Method for synthesizing atazanavir intermediate chlorohydrin by continuous flow biocatalysis
阅读说明：本技术 一种连续流生物催化合成阿扎那韦中间体氯醇的方法 (Method for synthesizing atazanavir intermediate chlorohydrin by continuous flow biocatalysis ) 是由 白云鹏 钱源益 张晓彦 于 2021-06-29 设计创作，主要内容包括：本发明涉及一种连续流生物催化合成阿扎那韦中间体氯醇的方法,使用羰基还原酶催化(S)-叔丁基(4-氯-3-羰基-1-苯丁基-2-基)氨基甲酸酯不对称还原制备光学活性叔丁基((2S,3R)-4-氯-3-羟基-1-苯丁基-2-基)氨基甲酸酯,葡萄糖脱氢酶提供辅酶循环,酶促反应是在连续流微反应器中发生。与现有技术相比,本发明提供了一种连续流生物催化合成阿扎那韦中间体氯醇的方法,可在连续流微反应器中立体选择性催化(S)-叔丁基(4-氯-3-羰基-1-苯丁基-2-基)氨基甲酸酯的不对称还原,生成相应的光学活性叔丁基((2S,3R)-4-氯-3-羟基-1-苯丁基-2-基)氨基甲酸酯,反应条件温和,催化效率高,转化率高,产品光学纯度好,ee值可高于98.3％,具有很好的工业应用前景。(The invention relates to a method for synthesizing atazanavir intermediate chlorohydrin by continuous flow biocatalysis, which uses carbonyl reductase to catalyze (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate to asymmetrically reduce to prepare optically active tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate, glucose dehydrogenase provides coenzyme circulation, and enzymatic reaction is carried out in a continuous flow microreactor. Compared with the prior art, the invention provides a method for synthesizing atazanavir intermediate chlorohydrin by continuous flow biocatalysis, which can be used for stereoselectively catalyzing asymmetric reduction of (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate in a continuous flow microreactor to generate corresponding optically active tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate, and has the advantages of mild reaction conditions, high catalytic efficiency, high conversion rate, good optical purity of products, and ee value higher than 98.3%, thereby having good industrial application prospect.)
1. Application of carbonyl reductase OdCR1 in catalyzing reduction of (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate to generate corresponding tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate.
2. The use according to claim 1, wherein the carbonyl reductase OdCR1 is derived from Saccharomyces cerevisiae S288C (Saccharomyces cerevisiae S288C) deposited at the china general microbiological culture collection center of the culture collection of microorganisms under the accession number: CGMCC No.22135, the preservation time is 2021, 04 and 06 days, and the preservation place is as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
3. The use according to claim 1, characterized in that the amino acid sequence of the carbonyl reductase OdCR1 is shown in SEQ ID No. 2.
4. A continuous flow biocatalytic method for synthesizing atazanavir intermediate chlorohydrin is characterized in that (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate is catalyzed by carbonyl reductase to prepare optically active tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate through asymmetric reduction, glucose dehydrogenase provides coenzyme circulation, and enzymatic reaction is carried out in a continuous flow microreactor.
5. The continuous flow biocatalytic synthesis of atazanavir intermediate chlorohydrin according to claim 4, wherein the carbonyl reductase has the amino acid sequence shown in SEQ ID No. 2.
6. The continuous flow biocatalytic synthesis of atazanavir intermediate chlorohydrin of claim 4, wherein carbonyl reductase, glucose dehydrogenase, glucose and NADP are added+Preparing solution I, preparing tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate into solution II, continuously introducing the solution I and the solution II into a continuous flow microreactor according to a proportion for reaction, carrying out a biocatalytic reaction, and carrying out cyclizationAnd purifying to obtain tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate.
7. The continuous flow biocatalytic synthesis of atazanavir intermediate chlorohydrin according to claim 4, wherein the ratio of the activities of carbonyl reductase and glucose dehydrogenase is 1: 1.5;
the molar ratio of glucose to (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate is 1.5: 1.
