Method for preparing chiral 2-chloro-3, 4-difluorophenethyl alcohol
阅读说明：本技术 一种制备手性2-氯-3,4-二氟苯乙醇的方法 (Method for preparing chiral 2-chloro-3, 4-difluorophenethyl alcohol ) 是由 周硕 赖敦岳 王瑞玲 张双玲 陈振明 于 2015-12-09 设计创作，主要内容包括：本发明公开了一种制备手性2-氯-3,4-二氟苯乙醇的方法,该方法以2-氯-3,4-二氟苯乙酮为底物,利用酮还原酶催化还原底物,生成手性2-氯-3,4-二氟苯乙醇,所述酮还原酶的氨基酸序列如SEQ ID NO.9所示。本发明以价廉易得的2-氯-3,4-二氟苯乙酮为底物,采用生物催化剂酮还原酶进行不对称还原反应,获得手性纯度高的2-氯-3,4-二氟苯乙醇,得率高、反应条件温和、操作简便,避免了化学还原法存在的反应条件苛刻、催化剂制备复杂、成本高、易燃和产物手性纯度不够高等问题,具有良好的实际工业应用价值。(The invention discloses a method for preparing chiral 2-chloro-3, 4-difluorophenyl ethanol, which takes 2-chloro-3, 4-difluorophenyl acetophenone as a substrate and utilizes ketoreductase to catalyze and reduce the substrate to generate the chiral 2-chloro-3, 4-difluorophenyl ethanol, wherein the amino acid sequence of the ketoreductase is shown as SEQ ID No. 9. The method takes cheap and easily-obtained 2-chloro-3, 4-difluoroacetophenone as a substrate, adopts a biocatalyst ketoreductase to carry out asymmetric reduction reaction to obtain 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 the product and the like in a chemical reduction method, and has good practical industrial application value.)
1. The method for preparing the chiral 2-chloro-3, 4-difluorophenethyl alcohol is characterized in that 2-chloro-3, 4-difluoroacetophenone is used as a substrate, the substrate is catalyzed and reduced by a ketoreductase, and the chiral 2-chloro-3, 4-difluorophenethyl alcohol is generated by reaction, wherein the amino acid sequence of the ketoreductase is shown as SEQ ID No. 9.
2. The method of claim 1, wherein the mass percent concentration of the substrate in the reaction system at the beginning of the reaction is 1% to 25%; the dosage of the ketoreductase is 1-30% of the mass of the substrate.
3. The method according to claim 2, wherein the substrate is present in the reaction system at the start of the reaction at a concentration of 1 to 20% by mass.
4. The method according to claim 2, wherein the ketoreductase is used in an amount of 5% to 20% by mass based on the mass of the substrate in the reaction system at the time of initiation of the reaction.
5. The process of claim 1, wherein the temperature of the reaction is from 10 ℃ to 45 ℃.
6. The process according to claim 5, wherein the reaction temperature is 20 ℃ to 30 ℃ and the reaction time is 20 hours to 25 hours.
7. The method according to claim 1, wherein the pH of the reaction solution is 6.0 to 10.0.
8. The method according to claim 7, wherein the pH of the reaction solution is 7.0 to 8.0.
9. The method of claim 1, wherein the reaction system further comprises a cofactor and a regeneration system thereof; the cofactor is NAD+NADH or NADP+/NADPH。
10. The method of claim 1, wherein the cofactor is present in an amount of from 0.02% to 2% by mass of the substrate.
Technical Field
The invention relates to the technical field of biological pharmacy and biochemical engineering, in particular to a method for preparing chiral 2-chloro-3, 4-difluorophenethyl alcohol.
Background
Ticagrelor (Ticagrelor) is a novel small molecule anticoagulant drug developed by AstraZeneca (AstraZeneca) corporation, usa. The medicine has obvious inhibition effect on platelet aggregation caused by ADP, and has quick effect after oral administration, thereby effectively improving the symptoms of patients with acute coronary heart disease. In the synthesis routes of ticagrelor (WO2008018822, WO2013150495), chiral 2-chloro-3, 4-difluorophenethyl alcohol (3) is a key intermediate. For example, one of the synthetic routes is as follows (journal of Chinese pharmaceutical industry, 2014, 45.4: 315-):
in the prior art, chiral 2-chloro-3, 4-difluorophenylethanol [ (S) -3] is synthesized by chemically asymmetric reduction of 2-chloro-3, 4-difluoroacetophenone, and the method is limited by factors such as insufficient optical purity, low yield, complex catalyst preparation, high cost, flammability and the like, so that the method is not favorable for practical production and utilization.
