CN113637713B - Method for preparing chiral 2-chloro-3, 4-difluorophenethyl alcohol - Google Patents

Method for preparing chiral 2-chloro-3, 4-difluorophenethyl alcohol Download PDF

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CN113637713B
CN113637713B CN202110939306.3A CN202110939306A CN113637713B CN 113637713 B CN113637713 B CN 113637713B CN 202110939306 A CN202110939306 A CN 202110939306A CN 113637713 B CN113637713 B CN 113637713B
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CN113637713A (en
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周硕
赖敦岳
王瑞玲
张双玲
陈振明
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Hangzhou Meiyi Biotechnology Co ltd
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract

The invention discloses a method for preparing chiral 2-chloro-3, 4-difluorophenethyl alcohol, which takes 2-chloro-3, 4-difluoroacetophenone as a substrate, and utilizes ketoreductase to catalyze and reduce the substrate to generate chiral 2-chloro-3, 4-difluorophenethyl alcohol, wherein the amino acid sequence of the ketoreductase is shown as SEQ ID NO. 9. The method takes the cheap and easily available 2-chloro-3, 4-difluoroacetophenone as a substrate, adopts biocatalyst ketoreductase to carry out asymmetric reduction reaction, obtains the 2-chloro-3, 4-difluorophenethyl alcohol with high chiral purity, has high yield, mild reaction conditions and simple and convenient operation, avoids the problems of harsh reaction conditions, complex catalyst preparation, high cost, flammability, insufficient chiral purity of products and the like in a chemical reduction method, and has good practical industrial application value.

Description

Method for preparing chiral 2-chloro-3, 4-difluorophenethyl alcohol
Technical Field
The invention relates to the technical field of biopharmaceuticals and biochemical engineering, in particular to a method for preparing chiral 2-chloro-3, 4-difluorophenethyl alcohol.
Background
Ticagrelor (Ticagrenor) is a novel small molecule anticoagulant developed by the company AstraZeneca, america. The medicine has obvious inhibition effect on platelet aggregation caused by ADP, and has rapid effect after oral administration, thereby effectively improving symptoms of patients with acute coronary heart disease. In the synthetic route of many ticagrelor (WO 2008018822, WO 2013150495), chiral 2-chloro-3, 4-difluorophenethyl alcohol (3) is a key intermediate. For example, one of the synthetic routes is as follows (journal of the Chinese medical industry, 2014, 45.4:315-321):
in the prior art, chiral 2-chloro-3, 4-difluorophenethyl alcohol [ (S) -3] is synthesized by chemical asymmetric reduction of 2-chloro-3, 4-difluoroacetophenone, and the method is often limited by factors such as insufficient optical purity of a product, low yield, complex preparation of a catalyst, high cost, combustibility and the like, so that the method is unfavorable for practical production and utilization.
The production of chiral secondary alcohol by using biocatalysis to realize reduction reaction is an efficient green catalysis technology. Through literature search, there is no example of a biological enzyme catalyzing the reduction reaction. Therefore, the ketoreductase capable of efficiently catalyzing the target reaction is obtained through wide screening, and the green production process of chiral 2-chloro-3, 4-difluorophenethyl alcohol with high yield and simple operation is established according to the ketoreductase, so that the ketoreductase has important significance.
Disclosure of Invention
The invention provides a method for preparing chiral 2-chloro-3, 4-difluorophenethyl alcohol, which has high product yield, good optical selectivity and environmental friendliness compared with the existing chemical synthesis process.
A method for preparing chiral 2-chloro-3, 4-difluorophenethyl alcohol uses 2-chloro-3, 4-difluoroacetophenone as a substrate, and utilizes Ketoreductase (KRED) to catalyze and reduce the substrate to generate chiral 2-chloro-3, 4-difluorophenethyl alcohol, wherein the amino acid sequence of the ketoreductase is shown as SEQ ID NO. 9.
The synthetic route of the reaction is as follows:
the amino acid sequences of the above ketoreductases can be prepared by commercial total gene synthesis.
The obtained product 2-chloro-3, 4-difluorophenethyl alcohol has chirality. When the amino acid sequence of the ketoreductase is shown as SEQ ID NO.9, 2-chloro-3, 4-difluorophenethyl alcohol generated by catalytic reduction of a substrate by the ketoreductase is mainly of an S type.
The ketoreductase can be prepared by adopting the conventional technical means in the field, and specifically comprises the following steps: connecting a gene fragment containing a ketoreductase gene with an enzyme digestion product of a pET28a plasmid, and transferring the gene fragment into competent E.coli BL21 (DE 3) to obtain a transformed recombinant; and (3) carrying out induced expression on the recombinants, crushing cells, centrifuging to obtain ketoreductase, and freeze-drying to obtain ketoreductase enzyme powder.