8. the continuous flow biocatalytic synthesis of atazanavir intermediate chlorohydrin according to claim 4, characterized in that in phosphate buffer of pH 5.5-7.5, in glucose dehydrogenase, glucose and NADP+In the presence of the carbonyl reductase, OdCR1, catalyzes the asymmetric reduction of the (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate.
9. The method for continuous flow biocatalytic synthesis of atazanavir intermediate chlorohydrin according to claim 4, characterized in that the concentration of substrate (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate in the reaction solution is 1-2 mmol/L.
10. The method for continuous flow biocatalytic synthesis of atazanavir intermediate chlorohydrin according to claim 4, wherein the amount of carbonyl reductase is 1-500U/L.
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a method for synthesizing atazanavir intermediate chlorohydrin by continuous flow biocatalysis.
Background
Atazanavir is an antiretroviral drug of the Protease Inhibitor (PI) class, which, like other antiretroviruses, can be used to treat Human Immunodeficiency Virus (HIV) infections. The drug is a commonly used anti-HIV drug recommended by the International anti-viral Association, the American expert group, for the treatment of adult HIV infections and is considered to be an effective once daily drug for treatment of both different types of patients. Wherein the intermediate of atazanavir is tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate, a compound with two chiral centers, which is used for the production of atazanavir after cyclization. The chemical synthesis route of atazanavir is shown in figure 1.
In 1997, Chen et al described a practical method for preparing alpha-N-BOC-epoxides from protected amino acid esters. The process can be easily carried out on a large scale without the use of hazardous reagents and allows the preparation of kilogram grades of alpha-N-BOC-epoxide. In this reaction step, the chlorohydrin, an intermediate product thereof, is synthesized by a two-step chemical method of amino acid ester, the reaction conditions are relatively severe, and the reaction is carried out at-78 ℃ (Tetrahedron Lett,1997,38(18): 3175-3178). In 2009, Alanvert et al introduced a biocatalytic reduction of α -haloketones to the corresponding α -halohydrins using carbonyl reductases with high stereoselectivity. Tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate was prepared on a 10g scale for 21h with a conversion of greater than 98.5% and a de (2S,3R) > 98.5% (Tetrahedron-Asymmetry,2009,20(21): 2462-2466). In 2019, Wu et al studied the short-chain dehydrogenase NaSDR from Novosphingobium aromaticivorans, which showed excellent stereoselectivity for the substrate chlorone, but relatively low activity. Thus, the catalytic efficiency of the mutant muSDR (G141A/I195L) obtained by Iterative Saturation Mutagenesis (ISM) of the site on the NaSDR active pocket, whose k is the substrate chlorone, is significantly improvedcatIncreased to 4.11s-1Is WT (1.15 s)-1) 3.57 times of the total weight of the powder. The total volume of the preparation experiment reaction is 500mL, the concentration of the substrate chloroketone is 150g/L, and the successful catalysis is carried out after the reaction is carried out for 20 hoursReduction to give the product chlorohydrin, de (2S,3R)>99%, the purity of the product after recrystallization was 99.5%, the yield was 85.3% (obtaining 64.6g of chlorohydrin) (Appl Microbiol Biotechnol,2019,103(11): 4417-.
In conclusion, tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate can be obtained by a biocatalytic process with excellent diastereoselectivity; however, enzymatic reactions using mechanical agitation have been reported to occur with low efficiency.
Disclosure of Invention
Aiming at the current situation that the preparation efficiency of optically active chiral tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate by a plurality of biological methods is low at present, the invention provides a method for synthesizing the atazanavir intermediate chlorohydrin by continuous flow biocatalysis.
The invention uses carbonyl reductase from Saccharomyces cerevisiae S288C (Saccharomyces cerevisiae S288C) as a catalyst to catalyze the asymmetric reduction of (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate, the reaction is carried out in a microreactor, two advection pumps work simultaneously to prepare chiral tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate.
The purpose of the invention can be realized by the following technical scheme:
the invention provides application of carbonyl reductase OdCR1 in catalyzing reduction of (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate to generate corresponding tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate.
