阅读说明：本技术 一种l-硒-甲基硒代半胱氨酸的制备方法 (Preparation method of L-selenium-methyl selenocysteine ) 是由 冯书晓 杨春梅 于 2021-08-30 设计创作，主要内容包括：本发明提供了一种L-硒-甲基硒代半胱氨酸的制备方法,包括以下步骤：首先对L-丝氨酸的羧基和氨基分别进行甲酯化和N-Boc基团保护,然后对丝氨酸的羟基进行碘化、溴化和甲苯磺酸酯三种方式的活化,本发明提供以L-丝氨酸为起始原料制备L-硒-甲基硒代半胱氨酸的三种组合合成路线,三种活化中间体分别与原位制备的甲硒醇钠溶液发生甲硒化取代反应,最后在旋转蒸发仪的旋蒸瓶中“一锅”完成保护基脱除和溶剂回收,得到L-硒-甲基硒代半胱氨酸。本发明所述方法的原料经济易得,反应步骤简单,反应条件温和,产品不需要光学拆分,反应直接产率高,工艺耦合程度高,所提供的优选中间体方案的通用性和兼容性强,为工业化生产提供便利。(The invention provides a preparation method of L-selenium-methyl selenocysteine, which comprises the following steps: the method comprises the steps of performing methyl esterification and N-Boc group protection on carboxyl and amino of L-serine respectively, performing iodination, bromination and tosylate activation on hydroxyl of the serine, performing methyl selenocysteine synthesis reaction with L-serine as a starting raw material, performing methyl selenocysteine substitution reaction with three activated intermediates and sodium methyl selenol solution prepared in situ respectively, and performing protective group removal and solvent recovery in a rotary evaporation bottle of a rotary evaporator in one pot to obtain the L-selenium-methyl selenocysteine. The method has the advantages of economical and easily obtained raw materials, simple reaction steps, mild reaction conditions, no need of optical resolution of products, high direct reaction yield, high process coupling degree, strong universality and compatibility of the provided optimized intermediate scheme, and convenience for industrial production.)

1. A preparation method of L-selenium-methyl selenocysteine is characterized by comprising the following steps: the preparation method comprises the following preparation steps:

step one, protecting groups: reacting thionyl chloride with methanol to obtain an intermediate product, mixing the intermediate product with L-serine for reaction to obtain L-serine methyl ester hydrochloride, and mixing the L-serine methyl ester hydrochloride with Boc anhydride for reaction at room temperature to obtain N-Boc-L-serine methyl ester;

step two, activating hydroxyl groups: activating a functional group at a hydroxyl position on the N-Boc-L-serine methyl ester obtained in the step one to obtain a hydroxyl activated intermediate;

step three, methyl selenization substitution: carrying out nucleophilic substitution reaction on the hydroxyl activated intermediate prepared in the step two and sodium methylselenolate solution prepared in situ to prepare N-Boc-3-methylseleno-L-alanine methyl ester;

step four, deprotection: and step three, removing Boc protecting groups of the N-Boc-3-methylseleno-L-alanine methyl ester in a one-pot method under the acidic condition of HCl/ethyl acetate/dioxane/tetrahydrofuran/water, and simultaneously, breaking ester bonds to hydrolyze methyl ester to obtain the product L-selenium-methylselenocysteine.

2. The method of claim 1, wherein the L-selenium-methylselenocysteine is prepared by the following steps: in the first step, the thionyl chloride and the methanol are mixed for 25-35 min in an ice bath, and the temperature of the ice bath is 0-10 ℃; the temperature of the mixed reaction of the intermediate product and L-serine is 50-65 ℃, and the stirring reaction time is 1.5-2.5 h.

3. The method of claim 1, wherein the L-selenium-methylselenocysteine is prepared by the following steps: in the first step, L-serine methyl ester hydrochloride is mixed with Boc anhydride to react under the alkaline condition, and the reaction temperature is 20-30 ℃ at room temperature; the alkaline conditions were: uniformly mixing L-serine methyl ester hydrochloride with a dichloromethane solution containing triethylamine; wherein, L-serine: sulfoxide chloride: methanol: triethylamine: boc acid anhydride: the molar charge ratio of dichloromethane is 1: (1.1-1.2): (8-10): (2-3): (1.1-1.2): (8-10).

4. The method of claim 1, wherein the L-selenium-methylselenocysteine is prepared by the following steps: the hydroxyl activated intermediate in the second step is a bromo intermediate, and the preparation method comprises the following steps: uniformly mixing N-Boc-L-serine methyl ester, triphenylphosphine and tetrahydrofuran under an ice bath condition, continuously dropwise adding N-bromosuccinimide (NBS) for 0.6-1.2 h, reacting at room temperature for 10-14 h, filtering out insoluble substances, removing a solvent, adding ethyl acetate/petroleum ether (1/1) for pulping, filtering out impurities, and removing the solvent to obtain a light yellow solid N-Boc-3-bromo-L-alanine methyl ester intermediate; wherein, N-Boc-L-serine methyl ester: triphenylphosphine: tetrahydrofuran: the molar feed ratio of NBS (bromine) is 1: (1.1-1.2): (8-10): (1.1-1.2).

5. The method of claim 1, wherein the L-selenium-methylselenocysteine is prepared by the following steps: the hydroxyl activated intermediate in the second step is an iodo intermediate, and the preparation method comprises the following steps: uniformly mixing N-Boc-L-serine methyl ester, triphenylphosphine, a catalyst and dichloromethane in an ice bath, continuously adding an iodine simple substance for 0.6-1.2 h, continuously reacting at room temperature for 10-14 h, filtering out insoluble substances, removing the solvent, adding ethyl acetate/petroleum ether (1/1), pulping, filtering out impurities, and removing the solvent to obtain a colorless oily N-Boc-3-iodine-L-alanine methyl ester intermediate; wherein, N-Boc-L-serine methyl ester: triphenylphosphine: tetrahydrofuran: catalyst: the molar charge ratio of iodine is 1: (1.1-1.2): (0.2-0.3): (8-10): (1.1-1.2); the catalyst is DMAP (dimethylaminopyridine) or imidazole.

