阅读说明：本技术 一种制备(s)-2-氯-1-(3,4-二氟苯基)乙醇的方法 (Method for preparing (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol ) 是由 陶荣盛 潘震华 朱傅赟 原犇犇 沈正权 孙梁栋 沈青 郑云 胡海亮 于 2020-06-03 设计创作，主要内容包括：本发明提供了一种制备(S)-2-氯-1-(3,4-二氟苯基)乙醇的方法,包括如下步骤：使用固定化载体LX-1000ODS或者LXES-J420将酮基还原酶KRED固定化,得到固定化酶；用固定化酶催化2-氯-1-(3,4-二氟苯基)乙酮进行还原反应,得到目标化合物。本发明方法的能够高效、稳定地催化式底物反应生成高光学纯度的产物,适合于工业化应用。(The invention provides a method for preparing (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol, which comprises the following steps: immobilizing keto reductase KRED by using an immobilized carrier LX-1000ODS or LXES-J420 to obtain an immobilized enzyme; catalyzing 2-chloro-1- (3, 4-difluorophenyl) ethanone by using immobilized enzyme to perform reduction reaction to obtain a target compound. The method can efficiently and stably catalyze the substrate reaction to generate a product with high optical purity, and is suitable for industrial application.)

1. A process for preparing (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol comprising the steps of:

A. immobilizing keto reductase KRED by using an immobilized carrier LX-1000ODS or LXES-J420 to obtain an immobilized enzyme;

B. and D, catalyzing the compound 2-chloro-1- (3, 4-difluorophenyl) ethanone shown in the formula I by using the immobilized enzyme obtained in the step A to perform reduction reaction to obtain a compound (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol shown in a formula II:

2. the method of claim 1, wherein the immobilized carrier is LX-1000 ODS.

3. The method of claim 2, wherein the ketoreductase has the amino acid sequence of SEQ ID NO 1.

4. The method of claim 3, wherein the ketoreductase SEQ ID NO 1 is expressed by a microorganism.

5. The method of claim 4, wherein the ketoreductase expressed gene of SEQ ID NO 1 is SEQ ID NO 2.

6. The method of claim 5, wherein the microorganism is Escherichia coli.

7. The method of claim 4, wherein step A is: and (3) centrifuging the fermented microbial thallus, breaking the wall, centrifuging, taking the supernatant to obtain a crude enzyme solution, mixing the crude enzyme solution with an immobilized carrier in a phosphate buffer solution, and adsorbing to obtain the immobilized enzyme.

8. The method of claim 1, wherein the reaction system of step B is phosphate buffered saline at ph 7.5-8.5.

9. The method of claim 8, wherein isopropanol and coenzyme NADP + are added to the reaction system.

10. The process of claim 8, wherein the reaction temperature is from 30 to 45 ℃.

Technical Field

The invention belongs to the technical field of biocatalysis, and particularly relates to a method for preparing (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol.

Background

Ticagrelor, a great name of ticagrelor, is a novel selective anticoagulant developed by AstraZeneca (AstraZeneca) in england and is the first reversible binding type P2Y12 adenosine diphosphate receptor (ADP) antagonist. Compared with clopidogrel, ticagrelor has the advantage of obviously reducing the symptoms of myocardial infarction, stroke or cardiovascular death of patients. In 2011, the American FDA is approved to be marketed, is approved in 85 countries worldwide, is listed in medical insurance catalogues of 29 countries, enters 31 national patient self-payment catalogues, and has wide market prospect.

Currently, ticagrelor is generally synthesized by a chemical method, and various synthetic process routes of ticagrelor are disclosed in the patent. In the synthesis route of ticagrelor disclosed in US7250419, the compound (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol shown in formula II is one of the key intermediates, and has high economic value.

Another route reported in EP2589587A1 is the use of CBS catalyst, BH₃The reduction gives chiral chlorohydrin II, but the ee value is only 90-93%, the product needs to be refined to increase the ee value and the cost is high. Patent document CN109112166A reports that the catalyst has high catalytic activityThe Ketoreductase (KRED) and the method for preparing the compound II by using the free enzyme to catalyze the compound 2-chloro-1- (3, 4-difluorophenyl) ethanone shown in the formula II have ee value of more than 99 percent.

Ketoreductase is expensive, and free enzyme can only be used once and cannot be recycled, so that the cost of raw materials for preparing the compound II by the ketone asymmetric reduction reaction of the compound I is high, and the method becomes an obstacle to industrial application.

Disclosure of Invention

In order to overcome the defect of poor economy in the enzymatic preparation of the compound II in the prior art, the inventors studied the immobilization technology of ketoreductase KRED. It is found that the specific immobilized enzyme can efficiently and stably catalyze the asymmetric reduction reaction of the compound I, and the production cost of the compound II is reduced. Specifically, the present invention includes the following technical solutions.