The method for producing the chiral secondary alcohol by utilizing the biological catalysis to realize the reduction reaction is an efficient green catalysis technology. There are no examples of bio-enzymes catalyzing this reduction reaction by literature search. Therefore, the ketoreductase which can efficiently catalyze the target reaction is obtained through wide screening, and the green production process of the chiral 2-chloro-3, 4-difluorophenethyl alcohol with high yield and simple operation is established according to the ketoreductase, so that the method has important significance.
Disclosure of Invention
The invention provides a method for preparing chiral 2-chloro-3, 4-difluorophenethyl alcohol, which has the advantages of 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-difluorophenylethanol is characterized in that 2-chloro-3, 4-difluoroacetophenone is used as a substrate, and the substrate is catalytically reduced by Ketoreductase (KRED) to generate the chiral 2-chloro-3, 4-difluorophenylethanol, 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 ketoreductases described above can all 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, the 2-chloro-3, 4-difluorophenethyl alcohol generated by catalytic reduction of a substrate by the ketoreductase is mainly S-shaped.
The ketoreductase can be prepared by adopting the conventional technical means in the field, and specifically comprises the following steps: connecting the gene fragment containing the ketoreductase gene with the enzyme digestion product of pET28a plasmid, and transferring the gene fragment into competent Escherichia coli E.coli BL21(DE3) to obtain a converted recombinant; and performing induction expression on the recombinant, 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 a chemical synthesis method, and no report related to biocatalytic preparation exists, but the invention discovers that ketoreductase with an amino acid sequence shown as SEQ ID NO.9 can catalyze 2-chloro-3, 4-difluoroacetophenone to be reduced into chiral 2-chloro-3, 4-difluorophenethyl alcohol.
Preferably, in the reaction system at the beginning of the reaction, the mass percentage concentration of the substrate is 1-25% (w/v), more preferably, 1-20%; the dosage of the ketoreductase is 1-30% of the mass of the substrate, and more preferably 5-20%.
Preferably, the reaction temperature is 10-45 ℃, and more preferably, 20-30 ℃; the reaction time is 20-25 h.
Preferably, the pH value of the reaction solution is 6.0-10.0; more preferably, the pH value is 7.0 to 8.0.
The reaction system also comprises a cofactor and a regeneration system thereof; the cofactor is NAD+NADH or NADP+NADPH; the cofactor regeneration system is capable of regenerating the oxidized cofactor NAD+/NADP+Conversion to the reduced cofactor NADH/NADPH.
Preferably, the dosage of the cofactor is 0.02-2% of the mass of the substrate.
After the reaction is finished, the reaction solution needs to be subjected to post-treatment to obtain a finished product, wherein the post-treatment comprises the following steps: adding diatomite into the reaction solution, filtering, extracting the filtrate by adopting normal hexane, washing and concentrating the obtained organic phase to obtain the finished product of chiral 2-chloro-3, 4-difluorophenethyl alcohol.
Compared with the prior art, the invention has the following beneficial effects:
the method takes 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 high chiral purity, has mild reaction conditions and simple and convenient operation, avoids the problems of harsh reaction conditions, complex catalyst preparation, high cost, flammability, low chiral purity of the product and the like existing in the existing pure chemical synthesis method, and has good practical industrial application value.
Drawings
FIG. 1 is an HPLC chromatogram of a sample taken after 0h of reaction in example 3 of the present invention.
FIG. 2 is an HPLC chromatogram of a sample taken 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 specific examples, but the present invention is not limited to the following examples.
EXAMPLE 1 Shake flask production Process for Ketoreductase (KRED) enzyme powder
A gene fragment (synthesized by Shanghai Czeri bioengineering Co., Ltd.) encoding the amino acid sequence shown in SEQ ID NO.9 was ligated with the enzyme-digested product of pET28a plasmid, transferred into a competent E.coli BL21(DE3) strain, screened to obtain positive clones, and inoculated into 4mL of ampicillin-resistant liquid LB medium for activation overnight (37 ℃, 200 rpm).
