CN112359078A - Chiral resolution method of isobutyrate compound - Google Patents

Chiral resolution method of isobutyrate compound Download PDF

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CN112359078A
CN112359078A CN202110032518.3A CN202110032518A CN112359078A CN 112359078 A CN112359078 A CN 112359078A CN 202110032518 A CN202110032518 A CN 202110032518A CN 112359078 A CN112359078 A CN 112359078A
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isobutyrate
chiral resolution
substituted
resolution method
compound
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洪浩
詹姆斯·盖吉
肖毅
陈轶晖
张娜
李艳君
龚天保
黄鑫
朱柳杨
李响
崔毅
李招
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Asymchem Life Science Tianjin Co Ltd
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Abstract

The invention provides a chiral resolution method of isobutyrate compounds. The isobutyrate compound is racemic isobutyrate compound and has the following structural general formula
Figure DEST_PATH_IMAGE001
，R1Any one selected from the following substituted or unsubstituted groups: pyridine, pyrazole, pyrimidone, 4-hydroxyquinoline, quinazoline, quinazolinone, R2Selected from substituted or unsubstituted alkyl and aryl, the chiral resolution method comprises: step S1, carrying out hydrolysis treatment on the racemic isobutyrate compound by using hydrolytic enzyme to obtain a hydrolyzed system, wherein the hydrolytic enzyme is hydrolytic enzyme from Thermomyces sp, Rhizomucor miehei and Bacillus sp; step S2, acid and unreacted in the hydrolyzed systemSeparating the hydrolyzed isobutyrate ester compound. The hydrolase used in the method has good stability, good corresponding body selectivity, mild reaction conditions, low cost and simple operation.

Description

Chiral resolution method of isobutyrate compound
Technical Field
The invention relates to the technical field of chiral resolution, in particular to a chiral resolution method of isobutyrate compounds.
Background
Most drugs are composed of chiral molecules, two of which may have significantly different biological activities. The drug molecule must match the molecular geometry of the receptor (the reacting substance) in order to achieve the desired drug effect. Thus, often only one of the two isomers is effective, and the other is ineffective or even detrimental. The heterocyclic substituted isobutyrate derivatives are compounds with simple structure and wide application, and some compounds are intermediates of a plurality of new drugs in the current experimental and clinical stages.
The chemical synthesis method of the chiral pyrazole isobutyric acid derivative is reported in the literature (Bioorganic and Medicinal Chemistry Letters, 2015, vol.25, # 3, p.668-672), the reaction process relates to the processes of alkaline hydrolysis, chiral reagent resolution, column chromatography purification and separation and the like, the method has long steps, the chiral reagent is difficult to screen, the price is high, the economy is poor, and the industrial large-scale production of the chiral pyrazole isobutyric acid derivative is greatly limited. The chromatographic resolution method needs to use expensive special stationary phase, so the cost is high and the operation is complex.
Disclosure of Invention
The invention mainly aims to provide a chiral resolution method of an isobutyrate compound, which aims to solve the problems of high cost and complex operation of the chiral resolution of the isobutyrate compound in the prior art.
In order to achieve the above object, according to one aspect of the present invention, there is provided a chiral resolution method of an isobutyrate compound, wherein the isobutyrate compound is a racemic isobutyrate compound having the following structural formula
Figure 364723DEST_PATH_IMAGE001
Wherein R is1Selected from: any one of substituted or unsubstituted pyridine, substituted or unsubstituted pyrazole, substituted or unsubstituted pyrimidone, substituted or unsubstituted 4-hydroxyquinoline, substituted or unsubstituted quinazoline and substituted or unsubstituted quinazolinone, R2Is selected from C1~C7Substituted or unsubstituted alkyl of, C6~C10The substituted or unsubstituted aryl group of (1), the chiral resolution method comprising: step S1, carrying out hydrolysis treatment on the racemic isobutyrate compound by using hydrolytic enzyme to obtain a hydrolyzed system, wherein the hydrolytic enzyme is hydrolytic enzyme from Thermomyces sp, Rhizomucor miehei and Bacillus sp; step S2, separating the acid and the non-hydrolyzed isobutyrate ester compound in the hydrolyzed system.
