阅读说明：本技术 一种d-泛解酸内酯的制备方法 (Preparation method of D-pantoic acid lactone ) 是由 林文龙 徐新 丁亚梅 陈昀 李立标 郑爱 王清清 于 2020-12-16 设计创作，主要内容包括：本发明提供了一种D-泛解酸内酯的制备方法,所述制备方法包括如下步骤：(1)DL-泛解酸内酯在D-泛解酸内酯水解酶的作用下进行开环反应,并与氨水反应成盐,然后进行后处理,得到D-泛解酸盐；(2)D-泛解酸盐在酸性条件下成环,得到含有D-泛解酸内酯的反应液。本发明提供的制备方法简单方便,溶剂可以回收利用,成本低廉,同时产物纯度较高,收率较高。(The invention provides a preparation method of D-pantoic acid lactone, which comprises the following steps: (1) carrying out a ring-opening reaction on DL-pantoic acid lactone under the action of D-pantoic acid lactone hydrolase, reacting with ammonia water to form salt, and carrying out post-treatment to obtain D-pantoic acid salt; (2) d-pantoate forms a ring under the acidic condition to obtain a reaction solution containing D-pantoic acid lactone. The preparation method provided by the invention is simple and convenient, the solvent can be recycled, the cost is low, and meanwhile, the product purity and the yield are high.)

1. A preparation method of D-pantoic acid lactone is characterized by comprising the following steps:

(1) carrying out a ring-opening reaction on DL-pantoic acid lactone under the action of D-pantoic acid lactone hydrolase, reacting with ammonia water to form salt, and carrying out post-treatment to obtain D-pantoic acid salt;

(2) d-pantoate is subjected to cyclization reaction under an acidic condition to obtain a reaction solution containing D-pantoic acid lactone.

2. The production method according to claim 1, wherein the ring-opening reaction uses water as a solvent;

preferably, the DL-pantoic acid lactone is added to the reaction system in the form of an aqueous solution, and more preferably, the concentration of the DL-pantoic acid lactone aqueous solution is 50 to 60%.

3. The production method according to claim 1 or 2, wherein the temperature of the ring-opening reaction is 20 to 30 ℃.

4. The production method according to any one of claims 1 to 3, wherein the ammonia water is added in an amount to control the pH of the ring-opening reaction to 6.5 to 7.5.

5. The method for preparing according to any one of claims 1 to 4, wherein the post-treatment comprises: the L-pantoic acid lactone is extracted, separated and removed by an organic solvent.

6. The production method according to any one of claims 1 to 5, wherein the reaction temperature for the ring formation is 60 to 90 ℃, preferably the reaction time is 2 to 3 hours;

and/or, the reaction pH value of the ring formation is 1.0-3.0;

and/or, the cyclization reaction takes water as a solvent.

7. The production method according to any one of claims 1 to 6, further comprising subjecting the reaction solution containing D-pantolactone to extraction in this order, collecting the organic layer, and crystallizing to obtain the D-pantolactone;

preferably, the extraction is performed by using a non-polar organic solvent, preferably ethyl acetate and/or dichloromethane; and/or the extraction times are 3-4.

8. The method of claim 7, wherein the crystallizing comprises: removing the solvent, dissolving, cooling and crystallizing;

preferably, the solvent used for the redissolution is methanol; and/or the temperature of the cooling crystallization is-15 to-5 ℃.

9. The preparation method according to claim 8, characterized in that the temperature reduction crystallization is followed by a secondary dissolution and then a temperature reduction recrystallization;

preferably, the temperature of the solvent used for the secondary dissolution is 50-60 ℃; and/or the solvent adopted by the secondary dissolution is ethyl acetate; and/or the temperature of the temperature reduction recrystallization is-15 to-5 ℃.