The overnight culture was inoculated into 100mL of a liquid LB medium containing an ampicillin antibody at 1/100, and the medium was cultured with shaking at 200rpm at 37 ℃ until the OD600 reached 0.6, and IPTG was added to continue the culture at 30 ℃ overnight. The cells were collected by centrifugation, and the cells were suspended in 10mL of a phosphate buffer (2mM, pH 7.0). And (3) ultrasonically crushing the cell suspension in an ice bath for 10 minutes, centrifuging, pre-freezing the supernatant overnight, and freeze-drying for 36 hours to obtain freeze-dried powder of Ketoreductase (KRED) enzyme powder.
Example 2 milligram-scale reaction of ketoreductase (amino acid sequence shown in SEQ ID NO. 9)
In a 5mL reaction flask, 10mg of substrate 2-chloro-3, 4-difluoroacetophenone and 0.1mL of isopropanol were added, and after the substrate was completely dissolved, 1.2mL of TEA-HCl buffer (0.1M, pH7.0), 0.1mg of NAD were added+、0.1mgNADP+(dissolved in 0.1mL of buffer), 20mg of glucose and 2mg of glucose dehydrogenase were added, and finally 2mg of ketoreductase KRED enzyme powder obtained in example 1 (0.5U/mg in 0.1mL of buffer) was added, respectively, and the mixture was subjected to shake reaction at 30 ℃ for 20 hours.
The product after reaction was taken out for HPLC analysis, and the yield and ee value of the product were determined, and the results are shown in Table 1:
TABLE 1 yield and ee value of the product of the ketoreductase reaction
The yield markers indicate: + for 1% -20% yield, + for 20% -50% yield, + + for 50% -80% yield, + +++ for 80% -95% yield, + +++++ for > 95% yield;
ee value notation: the ee value is the enantiomeric excess (enantiomeric excess) of the product 2-chloro-3, 4-difluorophenylethanol, calculated according to the formula: eeS＝(CS-3-CR-3)/(CS-3+CR-3)*100％，CS-3Means the concentration of S-2-chloro-3, 4-difluorophenethyl alcohol in the sample, CR-3Refers to the concentration of R-2-chloro-3, 4-difluorophenethyl alcohol in the sample.
+ represents ee < 50%, + + represents ee 50% -80%, + + + + represents ee 80% -90%, + +++ represents ee 90% -99%, and +++++ represents ee > 99%.
EXAMPLE 3 hundred milligram scale preparation
150mg of the substrate 2-chloro-3, 4-difluoroacetophenone and 0.3mL of isopropanol were added to a 5mL reaction flask, and after the substrate was completely dissolved, 1.2mL of TEA-HCl buffer (0.1M, pH7.0), 7.5mg of ketoreductase powder (amino acid sequence shown in SEQ ID NO.9, dissolved in 0.1mL of buffer), 0.75mg of NAD were added+(dissolved in 0.1mL buffer), magnetic stirring reaction was performed at 30 ℃ setting two reaction times: 0h and 20 h.
After the reaction, samples were taken and subjected to HPLC analysis, and the results are shown in FIGS. 1 and 2;
FIG. 1 is the analysis result of the product obtained after 0 hour of the reaction, in which 7.67 minutes of the substance was the substrate;
FIG. 2 is an analysis of the product obtained after 20 hours of reaction, in which the material with a retention time of 6.47 minutes is the product and the final conversion is greater than 99.5%.
Example 4 reaction at different temperatures and pH
Adding 150mg of substrate 2-chloro-3, 4-difluoroacetophenone and 0.3mL of isopropanol into a 5mL reaction bottle, and after the substrate is completely dissolved, respectively adding 1.2mL of 0.1M TEA-HCl buffer solution with different pH values (6.0-10.0), 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+(dissolved in 0.1mL buffer solution), and reacting for 20h under magnetic stirring at different temperatures (10-45 ℃). Samples were taken after the reaction for HPLC analysis of the product. Yield results for each group are asThe following table:
TABLE 2 yield of reaction at different temperatures and pH
As can be seen from the above table: the temperature of the reaction system is 10-45 ℃, and preferably 20-30 ℃. The pH of the reaction solution is 6.0 to 10.0, preferably 7.0 to 8.0.
