-selenium methyl selenocysteine.

Preferably, step (1) is specifically:

(a) adding L-serine methyl ester hydrochloride into the reaction medium under the condition of stirring, and cooling to 0 ~ 5 ℃;

(b) adding acetylation reagent, stirring for 2 ~ 5 hr, adding chlorination reagent, stirring for 0.5 ~ 2 hr, heating to 5 ~ 50 deg.C, stirring for 2 ~ 5 hr, and cooling to-5 ~ 0 deg.C;

(c) adding a selenizing reagent, stirring for 2 ~ 5 hours, heating to room temperature, and continuously stirring for 10 ~ 15 hours to obtain a reaction mixed solution;

(d) adjusting the pH of the reaction mixed liquor obtained in the step (c) to be less than 4.0, extracting with ethyl acetate for 2 ~ 5 times, combining the extract liquor, washing with water, drying, filtering, and evaporating under reduced pressure to remove ethyl acetate to obtain a reaction substrate.

Furthermore, the molar ratio of the L-serine methyl ester hydrochloride, the acetylation reagent, the chlorination reagent and the methyl selenization reagent is 1.0 (1.0 ~ 1.2): (1.0 ~ 1.6): 1.0 ~ 1.5).

Preferably, the reaction medium is dichloromethane (CH)₂Cl₂) Trichloromethane (CHCl)₃) 1, 2-dichloroethane (ClCH)₂CH₂Cl), Tetrahydrofuran (THF), diethyl ether (Et)₂O), ethylene glycol dimethyl ether (CH)₃OCH₂CH₂OCH₃) Methyl tert-butyl ether ((CH)₃)₃COCH₃) Dioxane (C)₄H₈O₂) Acetonitrile (CH)₃CN), Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), or a mixture of two of them; the acetylation reagent is acetyl Chloride (CH)₃COCl), acetyl bromide (CH)₃COBr), acetic anhydride ((CH)₃CO)₂O), ethyl acetate (CH)₃CO₂Et), acetic acid (CH)₃COOH); the chlorinating agent is thionyl chloride (SOCl)₂) Carbon oxychloride (COCl)₂) Phosphorus oxychloride (POCl)₃) Phosphorus trichloride (PCl)₃) Phosphorus pentachloride (PCl)₅) Mixtures of triphenylphosphine and N-chlorosuccinimide (Ph)₃P + NCS), a mixture of triphenylphosphine and carbon tetrachloride (Ph)₃P+CCl₄) Trimethylchlorosilane (Me)₃SiCl); the chemical formula of the methyl selenide reagent is CH₃SeM, wherein M is selected from H, Na, K or Li.

Further, the reaction medium is tetrahydrofuran, the acetylation reagent is acetyl chloride, the chlorination reagent is thionyl chloride, and the methyl selenylation reagent is sodium methyl selenol.

Preferably, an antacid is added before the addition of the acetylation reagent in step (b) and before the addition of the methyl selenization reagent in step (c).

Further, the acid-binding agent is selected from triethylamine (Et)₃N), pyridine (C)₅H₅N), sodium methoxide (CH)₃ONa), sodium ethoxide (EtONa), potassium tert-butoxide (t-BuOK), sodium bicarbonate (NaHCO)₃) Potassium bicarbonate (KHCO)₃) Sodium carbonate (Na)₂CO₃) Potassium carbonate (K)₂CO₃) Sodium hydroxide (NaOH), potassium hydroxide (KOH).

Preferably, the step (2) is specifically that the reaction substrate obtained in the step (1) is dissolved in cobalt chloride aqueous solution, the pH is adjusted to 7.0 ~ 8.0.0, amino acid acylase is added, the mixture is stirred and reacted for 4 ~ 110 hours at 20 ~ 40 ℃, the pH is adjusted to 5.0 ~ 6.0.0, activated carbon is added, the mixture is stirred for 0.3 ~ 1.5.5 hours at 80 ~ 85 ℃, solid-liquid separation is carried out, the liquid is taken and subjected to reduced pressure concentration, separation and purification by strong acid cation exchange resin, and recrystallization by water-alcohol solution, so as to obtain the L-selenomethylselenocysteine.