A process for preparing (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol comprising the steps of:

A. immobilizing keto reductase KRED by using an immobilized carrier LX-1000ODS or LXES-J420 to obtain an immobilized enzyme;

B. and D, catalyzing the compound 2-chloro-1- (3, 4-difluorophenyl) ethanone shown in the formula I by using the immobilized enzyme obtained in the step A to perform reduction reaction, so as to obtain a compound (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol shown in the formula II as one of ticagrelor intermediates:

preferably, the above-mentioned immobilization carrier is LX-1000 ODS.

In a preferred embodiment, the ketoreductase is derived from Lactobacillus kefir (Lactobacillus kefir) and has the amino acid sequence of SEQ ID NO: 1:

MTDRLKGKVAIVTGGTLGIGLAIADKFVEEGAKVVITGRRADVGERAAKSIGGTDVIRFVQHDASDEAGWTKLFDTTEEAFGPVTTVVNNAGIGVVKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKNKGLGASIINMSSIFGMVGDPTVGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGPIKTPMLDDVEGAEEMWSQRTKTPMGHIGEPNDIAWVCVYLASGESKFATGAEFVIDGGWTAQ(SEQ ID NO:1)。

the ketoreductase SEQ ID NO 1 can be obtained by microbial expression.

The expression gene of the ketoreductase SEQ ID NO. 1 is preferably SEQ ID NO. 2:

ATGACTGATCGTTTAAAAGGCAAAGTAGCAATTGTAACTGGCGGTACCTTGGGAATTGGCTTGGCAATCGCTGATAAGTTTGTTGAAGAAGGCGCAAAGGTTGTTATTACCGGCCGTCGGGCTGATGTAGGTGAACGGGCTGCCAAATCAATCGGCGGCACAGACGTTATCCGTTTTGTCCAACACGATGCTTCTGATGAAGCCGGCTGGACTAAGTTGTTTGATACGACTGAAGAAGCATTTGGCCCAGTTACCACGGTTGTCAACAATGCCGGAATTGGGGTCGTAAAGAGTGTTGAAGATACCACAACTGAAGAATGGCGCAAGCTGCTCTCAGTTAACTTGGATGGTGTCTTCTTCGGTACCCGTCTTGGAATCCAACGTATGAAGAATAAAGGACTCGGAGCATCAATCATCAATATGTCATCTATCTTCGGTATGGTTGGTGATCCAACTGTGGGTGCATACAACGCTTCAAAAGGTGCTGTCAGAATTATGTCTAAATCAGCTGCCTTGGATTGCGCTTTGAAGGACTACGATGTTCGGGTTAACACTGTTCATCCAGGTCCTATCAAGACACCAATGCTTGACGATGTTGAAGGGGCAGAAGAAATGTGGTCACAGCGGACCAAGACACCAATGGGTCATATCGGTGAACCTAACGATATCGCTTGGGTCTGTGTTTACCTGGCATCTGGCGAATCTAAATTTGCCACTGGTGCAGAATTCGTTATCGATGGTGGATGGACTGCTCAATAA(SEQ ID NO:2)。

preferably, the microorganism is Escherichia coli.

In one embodiment, the step a is: and (3) centrifuging the fermented microbial thallus, breaking the wall, centrifuging, taking the supernatant to obtain a crude enzyme solution, mixing the crude enzyme solution with an immobilized carrier in a phosphate buffer solution, and adsorbing to obtain the immobilized enzyme.

The reaction system in the step B is a phosphate buffer solution having a pH of 7.5 to 8.5, preferably a pH of 7.8 to 8.2, more preferably a pH of about 8.0.

The reaction temperature in the step B is 30-45 ℃. Preferably 35-42 deg.C, more preferably 37-40 deg.C.

Preferably, isopropanol and coenzyme NADP + may be added to the reaction system. NADP + (nicotinamide adenine dinucleotide phosphate, coenzyme II) functions as an oxidant to scavenge electrons, and ketoreductase reduces NADP + to NADPH using isopropanol, producing sufficient NADPH as a biosynthetic reductant, thereby facilitating the reduction reaction.

The immobilized ketoreductase provided by the invention can catalyze the asymmetric reduction reaction of the substrate I with high activity to generate the ticagrelor intermediate II with high optical purity, has the advantages of good stability and low cost, and is beneficial to realizing industrial production.

Detailed Description

The enzyme is immobilized by blocking free enzyme on solid material or limiting in a certain area by physical or chemical means, and the enzyme can still play a catalytic role and can be recycled. Compared with free enzyme, the immobilized enzyme has the advantages of high stability, convenient recovery, easy control, repeated use, low cost and the like, and plays an important role in the aspects of biological industry, medical and clinical diagnosis, chemical analysis, environmental protection, energy development, basic research and the like.