EXAMPLE 5 reaction with varying amounts of enzyme and coenzyme
Adding 150mg of substrate 2-chloro-3, 4-difluoroacetophenone and 0.3mL of isopropanol into a 5mL reaction bottle, adding 1.2mL0.1M TEA-HCl buffer solution with pH7.0 after the substrate is completely dissolved, respectively adding different amounts (1-30% of the substrate amount) of ketoreductase powder (the amino acid sequence is shown as SEQ ID NO.9 and is dissolved in 0.1mL of buffer solution), and respectively adding different amounts (0.02-2% of the substrate amount) of NAD+(dissolved in 0.1mL of buffer) and reacted at 30 ℃ for 20 hours with magnetic stirring. Samples were taken after the reaction for HPLC analysis of the product. The results of the various groups of yields are given in the following table:
TABLE 3 reaction yields with different enzyme and coenzyme amounts
As can be seen from the above table: in the reaction system, the dosage of the ketoreductase is 1-30% of the mass of the substrate, preferably 5-20%. The dosage of the cofactor is 0.02-2% of the mass of the substrate.
EXAMPLE 6 different substrate concentration reactions
Respectively adding 15-375 mg of substrate 2-chloro-3, 4-difluoroacetophenone and 0.3mL of isopropanol into a 5mL reaction bottle, after the substrate is completely dissolved, adding 1.2mL0.1M TEA-HCl buffer solution with pH of 7.0, adding different amounts of ketoreductase powder (amino acid sequence is shown as SEQ ID NO.9 and dissolved in 0.1mL of buffer solution) according to 5% of the substrate amount, and adding 0.75mg of NAD+(dissolved in 0.1mL of buffer) and reacted at 30 ℃ for 20 hours with magnetic stirring. After the reaction, a sample was taken and the product was subjected to HPLCAnd (6) analyzing. The results of the various groups of yields are given in the following table:
TABLE 4 reaction yield without substrate concentration
As can be seen from the above table: in the reaction system, the mass percentage concentration of the substrate is 1-25% (w/v), preferably 1-20%.
EXAMPLE 7 hectogram scale preparation
Adding 450g of substrate 2-chloro-3, 4-difluoroacetophenone into a 20L reaction kettle, adding 1.5L of isopropanol, and stirring until the substrate is completely dissolved; adding 8.5L Tris-HCl buffer (0.1M, pH7.0), and stirring (450rpm) at 30 deg.C; then, 22.5g of ketoreductase powder (amino acid sequence shown in SEQ ID NO. 9) and 2.25g of NAD were sequentially added+Thereafter, the reaction was carried out. The reaction was stopped with 99.3% conversion of the product after 24h of reaction monitored by HPLC.
Adding 50g of diatomite into the reaction solution, stirring for 0.5h, filtering, extracting the filtrate with n-hexane (3.0L multiplied by 2), returning the filter cake to the reaction kettle, adding n-hexane, stirring, filtering, and combining organic phases; the organic phase was washed twice with saturated brine and concentrated to give 0.37kg of 2-chloro-3, 4-difluorophenethyl alcohol in 81.4% yield and 98.7% GC purity in eesThe value was 99.5%.
EXAMPLE 8 kg-scale preparation Process
Adding 1.35kg of substrate 2-chloro-3, 4-difluoroacetophenone into a 20L reaction kettle, adding 3L of isopropanol, and stirring until the substrate is completely dissolved; adding 7.0L Tris-HC buffer (0.1M, pH7.0), and stirring (450rpm) at 30 deg.C; then adding 67.5g of ketoreductase powder (amino acid sequence shown as SEQ ID NO. 9) and 6.75g of NAD in sequence+Thereafter, the reaction was carried out. The reaction was terminated after 24h of reaction with 99.2% conversion of the product as monitored by HPLC.
Adding 150g of diatomite into the reaction solution, stirring for 0.5h, filtering, extracting the filtrate with n-hexane (9.0L multiplied by 2), returning the filter cake to the reaction kettle, adding n-hexane, stirring, filtering, and combining organic phases; the organic phase was washed twice with saturated brine and concentrated to give 1.09kg of 2-chloro-3, 4-difluorophenethyl etherAlcohol, yield 80.1%, GC purity 99.2%, eesThe value was 99.5%.