6. The method of claim 1, wherein the L-selenium-methylselenocysteine is prepared by the following steps: the hydroxyl activated intermediate in the third step is a tosylate intermediate, and the preparation method comprises the following steps: uniformly mixing N-Boc-L-serine methyl ester, pyridine, p-toluenesulfonyl chloride and dichloromethane at the temperature of-5 ℃, reacting at room temperature for 36-72 h, filtering out insoluble substances, removing a solvent, adding ethyl acetate/petroleum ether (1/1), pulping, filtering out impurities, and removing the solvent to obtain a white solid N-Boc-3-OTs-L-alanine methyl ester intermediate, wherein the N-Boc-L-serine methyl ester: pyridine: p-toluenesulfonyl chloride: the molar charge ratio of dichloromethane is 1: (4-5): (1.3-1.5): (10-20).

7. The method of claim 1, wherein the L-selenium-methylselenocysteine is prepared by the following steps: the nucleophilic substitution reaction in the third step comprises the following specific steps: under the conditions of inert gas protection and ice bath, uniformly mixing dimethyl diselenide and a dry solvent, adding sodium borohydride and methanol to react violently, preparing a sodium methylselenolate solution in situ, then adding the hydroxyl activated intermediate prepared in the step two, reacting at 40-65 ℃ for 12-18 h, adjusting the pH value to 1-4 with hydrochloric acid, removing the solvent, adding ethyl acetate for extraction, filtering impurities, and removing the solvent to obtain a white waxy solid N-Boc-3-methylseleno-L-alanine methyl ester; wherein, dimethyl diselenide: drying the solvent: sodium borohydride: methanol: the molar charge ratio of the hydroxyl activated intermediate is 1: (10-30): (1.5-2.5): (5.5-6.5): (1.2-1.8); the drying solvent is any one of tetrahydrofuran, DMF, toluene and dioxane.

8. The method of claim 1, wherein the L-selenium-methylselenocysteine is prepared by the following steps: the specific steps in the fourth step are as follows: mixing N-Boc-3-methylseleno-L-alanine methyl ester with 5.5-6.5M hydrochloric acid according to the mass-volume ratio of 1: 10-20 of the solvent, mixing in an evaporator, controlling the water bath temperature to be 85-95 ℃, evaporating for 2-3 h without vacuum, and evaporating the solvent by opening vacuum; secondly, adding an organic solvent containing 5.5-6.5M hydrochloric acid with the same volume for the second time, controlling the temperature of a water bath to be 55-65 ℃, turning the water bath in a vacuum-free manner for 1-2 hours, then turning on the vacuum and evaporating the solvent again to obtain a white solid, and carrying out recrystallization by adopting ethanol/water and decoloring by using activated carbon to obtain L-selenium-methyl selenocysteine; wherein the organic solvent is any one of toluene, dioxane, tetrahydrofuran, methanol and ethanol.

9. The method of claim 1, wherein the L-selenium-methylselenocysteine is prepared by the following steps: the method for protecting the group in the first step can also be as follows: directly mixing L-serine with Boc acid anhydride for reaction to obtain N-Boc-L-serine.

Technical Field

The invention belongs to the technical field of food nutrition enhancer processing, and particularly relates to a preparation method of L-selenium-methyl selenocysteine.

Background

L-selenium-methyl selenocysteine is a naturally-occurring small molecular selenocysteine, and is a methylated derivative of the 21 st amino acid L-selenocysteine of a human body.It is widely found in allium and brassica plants as well as in yeast. In vegetables such as broccoli, radish, brussels sprouts, cabbage, garlic, onion, leek, morchella and milk vetch (Astragalus, Leguminosae), selenium in an amount of 80% or more of the total selenium is present in the form of L-selenium-methylselenocysteine. L-selenium-methyl selenocysteine can be easily absorbed by gastrointestinal tract, has high bioavailability, and can be converted into methyl selenol (CH) by beta-lyase in tissue₃SeH) and then converted to hydrogen selenide (H)₂Se), hydrogen selenide is also a key metabolite of inorganic selenium (selenite or selenate). Hydrogen selenide is used in vivo to produce specific selenoproteins, such as glutathione peroxidase. Excessive hydrogen selenide is methylated into monomethyl, dimethyl and trimethyl metabolites in turn, and is finally excreted in human urine and/or respiratory system, or converted into mercury selenide for excretion, so that the L-selenium-methyl selenocysteine ingested by food has the characteristics of safety and low toxicity, and is approved as a novel food nutrition enhancer by No. 11 notice of the national Weijian Commission 2009, and listed in national standards GB14880-2012, GB2760-2014 and GB 1903.12-2015.

The synthesis of L-selenium-methyl selenocysteine includes an enzymatic resolution method and a direct synthesis method. In some of them, 2, 3-dihalopropionitrile reacts with methylselenolate to obtain 2-halo-3-methylselenopropionitrile, which is then hydrolyzed with acid to obtain 2-halo-3-methylselenopropionic acid, which then reacts with ammonia water to obtain DL-selenium-methylselenocysteine. In the other patent, the beta-methylseleno-alpha-aminopropionic acid derivative is generated by the addition reaction of methylseleno or a methylseleno salt water solution and the alpha-aminoacrylic acid derivative, then the esterified substance in the beta-methylseleno-alpha-aminopropionic acid derivative is hydrolyzed and acidified by alkali, and then heated and hydrolyzed by acid to remove N-acyl to obtain the beta-methylseleno-alpha-aminopropionic acid salt, and finally the DL-selenium-methylselenocysteine is obtained by alkali neutralization. In another patent, dimethyl diselenide and methyl selenol salt are prepared, then methyl selenol salt reacts with haloacetaldehyde to generate methyl seleno acetaldehyde, then methyl seleno formyl urea is prepared, and finally DL-selenium-methyl selenocysteine is prepared. The above methods all need to further obtain D and L-selenium-methyl selenocysteine through racemization and enzymatic resolution steps, and have the disadvantages of complicated steps and high production cost.

