阅读说明：本技术 一种利用重组酰亚胺酶合成(r)-异丁基戊二酸单酰胺的方法 (Method for synthesizing (R) -isobutyl glutaric acid monoamide by using recombinant imide enzyme ) 是由 杨仲毅 姜礼进 蔡青峰 汪怡璐 方嘉怡 覃文平 于 2021-07-16 设计创作，主要内容包括：本发明涉及一种利用重组酰亚胺酶合成(R)-异丁基戊二酸单酰胺的方法,属于药物中间体合成技术领域。为了解决现在的反应不佳的问题,提供一种利用重组酰亚胺酶合成(R)-异丁基戊二酸单酰胺的方法,该方法包括使底物异丁基戊二酰亚胺在重组酰亚胺酶的催化作用下,转化反应生成产物(R)-异丁基戊二酸单酰胺；重组酰亚胺酶的基因序列如SEQ ID NO.1所示,重组酰亚胺酶的序列如SEQ ID NO.2所示。本发明通过采用酶法催化,实现具有高活性和反应位点的高选择性,能够优先催化环状亚胺结构的基团水解形成二羧基单酰胺的结构,且形成的产物具有高手性R型选择性和去对称化反应,从而也能够达到高手性产物收率的效果。(The invention relates to a method for synthesizing (R) -isobutyl glutaric acid monoamide by using recombinant imide enzyme, belonging to the technical field of synthesis of pharmaceutical intermediates. In order to solve the problem of poor reaction at present, the method for synthesizing (R) -isobutyl glutarate monoamide by using recombinant imidiase is provided, and the method comprises the steps of converting a substrate isobutyl glutarimide into a product (R) -isobutyl glutarate monoamide under the catalytic action of the recombinant imidiase; the gene sequence of the recombinant imide enzyme is shown as SEQ ID NO.1, and the sequence of the recombinant imide enzyme is shown as SEQ ID NO. 2. The method realizes high activity and high selectivity of reaction sites by adopting enzyme catalysis, can preferentially catalyze the hydrolysis of a group of a cyclic imine structure to form a dicarboxyl monoamide structure, and can form a product with high chiral R-type selectivity and a de-symmetry reaction, thereby achieving the effect of high chiral product yield.)

1. A method for synthesizing (R) -isobutylglutaric acid monoamide using a recombinant imidiase, comprising the steps of:

the substrate compound isobutyl glutarimide of formula I is converted to generate a product compound (R) -isobutyl glutaric acid monoamide of formula II under the catalysis of recombinant imidic enzyme;

the gene sequence of the recombinant imide enzyme is shown as SEQ ID NO. 1; the sequence of the recombinant imide enzyme is shown as SEQ ID NO. 2.

2. The method for synthesizing (R) -isobutylglutaric acid monoamide using recombinant imidiase as claimed in claim 1, wherein the conversion reaction is carried out in water.

3. The method for synthesizing (R) -isobutylglutaric acid monoamide using recombinant imidiase of claim 1 or 2, wherein the pH of the system for the conversion reaction is controlled to 6.0-9.0.

4. The method for synthesizing (R) -isobutylglutaramide according to claim 3, wherein the temperature of the conversion reaction is 20℃ -50℃.

5. The method for synthesizing (R) -isobutylglutaramide according to claim 1 or 2, wherein the host bacterium for the recombinant imidinase is BL21(DE 3).

6. The method for synthesizing (R) -isobutylglutaramide according to claim 1 or 2, wherein the recombinant imidinase is selected from the group consisting of recombinant imidinase-containing cells and enzyme solutions.

Technical Field

The invention relates to a method for synthesizing (R) -isobutyl glutaric acid monoamide by using recombinant imide enzyme, belonging to the technical field of synthesis of pharmaceutical intermediates.

