阅读说明：本技术 一种制备(r)-3-氨基-4-(2,4,5-三氟苯基)-丁酸的方法 (Method for preparing (R) -3-amino-4- (2,4, 5-trifluorophenyl) -butyric acid ) 是由 竺伟 张小飞 马斌祥 于 2020-06-28 设计创作，主要内容包括：本发明公开了一种制备(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸的方法。本发明的方法以3-羰基-4-(2,4,5-三氟苯基)-丁酸酯为底物,乙二醇为助溶剂,在弱碱性条件以及转氨酶的催化作用下,一锅法生成(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸。本发明一步得到目标产物,操作简单,反应条件温和,适合工业化生产。(The invention discloses a method for preparing (R) -3-amino-4- (2,4, 5-trifluorophenyl) -butyric acid. The method takes 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butyrate as a substrate and ethylene glycol as a cosolvent, and generates (R) -3-amino-4- (2,4, 5-trifluorophenyl) -butyrate by a one-pot method under the alkalescent condition and the catalytic action of transaminase. The method can obtain the target product in one step, is simple to operate, has mild reaction conditions, and is suitable for industrial production.)

1. A method for preparing (R) -3-amino-4- (2,4, 5-trifluorophenyl) -butyric acid (I) is characterized in that 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butyrate (II) is used as a substrate, ethylene glycol is used as a cosolvent, and under the weak alkaline condition and the catalytic action of transaminase, the (R) -3-amino-4- (2,4, 5-trifluorophenyl) -butyric acid is generated by a one-pot method.

The reaction process is as follows:

wherein R1 is an alkyl substituent of C1-C4.

2. The method of claim 1, wherein the substrate 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butyrate is present at a concentration of 50 to 300 mM.

3. The method according to claim 1, wherein the concentration of the ethylene glycol in the reaction system is 5 to 50% (V/V).

4. The method of claim 1, wherein the weakly alkaline conditions are a pH of 7.5 to 10.0.

5. The method of claim 1, wherein the transaminase is from Arthrobacter athromobacter sp.

6. The process of claim 1, wherein the reaction temperature is 30 to 60 ℃.

The technical field is as follows:

the invention belongs to the technical field of biocatalysis, and particularly relates to a method for preparing (R) -3-amino-4- (2,4, 5-trifluorophenyl) -butyric acid by one-step catalysis of transaminase.

Background art:

sitagliptin phosphate is a drug developed by merck corporation for the treatment of type ii diabetes, and is marketed under the trade name of carnivol (janivia) after approval by the FDA in the united states in 2006, and is approved for use in several countries around the world. The medicine is mainly used for controlling blood sugar level by inhibiting the activity of dipeptidyl peptidase-4 (DPP-4), protecting endogenous incretin and enhancing the action of the endogenous incretin. Clinical research shows that the medicine is orally taken effectively, has obvious blood sugar reducing effect when being used alone or together with metformin and pioglitazone, and has the advantages of safe oral taking, high tolerance, less untoward effect, etc. Due to the limitation of the patent to the product, a plurality of pharmaceutical companies are researching from important chiral amino intermediates for synthesizing the drug, wherein (R) -3-amino-4- (2,4, 5-trifluorophenyl) -butyric acid is an important chiral intermediate for synthesizing sitagliptin phosphate, and the synthetic route of the sitagliptin phosphate is shown as Scheme 1.

Around the enzymatic preparation of chiral amino intermediates, chinese patent CN105018440B discloses the conversion of 3-carbonyl-4- (2,4, 5-trifluorophenyl) -methyl butyrate by a new transaminase derived from Mycobacterium (Mycobacterium vanbalenii) PYR-1 to obtain a highly chiral (R) -3-amino-4- (2,4, 5-trifluorophenyl) -methyl butyrate product.

Chinese patent CN10728616A discloses a method for converting 3-carbonyl-4- (2,4, 5-trifluorophenyl) -hydroxy butyrate substrate by using transaminase derived from arthrobacter, obtaining corresponding (R) -3-amino-4- (2,4, 5-trifluorophenyl) -hydroxy butyrate, and obtaining (R) -3-amino-4- (2,4, 5-trifluorophenyl) -butyric acid intermediate by LiOH strong base hydrolysis.

Chinese patent CN108586346A discloses a method for reacting 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butyric acid amide substrate by using transaminase mutant from arthrobacter, to obtain corresponding (R) -3-amino-4- (2,4, 5-trifluorophenyl) -butyric acid amide product.

