阅读说明：本技术 一种（s）-2-氯-1-（3，4-二氟苯基）乙醇的生物合成方法 (Biosynthesis method of (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol ) 是由 曹金辉 宗匡 喻海亮 曾鹏 刘建明 于 2021-07-30 设计创作，主要内容包括：本发明公开了一种(S)-2-氯-1-(3,4-二氟苯基)乙醇的生物合成方法,其包括如下步骤：酮基还原酶野生型基因进行突变处理、添加酶切位点、制备重组质粒、导入菌株、菌株的分泌和表达、反应。本发明提供的生物合成方法有着成本更低、选择性更高的优点。(The invention discloses a biosynthesis method of (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol, which comprises the following steps: carrying out mutation treatment on the ketoreductase wild type gene, adding enzyme cutting sites, preparing recombinant plasmids, introducing a strain, and carrying out secretion, expression and reaction on the strain. The biosynthesis method provided by the invention has the advantages of lower cost and higher selectivity.)

1. A biosynthesis method of (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol, which is characterized by comprising the following steps: the method comprises the following steps:

carrying out mutation treatment on ketoreductase wild type genes: performing mutation treatment on the ketoreductase gene and the gene sequence of the ketoreductase as shown in Seq ID No.1 to obtain a ketoreductase mutant gene, wherein the gene sequence of the ketoreductase mutant is shown in Seq ID No. 2.

Adding enzyme cutting sites: inserting double enzyme cutting sites into a ketoreductase mutant gene;

preparing a recombinant plasmid: inserting the ketoreductase mutant gene into an expression vector to obtain a recombinant plasmid;

introducing a strain: introducing the recombinant plasmid with the ketoreductase gene into a strain to obtain a recombinant expression strain;

secretion and expression of the strains: inducing the recombinant expression strain in a culture solution for expression and then collecting an enzyme solution;

reaction: the enzyme solution is put into a water/organic biphase system to react to prepare (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol.

2. The process for the biosynthesis of (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol according to claim 1, characterized in that: in the mutation treatment of the ketoreductase wild type gene, the mutation treatment mode is error-prone PCR.

3. The process for the biosynthesis of (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol according to claim 1, characterized in that: in the added enzyme cutting sites, the double enzyme cutting sites are NdeI/HindIII.

4. The method for the biosynthesis of (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol as claimed in claim 1, wherein: in the preparation of the recombinant plasmid, the expression vector was pET21a (+) 22.

5. The method for the biosynthesis of (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol as claimed in claim 1, wherein: the introduced strain is BL21(DE 3).

6. The method for the biosynthesis of (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol as claimed in claim 1, wherein: in the secretion and expression of the strain, the culture solution is an LB culture medium.

7. (S) -2-chloro-1- (3, 4-difluorophenyl) as claimed in claim 1The biosynthesis method of ethanol is characterized by comprising the following steps: in the secretion and expression of the strain, induction culture is also included, when OD is in the culture medium₆₀₀After 1.0, 0.2 miptg was added and the temperature was maintained at 30 ℃.

8. The method for the biosynthesis of (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol as claimed in claim 1, wherein: in the reaction, the water/organic two-phase system is a water/organic two-way system formed by mutually dissolving 40% of water and 60% of ethanol by mass fraction.

9. The method for the biosynthesis of (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol as claimed in claim 1, wherein: in the reaction, the reaction system also comprises NADP.

10. The process for the biosynthesis of (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol according to claim 9, characterized in that: the concentration of NADP is maintained at 0.05-1.0 mM.

Technical Field

The invention relates to the technical field of biosynthesis, in particular to a biosynthesis method of (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol.