Background

(S) -pregabalin ((S) -3-aminomethyl-5-methylhexanoic acid) is a lipophilic aminobutyric acid analog developed from pyroxene and has excellent anxiolytic and neuropathic pain-treating effects. The early synthesis method of pregabalin mainly adopts chemical resolution, and the production cost is relatively high. In the last decade, enzymatic methods have been used instead of chemical methods for resolution. Compared with the chemical resolution, the enzymatic resolution has the advantages of short synthetic route, low production cost, less three wastes and the like. The enzymes for resolution are mainly lipase, nitrilase and the like. The one-time theoretical yield of the resolution method is only 50%, and the recent enzyme catalysis-based desymmetry method has the one-time yield of 100% theoretically and is closely concerned by production enterprises.

The ee value of the R-isobutylglutaric acid monoamide obtained by asymmetrically hydrolyzing isobutylglutaric acid diamide in the prior art is also higher. Compared with isobutylglutarimide, isobutylglutarimide is easier to synthesize and is more suitable as a starting compound for enzymatic processes.

In patent document CN 112368262 a, (R) -isobutylglutarimide is obtained by starting with isobutylglutarimide and performing enzymatic asymmetric hydrolysis, and the biological enzyme mainly used in the method is lipase or esterase. However, according to the general rule, lipases and esterases are difficult to catalyze the ring-opening reaction of imide compounds, and related technical applications are not reported or repeated so far. The solubility of the raw material isobutyl glutarimide in the aqueous solution is low, and the patent adopts water with the ratio of 1:1-1: 2: the ratio of organic solvent is used to increase the solubility of the raw material, and the enzyme activity is difficult to preserve in such high concentration of organic solvent such as ethanol and acetone.

In 2015, scholars reported that (R) -isobutylglutarimide was obtained by hydrolyzing isobutylglutarimide under the catalysis of imide enzyme (Applied Microbiology and Biotechnology 2015,99(23):9961-9969), but the reaction efficiency was very low, the product concentration was only about 3.5g/L, the reaction conversion was only 64.6%, and the reaction solution was not extracted to obtain the target product. The reaction concentration and the conversion rate are low, and the product completely loses the advantages of a route for preparing (R) -isobutyl glutaric acid monoamide by using an enzymatic method to asymmetrically catalyze isobutyl glutarimide.

Disclosure of Invention

The invention provides a method for synthesizing (R) -isobutyl glutaric acid monoamide by using recombinant imide enzyme, aiming at solving the problems in the prior art, namely how to improve the activity of the imide enzyme, improve the conversion rate and the concentration of the product after reaction and obtain the product with high chiral purity.

The invention aims to realize the technical scheme that the method for synthesizing (R) -isobutyl glutaric acid monoamide by using recombinant imide enzyme is characterized by comprising the following steps:

the substrate compound isobutyl glutarimide of formula I is converted to generate a product compound (R) -isobutyl glutaric acid monoamide of formula II under the catalysis of recombinant imidic enzyme;

the gene sequence of the recombinant imide enzyme is shown as SEQ ID NO.1, and the sequence of the recombinant imide enzyme is shown as SEQ ID NO. 2.

The enzyme method catalysis is carried out on the substrate by adopting the recombinant imide enzyme, the enzyme which has the gene sequence shown as SEQ ID NO.1 and is shown as SEQ ID NO.2 has high activity and high selectivity of reaction sites on the substrate, the group of the cyclic imine structure can be preferentially catalyzed to be hydrolyzed to form a dicarboxyl monoamide structure, and the formed product has high chiral R-type selectivity and has a de-symmetry reaction, so that the effect of high chiral product yield can be achieved; meanwhile, the chiral R-type selective compound has the advantage of high chiral R-type selectivity, so that the obtained product has a high ee value, does not need to be split, is more suitable for being used as an intermediate for synthesizing the pregabalin, and is also beneficial to the improvement of the yield and the purity of the pregabalin synthesis. By adopting the imide enzyme with the gene sequence shown in SEQ ID NO.1, the recombinant imide enzyme expressed by culture can have higher proportion of soluble protein, thus being more beneficial to the expression of the recombinant imide enzyme and realizing the effect of high reactivity. Compared with the literature report, the concentration of the reaction product can be improved by more than 10 times, thereby having industrial application value.