Further, the above R1Is selected from
Figure 650211DEST_PATH_IMAGE002
、
Figure 793748DEST_PATH_IMAGE003
、
Figure 51554DEST_PATH_IMAGE004
、
Figure 12556DEST_PATH_IMAGE005
、
Figure 926286DEST_PATH_IMAGE006
、
Figure 732568DEST_PATH_IMAGE007
、
Figure 110460DEST_PATH_IMAGE008
And
Figure 353615DEST_PATH_IMAGE009
wherein R is3Is H, methyl, halogen or alkoxy, R2Is any one of ethyl, butyl, propyl, isobutyl, phenyl and benzyl.
Further, the alkoxy group is any one selected from the group consisting of a methoxy group, an ethoxy group, an n-propoxy group, an isopropoxy group, an n-butoxy group, an isobutoxy group, a sec-butoxy group and a tert-butoxy group.
Further, the amount of the hydrolase to be used is 0.005 to 2.0g/g of a racemic isobutyrate compound.
Further, the amount of the hydrolase to be used is 0.005 to 0.50g/g of racemic isobutyrate compound.
Further, the step S1 includes: mixing an isobutyrate compound, a cosolvent, a hydrolase and a buffer solution to form a hydrolysis reaction system; and (4) carrying out heat preservation treatment on the hydrolysis reaction system to carry out hydrolysis reaction to obtain a hydrolyzed system.
Further, the buffer solution is selected from a phosphate buffer solution, an acetic acid-acetate buffer solution, and a tris-hydrochloric acid buffer solution.
Further, the pH value of the buffer solution is 5.0-9.0.
Further, the dissolution promoter is selected from one or more of ethyl acetate, dimethyl sulfoxide, polyethylene glycol, methanol, ethanol, acetonitrile, methyl tert-butyl ether, n-heptane, n-hexane, dichloromethane, tetrahydrofuran and n-butanol.
Further, the cosolvent is ethyl acetate, tetrahydrofuran, methyl tert-butyl ether, ethanol or isopropanol.
Further, the temperature of the hydrolysis reaction is 10-50 ℃.
Further, the temperature of the hydrolysis reaction is 20-40 ℃.
Further, the temperature of the hydrolysis reaction is 25-35 ℃.
Furthermore, the time of the hydrolysis reaction is 8-120 h.
Further, the time of the hydrolysis reaction is 8-24 hours.
Further, the step S2 includes: extracting and separating the hydrolyzed system to obtain a first organic phase containing the isobutyrate compounds and a first water phase containing the hydrolysis products; adjusting the pH value of the first water phase to be below 3, extracting the first water phase, carrying out phase separation to obtain a second organic phase and a second water phase, and concentrating the second organic phase to obtain a hydrolysate.
By applying the technical scheme of the invention, the racemic isobutyrate compounds are selectively hydrolyzed by using hydrolytic enzyme to obtain the isobutyrate derivatives with single configuration or the corresponding hydrolytic acid. The hydrolase used in the method has good stability and good selectivity of a counterpart, and compared with other resolution technologies, the technology in the invention has the advantages of mild reaction conditions, easily obtained raw materials and catalysts, low cost, simple operation, environmental friendliness and suitability for industrial production.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.
The term "substituted" means that one or more hydrogens on a given atom are replaced with the indicated group, where the substituent used for substitution may be one commonly used in the art, such as halogen, alkyl, hydroxy, nitro, alkoxy, etc., and if the normal valency of the indicated atom is not exceeded under the circumstances present, the substitution results in a stable compound.