Preferably, the concentration of the cobalt chloride solution is 0.3 ~ 1mmol/L, the adding amount of the reaction substrate in the cobalt chloride solution is 22.4 ~ 67.2.2 g/L, the adding amount of the aminoacylase is 1.9% of ~ 2.4.4% of the mass of the reaction substrate, and the adding amount of the activated carbon is 0.1 ~ 0.5.5% of the mass of the reaction substrate.

Further, when the strong acid cation exchange resin is used for separation and purification, water and 3 ~ 8% ammonia water are sequentially used for elution, the ammonia water eluent is taken and decompressed and evaporated to dryness, and then water-ethanol is used for recrystallization.

The raw materials used in the above method are all common commercial products, wherein acylamino acid amidase I (EC 3.5.1.14) is used as acylamino acid hydrolase (Aminoacylase), and 732 type strongly acidic cation exchange resin is used as strongly acidic cation exchange resin.

The green and efficient method for preparing the L-selenium methyl selenocysteine has the following synthetic route:

the invention adopts a chemical enzyme method to prepare the L-selenium methyl selenocysteine, combines the advantages of the chemical method and the enzyme method, firstly uses cheap and easily obtained L-serine methyl ester hydrochloride as a raw material, adopts a one-pot method in chemical synthesis to prepare an enzyme action substrate L-2-acetamido-3-methyl seleno propionic acid, and then carries out enzymatic hydrolysis to directly obtain the optically pure L-selenium methyl selenocysteine.

The method has the advantages that the reaction substrate is prepared by a one-pot method, so that the instability of an intermediate is overcome, and the complicated steps of intermediate separation, purification and the like are reduced; and the defects of environmental pollution, equipment corrosion, selenium ether bond breakage, product racemization and the like caused by strong acid hydrolysis can be avoided by adopting enzyme catalytic hydrolysis. The process has the characteristics of easily obtained raw materials, low price, convenient operation, mild reaction conditions, environmental protection, single product, easy separation, high yield and suitability for industrial production.

Drawings

FIG. 1 is an elemental analysis chart of L-selenomethylselenocysteine obtained in example 1;

FIG. 2 is an XRD pattern of L-selenomethylselenocysteine obtained in example 1;

FIG. 3 is a mass spectrum of L-selenomethylselenocysteine obtained in example 1;

FIG. 4 is an infrared spectrum of L-selenomethylselenocysteine obtained in example 1;

FIG. 5 shows the preparation of L-selenomethylselenocysteine obtained in example 1¹H nuclear magnetic resonance image;

FIG. 6 shows the preparation of L-selenomethylselenocysteine obtained in example 1¹³C nuclear magnetic resonance image.

Detailed Description

In order to make the technical purpose, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention are further described with reference to specific examples, which are intended to explain the present invention and are not to be construed as limiting the present invention, and those who do not specify a specific technique or condition in the examples follow the techniques or conditions described in the literature in the art or follow the product specification.

The apparatus used in the following examples was a conventional commercially available product. Wherein the L-serine methyl ester hydrochloride is purchased from a phyllobacterium (S20131-100 g), CAS number: 5680-80-8; aminoacylase was purchased from a leaf organism of origin (acylase, S51676-100 g), CAS No.: 9012-37-7, the enzyme activity is more than or equal to 30000 u/g; the sodium methylselenolate is prepared by adopting temporary preparation (in an ice bath and nitrogen atmosphere, tetrahydrofuran is used as a solvent, methanol is used as an activating agent, and sodium borohydride is used as a reducing agent and reacts with dimethyl diselenide), and specifically comprises the following steps: adding 300 mL of tetrahydrofuran, 30.0 g of dimethyl diselenide and 12.5 g of sodium borohydride into a three-neck flask provided with a stirrer and a nitrogen gas guide tube, starting stirring under ice bath cooling and nitrogen protection, slowly dropwise adding 60 mL of methanol, completing dropwise adding for about 0.5 h, and continuing stirring and reacting for 0.5 h after completing dropwise adding to obtain the tetrahydrofuran solution of the methylselenol sodium.