In one embodiment of the invention, the carbonyl reductase OdCR1 is derived from Saccharomyces cerevisiae S288C (Saccharomyces cerevisiae S288C). The Saccharomyces cerevisiae (Saccharomyces cerevisiae) S288C is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number as follows: CGMCC No.22135, the preservation time is 2021, 04 and 06 days, and the preservation place is as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North. The Saccharomyces cerevisiae S288C has high catalytic activity and excellent stereoselectivity.
In one embodiment of the invention, the carbonyl reductase OdCR1 is NADPH dependent. The amino acid sequence of the carbonyl reductase is shown as SEQ ID No. 2.
The invention also provides a carbonyl reductase gene, the nucleotide sequence of which is shown in SEQ ID No.1, and the protein coded by the gene is carbonyl reductase OdCR 1.
The invention provides a method for synthesizing atazanavir intermediate chlorohydrin by continuous flow biocatalysis, carbonyl reductase is used for catalyzing (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate to asymmetrically reduce to prepare optically active tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate, glucose dehydrogenase provides coenzyme circulation, and enzymatic reaction is carried out in a continuous flow microreactor.
In one embodiment of the invention, carbonyl reductase solution and substrate (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate solution are continuously fed into a continuous-flow microreactor according to a proportion to react, and after the reaction is biocatalytic, the reaction is cyclized and purified to obtain tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate.
In one embodiment of the invention, carbonyl reductase, glucose dehydrogenase, glucose and NADP are used+Preparing a solution I, preparing tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate into a solution II, continuously introducing the solution I and the solution II into a continuous flow microreactor according to a proportion to perform reaction, and performing cyclization and purification after a biocatalytic reaction to obtain the tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate.
In one embodiment of the invention, tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate is dissolved using dimethyl sulfoxide (DMSO).
In one embodiment of the present invention, a first solution participating in the reaction is prepared using a sodium phosphate salt buffer solution having a concentration of 100mM and a pH of 5.0 to 6.0.
In one embodiment of the invention, the ratio of the activity of the carbonyl reductase to the activity of the glucose dehydrogenase is 1: 1.5.
In one embodiment of the invention, the molar ratio of glucose to (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate is 1.5: 1.
the volume ratio of the dimethyl sulfoxide to the reaction solution is 1:6, and the reaction solution refers to the total solution formed by mixing the solution I and the solution II.
The concentration of NADP + in the reaction solution is 0.2mmol/L, and the reaction solution refers to the total solution obtained by mixing the solution I and the solution II.
In one embodiment of the invention, the continuous flow microreactor is a continuous flow microchannel reactor, the continuous flow microchannel reactor is formed by connecting 1-20 microreactor chips, and comprises two feed inlets and one discharge outlet, the two feed inlets are respectively used for injecting a raw material A and a raw material B to perform continuous mixing reaction, and the one discharge outlet is used for discharging after reaction.
In one embodiment of the present invention, the continuous flow microreactor is formed by connecting 3 microreactor chips, the 3 microreactor chips are respectively arranged in 3 layers, the first microreactor chip is provided with two feeding ports, the third microreactor chip is provided with a discharging port, the second microreactor chip is connected with the first microreactor chip and the third microreactor chip, each microreactor chip is provided with a plurality of mixing reaction chambers for liquid mixing reaction, after the raw material A and the raw material B respectively enter and mix from the two feeding ports, the raw material A and the raw material B sequentially pass through each mixing reaction chamber of the first microreactor chip, each mixing reaction chamber of the second microreactor chip and each mixing reaction chamber of the third microreactor chip and then flow out from the discharging port of the third microreactor chip, the first microreactor chip, the second microreactor chip, the third microreactor chip, and the third microreactor chip, And the mixing reaction chambers on the third layer of microreactor chips are all used as places for carrying out continuous mixing reaction.
In an embodiment of the present invention, the two feed inlets are connected to longer inlet channels, which are the feed channel 1 and the feed channel 2, respectively, wherein the feed channel 1 is divided into two parts, and forms a cross design with the feed channel 2, which is beneficial to improving the mixing efficiency of two streams of fluid.