As analyzed in the background of the present application, the prior artChiral heterocyclic substituted isobutyrate derivatives or corresponding acids in the art need chiral reagent resolution or chromatographic separation, wherein chiral reagents are difficult to screen, expensive and poor in economy; the chromatographic resolution method needs to use an expensive special stationary phase, so that the cost is too high, and the operation is also complex. In order to reduce the cost and simplify the operation, the application provides a chiral resolution method of an isobutyrate compound, and the isobutyrate compound has the following structural general formula
Figure 613695DEST_PATH_IMAGE010
Wherein R is1Selected from: any one of substituted or unsubstituted pyridine, substituted or unsubstituted pyrazole, substituted or unsubstituted pyrimidone, substituted or unsubstituted 4-hydroxyquinoline, substituted or unsubstituted quinazoline and substituted or unsubstituted quinazolinone, R2Is selected from C1~C7Substituted or unsubstituted alkyl of, C6~C10The isobutyrate ester compound has racemic isobutyrate ester compound, and the chiral resolution method comprises the following steps: step S1, carrying out hydrolysis treatment on the racemic isobutyrate compound by using hydrolytic enzyme to obtain a hydrolyzed system, wherein the hydrolytic enzyme is hydrolytic enzyme from Thermomyces sp, Rhizomucor miehei and Bacillus sp; and step S2, separating the non-hydrolyzed isobutyrate ester compound in the hydrolyzed system.
The method utilizes hydrolase to selectively hydrolyze racemic isobutyrate compounds to obtain isobutyrate derivatives with single configuration or corresponding hydrolysis acids. The hydrolase used in the method has good stability and good selectivity of a counterpart, and compared with other resolution technologies, the technology in the invention has the advantages of mild reaction conditions, easily obtained raw materials and catalysts, low cost, simple operation, environmental friendliness and suitability for industrial production. The hydrolase used in the application is commercial enzyme, and the screening of more than 100 commercial enzymes in the application shows that the chiral resolution effect is more prominent when the hydrolase is hydrolase derived from Thermomyces.
Inferring from the catalytic Activity of enzymesThe isobutyrate compounds with the structural general formula can be hydrolyzed and catalyzed by the hydrolase to realize chiral resolution, and particularly, the resolution efficiency is particularly remarkable when the substituent in the structural general formula is selected as follows, such as R1Is selected from
Figure 364614DEST_PATH_IMAGE002
、
Figure 393750DEST_PATH_IMAGE003
、
Figure 899817DEST_PATH_IMAGE004
、
Figure 256980DEST_PATH_IMAGE005
、
Figure 670644DEST_PATH_IMAGE006
、
Figure 429653DEST_PATH_IMAGE007
、
Figure 168939DEST_PATH_IMAGE008
And
Figure 341294DEST_PATH_IMAGE009
wherein R is3Is H, methyl, halogen or alkoxy, R2Is any one of ethyl, butyl, propyl, isobutyl, phenyl and benzyl.
The above halogens are fluorine, chlorine, bromine and iodine, preferably fluorine, chlorine and bromine, "alkoxy" means an alkyl ether group (O-alkyl), and non-limiting examples of the alkoxy group include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy and tert-butoxy, etc.
The hydrolytic enzyme is mainly used for hydrolyzing racemic isobutyrate compounds, so the dosage can be selected according to the content of the racemic isobutyrate compounds, and in order to improve the utilization rate of the hydrolytic enzyme and the hydrolysis catalytic efficiency, the dosage of the hydrolytic enzyme is preferably 0.005-2.0 g/g of the racemic isobutyrate compounds; more preferably 0.005 to 0.50g/g of racemic isobutyrate ester compound.
The hydrolysis of the present application requires only the contact-catalyzed hydrolysis of the hydrolase and the isobutyrate ester compound dissolved in a solvent, and in order to improve the hydrolysis efficiency, the step S1 includes: mixing an isobutyrate compound, a cosolvent, a hydrolase and a buffer solution to form a hydrolysis reaction system; and (4) carrying out heat preservation treatment on the hydrolysis reaction system to carry out hydrolysis reaction to obtain a hydrolyzed system.