In the above-mentioned method for synthesizing (R) -isobutylglutaric acid monoamide using recombinant imidinase, the conversion reaction is carried out in water. Organic solvents are not needed, so that the environment is protected; meanwhile, water is used as a solvent, so that the compound of the formula I, namely isobutyl glutarimide (IBI), is in a heterogeneous state in a reaction system, and the method is more favorable for improving the substrate conversion rate of the reaction and improving the yield, purity and quality effects of the product. When water is used as the solvent, the concentration of the compound isobutyl glutarimide of the formula I is preferably 30-40 g/L. Meanwhile, the water used as the solvent also has the advantage of environmental friendliness, and is more beneficial to reducing the influence on environmental pollution.

In the above method for synthesizing (R) -isobutylglutaric acid monoamide using a recombinant imidiase, preferably, the pH of the system for the conversion reaction is controlled to 6.0 to 9.0. The control of the pH value of the reaction system is more favorable for maintaining the activity of the recombinant imide enzyme and improving the reaction efficiency and the conversion rate. If the pH is too high, the substrate is decomposed and unnecessary waste is caused, and therefore, it is preferable to carry out the reaction in the above-mentioned range, whereby the conversion of the substrate can be facilitated and the influence of unnecessary impurities due to the decomposition of the substrate and the like can be reduced.

In the above method for synthesizing (R) -isobutylglutaric acid monoamide using recombinant imidiase, preferably, the temperature of the conversion reaction is 20 ℃ to 50 ℃. By adopting the recombinant imide enzyme to carry out catalytic reaction, the method has the advantage of mild reaction temperature, is more favorable for ensuring the activity of the imide enzyme in the temperature range, and ensures the effects of high selectivity and high conversion rate of the reaction on chirality. As a further preference, the temperature of the conversion reaction is between 35 ℃ and 45 ℃.

In the above-mentioned method for synthesizing (R) -isobutylglutaramide using recombinant imidinase, the recombinant imidinase gene can be synthesized by techniques well known in the art, such as PCR amplification, gene synthesis, etc. For example, the gene engineering bacteria can be obtained by cloning the gene containing the recombinant imide enzyme into an expression vector and then transforming host cells. For optimizing a prokaryotic expression system and a yeast expression system of the expression system, PET is preferably adopted for expression, and the host cell adopts Escherichia coli. Preferably, the host strain of the recombinant imide enzyme is BL21(DE 3). After the fermentation of the genetic engineering bacteria, soluble protein accounts for more than 50% of total target protein, which is more beneficial to gene expression, and the imide enzyme with high activity in the enzyme solution is realized, thereby achieving the advantage of more facilitating the enzyme solution to catalyze the R-type high-selectivity enzyme of the substrate to form a corresponding R-type high-chiral-purity product.

In the method for synthesizing (R) -isobutylglutaric acid monoamide using a recombinant imidiase, the recombinant imidiase is preferably selected from the group consisting of an imidiase-containing cell and an enzyme solution.

The reaction equation of the above-described method for synthesizing (R) -isobutylglutaric acid monoamide using recombinant imidinase of the present invention can be expressed as follows:

the raw material isobutyl glutarimide (IBI) in the method for synthesizing (R) -isobutyl glutarimide by utilizing recombinant imidinase can also be synthesized by adopting IBA as a raw material, then adopting the enzymatic method of the invention to catalyze and synthesize IBM, and then synthesizing the corresponding (S) -pregabalin ((S) -3-aminomethyl-5-methylhexanoic acid). Can be represented by the following reaction equation:

in summary, compared with the prior art, the invention has the following advantages:

1. by adopting an enzyme method and adopting the recombinant imide enzyme to carry out enzyme catalysis, the method realizes high activity and high selectivity of reaction sites, can preferentially catalyze the hydrolysis of groups of a cyclic imine structure to form a dicarboxyl monoamide structure, and the formed product has high chiral R-type selectivity and a de-symmetry reaction, thereby also achieving the effect of high chiral product yield.