In the invention, each micro-reactor chip is provided with a plurality of mixing reaction chambers for liquid mixing reaction, different mixing reaction chambers are communicated through pipelines, and the structural design is favorable for forming backflow on local fluid in the mixing reaction chambers, so that the back mixing degree of different reaction liquids is improved, and the mixing and mass transfer efficiency of reactants and catalysts is improved. In the invention, the mixed reaction chambers cascaded layer by layer further improve the micro-mixing intensity of reactants and are beneficial to strengthening the reaction process.
In one embodiment of the present invention, the microreactor chip is integrally arranged in a serpentine arrangement form, a zigzag arrangement form or a zigzag arrangement form, the mixing reaction chambers on the microreactor chip are in a drop-shaped structure, a lantern-shaped structure or a heart-shaped structure, and the mixing reaction chambers connected on the microreactor chip are connected with each other by a micro-tubular structure or a groove-shaped structure.
In one embodiment of the present invention, the material of the micro reactor chip is one of glass, ceramic, corrosion-resistant alloy or fluoropolymer.
In one embodiment of the invention, the carbonyl reductase is derived from Saccharomyces cerevisiae S288C (Saccharomyces cerevisiae S288C). The Saccharomyces cerevisiae (Saccharomyces cerevisiae) S288C is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number as follows: CGMCC No.22135, the preservation time is 2021, 04 and 06 days, and the preservation place is as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North. The Saccharomyces cerevisiae S288C has high catalytic activity and excellent stereoselectivity.
The invention also provides a method for obtaining the carbonyl reductase:
through large-scale screening of microorganisms in nature and strains of microorganisms deposited in laboratories, it was found that s.cerevisiae S288C can catalyze the reduction of (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate to the corresponding tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate. The carbonyl reductase catalyzing the corresponding reaction in S288C, designated carbonyl reductase OdCR1, was obtained by shotgun cloning, and is NADPH dependent. The amino acid sequence of the carbonyl reductase is shown as SEQ ID No. 2.
Saccharomyces cerevisiae S288C (Saccharomyces cerevisiae S288C) and carbonyl reductase OdCR1 can catalyze the reduction of (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate to produce the corresponding tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate.
The invention also provides application of the carbonyl reductase OdCR1 in asymmetric reduction of (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate. Wherein the chemical structure of the (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate is shown as follows:
in one embodiment of the present invention, the asymmetric reduction of the (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate can be carried out as follows: in a phosphate buffer solution with pH of 5.5-7.5, in the presence of glucose dehydrogenase, glucose and NADP+In the presence of the carbonyl reductase, OdCR1, catalyzes the asymmetric reduction of the (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate.
In one embodiment of the invention, in the application, the concentration of the substrate in the reaction solution can be 1-2 mmol/L. According to the adopted reaction system, the dosage of the carbonyl reductase can be 1-500U/L.
Enzymatic asymmetric reduction of (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate, oxidation of the coenzyme NADPH to NADP+In order to perform cyclic regeneration of coenzyme NADPH, glucose and glucose dehydrogenase derived from Bacillus megaterium (J Ind Microb Biotechnol,2011,38: 633-641) were additionally added to the reaction system.
The conversion of the reaction and the enantiomeric excess (ee) of the product can be analyzed by liquid chromatography, preferably using a C18 column (4.6 mm. times.250 mm) with a mobile phase of acetonitrile and phosphoric acid solution (0.1%) in a ratio of 7: 3. The ultraviolet wavelength of the detector is 210nm, and the column temperature is constant at 40 ℃.
In one embodiment of the present invention, after completion of the enzymatic reaction, the reaction solution is cooled to room temperature, extracted with an equal amount of a water-insoluble organic solvent which is conventional in the art, such as ethyl acetate, butyl acetate, toluene, methylene chloride, chloroform, isopropyl ether, methyl t-butyl ether, etc., repeated twice, the extracts are combined, washed with saturated brine, and dried overnight with anhydrous sodium sulfate. The solvent is removed by rotary evaporation, and the corresponding crude product of optically active tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate can be obtained.