At present, the direct synthesis method for preparing the L-selenium-methyl selenocysteine has obvious technical advantages. However, the early direct chemical synthesis method has the problems of difficult acquisition of reagents, high price, harsh reaction conditions, long reaction time and the like, for example, the synthesis method of the chloropropionic acid sodium diselenide needs to be carried out at the temperature of-70 ℃, has strict requirements on equipment and is difficult to put into large-scale production; for example, the synthesis method of the tert-butoxyacyl protected serine has longer reaction time and higher prices of the related raw materials and the protective agent; for example, the synthesis method of substituting the sodium methylselenolate for the chloropropionic acid has high cost of the used chloropropionic acid. In recent years, a method for directly preparing optically pure L-selenium-methyl selenocysteine by taking N-acetyl-3-chlorine-L-serine methyl ester as a starting material is patented, wherein N-acetyl-3-chlorine-L-serine methyl ester and methyl selenol salt are mixed to obtain N-acetyl-3-methyl seleno-L-serine methyl ester, then the N-acetyl-3-methyl seleno-L-serine methyl ester is mixed with hydrochloric acid solution to carry out hydrolysis reaction to obtain selenium-methyl selenocysteine, and finally the optically pure L-selenium-methyl selenocysteine is obtained through crystallization and purification. The other patent discloses a preparation method of L-selenium-methyl selenocysteine by using N-acetyl-3-chlorine-L-serine methyl ester as a starting material, which comprises the steps of firstly carrying out nucleophilic substitution reaction on methyl selenol sodium and N-acetyl-3-chlorine-L-serine methyl ester to obtain N-acetyl-3-selenium methyl-L-selenocysteine methyl ester; then hydrolyzing in hydrochloric acid solution to obtain the L-selenium-methyl selenocysteine. Although the two methods preliminarily solve the problem of harsh reaction conditions faced by the early direct chemical synthesis method, the problems of expensive and difficult acquisition of N-acetyl-3-chloro-L-serine methyl ester raw materials still exist, and the problems of difficult deprotection of N-acetyl and easy racemization of deacetylation protecting groups still exist. In order to solve the racemization problem, a patent discloses a method for preparing L-selenium-methyl selenocysteine by taking L-serine methyl ester hydrochloride as an initial raw material. In addition, the patent also discloses a method for preparing (R) -selenomethylselenocysteine by taking L-serine as a starting material, which comprises the steps of preparing (3S, 6S) -3, 6-dichloromethyl-2, 5-diketopiperazine through esterification, cyclization and chlorination in sequence, preparing sodium methylselenolate, generating (3S, 6S) -3, 6-dimethylselenomethyl-2, 5-diketopiperazine through nucleophilic substitution reaction, and finally hydrolyzing to generate (R) -selenomethylselenocysteine. The process has the disadvantages of complicated steps, need of using dangerous chemical sodium metal, low yield (52.3%) in the cyclization step and high post-treatment cost. Therefore, the prior technical efforts still leave more technical problems, and the preparation technology for directly synthesizing the L-selenium-methylselenocysteine still needs to overcome the problems of easy availability of raw materials, high price, harsh reaction conditions and racemization by-products.

Disclosure of Invention

In order to solve the existing problems, the invention provides a preparation method of L-selenium-methyl selenocysteine, and the synthesis method has the characteristics of easily available and cheap raw materials, mild reaction conditions, simplicity, directness and practicability, avoidance of racemization byproducts, convenient post-treatment, no need of a splitting step and suitability for large-scale production.

The technical scheme adopted by the invention for solving the technical problems is as follows: a preparation method of L-selenium-methyl selenocysteine comprises the following preparation steps:

step one, protecting groups: reacting thionyl chloride with methanol to obtain an intermediate product, mixing the intermediate product with L-serine for reaction to obtain L-serine methyl ester hydrochloride, and mixing the L-serine methyl ester hydrochloride with Boc anhydride for reaction at room temperature to obtain N-Boc-L-serine methyl ester;

step two, activating hydroxyl groups: activating a functional group at a hydroxyl position on the N-Boc-L-serine methyl ester obtained in the step one to obtain a hydroxyl activated intermediate;

step three, methyl selenization substitution: carrying out nucleophilic substitution reaction on the hydroxyl activated intermediate prepared in the step two and sodium methylselenolate solution prepared in situ to prepare N-Boc-3-methylseleno-L-alanine methyl ester;

step four, deprotection: and step three, removing Boc protecting groups of the N-Boc-3-methylseleno-L-alanine methyl ester in a one-pot method under the acidic condition of HCl/ethyl acetate/dioxane/tetrahydrofuran/water, and simultaneously, breaking ester bonds to hydrolyze methyl ester to obtain the product L-selenium-methylselenocysteine.

Further, in the first step, the thionyl chloride and the methanol are mixed for 25-35 min in an ice bath, and the temperature of the ice bath is 0-10 ℃; the temperature of the mixed reaction of the intermediate product and L-serine is 50-65 ℃, and the stirring reaction time is 1.5-2.5 h.

Further, in the first step, L-serine methyl ester hydrochloride is mixed with Boc anhydride to react under the alkaline condition, and the reaction temperature is 20-30 ℃ at room temperature; the alkaline conditions were: uniformly mixing L-serine methyl ester hydrochloride with a dichloromethane solution containing triethylamine; wherein, L-serine: sulfoxide chloride: methanol: triethylamine: boc acid anhydride: the molar charge ratio of dichloromethane is 1: (1.1-1.2): (8-10): (2-3): (1.1-1.2): (8-10).

Further, the hydroxyl activated intermediate in the second step is a bromo intermediate, and the preparation method comprises the following steps: uniformly mixing N-Boc-L-serine methyl ester, triphenylphosphine and tetrahydrofuran under an ice bath condition, continuously dropwise adding N-bromosuccinimide (NBS) for 0.6-1.2 h, reacting at room temperature for 10-14 h, filtering out insoluble substances, removing a solvent, adding ethyl acetate/petroleum ether (1/1) for pulping, filtering out impurities, and removing the solvent to obtain a light yellow solid N-Boc-3-bromo-L-alanine methyl ester intermediate; wherein, N-Boc-L-serine methyl ester: triphenylphosphine: tetrahydrofuran: the molar feed ratio of NBS (bromine) is 1: (1.1-1.2): (8-10): (1.1-1.2).