In the currently reported enzymatic synthesis of sitagliptin phosphate intermediate, chiral amino ester products are still used as the main raw materials, and then the raw materials are treated by strong alkali and hydrolyzed into beta-amino acid final products; in addition, the conversion efficiency of the reaction system using DMSO as a cosolvent is not high, and the reaction process is also inhibited by a substrate, so that the production efficiency and the cost of the intermediate are influenced by the practical problems. Therefore, further improvements and enhancements are still needed for the enzymatic preparation of sitagliptin phosphate intermediate (I).

The invention content is as follows:

the invention aims to provide a preparation method of (R) -3-amino-4- (2,4, 5-trifluorophenyl) -butyric acid, which is simple to operate and easy to industrialize aiming at the defects of the prior art.

The technical scheme adopted by the invention is as follows:

wherein R1 is an alkyl substituent of C1-C4.

The invention provides a method for preparing (R) -3-amino-4- (2,4, 5-trifluorophenyl) -butyric acid, which comprises the following steps: the (R) -3-amino-4- (2,4, 5-trifluorophenyl) -butyric acid is generated by a one-pot method under the catalysis of transaminase and the weak alkaline condition by using 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butyric acid ester as a substrate and ethylene glycol as a cosolvent.

Further, the operation steps for preparing (R) -3-amino-4- (2,4, 5-trifluorophenyl) -butyric acid by the one-pot enzyme method are as follows: dissolving a 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butyrate substrate by using ethylene glycol as a cosolvent to prepare a certain concentration, and adding a reaction system within 2 h; preparing a buffer solution with a certain concentration as a reaction medium, and adding a certain amount of isopropylamine, pyridoxal phosphate, ethylene glycol and transaminase into the reaction medium; stirring and reacting at a certain temperature, and controlling the pH value in the reaction process by utilizing isopropylamine with a certain concentration; the conversion and the optical purity of the product were analyzed by high performance liquid chromatography at the end of the reaction.

Further, the concentration of the substrate 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butyrate is 50-300 mM, and preferably 100 mM.

Further, the 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butanoate-based substrate is methyl 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butanoate, ethyl 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butanoate, isopropyl 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butanoate, tert-butyl 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butanoate, etc., preferably ethyl 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butanoate.

Further, the concentration of the ethylene glycol in the reaction system is 5-50% (V/V), and the preferable concentration of the reaction system is 20%

Further, the weak alkaline condition is pH 7.5 to 10.0, preferably pH 9.0.

Further, the transaminase is from Arthrobacter arthromobacter sp.

Further, the reaction is carried out at a reaction temperature of 30 to 60 ℃, preferably 50 ℃.

The invention has the beneficial effects that through optimization of an organic solvent of the transaminase, ethylene glycol is found as a cosolvent, so that the catalytic efficiency of the transaminase on 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butyrate is improved, and simultaneously after the reaction is finished, the (R) -3-amino-4- (2,4, 5-trifluorophenyl) -butyrate product can be directly obtained, the optical purity is more than 99%, and the conversion rate is more than 96%.

Drawings

FIG. 1 protein electrophoretogram of transaminase

Detailed Description

The technical content of the present invention is further described below with reference to specific examples for better understanding of the content of the present invention, but the scope of the present invention is not limited thereto.

Example 1 expression of Arthrobacter transaminase protein in E.coli

Obtaining a wild type gene sequence of transaminase by means of NCBI website, artificially designing and synthesizing the whole gene after codon optimization, respectively introducing NdeI and XhoI enzyme cutting sites at two ends of the gene, and cloning the enzyme cutting sites to pET-24a vector. The constructed recombinant plasmid is transformed into DH5 alpha competent cell containing Kan by chemical transformation method⁺Resistant LB plates were cultured overnight in an inverted culture at 37 ℃. Selecting positive monoclone to carry out gene sequencing, after the sequence is determined to be correct, transferring the recombinant expression vector into escherichia coli BL21(DE3) competent cells, extracting the monoclone cells, and obtaining the arthrobacter transaminase gene engineering strain capable of inducing expression.

BL21(DE3) cells containing the target gene were inoculated into a cell line containing Kan⁺The mixture was cultured overnight at 37 ℃ in a resistant LB tube to obtain a primary seed culture. Transferring the seed culture solution into a 2YT liquid culture medium containing resistance according to the inoculation ratio of 1%, placing the culture medium in a shaking table, culturing for 3-5h at 37 ℃ and 200rpm, cooling to 20 ℃ when OD reaches 0.6-0.8, adding IPTG (isopropyl-beta-thiogalactoside) with the concentration controlled at 0.2mM, and performing overnight induction expression. Centrifuging the fermentation liquid, collecting thallus, dissolving the collected thallus with phosphate buffer solution, crushing the cell in a high-pressure homogenizer, centrifuging at 12000rpm for 10min again to obtain supernatant as transaminase protein, and observing the protein expression by electrophoresis, as shown in figure 1.