10. The method for preparing according to any one of claims 1 to 9, comprising the steps of:

(1) carrying out a ring-opening reaction on a DL-pantoic acid lactone aqueous solution with the concentration of 50-60% under the action of immobilized D-pantoic acid lactone hydrolase, reacting with ammonia water to form salt, and then extracting by using a non-polar organic solvent to remove L-pantoic acid lactone to obtain a D-pantoic acid salt aqueous solution;

(2) performing cyclization reaction on the D-pantoic acid salt aqueous solution for 2-3h under the conditions that the pH value is 1.0-3.0 and the temperature is 60-90 ℃ to obtain a reaction solution containing D-pantoic acid lactone;

(3) adding a nonpolar solvent into the reaction solution for extraction, concentrating to remove the solvent, adding methanol for redissolution, and cooling and crystallizing to obtain a D-pantolactone crude product;

(4) and dissolving the D-pantolactone crude product in ethyl acetate at 50-60 ℃, and cooling and crystallizing to obtain the D-pantolactone.

Technical Field

The invention belongs to the technical field of biochemical engineering, relates to a preparation method of D-pantolactone, and particularly relates to a method for preparing D-pantolactone by taking DL-pantolactone as a raw material.

Background

D-pantoate is the precursor of D-calcium pantothenate, which is a vitamin B drug and is a component of coenzyme A, participates in the metabolism of protein, fat and sugar in vivo, and can also be used for vitamin B deficiency, peripheral neuritis and intestinal colic after operation; can be used for treating disseminated lupus erythematosus with vitamin C; the method is also widely applied to the food industry and livestock breeding.

At present, the main technique for producing D-pantoic acid lactone consists in chiral resolution of the racemate. The traditional method is to use a chemical resolution method to resolve racemic pantoic acid lactone, and has the disadvantages of high resolution cost, complex process, high resolution difficulty and difficult separation. There is also a report on a method for removing L ester by hydrolyzing L ester with L-pantoic lactone hydrolase, but the hydrolysis is difficult to complete and the product purity is difficult to meet the requirement. Meanwhile, the D-pantoic acid lactone hydrolase is used for hydrolyzing the D ester, but the chiral separation, cyclization, crystallization and refining processes of a hydrolyzed product are not specifically described.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a preparation method of D-pantoic acid lactone, and the invention provides a method for preparing D-pantoic acid lactone from DL-pantoic acid lactone.

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

the invention provides a preparation method of D-pantoic acid lactone, which comprises the following steps:

(1) carrying out a ring-opening reaction on DL-pantoic acid lactone under the action of D-pantoic acid lactone hydrolase, reacting with ammonia water to form salt, and carrying out post-treatment to obtain D-pantoic acid salt;

(2) d-pantoate is subjected to cyclization reaction under an acidic condition to obtain a reaction solution containing D-pantoic acid lactone.

According to the invention, on the basis of carrying out ring-opening reaction on D-pantoic acid lactone by using D-pantoic acid lactone hydrolase, ammonia water is selected to react with D-pantoic acid, so that the solubility of D-pantoic acid in water is increased, and the D-pantoic acid is insoluble in an organic solvent, so that the L-pantoic acid lactone can be extracted, separated and recycled by using the organic solvent, and the organic solvent used for extraction can be recycled by using simple normal pressure distillation.

The invention adopts D-pantoic acid lactone hydrolase to ensure that all D-pantoic acid salt is dissolved in water, avoids the phenomenon that the L-pantoic acid lactone is not hydrolyzed completely by using the L-pantoic acid lactone hydrolase at present, and the residue can not be separated completely, and has good industrial prospect.

The ring-opening reaction of the present invention uses water as a solvent.

In a preferred embodiment of the present invention, the DL-pantolactone is added to the reaction system in the form of an aqueous solution, and the concentration of the aqueous solution of DL-pantolactone is more preferably 50 to 60%, for example, 52%, 55%, 58%.

The invention preferably adopts DL-pantoic acid lactone solution with higher concentration, can accelerate the reaction speed, simultaneously saves the step of reaction liquid concentration before extraction operation, reduces the using amount of organic solvent in the extraction process, shortens the post-treatment time and difficulty, and simultaneously avoids the increase of impurities in the process of long-time high-temperature concentration of the split reaction liquid. If the concentration of the DL-pantoic acid lactone is too low, the reaction solution needs to be concentrated or the amount of the organic reagent needs to be doubled after the reaction, so that the same post-treatment effect as the invention can be achieved, and if the concentration of the reaction solution is too high, the ring-opening reaction is incomplete and the yield is reduced.