The invention provides a method for synthesizing atazanavir intermediate chlorohydrin by continuous flow biocatalysis, which has high catalytic efficiency compared with the traditional mechanical stirring preparation. The carbonyl reductase used is derived from Saccharomyces cerevisiae S288C (Saccharomyces cerevisiae S288C), and has high catalytic activity and excellent stereoselectivity. The invention also discloses a method for carrying out enzymatic reaction in the continuous flow microreactor by utilizing the advection pump to sample into the continuous flow microreactor and injecting different substances into the two pumps.
The positive progress effects of the invention are as follows: the invention provides a method for synthesizing atazanavir intermediate chlorohydrin by continuous flow biocatalysis, which can be used for stereoselectively catalyzing asymmetric reduction of (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate in a continuous flow microreactor to generate corresponding optically active tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate, and has the advantages of mild reaction conditions, high catalytic efficiency, high conversion rate, good optical purity of products, ee value higher than 98.3 percent and good industrial application prospect.
Drawings
FIG. 1 is a chemical synthesis route of atazanavir.
Detailed Description
The invention provides a method for synthesizing atazanavir intermediate chlorohydrin by continuous flow biocatalysis, carbonyl reductase is used for catalyzing (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate to asymmetrically reduce to prepare optically active tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate, glucose dehydrogenase provides coenzyme circulation, and enzymatic reaction is carried out in a continuous flow microreactor.
In one embodiment of the invention, carbonyl reductase solution and substrate (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate solution are continuously fed into a continuous-flow microreactor according to a proportion to react, and after the reaction is biocatalytic, the reaction is cyclized and purified to obtain tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate.
In one embodiment of the invention, carbonyl reductase, glucose dehydrogenase, glucose and NADP are used+Preparing a solution I, preparing tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate into a solution II, continuously introducing the solution I and the solution II into a continuous flow microreactor according to a proportion to perform reaction, and performing cyclization and purification after a biocatalytic reaction to obtain the tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate.
In one embodiment of the invention, tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate is dissolved using dimethyl sulfoxide (DMSO).
In one embodiment of the present invention, a first solution participating in the reaction is prepared using a sodium phosphate salt buffer solution having a concentration of 100mM and a pH of 5.0 to 6.0.
In one embodiment of the invention, the ratio of the activity of the carbonyl reductase to the activity of the glucose dehydrogenase is 1: 1.5.
In one embodiment of the invention, the molar ratio of glucose to (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate is 1.5: 1.
the volume ratio of the dimethyl sulfoxide to the reaction solution is 1:6, and the reaction solution refers to the total solution formed by mixing the solution I and the solution II.
The concentration of NADP + in the reaction solution is 0.2mmol/L, and the reaction solution refers to the total solution obtained by mixing the solution I and the solution II.
In one embodiment of the invention, the microreactor device is formed by connecting 3 microreactor chips, the 3 microreactor chips are respectively arranged into 3 layers, the first microreactor chip is provided with two feeding ports, the third microreactor chip is provided with a discharging port, the second microreactor chip is connected with the first microreactor chip and the third microreactor chip, each microreactor chip is provided with a plurality of mixing reaction chambers for liquid mixing reaction, after respectively entering and mixing from the two feeding ports, raw material A and raw material B sequentially pass through each mixing reaction chamber of the first microreactor chip, each mixing reaction chamber of the second microreactor chip and each mixing reaction chamber of the third microreactor chip and then flow out from the discharging port of the third microreactor chip, the first microreactor chip, the second microreactor chip, the third microreactor chip and the mixing reaction chambers of the third microreactor chip are connected with one another, And the mixing reaction chambers on the third layer of microreactor chips are all used as places for carrying out continuous mixing reaction.
Two feed inlets all are connected with longer inlet channel, are feedstock channel 1 and feedstock channel 2 respectively, and wherein, feedstock channel 1 is divided into two, forms the crisscross design with feedstock channel 2, is favorable to improving the mixing efficiency of two strands of fluid.
In the invention, each micro-reactor chip is provided with a plurality of mixing reaction chambers for liquid mixing reaction, different mixing reaction chambers are communicated through pipelines, and the structural design is favorable for forming backflow on local fluid in the mixing reaction chambers, so that the back mixing degree of different reaction liquids is improved, and the mixing and mass transfer efficiency of reactants and catalysts is improved. In the invention, the mixed reaction chambers cascaded layer by layer further improve the micro-mixing intensity of reactants and are beneficial to strengthening the reaction process.