Further, the hydroxyl activated intermediate in the second step is an iodo intermediate, and the preparation method comprises the following steps: uniformly mixing N-Boc-L-serine methyl ester, triphenylphosphine, a catalyst and dichloromethane in an ice bath, continuously adding an iodine simple substance for 0.6-1.2 h, continuously reacting at room temperature for 10-14 h, filtering out insoluble substances, removing the solvent, adding ethyl acetate/petroleum ether (1/1), pulping, filtering out impurities, and removing the solvent to obtain a colorless oily N-Boc-3-iodine-L-alanine methyl ester intermediate; wherein, N-Boc-L-serine methyl ester: triphenylphosphine: tetrahydrofuran: catalyst: the molar charge ratio of iodine is 1: (1.1-1.2): (0.2-0.3): (8-10): (1.1-1.2); the catalyst is DMAP (dimethylaminopyridine) or imidazole.

Further, the hydroxyl activated intermediate in the third step is a tosylate intermediate, and the preparation method comprises the following steps: uniformly mixing N-Boc-L-serine methyl ester, pyridine, p-toluenesulfonyl chloride and dichloromethane at the temperature of-5 ℃, reacting at room temperature for 36-72 h, filtering out insoluble substances, removing a solvent, adding ethyl acetate/petroleum ether (1/1), pulping, filtering out impurities, and removing the solvent to obtain a white solid N-Boc-3-OTs-L-alanine methyl ester intermediate, wherein the N-Boc-L-serine methyl ester: pyridine: p-toluenesulfonyl chloride: the molar charge ratio of dichloromethane is 1: (4-5): (1.3-1.5): (10-20).

Further, the nucleophilic substitution reaction in step three comprises the following specific steps: under the conditions of inert gas protection and ice bath, uniformly mixing dimethyl diselenide and a dry solvent, adding sodium borohydride and methanol to react violently, preparing a sodium methylselenolate solution in situ, then adding the hydroxyl activated intermediate prepared in the step two, reacting at 40-65 ℃ for 12-18 h, adjusting the pH value to 1-4 with hydrochloric acid, removing the solvent, adding ethyl acetate for extraction, filtering impurities, and removing the solvent to obtain a white waxy solid N-Boc-3-methylseleno-L-alanine methyl ester; wherein, dimethyl diselenide: drying the solvent: sodium borohydride: methanol: the molar charge ratio of the hydroxyl activated intermediate is 1: (10-30): (1.5-2.5): (5.5-6.5): (1.2-1.8); the drying solvent is any one of tetrahydrofuran, DMF, toluene and dioxane.

Further, the specific steps in the fourth step are as follows: mixing N-Boc-3-methylseleno-L-alanine methyl ester with 5.5-6.5M hydrochloric acid according to the mass-volume ratio of 1: 10-20 of the solvent, mixing in an evaporator, controlling the water bath temperature to be 85-95 ℃, evaporating for 2-3 h without vacuum, and evaporating the solvent by opening vacuum; secondly, adding an organic solvent containing 5.5-6.5M hydrochloric acid with the same volume for the second time, controlling the temperature of a water bath to be 55-65 ℃, turning the water bath in a vacuum-free manner for 1-2 hours, then turning on the vacuum and evaporating the solvent again to obtain a white solid, and carrying out recrystallization by adopting ethanol/water and decoloring by using activated carbon to obtain L-selenium-methyl selenocysteine; wherein the organic solvent is any one of toluene, dioxane, tetrahydrofuran, methanol and ethanol.

Further, the method for protecting the group in the step one can also be: directly mixing L-serine with Boc acid anhydride for reaction to obtain N-Boc-L-serine.

The invention has the beneficial effects that:

1. the invention relates to a novel method for preparing L-selenium-methyl selenocysteine by taking L-serine as an initial raw material, wherein the L-serine raw material adopted by a novel synthesis process route is economic and easy to obtain, and the preparation of the L-selenium-methyl selenocysteine can be completed only by four simple steps of group protection, hydroxyl activation, methyl selenylation reaction and de-protection group;

2. the group protection step adopts a one-pot method to finish the protection of amino and carboxyl of the L-serine, and the operation is simple and convenient;

3. three different intermediates (a bromo intermediate, an iodo intermediate and a tosylate intermediate) are respectively prepared in the hydroxyl activation step, so that the advantages of a process route, the universality and the compatibility of the method which takes the L-serine as a starting material are fully embodied;

4. the mesylation substitution step adopts an in-situ method to prepare a sodium methylselenolate solution, three different intermediates (a bromo intermediate, an iodo intermediate and a tosylate intermediate) can be efficiently compatible, and a homogeneous reaction system of the in-situ solution provides a suitable chemical environment for the smooth proceeding of nucleophilic substitution reaction (the reaction mechanisms of the three intermediates are the same);

5. the step of removing the protecting group does not need to be finished in a reaction kettle, the reaction process is coupled with the reduced pressure distillation process, the removal of the protecting group is finished by adopting a rotary evaporator in steps, the process is simple, the operation is easy, and the dual purposes of consumption reduction and efficiency improvement are achieved;

6. the method has the advantages of economic and easily obtained raw materials, easy preparation of the intermediate, simple and convenient operation, energy conservation and consumption reduction due to process coupling, and suitability for industrial production.

Detailed Description

The embodiments of the present invention are described in detail with reference to specific embodiments, and the embodiments and specific operations are provided in the present embodiment on the premise of the technical solution of the present invention, but the scope of the present invention is not limited to the following embodiments.