2. The recombinant imide enzyme has high chiral selectivity and conversion rate by adopting the enzymatic catalysis, the ee value of the product reaches more than 99%, and the conversion rate can reach more than 90%; the intermediate is more favorable for improving the quality of products when being used as the synthetic intermediate of pregabalin.

Drawings

FIG. 1 is an SDS-PAGE analysis of the expression of the imide enzyme-engineered bacteria of example 1 of the present invention.

FIG. 2 is an SDS-PAGE analysis of the expression of the imide enzyme engineering bacteria of example 2 of the present invention in TB medium.

FIG. 3 is a graph of the reaction time versus system product concentration change analysis for the conversion of imide-catalyzed IBI to IBM reaction process in example 4 of the present invention.

FIG. 4 is a chart of HPLC (A) and optical purity (B) analysis of the (R) -IBM product obtained in example 5 of the present invention.

FIG. 5 is a mass spectrum of the (R) -IBM standard (A) and the product (B) extracted from the imidizing enzyme-catalyzed reaction solution according to the present invention.

Detailed Description

The technical solutions of the present invention will be further specifically described below with reference to specific examples and drawings, but the present invention is not limited to these examples.

Example 1

The recombinant imide enzyme engineering bacteria are obtained by synthesizing and cloning imide enzyme gene BpIH into PET30a NdeI and HindIII sites by Kinsley company, and then transforming the imide enzyme gene BpIH into recombinant imide enzyme engineering bacteria pT67/BL21(DE 3).

For the specific cloning method, the cloning method is conventional in the art, preferably expressed by PET, the host cell is Escherichia coli, the gene sequence of the recombinant imide enzyme engineering bacterium (pT67/BL21(DE3) is shown as SEQ ID NO.1, and the protein sequence of the recombinant imide enzyme is shown as SEQ ID NO. 2.

A single colony of the above recombinant imide enzyme engineering bacterium pT67/BL21(DE3) growing on an LB plate containing 50. mu.g/mL kanamycin is picked up, inoculated into 4mL LB liquid medium containing 50. mu.g/mL kanamycin, cultured at a temperature of 37 ℃ and a rotation speed of 200rpm until the OD600 is about 0.6-0.8, then IPTG with a final concentration of 0.5mmol/L is added, and then the culture is continued for 16h at 15 ℃ or 4h at 37 ℃, 450. mu.l of culture solution is respectively centrifuged to collect corresponding bacteria, 300. mu.l of lysis buffer (50mM Tris,150mM NaCl, 5% glycerol pH8.0) is respectively added, and then the ultrasonic cell wall breaking is carried out for 1 min, and the corresponding lysis solution is respectively collected.

Mu.l of each lysate was centrifuged at 15000rpm for 10min, the pellet was resuspended in 150. mu.l of 5 × loading buffer, and the lysate, lysate supernatant and lysate supernatant, respectively, were centrifuged to pellet resuspension and analyzed by SDS-PAGE.

Wherein in FIG. 1, the M-protein is labeled; PC (personal computer)₁-BSA(1μg)；PC₂-BSA(1μg)； NC₁-non-induced cell lysate; 1-cell lysate, induced at 15 ℃ for 16 hours; 2-cell lysate, induced at 37 ℃ for 4 hours; NC (numerical control)₂-supernatant of a lysate without induced cells; 3-supernatant of cell lysate, induced for 16 hours at 15 ℃; 4-cell lysate supernatant, induced at 37 ℃ for 4 hours; NC 3: uninduced cell lysate debris; inducing cell lysate debris at 5-15 deg.C for 16 h; 6-cell lysate debris, induced at 37 ℃ for 4 hours.