In the prior art, chiral 2-chloro-3, 4-difluorophenethyl alcohol is prepared by adopting a chemical synthesis method, no relevant report on biocatalysis preparation exists, and the invention discovers that ketoreductase with an amino acid sequence shown as SEQ ID NO.9 can catalyze 2-chloro-3, 4-difluoroacetophenone to reduce into chiral 2-chloro-3, 4-difluorophenethyl alcohol.
Preferably, the mass percentage concentration of the substrate in the reaction system at the beginning of the reaction is 1-25% (w/v), more preferably 1-20%; the amount of the ketoreductase to be used is 1 to 30% by mass of the substrate, more preferably 5 to 20%.
Preferably, the temperature of the reaction is 10 to 45 ℃, more preferably 20 to 30 ℃; the reaction time is 20-25 h.
Preferably, the pH of the reaction solution is 6.0 to 10.0; more preferably, the pH is 7.0 to 8.0.
The reaction system also comprises cofactors and a regeneration system thereof; the cofactor is NAD + NADH or NADP + NADPH; the cofactor regeneration system is capable of oxidizing the cofactor NAD + /NADP + A process of conversion to the reduced cofactor NADH/NADPH.
Preferably, the cofactor is used in an amount of 0.02% to 2% of the mass of the substrate.
After the reaction is finished, the reaction liquid is subjected to post-treatment to obtain a finished product, wherein the post-treatment is as follows: adding diatomite into the reaction liquid, filtering, extracting filtrate by adopting normal hexane, and washing and concentrating an obtained organic phase to obtain the finished chiral 2-chloro-3, 4-difluorophenethyl alcohol.
Compared with the prior art, the invention has the following beneficial effects:
the invention uses 2-chloro-3, 4-difluoroacetophenone as a substrate, adopts specific ketoreductase to carry out asymmetric reduction reaction, obtains the 2-chloro-3, 4-difluorophenethyl alcohol with high yield and chiral purity, has mild reaction conditions and simple and convenient operation, avoids the problems of harsh reaction conditions, complex catalyst preparation, high cost, inflammability, insufficient chiral purity of products and the like in the existing pure chemical synthesis method, and has good practical industrial application value.
Drawings
FIG. 1 is an HPLC chart of a sample obtained by sampling after 0h of reaction in example 3 of the present invention.
FIG. 2 is an HPLC chart of a sample obtained by sampling after 20 hours of reaction in example 3 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples, but the present invention is not limited to the following examples.
Example 1 Process for the shake flask production of Ketoreductase (KRED) enzyme powder
The gene fragment encoding the amino acid sequence shown in SEQ ID No.9 (synthesized by Shanghai JieRui bioengineering Co., ltd.) was ligated with the digested product of pET28a plasmid, transferred into competent E.coli BL21 (DE 3) strain, screened to obtain positive clones, inoculated into 4mL ampicillin-resistant liquid LB medium and activated overnight (37 ℃ C., 200 rpm).
The overnight cultures were transferred to 100mL of ampicillin-antibody-containing liquid LB medium at an inoculum size of 1/100, cultured with shaking at 37℃and 200rpm until OD600 reached 0.6, and IPTG was added to continue the culture overnight at 30 ℃. Cells were collected by centrifugation and suspended in 10mL of phosphate buffer (2 mM, pH 7.0). Placing the cell suspension in ice bath, ultrasonic crushing for 10 min, centrifuging, pre-freezing supernatant overnight, and lyophilizing for 36h to obtain lyophilized powder Ketoreductase (KRED) enzyme powder.
EXAMPLE 2A ketoreductase (amino acid sequence shown in SEQ ID NO. 9) milligram-scale reaction
In a 5mL reaction flask, 10mg of 2-chloro-3, 4-difluoroacetophenone as a substrate and 0.1mL of isopropanol were added, and after the substrate was completely dissolved, 1.2mL of TEA-HCl buffer (0.1M, pH 7.0) and 0.1mg of NAD were added + 、0.1mgNADP + (in 0.1mL of buffer), 20mg of glucose and 2mg of glucose dehydrogenase were added, and finally, 2mg of the ketoreductase KRED enzyme powder (0.5U/mg, in 0.1mL of buffer) obtained in example 1 was added, respectively, and reacted at 30℃for 20 hours by shaking.
The reaction products were analyzed by HPLC, and the yields and ee values of the products were measured, and the results are shown in Table 1:
TABLE 1 yields and ee values of the products of ketoreductase reactions
Yield labeling indicates: + represents the yield of 1% -20%, ++ represents the yield of 20% -50%, +++ represents yield 50% -80%, +++++ represents the yield is 80-95%, yield 80% 95%;
ee value labeling indicates: the ee value refers to the enantiomeric excess (enantiomeric excess) of the product 2-chloro-3, 4-difluorophenethyl alcohol as follows: ee (ee) S ＝(C S-3 -C R-3 )/(C S-3 +C R-3 )*100％，C S-3 Refers to the concentration of S-2-chloro-3, 4-difluorophenethyl alcohol, C in a sample R-3 Refers to the concentration of R-2-chloro-3, 4-difluorophenethyl alcohol in the sample.