In one embodiment of the present invention, the microreactor chip is integrally arranged in a serpentine arrangement form, a zigzag arrangement form or a zigzag arrangement form, the mixing reaction chambers on the microreactor chip are in a drop-shaped structure, a lantern-shaped structure or a heart-shaped structure, and the mixing reaction chambers connected on the microreactor chip are connected with each other by a micro-tubular structure or a groove-shaped structure.
In one embodiment of the present invention, the material of the micro reactor chip is one of glass, ceramic, corrosion-resistant alloy or fluoropolymer.
In one embodiment of the invention, the carbonyl reductase is derived from Saccharomyces cerevisiae S288C (Saccharomyces cerevisiae S288C). The Saccharomyces cerevisiae (Saccharomyces cerevisiae) S288C is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number as follows: CGMCC No.22135, the preservation time is 2021, 04 and 06 days, and the preservation place is as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North. The Saccharomyces cerevisiae S288C has high catalytic activity and excellent stereoselectivity.
The invention also provides a method for obtaining the carbonyl reductase:
through large-scale screening of microorganisms in nature and strains of microorganisms deposited in laboratories, it was found that s.cerevisiae S288C can catalyze the reduction of (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate to the corresponding tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate. The carbonyl reductase catalyzing the corresponding reaction in S288C, designated carbonyl reductase OdCR1, was obtained by shotgun cloning, and is NADPH dependent. The amino acid sequence of the carbonyl reductase is shown as SEQ ID No. 2.
Saccharomyces cerevisiae S288C (Saccharomyces cerevisiae S288C) and carbonyl reductase OdCR1 can catalyze the reduction of (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate to produce the corresponding tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate.
The invention also provides application of the carbonyl reductase OdCR1 in asymmetric reduction of (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate. Wherein the chemical structure of the (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate is shown as follows:
in one embodiment of the present invention, the (S) -tert-butyl groupThe asymmetric reduction of the (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate can be carried out in the following exemplary manner: in a phosphate buffer solution with pH of 5.5-7.5, in the presence of glucose dehydrogenase, glucose and NADP+In the presence of the carbonyl reductase, OdCR1, catalyzes the asymmetric reduction of the (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate.
In one embodiment of the invention, in the application, the concentration of the substrate in the reaction solution can be 1-2 mmol/L. According to the adopted reaction system, the dosage of the carbonyl reductase can be 1-500U/L.
Enzymatic asymmetric reduction of (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate, oxidation of the coenzyme NADPH to NADP+In order to perform cyclic regeneration of coenzyme NADPH, glucose and glucose dehydrogenase derived from Bacillus megaterium (J Ind Microb Biotechnol,2011,38: 633-641) were additionally added to the reaction system.
The conversion of the reaction and the enantiomeric excess (ee) of the product can be analyzed by liquid chromatography, preferably using a C18 column (4.6 mm. times.250 mm) with a mobile phase of acetonitrile and phosphoric acid solution (0.1%) in a ratio of 7: 3. The ultraviolet wavelength of the detector is 210nm, and the column temperature is constant at 40 ℃.
In one embodiment of the present invention, after completion of the enzymatic reaction, the reaction solution is cooled to room temperature, extracted with an equal amount of a water-insoluble organic solvent which is conventional in the art, such as ethyl acetate, butyl acetate, toluene, methylene chloride, chloroform, isopropyl ether, methyl t-butyl ether, etc., repeated twice, the extracts are combined, washed with saturated brine, and dried overnight with anhydrous sodium sulfate. The solvent is removed by rotary evaporation, and the corresponding crude product of optically active tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate can be obtained.
The invention is described in detail below with reference to the figures and specific embodiments.
The individual reaction or detection conditions described in the summary of the invention may be combined or modified according to common general knowledge in the art and may be verified experimentally. The present invention is further illustrated by the following examples, and it should be understood that the examples, while indicating preferred embodiments of the invention, are given by way of illustration only, and are not intended to limit the invention to the examples so described.