The method selects the DL-pantoic acid lactone aqueous solution with the concentration of 50-60%, can obtain the D-pantoic acid salt only by simple extraction and separation, has simple operation and low cost, and can repeatedly recycle the solvent.

In order to improve the efficiency of the ring-opening reaction, the temperature of the ring-opening reaction is 20-30 ℃, such as 22 ℃, 25 ℃, 28 ℃ and the like.

In the present invention, ammonia water is used to form a salt with D-pantoic acid to increase water solubility, and ammonia water is used to adjust the pH of the reaction, and the amount of ammonia water is controlled to control the pH of the ring-opening reaction to 6.5 to 7.5, for example, 7. Preferably, the aqueous ammonia is added to the reaction system in the form of dropwise addition.

The post-treatment of the invention comprises: the L-pantoic acid lactone is extracted, separated and removed by an organic solvent.

The reaction temperature of the cyclization is 60-90 ℃, such as 65 ℃, 70 ℃, 75 ℃, 80 ℃, 85 ℃, and the preferred reaction time is 2-3h, such as 2.2h, 2.5h, 2.8h and the like.

The reaction pH for the cyclization according to the invention is from 1.0 to 3.0, for example 2.0.

The cyclization reaction of the invention takes water as a solvent.

As a preferable technical scheme, the preparation method further comprises the steps of sequentially extracting the reaction liquid containing the D-pantoic acid lactone, collecting the organic layer, and crystallizing to obtain the D-pantoic acid lactone.

As a preferred technical scheme of the invention, a non-polar organic solvent is selected for extraction, preferably ethyl acetate and/or dichloromethane; and/or the extraction times are 3-4.

The crystallization of the invention comprises: removing the solvent, dissolving, cooling and crystallizing.

The solvent adopted by the redissolution is methanol; and/or the temperature of the temperature-reducing crystallization is-15 to-5 ℃, such as-12 ℃, 10 ℃ and 8 ℃.

As a preferable technical scheme of the invention, the secondary dissolution is carried out after the temperature reduction crystallization, and then the temperature reduction recrystallization is carried out.

The temperature of the solvent adopted for the secondary dissolution is 50-60 ℃; and/or the solvent adopted by the secondary dissolution is ethyl acetate; and/or the temperature of the temperature-reducing recrystallization is-15 to-5 ℃, such as-12 ℃, 10 ℃ and 8 ℃.

As a specific embodiment of the present invention, the preparation method comprises the steps of:

(1) carrying out a ring-opening reaction on a DL-pantoic acid lactone aqueous solution with the concentration of 50-60% under the action of immobilized D-pantoic acid lactone hydrolase, reacting with ammonia water to form salt, and then extracting by using a non-polar organic solvent to remove L-pantoic acid lactone to obtain a D-pantoic acid salt aqueous solution;

(2) performing cyclization reaction on the D-pantoic acid salt aqueous solution for 2-3h under the conditions that the pH value is 1.0-3.0 and the temperature is 60-90 ℃ to obtain a reaction solution containing D-pantoic acid lactone;

(3) adding a nonpolar solvent into the reaction solution for extraction, concentrating to remove the solvent, adding methanol for redissolution, and cooling and crystallizing to obtain a D-pantolactone crude product;

(4) and dissolving the D-pantolactone crude product in ethyl acetate at 50-60 ℃, and cooling and crystallizing to obtain the D-pantolactone.

Compared with the prior art, the invention has the following beneficial effects:

(1) the preparation method provided by the invention selects D-pantoic acid lactone hydrolase, and is matched with ammonia water for use, and the ammonia water can increase the water solubility of D-pantoic acid on one hand and control the pH value of the solution on the other hand, so that the pH value of the solution is stabilized at the optimal activity condition of hydrolase;

(2) in the invention, the DL-pantoic acid lactone aqueous solution with the concentration of 50-60% is preferably selected, so that the reaction steps can be reduced, the solvent can be saved, and the increase of impurities can be avoided;

(3) the preparation method provided by the invention is simple to operate, low in cost, high in product purity, high in yield, green and environment-friendly.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the specific embodiments are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Example 1

This example provides a process for the preparation of D-pantolactone.