+ represents ee value <50%, ++ represents ee value 50% -80%, +++ stands for ee the value is 80% -90%, ++++ represents ee the value is 90% -99%, value 90% -99%.
Example 3 hundred milligram grade preparation Process
In a 5mL reaction flask, 150mg of 2-chloro-3, 4-difluoroacetophenone as a substrate and 0.3mL of isopropanol were added, and after the substrate was completely dissolved, 1.2mL of TEA-HCl buffer (0.1M, pH 7.0) and 7.5mg of ketoreductase powder (amino acid sequence shown in SEQ ID NO.9 were addedDissolved in 0.1mL buffer), 0.75mgNAD + (in 0.1mL buffer), the reaction was magnetically stirred at 30deg.C, setting two reaction times: 0h and 20h.
Samples were taken after the reaction and the products were analyzed by HPLC, the results are shown in fig. 1 and 2;
FIG. 1 shows the results of analysis of the product obtained after 0 hours of reaction, the 7.67 minute substance being the substrate;
FIG. 2 shows the results of analysis of the product obtained after 20 hours of reaction, in which the material with a retention time of 6.47 minutes was the product, with a final conversion of greater than 99.5%.
Example 4 reactions at different temperatures and pH
150mg of substrate 2-chloro-3, 4-difluoroacetophenone and 0.3mL of isopropanol are added into a 5mL reaction bottle, and after the substrate is completely dissolved, 1.2mL of TEA-HCl buffer solution with different pH values (6.0-10.0) and 7.5mg of ketoreductase powder (the amino acid sequence is shown as SEQ ID NO.9 and dissolved in 0.1mL of buffer solution) and 0.75mg of NAD are respectively added + (dissolved in 0.1mL buffer solution) and reacted for 20h under magnetic stirring at different temperatures (10-45 ℃). Samples were taken after the reaction and the product was analyzed by HPLC. The results of each set of yields are shown in the following table:
TABLE 2 yields of reactions at different temperatures and pH
From the table above, it can be seen that: the temperature of the reaction system is 10 to 45 ℃, preferably 20 to 30 ℃. The pH of the reaction solution is 6.0 to 10.0, preferably 7.0 to 8.0.
Example 5 reactions with different enzyme and coenzyme amounts
150mg of substrate 2-chloro-3, 4-difluoroacetophenone and 0.3mL of isopropanol are added into a 5mL reaction bottle, after the substrate is completely dissolved, 1.2mL of 0.1M TEA-HCl buffer solution with pH of 7.0 is added, different amounts (1-30% of the substrate amount) of ketoreductase powder (the amino acid sequence is shown as SEQ ID NO.9 and dissolved in 0.1mL buffer solution) are respectively added, and different amounts (0.02% -2% of the substrate amount) of NAD are respectively added + (in 0.1mL buffer), inThe reaction was carried out for 20h with magnetic stirring at 30 ℃. Samples were taken after the reaction and the product was analyzed by HPLC. The results of each set of yields are shown in the following table:
TABLE 3 reaction yields at different enzyme and coenzyme levels
From the table above, it can be seen that: in the reaction system, the dosage of the ketoreductase is 1-30% of the mass of the substrate, preferably 5-20%. The consumption of the cofactor is 0.02% -2% of the mass of the substrate.
EXAMPLE 6 reaction at different substrate concentrations
In a 5mL reaction flask, adding (15-375 mg) substrate 2-chloro-3, 4-difluoroacetophenone and 0.3mL isopropanol respectively, after the substrate is completely dissolved, adding 1.2mL of 0.1M TEA-HCl buffer solution with pH of 7.0, adding different amounts of ketoreductase powder (amino acid sequence shown as SEQ ID NO.9 and dissolved in 0.1mL buffer solution) according to 5% of the substrate amount, and adding 0.75mg NAD + (in 0.1mL buffer) and was reacted for 20 hours under magnetic stirring at 30 ℃. Samples were taken after the reaction and the product was analyzed by HPLC. The results of each set of yields are shown in the following table:
TABLE 4 reaction yield at no substrate concentration
From the table above, it can be seen that: the mass percentage concentration of the substrate in the reaction system is 1% -25% (w/v), preferably 1% -20%.