EXAMPLE 2 transamination of transaminase cells in different organic solvents

First, 0.26g of each 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butyric acid ethyl ester substrate was dissolved in 1mL of different organic solvents to prepare a substrate concentration of 100 mM. All reaction systems were controlled to 10mL, and 1mL of 1M triethanolamine buffer pH 9.0, 1mL of 10mM pyridoxal phosphate, 1mL of 3M isopropylamine, and 0.5g of enzyme cells were added to each reaction vessel in sequence, 1mL each for a different organic solvent. The reaction was run at 50 ℃ with the pH maintained at 9.0 by controlling the reaction with 3M isopropylamine. The substrate is added into the reaction system within 2h in a constant speed dropwise adding mode, and the final concentration of the organic solvent in each reaction system is 20% (V/V). After 24h of reaction, samples were taken and centrifuged respectively, and liquid phase analysis was performed to determine the conversion rate and the chiral purity of the product under different conditions, and the specific results are shown in table 1.

TABLE 1 Effect of different organic solvents on the reaction System

Organic solvent	Conversion rate	(R) chiral purity of the product
			20% ethanol	75.1％	98.2％
20％DMSO	84.3％	97.8％
			20% ethylene glycol	96.4％	99.1％
20% methanol	75.1％	94.2％
			20% tetrahydrofuran	73.1％	98.1％
20% acetonitrile	85.6％	90.5％
			20％MTBE	82.9％	97.9％
20% toluene	49.5％	98.0％
			20% isopropyl alcohol	81.7％	98.3％
20% tert-butanol	66.5％	95.2％
			20% n-butanol	57.2％	/
20% Ethyl acetate	70.3％	96.8％
			20% butyl acetate	25.3％	/
20％DMF	21％	61％
			20% n-hexane	25.4％	97.5％
20% n-heptane	19.7％	95.4％
			20% cyclohexane	31.8％	98.2％

Example 3 efficiency of transaminase cell conversion to various concentrations of ethylene glycol

A substrate concentration of 300mM was prepared by dissolving 0.78g each of 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butyric acid ethyl ester substrate in ethylene glycol as an organic solvent. Each reaction system was controlled to 10mL, and 1mL of 1M triethanolamine buffer pH 9.0, 1mL of 10mM pyridoxal phosphate, 1mL of 3M isopropylamine and 0.5g of enzyme cells were sequentially added to the reaction vessels, and different amounts of ethylene glycol were added to each reaction vessel, to maintain the ratios of ethylene glycol in the reaction systems at 5%, 10%, 20%, 30% and 50%, respectively. The reaction was run at 50 ℃ with the pH maintained at 7.5 by controlling the reaction with 3M isopropylamine. The substrate is added into the reaction system within 2h in a constant-speed dropwise adding mode. After 24h of reaction, samples were taken and centrifuged respectively, and liquid phase analysis was performed to determine the conversion rate and the chiral purity of the product under different conditions, and the specific results are shown in Table 2.

TABLE 2 Effect of different amounts of ethylene glycol on the reaction System

Organic solvent	Conversion rate	(R) chiral purity of the product
			5% ethylene glycol	89.3％	98.8％
10% ethylene glycol	89.8％	98.8％
			20% ethylene glycol	96.4％	99.1％
30% ethylene glycol	87.2％	98.6％
			50% ethylene glycol	71.6％	99.0％

Example 4 conversion efficiency of different ester substrates

Substrate concentrations of 0.24g, 0.26g, 0.27g and 0.29g of 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butyric acid methyl ester, 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butyric acid ethyl ester, 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butyric acid isopropyl ester and 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butyric acid tert-butyl ester substrate were dissolved with ethylene glycol to 100mM, respectively. Each reaction system was controlled to 10mL, and 1mL of 1M triethanolamine buffer pH 9.0, 1mL of 10mM pyridoxal phosphate, 1mL of 3M isopropylamine and 0.5g transaminase cells, 1mL of ethylene glycol as a co-solvent were added to the reaction vessel in this order, maintaining the proportion of ethylene glycol in the reaction system at 20%. The reaction was run at 50 ℃ with the pH maintained at 9.0 by controlling the reaction with 3M isopropylamine. The substrate is added into the reaction system within 2h in a constant-speed dropwise adding mode. After 24h of reaction, samples were taken and centrifuged respectively, and liquid phase analysis was performed to determine the conversion rate and the chiral purity of the product under different conditions, and the specific results are shown in Table 3.

TABLE 3 Effect of different substrates on the reaction System