(1) Adding 150g of DL-pantoic acid lactone into a 500mL three-necked bottle, adding 150g of water for dissolving, adding 150mL of immobilized D-pantoic acid lactone hydrolase, controlling the temperature to be 25 ℃, dropwise adding ammonia water to control the pH to be within the range of 6.5-7.5 until the reaction is stopped, extracting and separating for 4 times by using 300g of ethyl acetate, collecting a water phase, sampling after each extraction, and detecting the peak area of the lactone in the water phase by using an HPLC method, wherein the peak area is shown in Table 1;

TABLE 1

Sample (I)	Relative residual amount of lactone/%)
		Before extraction	100
After one-time extraction	26.73
		After the second extraction	7.46
After three times of extraction	0.18
		After four times of extraction	Not detected out

(2) Adjusting the pH value of the obtained D-pantoic acid salt aqueous solution to 1.0 by using sulfuric acid, heating to 75 ℃, carrying out cyclization reaction for 2 hours, cooling, adjusting the pH value to 4.5-5.0, extracting for 3 times by using 250g of ethyl acetate, concentrating to remove a solvent to obtain D-pantoic acid lactone solid, adding a small amount of anhydrous methanol, stirring for dissolving, cooling and crystallizing to obtain 53.4g of D-pantoic acid lactone crude product with specific rotation of 45.82 degrees;

(3) the crude crystals were dissolved in 13g of ethyl acetate at 55 ℃ and then recrystallized by cooling to give 47.8g of D-pantolactone with a specific rotation of 49.83 °.

Example 2

This example provides a process for the preparation of D-pantolactone.

(1) Adding 200g of DL-pantolactone into a 500mL three-necked bottle, adding 190g of water for dissolving, adding 250mL of immobilized D-pantolactone hydrolase, controlling the temperature to be 20 ℃, dropwise adding ammonia water to control the pH value to be within the range of 6.5-7.5 until the reaction is stopped, then extracting and separating for 3 times by using 450g of ethyl acetate, and collecting a water phase;

(2) adjusting the pH value of the obtained D-pantoic acid salt aqueous solution to 3.0 by using sulfuric acid, heating to 60 ℃, carrying out cyclization reaction for 3 hours, cooling, adjusting the pH value to 4.5-5.0, extracting for 3 times by using 400g of ethyl acetate, concentrating to remove a solvent to obtain D-pantoic acid lactone solid, adding a small amount of anhydrous methanol, stirring for dissolving, cooling and crystallizing to obtain 69.5g of D-pantoic acid lactone crude product with the specific rotation degree of 45.30 degrees;

(3) the crude crystals were dissolved in 15g of ethyl acetate at 60 ℃ and then recrystallized by cooling to give 61.8g of D-pantolactone with a specific rotation of 49.24 °.

Example 3

This example provides a process for the preparation of D-pantolactone.

(1) Adding 100g of DL-pantolactone into a 500mL three-necked flask, adding 100g of water for dissolving, adding 150mL of immobilized D-pantolactone hydrolase, controlling the temperature to be 30 ℃, dropwise adding ammonia water to control the pH value to be within the range of 6.5-7.5 until the reaction is stopped, then extracting and separating by using 300g of ethyl acetate for 3 times, and collecting a water phase;

(2) adjusting the pH value of the obtained D-pantoic acid salt aqueous solution to 3.0 by using sulfuric acid, heating to 90 ℃, carrying out cyclization reaction for 2 hours, cooling, adjusting the pH value to 4.5-5.0, extracting for 3 times by using 200g of ethyl acetate, concentrating to remove a solvent to obtain D-pantoic acid lactone solid, adding a small amount of anhydrous methanol, stirring for dissolving, cooling and crystallizing to obtain 36.7g of D-pantoic acid lactone crude product with the specific rotation degree of 45.72 degrees;

(3) the crude crystals were dissolved in 9g of ethyl acetate at 60 ℃ and then recrystallized by cooling to give 31.2g of D-pantolactone with a specific rotation of 49.65 °.