The results of the analysis are shown in FIG. 1:

as can be seen from FIG. 1, compare lane NC₁1 and 2, the target protein is expressed, the molecular weight of the target protein is between 35 and 40kDa and is consistent with a theoretical value of 37.29 kD; the expression level of the target protein is obviously higher than that of the target protein induced at 37 ℃ for 4h than that induced at 15 ℃ for 16h, and is about 10 mg/L. Comparison of lanes 1-6 in FIG. 1 shows that the target protein is solubleRelatively poor, soluble protein accounts for about 20% of the total target protein induced at 37 ℃ for 4 h.

Example 2

The recombinant imide enzyme engineering bacteria are obtained by synthesizing and cloning imide enzyme gene BpIH into PET30a NdeI and HindIII sites by Kinsley company, and then transforming the imide enzyme gene BpIH into recombinant imide enzyme engineering bacteria pT67/BL21(DE 3).

For the specific cloning method, the conventional cloning method in the field, preferably the expression PET, is adopted, the host cell adopts Escherichia coli, the gene sequence of the recombinant imide enzyme engineering bacterium pT67/BL21(DE3) is shown as SEQ ID NO.1, and the protein sequence of the recombinant imide enzyme is shown as SEQ ID NO. 2.

A single colony of the above recombinant imide enzyme engineering bacterium (pT67/BL21(DE3)) grown on an LB plate containing 50. mu.g/mL kanamycin was picked up, inoculated into 4mL of LB liquid medium containing 50. mu.g/mL kanamycin, cultured overnight at 37 ℃ and 200rpm, 0.5mL of the resulting culture was inoculated into a 250mL Erlenmeyer flask containing 50mL of TB medium, cultured at 37 ℃ and 200rpm for 4 hours, induced with 0.5mmol/L IPTG, cultured at 16 ℃, 25 ℃ and 37 ℃ for 12 hours, and the corresponding cells were collected and examined by SDS-PAGE.

Wherein in FIG. 2, the M-protein is labeled; 1. 2, 3-cell lysate, supernatant of cell lysate and fragments of cell lysate, and inducing for 12 hours at 16 ℃; 4. 5, 6-cell lysate, supernatant of cell lysate and fragments of cell lysate, and inducing for 12 hours at 25 ℃; 7. 8, 9-cell lysate, supernatant of cell lysate, debris of cell lysate, and induced at 37 ℃ for 12 hours.

The specific test results are shown in fig. 2:

as can be seen from fig. 2, the ratio of the target protein to the total protein and the ratio of the target protein to the target protein in the TB medium were increased compared to the LB medium. When the protein is induced by 0.5mmol/L IPTG at 25 ℃, the content of soluble protein is the highest, which accounts for about 75 percent of the total target protein, and the expression amount is about 100 mg/L.

Example 3

1mL of LB culture solution obtained after overnight culture in example 2 was inoculated into 50mL of TB medium, cultured at 37 ℃ for 4 hours at 200rpm, 25mL of LB culture solution was inoculated into 2.0L of TB medium, cultured at 250rpm for 4 hours at 37 ℃ and then added with 0.5mmol/L IPTG to continue culturing for 12 hours, and the stirring speed was adjusted to maintain DO > 20% during fermentation. Centrifugally collecting thalli after fermentation; the thalli is frozen at-20 ℃ for standby, or the thalli is resuspended by deionized water with the weight of 4 times and then is homogenized at 900bar for wall breaking to obtain corresponding enzyme liquid, and the enzyme liquid is stored at-20 ℃ for standby.

Example 4

Adding 180mL of deionized water and 25g of isobutyl glutarimide (IBI) into a clean reaction bottle, uniformly stirring, adjusting the pH of the system to 8.0 by using 1mol/L NaOH aqueous solution, adding 320mL of enzyme solution obtained in example 3, controlling the temperature to 35 ℃, reacting at the rotation speed of 250rpm, maintaining the pH of the reaction system to be about 8.0 by using 1mol/L NaOH aqueous solution during the reaction, fully reacting until the reaction is finished to obtain corresponding reaction solution, wherein the concentration of a product (R) -isobutyl glutaric acid monoamide (IBM) reaches 49.11g/L, the conversion rate reaches 90.9 percent, and the formation of IBM hydrolysate IBA is not detected.