The material sources in the following examples are:
a microreactor.
Advection pumps were purchased from Beijing Star science and technology development Inc.
The vector pUC118 and the restriction enzyme Sau3AI were purchased from Takara Bio Inc.
Expression plasmid pET28a was purchased from Novagen.
Coli DH5 α and E.coli BL21(DE3) competent cells, 2 XTAQA PCR MasterMix, agarose gel DNA recovery kits were purchased from Beijing Tiangen Biochemical technology Ltd.
Both restriction enzymes EcoR I and Hind III are commercially available from New England Biolabs (NEB).
Unless otherwise indicated, specific experiments in the following examples were performed according to methods and conditions conventional in the art, or according to the commercial instructions of the kits.
In the embodiment, the microreactor device is formed by connecting 3 microreactor chips, the 3 microreactor chips are respectively arranged into 3 layers, the first microreactor chip is provided with two feeding holes, the third microreactor chip is provided with a discharging hole, the second microreactor chip is connected with the first microreactor chip and the third microreactor chip, each microreactor chip is provided with a plurality of mixing reaction chambers for liquid mixing reaction, after a raw material A and a raw material B respectively enter and are mixed from the two feeding holes, and the mixed reaction chambers sequentially penetrate through each mixed reaction chamber of the first layer of microreactor chip, each mixed reaction chamber of the second layer of microreactor chip and each mixed reaction chamber of the third layer of microreactor chip and then flow out from a discharge port of the third layer of microreactor chip, and the mixed reaction chambers on the first layer of microreactor chip, the second layer of microreactor chip and the third layer of microreactor chip are all used as places for carrying out continuous mixed reaction.
Two feed inlets all are connected with longer inlet channel, are feedstock channel 1 and feedstock channel 2 respectively, and wherein, feedstock channel 1 is divided into two, forms the crisscross design with feedstock channel 2, is favorable to improving the mixing efficiency of two strands of fluid.
Sodium phosphate buffer solution (100mM, pH 6.0) make-up solution I (carbonyl reductase, glucose dehydrogenase, glucose and NADP)+) And a solution of bis ((S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate (DMSO cosolvent)) respectively enters a micro-reactor mixing zone through a feeding flow channel 1 and a feeding flow channel 2, and is instantly and uniformly mixed, and two different materials are ensured not to be contacted with each other before entering the micro-reactor mixing zone; after the enzyme solution and the substrate solution enter the microchannel reactor, the enzyme solution and the substrate solution can efficiently and rapidly perform catalytic reaction in the tube pass of the microreactor through homogenization and microcosmic control of temperature and concentration in each unit reactor, and finally, a product tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate is generated and discharged into 1mol/L sulfuric acid solution from a discharge port to terminate the reaction.
After the reaction is finished, extracting twice with equal volume of ethyl acetate, combining extracts, removing the ethyl acetate solvent by using a rotary evaporator, purifying the crude product by using a silica gel chromatographic column (the particle size is 200-300 meshes), and eluting the target product by using a mobile phase of ethyl acetate and petroleum ether (1:20) to obtain the pure tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate.
EXAMPLE 1 Synthesis of chiral tert-butyl ((2S,3R) -4-chloro-3-hydroxy-1-phenylbutyl-2-yl) carbamate by continuous flow biocatalysis of (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate
The whole enzymatic experiment is carried out in a microreactor, two advection pumps are needed to be communicated into the microreactor, a pump 1 injects (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate (dissolved by DMSO) into the microreactor, and a pump 2 injects carbonyl reductase, glucose dehydrogenase, glucose and NADP into the microreactor+And the liquid of the two pumps is converged in the microreactor, so that the enzyme catalysis reaction is carried out.