A preparation method of L-selenium-methyl selenocysteine comprises the following preparation steps:

step one, protecting groups: taking L-serine as a starting material, and sequentially carrying out thionyl chloride/methanol esterification reaction and N-tert-butyloxycarbonyl (N-Boc) protecting group reaction of introducing Boc anhydride into amino acid under alkaline condition to prepare N-Boc-L-serine methyl ester;

step two, activating hydroxyl groups: and (3) carrying out functional group activation of a hydroxyl position on the N-Boc-L-serine methyl ester obtained in the step one in a second mode. One is bromine (I) catalyzed by triphenylphosphine/imidazole/DMAP₂) Or halogenating reagents such as N-bromosuccinimide (NBS) and the like carry out bromination (iodo) replacement on hydroxyl to prepare an intermediate of N-Boc-3-bromo (iodo) -L-alanine methyl ester; secondly, in the presence of pyridine, p-toluenesulfonyl chloride (TsCl) is adopted to esterify hydroxyl to prepare an N-Boc-3-OTs-L-alanine methyl ester intermediate;

step three, methyl selenization substitution: respectively carrying out nucleophilic substitution reaction on the two hydroxyl activated intermediates prepared in the second step and a sodium methylselenolate solution prepared in situ, and respectively substituting bromine (iodine) or OTs groups with methylseleno groups to prepare N-Boc-3-methylseleno-L-alanine methyl ester;

step four, deprotection: and step three, removing Boc protecting groups of the N-Boc-3-methylseleno-L-alanine methyl ester in a one-pot method under the acidic condition of HCl/ethyl acetate/dioxane/tetrahydrofuran/water, and simultaneously, breaking ester bonds to hydrolyze methyl ester to obtain the product L-selenium-methylselenocysteine.

Further, in the first step, firstly, the thionyl chloride and dry methanol are mixed for half an hour in an ice bath, then the mixture is mixed with the L-serine and stirred for 2 hours, after the reaction is carried out for 2 hours at the temperature of not more than 65 ℃, the solution is removed by reduced pressure distillation, and white solid L-serine methyl ester hydrochloride is obtained; continuously and uniformly mixing the white solid with dichloromethane solution containing triethylamine without purification and pot rotation, then mixing with Boc anhydride, reacting overnight at room temperature, and removing the solvent by reduced pressure distillation to obtain white viscous solid N-Boc-L-serine methyl ester;

further, in step one, L-serine: sulfoxide chloride: methanol: triethylamine: boc acid anhydride: the molar charge ratio of dichloromethane is 1: (1.1-1.2): (8-10): (2-3): (1.1-1.2): (8-10); the ice bath condition is 0-10 ℃, the room temperature condition is 20-30 ℃, and the same is carried out below.

Further, the hydroxyl activated intermediate in the second step is a bromo intermediate, and the preparation method comprises the following steps: uniformly mixing N-Boc-L-serine methyl ester, triphenylphosphine and tetrahydrofuran under an ice bath condition, continuously dropwise adding NBS (bromine) within one hour, reacting at room temperature for 12 hours after dropwise adding, filtering out insoluble substances, removing the solvent under reduced pressure, adding ethyl acetate/petroleum ether (1/1) with volume being three times that of tetrahydrofuran, pulping, filtering out impurities, and removing the solvent through reduced pressure distillation to obtain a light yellow solid N-Boc-3-bromine-L-alanine methyl ester intermediate; wherein, N-Boc-L-serine methyl ester: triphenylphosphine: tetrahydrofuran: the molar feed ratio of NBS (bromine) is 1: (1.1-1.2): (8-10): (1.1-1.2).

Further, the hydroxyl activated intermediate in the second step is an iodo intermediate, and the preparation method comprises the following steps: uniformly mixing N-Boc-L-serine methyl ester, triphenylphosphine, a catalyst and dichloromethane under an ice bath condition, continuously adding an iodine simple substance within one hour, continuously reacting at room temperature for 12 hours, filtering out insoluble substances, removing the solvent under reduced pressure, adding ethyl acetate/petroleum ether (1/1) with the volume being three times that of the dichloromethane, pulping, filtering out impurities, and removing the solvent through reduced pressure distillation to obtain a colorless oily N-Boc-3-iodine-L-alanine methyl ester intermediate; wherein, N-Boc-L-serine methyl ester: triphenylphosphine: tetrahydrofuran: catalyst: the molar charge ratio of iodine is 1: (1.1-1.2): (0.2-0.3): (8-10): (1.1-1.2); the catalyst is DMAP (dimethylaminopyridine) or imidazole.

Further, the hydroxyl activated intermediate in the third step is a tosylate intermediate, and the preparation method comprises the following steps: uniformly mixing N-Boc-L-serine methyl ester with pyridine, p-toluenesulfonyl chloride and dichloromethane at 0 ℃, reacting at room temperature for 48 hours, filtering out insoluble substances, removing the solvent under reduced pressure, adding ethyl acetate/petroleum ether (1/1) with volume being three times of that of the dichloromethane, pulping, filtering out impurities, and removing the solvent through reduced pressure distillation to obtain a white solid N-Boc-3-OTs-L-alanine methyl ester intermediate; wherein, N-Boc-L-serine methyl ester: pyridine: p-toluenesulfonyl chloride: the molar charge ratio of dichloromethane is 1: (4-5): (1.3-1.5): (10-20).

The three intermediates of bromo, iodo and tosylate are respectively selected, three synthetic processes can be combined, and the preferred sequence is that the tosylate intermediate is superior to the bromo intermediate and the iodo intermediate. The tosylate intermediate is a preferred scheme of the invention, and the tosylate intermediate has the advantages of mild reaction conditions, cheap and easily-obtained reagents, simple purification treatment steps, and 100% conversion to iodo (bromo) intermediate under mild conditions (room temperature) especially when the tosylate intermediate is mixed with an acetone solution containing 3-5% of sodium iodide (sodium bromide).

Further, in the third step, the nucleophilic substitution reaction specifically comprises the steps of uniformly mixing dimethyl diselenide and a drying solvent under the protection of inert gas and under the ice bath condition, adding sodium borohydride and methanol for violent reaction, preparing a sodium methylselenolate solution in situ, then adding the bromo intermediate or iodo intermediate or tosylate intermediate prepared in the step (2), reacting for 12-18 hours at a temperature of no more than 65 ℃, adjusting the pH value to 1-4 with concentrated hydrochloric acid, evaporating the solvent under reduced pressure, adding ethyl acetate for extraction for 3-5 times, washing ethyl acetate extract liquid with saturated saline solution, drying with anhydrous sodium sulfate, and evaporating ethyl acetate under reduced pressure to obtain a white waxy solid N-Boc-3-methylseleno-L-alanine methyl ester; wherein, dimethyl diselenide: drying the solvent: sodium borohydride: methanol: the molar charge ratio of the hydroxyl activated intermediate is 1: (10-30): (1.5-2.5): (5.5-6.5): (1.2-1.8); the drying solvent is any one of tetrahydrofuran, DMF, toluene and dioxane.