Background

Ticagrelor (also known as ticagrelor) is a new generation of anticoagulant drug developed by astrazepam, is used for antithrombotic treatment after acute coronary syndrome and stent placement, and is approved by FDA in the united states for marketing in 2011. Compared with the current first-line clinical medicine clopidogrel, ticagrelor has the advantages that the ticagrelor can directly take effect after being taken, and the platelet function can be quickly recovered and the cardiovascular death and myocardial infarction can be reduced after the medicine is stopped, so that the clopidogrel has good market prospect. The synthesis route of the drug is reported in the U.S. Pat. No. 5,7250419, and an intermediate 10 (shown in the following figure) is one of three key intermediates for synthesizing ticagrelor, and has high economic value. As described in US7067663, intermediate 10 can be synthesized by asymmetric cyclopropanation of menthol as a chiral prosthetic group, but at high cost. There is also a patent (EP2589587a1) which reports a process for the preparation of intermediate 10 from nitroketones, with ketoreductases as catalysts, but with low product yields and chirality values of only 80%. Another route reported in EP2589587a1 is to obtain (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol (chiral chlorohydrin) by CBS catalytic reduction, but the chiral value is only 90-93%, repeated purifications are required, and the catalyst is expensive. CN109112166A reports that the use of a bio-enzyme catalyst instead of CBS for synthesizing chiral chlorohydrin has mild reaction and reduced cost, but the concentration of a substrate is still not high enough (50-100 g/L), the reaction time is long, and the chiral value still needs to be improved (generally, the chiral product needs to be more than or equal to 99.5%). In summary, there is a need in the art for a less costly and more selective method for synthesizing chiral chlorohydrins.

Disclosure of Invention

This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.

The invention is proposed in view of the above and/or problems existing in the existing ticagrelor intermediate preparation methods.

Therefore, one of the purposes of the present invention is to overcome the defects of the existing ticagrelor intermediate preparation method, and provide a biosynthesis method of (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol.

To solve the above technical problem, according to an aspect of the present invention, the present invention provides the following technical solutions: a method for the biosynthesis of (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol comprising the steps of:

carrying out mutation treatment on ketoreductase wild type genes: performing mutation treatment on the ketoreductase gene and the gene sequence of the ketoreductase as shown in Seq ID No.1 to obtain a ketoreductase mutant gene, wherein the gene sequence of the ketoreductase mutant is shown in Seq ID No. 2.

Adding enzyme cutting sites: inserting double enzyme cutting sites into a ketoreductase mutant gene;

preparing a recombinant plasmid: inserting the ketoreductase mutant gene into an expression vector to obtain a recombinant plasmid;

introducing a strain: introducing the recombinant plasmid with the ketoreductase gene into a strain to obtain a recombinant expression strain;

secretion and expression of the strains: inducing the recombinant expression strain in a culture solution for expression and then collecting an enzyme solution;

reaction: the enzyme solution is put into a water/organic biphase system to react to prepare (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol.

The present invention provides a method for biosynthesis of (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol, wherein: in the mutation treatment of the ketoreductase wild-type gene, the mutation treatment is carried out by error-prone PCR.

The present invention provides a method for biosynthesis of (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol, wherein: among the added restriction sites, the double restriction sites were NdeI/HindIII.

The present invention provides a method for biosynthesis of (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol, wherein: in preparing the recombinant plasmid, the expression vector was pET21a (+) 22.

The present invention provides a method for biosynthesis of (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol, wherein: introduced into a strain BL21(DE 3).

The present invention provides a method for biosynthesis of (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol, wherein: in the secretion and expression of the strain, the culture solution is an LB culture medium.

The present invention provides a method for biosynthesis of (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol, wherein: in the secretion and expression of the strain, induction culture is also included, when OD is in the culture medium₆₀₀After 1.0, 0.2mM IPTG was added and the temperature was maintained at 30 ℃.

The present invention provides a method for biosynthesis of (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol, wherein: in the reaction, the water/organic two-phase system is a water/organic two-way system formed by mutually dissolving 40% of water and 60% of ethanol by mass fraction.

The present invention provides a method for biosynthesis of (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol, wherein: in the reaction, NADP is also included in the reaction system.

The present invention provides a method for biosynthesis of (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol, wherein: the concentration of NADP is maintained at 0.05-1.0 mM.

The invention provides a biosynthesis method of (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol, which has the advantages of lower cost and better selectivity.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:

fig. 1 is a process diagram of synthesizing a key chiral intermediate (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol of ticagrelor in the present invention.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below with reference to examples of the specification.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.

Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

Example 1

The specific principle of the design of the invention is that ketoreductase mutator is used for synthesizing ticagrelor key chiral neutral (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol, the specific reaction process is shown in figure 1, the ketoreductase mutator is derived from wild ketomutator, and the specific synthesis method comprises the following steps: the method comprises the steps of adding enzyme cutting sites NdeI/HindIII into a ketoreductase wild gene shown as Seq ID No.1 or a ketoreductase mutant gene shown as Seq ID No.2 at two points, carrying out DNA enzyme cutting purification, inserting the product into an expression vector pET21a (+)22 to obtain a recombinant plasmid, and transforming the recombinant plasmid into BL21(DE3) to construct a recombinant expression strain. Culturing the recombinant strain in LB culture medium to OD₆₀₀After 1.0, 0.2mM IPTG was added and induced culture was carried out at 30 ℃ for 4 to 5 hours, and the cells were collected by centrifugation and disrupted by sonication to give a crude enzyme solution of wild type or mutant. Adding the crude enzyme solution into a water/organic two-way system in which 40% of water and 60% of ethanol are mutually soluble in mass fraction, and simultaneously maintaining the NADP concentration of 0.05-1.0 mM in the system for reaction.

Example 2

The preparation method of the wild type keto mutant comprises the following steps:

as shown in Seq ID No.1, a ketoreductase wild gene sequence of Saccharomyces cerevisiae (Saccharomyces cerevisiae) is introduced into random mutation and/or introduced point saturation mutation based on enzyme protein structure/simulated substrate docking in an error-prone PCR mode, and then high-throughput screening is carried out to obtain a wild type ketomutator shown in Seq ID No.2 in the invention.

A protein sequence obtained by transcription and translation of a ketoreductase wild gene of Saccharomyces cerevisiae (Saccharomyces cerevisiae) is shown as Seq ID No.3, a protein sequence obtained by translation of a wild type ketomutant is shown as Seq ID No.4, compared with the ketoreductase wild gene, the wild type ketomutant has the difference that phenylalanine at position 87 is mutated into tyrosine, cysteine at position 130 is mutated into threonine or leucine or methionine, isoleucine at position 134 is mutated into methionine, tyrosine at position 165 is mutated into isoleucine, lysine at position 171 is mutated into arginine or histidine, leucine at position 198 is mutated into tryptophan or histidine, and Seq ID No.4 shows the possibility of one protein sequence, but the possibility of mutation of other various protein sequences has the same performance.

Except for Seq ID No.2, the wild type ketone group mutant gene is changed into TAT at 259-261, CTA or ATG at 388-390, ATG at 400-402, ATT at 498-599, CGT or CAT at 211-513, TGG or CAT at 598-600, and any change of any site is also included.

Example 3

Adding a ketoreductase mutant enzyme solution obtained by induced culture into a water/organic phase two-phase system, rechecking the generation process in the example 1 under the assistance of a high-efficiency coenzyme regeneration system consisting of glucose dehydrogenase/glucose, and measuring the obtained substrate conversion rate and the product chiral rate, wherein the obtained substrate conversion rate is more than 99.5 percent after multiple experiments, the highest conversion rate is 99.9 percent, the chiral value of the product is more than 99.5 percent after multiple experiments, and the percentage of the highest chiral value is more than 99.5 percent.

Example 4

The substrate conversion during the reaction was measured as follows: taking 200 microliter to 800 microliter of solvent from the reaction solution, oscillating, mixing, centrifuging, continuously diluting the supernatant in acetonitrile, and carrying out sample injection analysis. Using Agilent 5TC-C18 (250X 4.6mm), the detection wavelength was 264nm, the flow rate was 1.0mL/min, the column temperature was 15 ℃, and the mobile phase was acetonitrile: water 45: 55 (V/V). The substrate peak time was 8.20 minutes and the product peak time was 11.63 minutes. The chiral value of the product was measured as follows: the product was analyzed by making a 0.1mg/ml sample with mobile phase, using CHIRALPAK IA (150X 4.6mm, 5 μm) chiral column, measuring wavelength 264nm, flow rate 1.0ml/min, column temperature 15 ℃, mobile phase n-hexane: ethanol 98: 2 (V/V). The R-isomer peak time was 13.52 minutes and the S-isomer peak time was 11.73 minutes.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

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