Solution one contains glucose and NADP+The concentration of (A) is 1.5mmol/L and 0.2mmol/L respectively; solution two was made up with DMSO to give (S) -tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate in a final concentration of 10 mmol/L. Reacting at 40 deg.C with the flow rates of solution one and solution two being 0.36mL/min and 0.06mL/min respectively, and measuring substrate conversion rate by liquid chromatography>99% and ee value can be higher than 98.3%.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Sequence listing
<110> university of east China's college of science
<120> method for synthesizing atazanavir intermediate chlorohydrin by continuous flow biocatalysis
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 771
<212> DNA
<213> Saccharomyces cerevisiae
<400> 1
atgaacacca gcagccgtat cacctacttc atcattggcg gtagccgtgg tatcggcttc 60
aacctggtga agattctgag cgcgagcacc ggtaacaccg ttatcaccag cattcgtggt 120
agcccgagcc tgccgaaaaa caagcaggtg gaagacctgg cgaaaatccg taagaacatt 180
cacatcgtgc agctggatct gaccaaggac gaaagcatcg gtaacatcgc ggatgagatc 240
aaaaagaccc cgtttttcct gggtatcgac attttcatcg cgtgcagcgc ggttagcgac 300
agctattaca aggttctgga gaccccgaaa agcgtttggc tgaaccacta cagcaccaac 360
gcgctgggcc cgattctggc gctgcaaaag gtgtacccgc tgctgctgct gaagaaaacc 420
cgtaagatct ttttcattag cagcgttgcg ggtagcatta acgcgttcgt tccgctgagc 480
gttagcgcgt atggtcagag caaagcggcg ctgaactacg cggttaaaac cctgagcttt 540
gagctgaaac cggagggttt taccgtggtt gcgtttcacc cgggtatggt tagcaccgac 600
atgggtcaat acggtctgga ccacttcaaa gaaaagaaca tcgacattag cggtgttaac 660
atcattaccc cggaagagag cgcgagcgcg ctgatcgacg ttttccgtaa gatcctgccg 720
gaggataacg gcaagttttt caactacgat ggtagcgaag gcgttttcta a 771
<210> 2
<211> 256
<212> PRT
<213> Saccharomyces cerevisiae
<400> 2
Met Asn Thr Ser Ser Arg Ile Thr Tyr Phe Ile Ile Gly Gly Ser Arg
1 5 10 15
Gly Ile Gly Phe Asn Leu Val Lys Ile Leu Ser Ala Ser Thr Gly Asn
20 25 30
Thr Val Ile Thr Ser Ile Arg Gly Ser Pro Ser Leu Pro Lys Asn Lys
35 40 45
Gln Val Glu Asp Leu Ala Lys Ile Arg Lys Asn Ile His Ile Val Gln
50 55 60
Leu Asp Leu Thr Lys Asp Glu Ser Ile Gly Asn Ile Ala Asp Glu Ile
65 70 75 80
Lys Lys Thr Pro Phe Phe Leu Gly Ile Asp Ile Phe Ile Ala Cys Ser
85 90 95
Ala Val Ser Asp Ser Tyr Tyr Lys Val Leu Glu Thr Pro Lys Ser Val
100 105 110
Trp Leu Asn His Tyr Ser Thr Asn Ala Leu Gly Pro Ile Leu Ala Leu
115 120 125
Gln Lys Val Tyr Pro Leu Leu Leu Leu Lys Lys Thr Arg Lys Ile Phe
130 135 140
Phe Ile Ser Ser Val Ala Gly Ser Ile Asn Ala Phe Val Pro Leu Ser
145 150 155 160
Val Ser Ala Tyr Gly Gln Ser Lys Ala Ala Leu Asn Tyr Ala Val Lys
165 170 175
Thr Leu Ser Phe Glu Leu Lys Pro Glu Gly Phe Thr Val Val Ala Phe
180 185 190
His Pro Gly Met Val Ser Thr Asp Met Gly Gln Tyr Gly Leu Asp His
195 200 205
Phe Lys Glu Lys Asn Ile Asp Ile Ser Gly Val Asn Ile Ile Thr Pro
210 215 220
Glu Glu Ser Ala Ser Ala Leu Ile Asp Val Phe Arg Lys Ile Leu Pro
225 230 235 240
Glu Asp Asn Gly Lys Phe Phe Asn Tyr Asp Gly Ser Glu Gly Val Phe
245 250 255
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