As is well known in the field of biological catalysis, compared with a free enzyme method, the application of an immobilized enzyme technology has the advantages of simplified production process, improved production efficiency and the like. Meanwhile, the enzyme can be used for multiple times, and the stability of the enzyme is improved, so that the productivity of unit enzyme is effectively improved; and secondly, the immobilized enzyme is easily separated from the substrate and the product, the purification process is simplified, the yield is high, and the product quality is good. However, enzyme immobilization also has a number of disadvantages, enzyme activity is usually lost during immobilization, and the chiral purity of the catalytic reaction product sometimes changes, which often makes it difficult to ensure high chiral purity.

In order to study immobilization of ketoreductase KRED, the inventors have tried various types of immobilization methods, including adsorption, crosslinking, embedding, carrier coupling (also referred to as covalent binding), and the like. In the selection of carriers for adsorption/carrier coupling, the ion exchange resins are considered with emphasis, including commercial amino-immobilized carriers such asEC-HA and EC-EA of EC series; seplite LX-1000HA and Seplite LX-1000NH of Xian blue Xiao Tech Co., Ltd; relizyme^TMThe series HA403, EA 403; commercial epoxy-based immobilization supports such asEC-EP, EC-HFA of the EC series; seplite LX-1000EP, Seplite LX-1000HFA, LX-1000ODS, LXES-J420, LX-Q650C, LX-T300, LX-G20 from Xian blue-Xiao science and technology Co., Ltd; relizyme^TMSeries EP403, HFA403, etc. See table 1 below.

Experiments show that the ketoreductase KRED, particularly the ketoreductase with the amino acid sequence of SEQ ID NO. 1, can be ideally matched with two immobilized carriers LX-1000ODS and LXES-J420, particularly LX-1000ODS, and after being immobilized by an adsorption method, the ketoreductase KRED can efficiently and stably catalyze the compound shown in the formula I to carry out chiral reduction to obtain the compound shown in the formula II with high optical activity, so that the bottleneck existing in the existing enzymatic production process is solved, the production cost is reduced, and a foundation is laid for realizing the large-scale production of the compound shown in the formula II by the enzymatic method.

Surprisingly, the optical purity (ee value) of the product of the reaction catalyzed by the immobilized KRED (SEQ ID NO:1) on the carrier LX-1000ODS exceeded that of the product of the reaction catalyzed by the free enzyme, suggesting that the stereoselectivity of the immobilized KRED enzyme LX-1000ODS is enhanced for reasons to be further investigated. It is possible that the ketoreductase SEQ ID NO 1 is adsorbed by the carrier LX-1000ODS, and then three-dimensional conformation change or space change of an enzyme active site occurs, so that correct folding of enzyme protein is promoted, enzyme activity is enhanced, and selectivity of a substrate is improved.

As used herein, the terms "compound of formula II", "compound II" and "product compound II" have the same meaning and refer to the desired compound (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol. Similarly, the terms "compound of formula I", "substrate I" and "compound I" mean the same and refer to 2-chloro-1- (3, 4-difluorophenyl) ethanone as substrate for the enzymatic reaction.

To further reduce the reaction raw material cost, ketoreductase SEQ ID NO 1 can be produced by itself, such as by microbial fermentation expression. In this case, the ketoreductase SEQ ID NO 1 to be immobilized can be crude enzyme liquid, such as liquid obtained after cell breaking of fermentation thalli, so that complicated steps of enzyme extraction, purification and the like can be omitted, the de novo preparation of immobilized enzyme is realized, and the price of the used commercial enzyme is greatly reduced.

The above microorganisms may be any suitable microorganisms for expressing ketoreductase SEQ ID NO 1, including bacteria and fungi. Coli having a rapid growth is preferred, and E.coli BL21(DE3) is more preferred.

The ketoreductase SEQ ID NO. 1 used in the present invention has a definite structure, and thus a person skilled in the art can easily obtain the genes encoding it, expression cassettes and plasmids containing the genes, and transformants containing the plasmids. These genes, expression cassettes, plasmids, and transformants can be obtained by genetic engineering construction means well known to those skilled in the art.

The expressed gene of ketoreductase SEQ ID NO. 1 can be codon optimized depending on the transformant, i.e., the expressing microorganism. Different organisms often show a special preference for one of several codons encoding the same amino acid due to mutation tendencies and natural selection. For example, in rapidly growing microorganisms such as E.coli, the optimized codons reflect the composition of their respective pools of genomic tRNA's. In order to express ketoreductase SEQ ID NO 1 in Escherichia coli, the expression gene SEQ ID NO 2 thereof was designed and synthesized.

Expression of the ketoreductase is achieved by cloning the gene, e.g., SEQ ID NO:2, of the enzyme into an expression vector and introducing into E.coli. Then the crude enzyme solution and the immobilized carrier are fixed according to a certain proportion. The obtained immobilized enzyme can effectively realize the biotransformation of the substrate 2-chloro-1- (3, 4-difluorophenyl) ethanone.

The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.