Example 7 hundred gram grade preparation Process
450g of substrate 2-chloro-3, 4-difluoroacetophenone is added into a 20L reaction kettle, 1.5L isopropanol is added, and the mixture is stirred until the mixture is completely dissolved; 8.5L Tris-HCl buffer (0.1M, pH 7.0) was added thereto, and stirred at 30℃uniformly (450 rpm); then 22.5g ketoreductase powder (amino acid sequence shown as SEQ ID NO. 9) and 2.25g NAD are added in turn + After that, the reaction was carried out. After 24h of reaction, the conversion of the product was 99.3% monitored by HPLC, and the reaction was terminated.
50g of diatomite is added into the reaction solution and stirred for 0.5h, the mixture is filtered, the filtrate is extracted by n-hexane (3.0L multiplied by 2), a filter cake returns to the reaction kettle, the n-hexane is added, stirred and filtered, and the organic phases are combined; the organic phase was washed twice with saturated brine and concentrated to give 0.37kg of 2-chloro-3, 4-difluorophenethyl alcohol in a yield of 81.4%, GC purity of 98.7%, ee s The value was 99.5%.
Example 8 kilogram level preparation Process
1.35kg of substrate 2-chloro-3, 4-difluoroacetophenone is added into a 20L reaction kettle, 3L isopropanol is added, and stirring is carried out until complete dissolution; 7.0L Tris-HC buffer (0.1M, pH 7.0) was added thereto, and stirred at 30℃uniformly (450 rpm); then 67.5g ketoreductase powder (amino acid sequence shown as SEQ ID NO. 9) and 6.75g NAD are added in turn + After that, the reaction was carried out. After 24h of reaction, the conversion of the product was 99.2% by HPLC and the reaction was terminated.
150g of diatomite is added into the reaction solution and stirred for 0.5h, the mixture is filtered, the filtrate is extracted by n-hexane (9.0L multiplied by 2), the filter cake returns to the reaction kettle, the n-hexane is added, stirred and filtered, and the organic phases are combined; the organic phase was washed twice with saturated brine, and concentrated to give 1.09kg of 2-chloro-3, 4-difluorophenethyl alcohol in 80.1% yield, 99.2% GC purity and ee s The value was 99.5%.
Comparative example 1
In a 5mL reaction flask, 10mg of 2-chloro-3, 4-difluoroacetophenone as a substrate and 0.1mL of isopropanol were added, and after the substrate was completely dissolved, 1.2mL of TEA-HCl buffer (0.1M, pH 7.0) and 0.1mg of NAD were added + 、0.1mgNADP + (in 0.1mL buffer solution), and finally, 2mg of ketoreductase KRED enzyme powder (the amino acid sequence is shown as SEQ ID NO.22, and dissolved in 0.1mL buffer solution) is added respectively, and the mixture is subjected to shaking reaction for 20h at 30 ℃.
HPLC analysis is carried out on the product after the reaction, and the yield and ee value of the product are detected, so that no 2-chloro-3, 4-difluorophenethyl alcohol is produced.
Comparative example 2
In a 5mL reaction flask, 10mg of 2-chloro-3, 4-difluoroacetophenone as a substrate and 0.1mL of isopropanol were added, and after the substrate was completely dissolved, 1.2mL of TEA-HCl buffer (0.1M, pH 7.0) and 0.1mg of NAD were added + 、0.1mgNADP + (in 0.1mL buffer), and finally, 2mg of ketoreductase KRED enzyme powder (the amino acid sequence is shown as SEQ ID NO.23, and dissolved in 0.1mL buffer) was added, and the mixture was subjected to shaking reaction at 30℃for 20 hours.
HPLC analysis is carried out on the product after the reaction, and the yield and ee value of the product are detected, so that no 2-chloro-3, 4-difluorophenethyl alcohol is produced.
The above embodiments are provided to illustrate the technical concept and features of the present invention and are intended to enable those skilled in the art to understand the content of the present invention and implement the same, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made in accordance with the spirit of the present invention should be construed to be included in the scope of the present invention.
Sequence listing
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Claims (2)

1. A method for preparing S-type chiral 2-chloro-3, 4-difluorophenethyl alcohol is characterized in that 2-chloro-3, 4-difluoroacetophenone is used as a substrate, ketoreductase is utilized to catalyze and reduce the substrate, the S-type chiral 2-chloro-3, 4-difluorophenethyl alcohol is generated by reaction, and the amino acid sequence of the ketoreductase is shown as SEQ ID NO. 9;
the reaction temperature is 30 ℃, and the pH value of the reaction solution is 6.0-8.0; in a reaction system at the beginning of the reaction, the mass percentage concentration of the substrate is 1% -20%; the dosage of the ketoreductase is 5% -20% of the mass of the substrate; the reaction time is 20-25 h;
the reaction system also comprises a cofactor and a regeneration system thereof, wherein the cofactor is NAD + NADH or NADP + NADPH; the consumption of the cofactor is 0.02% -2% of the mass of the substrate.
2. The method of claim 1, wherein the pH of the reaction solution is 7.0 to 8.0.
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