Example 5

Taking 400mL of the reaction solution obtained by the method of the embodiment 4, wherein the reaction solution contains 19.64g of the product IBM, centrifuging the reaction solution at 10000rpm for 15min, adjusting the pH of the clear solution to 3.0 by using 1mol/L HCl, filtering to obtain 25g of IBM wet crude product, dissolving the obtained crude product by using 200mL of anhydrous ethanol, adding 2.0g of activated carbon, filtering, concentrating under reduced pressure to separate out a solid, filtering to obtain a solid product, and drying the obtained solid product by using hot air at 40 ℃ for 4h to obtain 13.5g of the finished product (R) -isobutyl glutaric acid monoamide. The product (R) -isobutylglutaric acid monoamide (IBM) was 98.27% pure by HPLC and 99.96% pure by HPLC (fig. 5A); the optical purity was 99.98%, the ee value was 99.96% (FIG. 5B), and the extraction yield was 68.74%.

The mass spectrograms of the product and the standard are shown in FIG. 5, the two have the same molecular mass, and the fragment ion peaks are matched, thus proving that the product is (R) -isobutyl glutaric acid monoamide (IBM).

Example 6

Adding 180mL of deionized water and 25g of isobutyl glutarimide (IBI) into a clean reaction bottle, uniformly stirring, adjusting the pH of the system to 8.5 by using 1mol/L NaOH aqueous solution, adding 320mL of the enzyme solution obtained in the example 3 after wall breaking, then, controlling the temperature to 35 ℃, reacting at the rotating speed of 250rpm, maintaining the pH of the reaction system to be about 8.5 by using 1mol/L NaOH aqueous solution during the reaction, tracking and detecting by HPLC until the reaction is sufficient, obtaining the corresponding reaction solution after the reaction is finished, wherein the concentration of a product (R) -isobutyl glutaric acid monoamide (IBM) reaches 49.85g/L, and the formation of an IBM hydrolysate IBA is not detected.

Example 7

Taking 400mL of the reaction solution obtained by the method of the embodiment 6, using the reaction solution (IBM concentration is 49.85g/L) to contain 19.94g of the product IBM in total, centrifuging the reaction solution at 10000rpm for 15min, adjusting the pH of clear solution to 3.0 by using 1mol/L HCl, filtering to obtain IBM wet crude product, dissolving the obtained wet crude product by using 200mL of absolute ethyl alcohol, adding 2.0g of activated carbon, filtering, concentrating under reduced pressure, precipitating solid, filtering to obtain a solid product, and drying the obtained solid product by hot air at 40 ℃ for 4h to obtain 14.3g of finished product (R) -isobutyl glutaric acid monoamide (IBM). The chromatographic purity of the product (R) -isobutylglutaric acid monoamide (IBM) was 98.85% and the content was 99.96% by HPLC; the optical purity was 99.98%, the ee value was 99.95%, and the extraction yield was 71.7%.

The mass spectrograms of the obtained product and the standard product are consistent with those shown in FIG. 5, the two products have the same molecular mass, and the fragment ion peaks are identical, which shows that the product with high chiral purity is IBM.

Example 8

Adding 180mL of deionized water and 25g of isobutyl glutarimide (IBI) into a clean reaction bottle, uniformly stirring, adjusting the pH of the system to 7.5 by using 1mol/L NaOH aqueous solution, adding 320mL of the enzyme solution obtained in the example 3 after wall breaking, then, controlling the temperature to be 45 ℃, reacting at the rotating speed of 250rpm, maintaining the pH of the reaction system to be about 7.5 by using 1mol/L NaOH aqueous solution during the reaction, tracking and detecting by HPLC until the reaction is sufficient, obtaining the corresponding reaction solution after the reaction is finished, wherein the concentration of a product (R) -isobutyl glutaric acid monoamide (IBM) reaches 49.23g/L, and the formation of an IBM hydrolysate IBA is not detected.