Further, the specific steps in the fourth step are as follows: mixing N-Boc-3-methylseleno-L-alanine methyl ester with 6M hydrochloric acid according to the mass-volume ratio of 1: 10-20 of the weight percentage is placed in a rotary evaporator bottle to be mixed, the temperature of a water bath is set to be 85-95 ℃, after the vacuum rotation is not started for 2-3 hours, the vacuum is started to evaporate the solvent; secondly, adding an organic solvent containing 6M hydrochloric acid with the same volume for the second time, setting the water bath temperature to be 55-65 ℃, turning for 1-2 hours without vacuum, opening the vacuum, evaporating the solvent again, completely removing a protecting group to obtain a deprotected product which is a white solid, recrystallizing by using ethanol/water, and decolorizing by using activated carbon to obtain a pure L-selenium-methyl selenocysteine product; wherein the organic solvent is any one of toluene, dioxane, tetrahydrofuran, methanol and ethanol.

Further, the method for protecting the group in the step one can also be: directly mixing L-serine with Boc acid anhydride for reaction to obtain N-Boc-L-serine.

The first embodiment is as follows:

the new method for preparing the L-selenium-methyl selenocysteine by taking the L-serine as the starting raw material comprises the following steps:

(1) preparation of N-Boc-L-serine methyl ester

500mL of anhydrous methanol is poured into a three-neck flask, the three-neck flask is placed in an ice bath environment at 0-10 ℃, 60.25mL of thionyl chloride is slowly added into the three-neck flask by a constant-pressure dropping funnel, and the temperature of reactants in the three-neck flask is kept at 0-5 ℃ during dropping. After the thionyl chloride is added, the reaction is continued for one hour at the temperature of 0-5 ℃, then 50.0g L-serine hydrochloride (0.35 mol) is added, and the reaction temperature is raised to 65 ℃ for stirring reaction. After the reaction was carried out for 2 hours, the reaction solution was concentrated under reduced pressure to obtain a crude product, which was washed several times with anhydrous methanol in a small amount to remove impurities such as hydrogen chloride contained therein, and weighed to obtain 60.7g L-serine methyl ester hydrochloride in a yield of 91.36%. The three-necked flask is continuously placed in an ice bath environment at 0-10 ℃ without rotating a pot, 200mL of dichloromethane and 75mL of triethylamine are added, 75mL of Boc anhydride is dropwise added, the ice bath is stopped after the dropwise addition is finished, the temperature is slowly raised to 20 ℃ for continuous reaction for 12 hours, the solvent is removed by reduced pressure distillation, the residue is extracted for 3 times by 200mL of ethyl acetate, the ethyl acetate solution is collected and washed for three times by 100mL of saturated sodium bicarbonate solution and 100mL of saturated saline solution respectively, anhydrous sodium sulfate is dried, and 73.9g of colorless oily viscous N-Boc-L-serine methyl ester is obtained after the ethyl acetate is removed under reduced pressure, and the yield is 95.8%.

Analysis and detection results of serine methyl ester hydrochloride: a white solid. Melting point 163-. The structure was analyzed by hydrogen and carbon Nuclear Magnetic Resonance (NMR) and the results were as follows:¹HNMR(400 MHz，DMSO) δ8.60 (s，3H，-NH₂·HCl)，5.66(t，J=4.6 Hz，1H，-OH)，4.10(t，J=3.4Hz，1H,-CH)，3.83 (t，J=7.4Hz，2H,-CH₂)，3.74 (s，3H,-CH₃)。¹³CNMR(101MHz，DMSO) δ 168.95，59.89，54.80，53.21。

analysis and detection results of serine methyl ester: a colorless oil. R_f=0.3 (petroleum ether/ethyl acetate = 1/1). The structure was analyzed by hydrogen and carbon Nuclear Magnetic Resonance (NMR) and the results were as follows:¹HNMR(400 MHz，DMSO) δ 5.47 (br，1H)，4.39 (br，1H)，4.00~3.87 (m，2H)，3.79 (s，3H)，2.45 (br，1H)，1.45 (s，9H)。¹³CNMR(101MHz，DMSO) δ 171.42，155.75，80.23，63.21, 55.74, 52.55, 28.28。

(2) preparation of N-Boc-3-iodo-L-alanine methyl ester

Under the protection of inert gas, 108g of triphenylphosphine and 27g of imidazole are dissolved in 1200mL of dichloromethane at 0 ℃, 105g of iodine is added in three portions and then stirred for half an hour, N-Boc-L-serine methyl ester prepared in the step (1) is slowly added in portions, the stirring is continued for 1 hour at 0 ℃, the stirring is continued for 2 hours at room temperature, the solvent is removed by reduced pressure distillation, 300mL of petroleum ether/ethyl acetate (1/1) is added and pulped, the brown viscous liquid is filtered and then washed with 1500mL of petroleum ether/ethyl acetate (1/1), the filtrate is collected and concentrated in vacuum to obtain 93g of gray yellow oily liquid N-Boc-3-iodine-L-alanine methyl ester, and the yield is 84%.

Analytical detection results of iodo-L-alanine methyl ester: a colorless oil. The structure was analyzed by hydrogen and carbon Nuclear Magnetic Resonance (NMR) and the results were as follows:¹H NMR (400 MHz, CDCl₃) δ 5.37 (br s, 1H), 4.52 (br s, 1H), 3.80 (s, 3H), 3.61-3.52 (m, 2H), 1.46 (s, 9H)。¹³C NMR (100 MHz, CDCl₃) δ 170.01, 154.84, 80.45,53.62, 53.05, 28.27, 7.81。

(3) preparation of N-Boc-3-methylseleno-L-alanine methyl ester

Under the protection of inert gas and at the temperature of <10 ℃, 30mL of dimethyl diselenide and 600mL of dry tetrahydrofuran are mixed uniformly, 30g of sodium borohydride and 180mL of methanol are added for violent reaction, then a methyl selenol sodium solution is prepared in situ, N-Boc-3-iodine-L-alanine methyl ester prepared in the step (2) is added, after the reaction is carried out for 12-18 hours at the temperature of 25 ℃, concentrated hydrochloric acid is used for adjusting the pH value to be 1-4, the solvent is evaporated to dryness under reduced pressure, 900mL of ethyl acetate is added for extraction for 3 times, ethyl acetate extract liquid is washed with 450mL of saturated saline solution for 2 times respectively, anhydrous sodium sulfate is used for drying, ethyl acetate is evaporated to dryness under reduced pressure, 65g of light yellow solid N-Boc-3-methyl seleno-L-alanine methyl ester is obtained, and the yield is 98%.