Example 4

This example provides a process for the preparation of D-pantolactone.

(1) Adding 150g of DL-pantolactone into a 500mL three-necked bottle, adding 350g of water for dissolving, adding 150mL of immobilized D-pantolactone hydrolase, controlling the temperature to be 25 ℃, dropwise adding ammonia water to control the pH to be within the range of 6.5-7.5 until the reaction is stopped, extracting and separating for 3 times by using 300g of ethyl acetate, collecting a water phase, extracting and sampling after each extraction, and detecting the peak area of the lactone in the water phase by an HPLC method, wherein the peak area is shown in Table 2;

TABLE 2

Sample (I)	Relative residual amount of lactone/%)
		Before extraction	100
After the second extraction	48.97
		After three times of extraction	33.98

(2) Adjusting the pH value of the obtained D-pantoic acid salt aqueous solution to 3.0 by using sulfuric acid, heating to 75 ℃, carrying out cyclization reaction for 2 hours, cooling, adjusting the pH value to 4.5-5.0, extracting for 3 times by using 250g of ethyl acetate, concentrating to remove the solvent to obtain D-pantoic acid lactone solid, adding a small amount of anhydrous methanol, stirring for dissolving, cooling and crystallizing to obtain a D-pantoic acid lactone crude product with the specific rotation degree of-28.66 degrees.

Example 5

Compared with example 1, the difference is only that: the organic solvent for extraction in step (2) was replaced from ethyl acetate to dichloromethane.

46.9g of D-pantolactone is finally obtained, and the specific rotation is 49.35 degrees.

Example 6

Compared with example 1, the difference is only that: and (3) replacing the redissolving solvent in the step (2) with ethyl acetate from methanol.

63.2g of D-pantolactone is finally obtained, the specific rotation is 48.54 degrees.

Example 7

Compared with example 1, the difference is only that: and (4) replacing the solvent adopted in the secondary dissolution in the step (3) by methanol from ethyl acetate.

39.7g of D-pantolactone is finally obtained, the specific rotation is 49.78 degrees.

Comparative example 1

The comparative example provides a chemical resolution preparation method for chemically resolving D-pantolactone.

(1) Adding 50g of guanidine carbonate into a 1000mL three-necked bottle, adding 500g of purified water, stirring for dissolving, adding 45g of calcium hydroxide, reacting at 60 ℃ for 1h, filtering, transferring the filtrate into the 1000mL three-necked bottle, adding 65g of DL-pantoic acid lactone, continuing to react at 60 ℃ for 1h, concentrating under reduced pressure to be viscous, adding a proper amount of acetone, evaporating to dryness, adding acetone, stirring for dissolving, cooling to-20 ℃ for crystallization, filtering, drying to obtain 80.2g of DL-pantoic acid guanidine, and preparing D-pantoic acid guanidine and L-pantoic acid guanidine by the same method;

(2) adding 95g of absolute methanol and 48g of DL-guanidine pantoate into a 250mL single-mouth bottle, heating to 50 ℃, stirring and dissolving, cooling to 10 ℃, adding a small amount of D-guanidine pantoate seed crystal, stirring and crystallizing, filtering and drying to obtain 4.1g of D-guanidine pantoate, supplementing 4.1g of DL-ester, repeating the above operations, adding a small amount of L-guanidine pantoate to obtain 4.0g of L-guanidine pantoate, and repeating the above operations to obtain D-guanidine pantoate and L-guanidine pantoate;

(3) 30g of D-guanidine pantoate and a proper amount of water are added into a 250mL single-neck bottle and stirred to be dissolved, the pH value is adjusted to be below 1.5 by concentrated sulfuric acid, and the reaction is carried out for 2.5h at 90 ℃. Cooling, extracting with dichloromethane, concentrating under reduced pressure to remove dichloromethane and obtain crude product of D-pantolactone 26.2g, adding appropriate amount of ethyl acetate, and recrystallizing to obtain D-pantolactone 23.58g with specific rotation degree of-49.02.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.