Example 9

Taking 400mL of the reaction solution obtained by the method of the embodiment 8, using 19.69g of the product (R) -isobutylglutaric acid monoamide (IBM) which is contained in the reaction solution (the concentration of the IBM is 49.23g/L), centrifuging the reaction solution for 15min at 10000rpm, adjusting the pH of clear liquid to 3.0 by using HCl with the concentration of 1mol/L, filtering to obtain IBM wet crude product, dissolving the obtained wet crude product by using 200mL of absolute ethyl alcohol, adding 2.0g of activated carbon, filtering, concentrating under reduced pressure to precipitate solid, filtering to obtain solid product, drying the obtained solid product by hot air at 40 ℃ for 4h to obtain 14.1g of finished product (R) -isobutylglutaric acid monoamide (IBM). The chromatographic purity of the product IBM is 98.88 percent and the content is 99.97 percent by HPLC; the optical purity was 99.98%, the ee value was 99.96%, and the extraction yield was 71.61%.

The mass spectrograms of the obtained product and the standard product are consistent with those shown in FIG. 5, the two products have the same molecular mass, and the fragment ion peaks are identical, which shows that the product with high chiral purity is IBM.

Example 10

Adding 180mL of deionized water and 25g of isobutyl glutarimide (IBI) into a clean reaction bottle, uniformly stirring, adjusting the pH of the system to 6-9 by using 1mol/L NaOH aqueous solution, adding 320mL of the enzyme solution obtained in the example 3 after wall breaking, then, controlling the temperature to be 20-50 ℃, reacting at the rotating speed of 250rpm, maintaining the pH of the reaction system to be 6-9 by using 1mol/L NaOH aqueous solution during the reaction, tracking and detecting by HPLC (high performance liquid chromatography) until the reaction is sufficient, obtaining corresponding reaction liquid after the reaction is finished, enabling the concentration of a product (R) -isobutyl glutaric acid monoamide (IBM) to reach more than 49g/L, and not detecting the formation of IBM hydrolysate IBA.

Taking 400mL of the reaction solution obtained by the method, taking 19.6g of the product (R) -isobutyl glutaric acid monoamide (IBM) which is contained in the reaction solution (the concentration of the IBM is 49 g/L), centrifuging the reaction solution at 10000rpm for 15min, adjusting the pH of clear solution to 3.0 by using HCl with the concentration of 1mol/L, filtering to obtain an IBM wet crude product, dissolving the obtained wet crude product by using 200mL of absolute ethyl alcohol, adding 2.0g of activated carbon, filtering, concentrating under reduced pressure, precipitating a solid, filtering to obtain a solid product, and drying the obtained solid product by hot air at 40 ℃ for 4h to obtain 14.1g of the finished product (R) -isobutyl glutaric acid monoamide (IBM). HPLC determination shows that the chromatographic purity of the product IBM is more than 98.85 percent, and the content is more than 99.95 percent; the optical purity is above 99.97%, the ee value is above 99.95%, and the extraction yield is 71.94%.

The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

Sequence listing

<110> Taizhou college

Taizhou Dachen pharmaceutical Co Ltd

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His Phe Phe Thr Tyr Leu Arg Asp Ala Phe Asp Val Leu Tyr Glu Glu

245 250 255

Gly Asp Glu Ala Pro Lys Met Leu Ser Ile Gly Met His Cys Arg Leu

260 265 270

Leu Gly Arg Pro Gly Arg Phe Arg Ala Leu Gln Arg Phe Leu Asp His

275 280 285

Ile Glu Gln His Asp Arg Val Trp Val Thr Arg Arg Val Asp Ile Ala

290 295 300

Arg His Trp Arg Glu His His Pro Tyr Gln Gln Asn Asn Arg Gly Ala

305 310 315 320

Ala Ala