The analysis and detection result of the methylseleno-L-alanine methyl ester is as follows: light yellow solid. The melting point is 92-93 ℃. The structure was analyzed by hydrogen and carbon Nuclear Magnetic Resonance (NMR) and the results were as follows:¹H NMR (400 MHz, CDCl₃) δ 5.38 (d, 1H), 3.69 (s, 3H), 3.32 (m, 1H), 3.07 (m, 2H), 2.06 (s, 3H), 1.38 (s, 9H)。¹³C NMR (100 MHz, CDCl₃) δ 171.11, 154.84, 80.05, 53.62, 52.45, 32.42, 25.67, 7.81, 6.03。

(4) preparation of L-selenium-methyl selenocysteine

Transferring the light yellow solid of the N-Boc-3-methylseleno-L-alanine methyl ester prepared in the step (4) into a 2L rotary evaporation bottle of a rotary evaporator, adding 1300mL of 6mol/L hydrochloric acid solution, setting the rotary evaporation temperature to be 95 ℃, setting the vacuum degree to be one atmospheric pressure (1 atm), starting to rotate for 2 hours, setting the vacuum degree to be 0.5 atmospheric pressure (0.5 atm), rotating for 1 hour, rotating for 2 hours under 0.02 atmospheric pressure (0.02 atm) until the mixture is dried, and removing the hydrochloric acid solution under reduced pressure; adding 650mL of ethyl acetate and 100mL of concentrated hydrochloric acid again, setting the rotary evaporation temperature to be 55 ℃, setting the vacuum degree to be one atmosphere (1 atm), starting to rotate for 2 hours, setting the vacuum degree to be 0.5 atmosphere (0.5 atm), rotating for 1 hour, rotating for 2 hours until the mixture is dried, and collecting a solid product; washing the solid product with water at 100 ℃, collecting the water solution, adding active carbon, filtering the active carbon after decoloration, adding pyridine to adjust the pH value to 6-7, adding ethanol, standing below 10 ℃ for crystallization for 12h, filtering and drying to obtain 30g of L-selenium-methyl selenocysteine white solid (yield is 93%).

The analysis and detection result of the selenium-methyl selenocysteine is as follows: a white-like solid. Melting point 165-; optical rotation [ alpha ]]_D ²⁰ =-13.6 (c=10mg/mL, H₂O). The purity of the L-configuration (HPLC chiral chromatography) was 98.2%. The structure was analyzed by hydrogen and carbon Nuclear Magnetic Resonance (NMR) and the results were as follows:¹H-NMR (D₂O) δ (ppm) 4.13 (t, 1H) , 3.07 (dd, 2H), 2.05 (s, 3H); ¹³C-NMR (D₂O) δ (ppm) 173.45, 54.56, 25.65, 6.03。

example two:

the new method for preparing the L-selenium-methyl selenocysteine by taking the L-serine as the starting raw material comprises the following steps:

(1) preparation of N-Boc-L-serine methyl ester As in step (1) of example or using commercially available starting material

(2) Preparation of N-Boc-3-bromo-L-alanine methyl ester

Under the condition of ice bath at 0-5 ℃, 73g of N-Boc-L-serine methyl ester prepared in the step (1) or sold on the market and 110g of triphenylphosphine are dissolved in 900mL of dry tetrahydrofuran, 75g N-bromosuccinimide (NBS reagent) is added in batches within one hour, the solution is dark brown, the temperature is slowly raised to 20-30 ℃, the stirring is continued for 12 hours, insoluble substances are removed by filtration, the solvent is removed by distillation under reduced pressure, 500mL of petroleum ether/ethyl acetate (1/1) is added for pulping, the brown suspension liquid is filtered, then 500mL of petroleum ether/ethyl acetate (1/1) is used for further washing, the filtrate is collected and concentrated in vacuum, 84g of gray yellow oily liquid N-Boc-3-bromo-L-alanine methyl ester is obtained, and the yield is 89%.

Analysis and detection results of bromo-L-alanine methyl ester: a pale yellow solid. Melting point 55-56 ℃. The structure was analyzed by hydrogen and carbon Nuclear Magnetic Resonance (NMR) and the results were as follows:¹HNMR(400 MHz，DMSO) δ 5.31 (br, 1H), 4.57 (m, 1H), 3.65 (dd, 1H), 3.63 (s, 3H), 3.54 (dd, 1H), 1.29 (9H, s); ¹³CNMR(101MHz，DMSO) δ 169.63, 154.90, 80.52, 53.91, 52.94, 34.02, 28.31.

(3) preparation of N-Boc-3-methylseleno-L-alanine methyl ester As in example step (3)

In a specific embodiment, the N-Boc-3-iodo-L-alanine methyl ester intermediate is replaced with an N-Boc-3-bromo-L-alanine methyl ester intermediate.

(4) Preparation of L-Se-methylselenocysteine the same procedure as in example (4)

Example three:

the new method for preparing the L-selenium-methyl selenocysteine by taking the L-serine as the starting raw material comprises the following steps:

(1) preparation of N-Boc-L-serine methyl ester As in step (1) of example or using commercially available starting material

(2) Preparation of N-Boc-3-OTs-L-alanine methyl ester

Under the protection of inert gas and under the condition of ice bath at 0 ℃, 73g of N-Boc-L-serine methyl ester prepared in the step (1) or sold on the market is dissolved in 1500mL of dichloromethane, 105mL of pyridine is added into the solution continuously, stirring is carried out for half an hour, 91g of p-toluenesulfonyl chloride (TsCl reagent) is added in batches within one hour, the temperature is maintained between 0 ℃ and 5 ℃, stirring is carried out continuously for 48 hours, insoluble substances are removed by filtration, the solvent is removed by distillation under reduced pressure, 500mL of petroleum ether/ethyl acetate (1/1) are added for pulping, a brown suspension liquid is filtered, then 500mL of petroleum ether/ethyl acetate (1/1) is used for washing continuously, and the filtrate is collected and concentrated in vacuum to obtain 84g of yellow solid N-Boc-3-OTs-L-alanine methyl ester with the yield of 75%.