Comparative example 1
In a 5mL reaction flask, 10mg of substrate 2-chloro-3, 4-difluoroacetophenone and 0.1mL of isopropanol were added, and after the substrate was completely dissolved, 1.2mL of TEA-HCl buffer (0.1M, pH7.0), 0.1mg of NAD were added+、0.1mgNADP+(dissolved in 0.1mL buffer), and finally, 2mg ketoreductase KRED enzyme powder (amino acid sequence shown in SEQ ID NO.22, dissolved in 0.1mL buffer) was added, respectively, and the mixture was subjected to shake reaction at 30 ℃ for 20 hours.
HPLC analysis of the reacted product was carried out, and the yield of the product and the ee value of the product were determined, as a result, no 2-chloro-3, 4-difluorophenethyl alcohol was produced.
Comparative example 2
In a 5mL reaction flask, 10mg of substrate 2-chloro-3, 4-difluoroacetophenone and 0.1mL of isopropanol were added, and after the substrate was completely dissolved, 1.2mL of TEA-HCl buffer (0.1M, pH7.0), 0.1mg of NAD were added+、0.1mgNADP+(dissolved in 0.1mL buffer), and finally, 2mg ketoreductase KRED enzyme powder (amino acid sequence shown in SEQ ID NO.23, dissolved in 0.1mL buffer) was added, respectively, and the mixture was subjected to shake reaction at 30 ℃ for 20 hours.
HPLC analysis of the reacted product was carried out, and the yield of the product and the ee value of the product were determined, as a result, no 2-chloro-3, 4-difluorophenethyl alcohol was produced.
The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Sequence listing
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Tyr Ala Leu Lys Leu Pro Gly Ile Ile His Ile Asp Ala Ala Glu Ile
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Met Ser Asp Ser Pro Ala Glu Gly Leu Asp Leu Ala Leu Lys Lys Met
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Gly Thr Asp Tyr Val Asp Leu Tyr Leu Leu His Ser Pro Phe Val Ser
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Gln Leu Tyr Lys Ser Gly Lys Ala Lys Asn Ile Gly Val Ser Asn Phe
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Gln Val Asn Gln Ile Glu Phe Ser Pro Phe Leu Gln Asn Gln Thr Pro
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Glu Asn Asp Ala Tyr Lys Ala Val Leu Thr Ala Leu Lys Asp Gly Tyr
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Arg His Ile Asp Thr Ala Ala Ile Tyr Arg Asn Glu Asp Gln Val Gly
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Gln Ala Ile Lys Asp Ser Gly Val Pro Arg Glu Glu Ile Phe Val Thr
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Gln Ser Leu Lys Arg Leu Gly Leu Asp Tyr Val Asp Leu Tyr Leu Met
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His Trp Pro Ala Arg Leu Asp Pro Ala Tyr Ile Lys Asn Glu Asp Ile
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Leu Ser Val Pro Thr Lys Lys Asp Gly Ser Arg Ala Val Asp Ile Thr
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Leu Ile Asn Ile Thr Gln Ala Val Leu Pro Ile Phe Gln Ala Lys Asn
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Ser Gly Asp Ile Val Asn Leu Gly Ser Ile Ala Gly Arg Asp Ala Tyr
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Pro Thr Gly Ser Ile Tyr Cys Ala Ser Lys Phe Ala Val Gly Ala Phe
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Thr Asp Ser Leu Arg Lys Glu Leu Ile Asn Thr Lys Ile Arg Val Ile
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Leu Ile Ala Pro Gly Leu Val Glu Thr Glu Phe Ser Leu Val Arg Tyr
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Arg Gly Asn Glu Glu Gln Ala Lys Asn Val Tyr Lys Asp Thr Thr Pro
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Leu Met Ala Asp Asp Val Ala Asp Leu Ile Val Tyr Ala Thr Ser Arg
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Lys Gln Asn Thr Val Ile Ala Asp Thr Leu Ile Phe Pro Thr Asn Gln
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Ala Ser Pro His His Ile Phe Arg Gly
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