Analysis and detection results of alanine methyl ester: white solid, melting point 74-75 ℃. The structure was analyzed by hydrogen and carbon Nuclear Magnetic Resonance (NMR) and the results were as follows:¹HNMR(400 MHz，DMSO) δ 7.75 (d,2H) , 7.34 (d,2H) , 5.34 (d,1H),4.5 (m,1H) , 4.37 (dd, 1H) , 4.26 (dd, 1H) , 3.68 (s,3H) , 2.43 (s,3H), 1.44 (s, 9H)。¹³CNMR(101MHz，DMSO) δ 182.83, 168.82, 145.01, 132.21, 129.74, 127.72, 80.12, 69.32, 52.84, 52.61, 28.01, 21.43。

(3) preparation of N-Boc-3-methylseleno-L-alanine methyl ester As in example step (3)

In a specific embodiment, the N-Boc-3-iodo-L-alanine methyl ester intermediate is replaced with N-Boc-3-OTs-L-alanine methyl ester.

(4) Preparation of L-Se-methylselenocysteine the same procedure as in example (4)

Example four:

the novel method for preparing the L-selenium-methyl selenocysteine by taking the L-serine as the starting raw material comprises the following simplified steps:

(1) preparation of N-Boc-L-serine

Adding 50g L-serine into a three-neck flask, placing the three-neck flask in an ice bath environment at 0-10 ℃, adding 360mL of dioxane and 40mL of water, dropwise adding 75mL of Boc anhydride, stopping the ice bath after the dropwise adding is finished, slowly heating to 20 ℃, continuing to react for 12 hours, carrying out reduced pressure distillation to remove the solvent, extracting the residue with 200mL of ethyl acetate for 3 times, collecting an ethyl acetate solution, washing with 100mL of saturated sodium bicarbonate solution and 100mL of saturated saline water for three times respectively, drying with anhydrous sodium sulfate, and removing the ethyl acetate under reduced pressure to obtain the N-Boc-L-serine with the yield of 90%.

(2) Preparation of N-Boc-3-OTs-L-alanine

Dissolving the N-Boc-L-serine prepared in the step (1) into 200mL of dry pyridine under an ice bath condition at-10 ℃, stirring for half an hour, adding 91g of p-toluenesulfonyl chloride (TsCl reagent) in batches within one hour, keeping the temperature between 0 and 5 ℃, continuing stirring for 48 hours, pouring the obtained product into 1000mL of ice water after the reaction is finished, filtering to obtain white solid N-Boc-3-OTs-L-alanine, and recrystallizing with ethanol to obtain colorless needle crystals with the yield of 68%.

(3) Preparation of N-Boc-3-methylseleno-L-alanine As in example step (3)

In a specific embodiment, the N-Boc-3-iodo-L-alanine methyl ester intermediate is replaced with N-Boc-3-OTs-L-alanine.

(4) Preparation of L-Se-methylselenocysteine the same procedure as in example (4)

The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The applicant selects L-serine with high optical purity and more economical and easily available as a starting material, and has greatly reduced synthesis cost and market price of N-Boc-L-serine methyl ester which is a downstream raw material of L-serine due to the mature progress and scale-up of amino acid synthesis technology, protein peptide synthesis technology in recent years, and has higher competitiveness from the economic cost perspective. On the other hand, from the technical process, compared with the N-acetyl protecting group, the N-Boc protecting group is easier to deprotect, has low requirements on the acidity of an acidolysis solution and the reaction temperature and the reaction time, and avoids the problem of racemization by-products caused by strong acid, high temperature and long-time reaction.

The invention aims to provide a synthetic method for preparing L-selenium-methyl selenocysteine by using L-serine or N-Boc-L-serine methyl ester as a starting material or by using the L-serine raw material through N-Boc-L-serine methyl ester. The synthesis method has the characteristics of easily available and cheap raw materials, mild reaction conditions, simplicity, directness and practicality, avoids the generation of racemization byproducts, is convenient for post-treatment, does not need a resolution step, and is suitable for large-scale production.

The invention relates to a novel method for preparing L-selenium-methyl selenocysteine by taking L-serine as a starting raw material, wherein the L-serine raw material adopted by a novel synthesis process route is economical and easy to obtain, and the preparation of the L-selenium-methyl selenocysteine can be completed only by four simple steps of group protection, hydroxyl activation, methyl selenylation reaction and de-protection group. The group protection step adopts a one-pot method to finish the protection of amino and carboxyl of the L-serine, and the operation is simple and convenient. Three different intermediates (a bromo intermediate, an iodo intermediate and a tosylate intermediate) are respectively prepared in the hydroxyl activation step, and the advantages of a process route using L-serine as a starting material, the universality and the compatibility of the method are fully embodied. The mesylation substitution step adopts an in-situ method to prepare a sodium methylselenolate solution, can be efficiently compatible with three different intermediates (a bromo intermediate, an iodo intermediate and a tosylate intermediate), and a homogeneous reaction system of the in-situ solution provides a suitable chemical environment for the smooth implementation of nucleophilic substitution reaction (the reaction mechanisms of the three intermediates are the same). The step of removing the protecting group is not required to be completed in a reaction kettle, the reaction process is coupled with the reduced pressure distillation process, the removal of the protecting group is completed by adopting a rotary evaporator in steps, the process is simple, the operation is easy, and the dual purposes of consumption reduction and efficiency improvement are achieved. In a word, the method has the advantages of economical and easily obtained raw materials, easy preparation of the intermediate, simple and convenient operation, energy conservation and consumption reduction due to process coupling, and suitability for industrial production.

It should be noted that while the invention has been described in terms of the above-mentioned embodiments, other embodiments are also possible. It will be apparent to those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention, and it is intended that all such changes and modifications be covered by the appended claims and their equivalents.