The method comprises the steps of mixing an isobutyrate compound, a cosolvent and hydrolase to form a uniform reaction system, providing a pH value environment suitable for the activity of the hydrolase for the hydrolysis reaction system by adopting a buffer solution, and then carrying out hydrolysis reaction at a temperature suitable for the activity of the hydrolase, thereby realizing high activity exertion of the hydrolase as far as possible.
When the isobutyrate ester compound is added, the isobutyrate ester compound may be dissolved in the dissolution accelerator to form a solution, and then the isobutyrate ester compound solution may be mixed with the hydrolase and the buffer solution.
Any organic solvent having high solubility for the isobutyrate compounds can be used for forming the dissolution promoter, such as ethyl acetate, dimethyl sulfoxide, polyethylene glycol, methanol, ethanol, acetonitrile, methyl tert-butyl ether, n-heptane, n-hexane, dichloromethane, tetrahydrofuran, n-butanol, and other reagents commonly used in the art, and the dissolution promoter can improve the conversion rate of the isobutyrate compounds and/or the e.e. value of the product. Moreover, different types of solvents have different effects on the conversion and the e.e. value of the product. Preferably the aforementioned co-solvent is ethyl acetate, tetrahydrofuran, methyl tert-butyl ether, ethanol or isopropanol, thereby increasing the conversion and the product e.e. value.
The buffer solution used in the present application may be a buffer solution commonly used for enzymes, and in order to save cost, the buffer solution is preferably selected from a phosphate buffer solution, an acetic acid-acetate buffer solution, and a tris-hcl buffer solution, and the pH of the buffer solution is preferably 5.0 to 9.0. The phosphate buffer solution may be potassium phosphate buffer solution, sodium phosphate buffer solution, etc. commonly used in the art.
In the reaction process, the pH value of the hydrolysis reaction system changes with the progress of hydrolysis, and in order to maintain high activity of the hydrolase, the pH value of the hydrolysis reaction system is preferably maintained at 7.0 to 8.0 during the hydrolysis reaction, and is preferably adjusted by phosphoric acid, acetic acid, or hydrochloric acid.
The hydrolysis reaction temperature in the present application may be selected within a temperature range in which the hydrolase activity is exhibited, and in order to make the hydrolase have as high an activity as possible, the hydrolysis reaction temperature is preferably 10 to 50 ℃, more preferably 20 to 40 ℃, and still more preferably 25 to 35 ℃.
The hydrolysis reaction catalyzed by the hydrolase is relatively mild, so that the hydrolysis reaction time can be properly prolonged in order to improve the hydrolysis conversion rate, and can be selected according to the content of the racemate in the substrate, wherein the hydrolysis reaction time is preferably 8-120 h, and is preferably 8-24 h.
In another embodiment of the present application, the step S2 includes: extracting and separating the hydrolyzed system to obtain a first organic phase containing the isobutyrate compounds and a first water phase containing the hydrolysis products; adjusting the pH value of the first water phase to be below 3, extracting the first water phase, separating the phases to obtain a second organic phase and a second water phase, and concentrating the second organic phase to obtain a hydrolysate.
After the hydrolysis is completed, the substances in the obtained hydrolyzed system have different solubilities, so that the separation can be completed by adopting the traditional extraction method. Wherein the extractant used for the extraction of the system after hydrolysis and the extractant used for the extraction of the first aqueous phase may be the same extractant.
The advantageous effects of the present application will be further described below with reference to examples and comparative examples.
Example 1
Adding 0.1g of substrate ester into 5 mL of 0.3M potassium phosphate buffer solution in a 10 mL reaction bottle, adjusting the pH to 7.0-7.5, adding 0.005-0.1 g of hydrolase (a freeze-dried powder preparation or an enzyme solution, specifically shown in Table 1) to form a hydrolysis reaction system, controlling the pH of the hydrolysis reaction system to 7.0-7.5, and stirring at the constant temperature of 30 +/-3 ℃ for a certain time to obtain a hydrolyzed system. Stopping reaction by using 900 mu L of 50% acetonitrile after 100 mu L of hydrolyzed system, fully shaking and uniformly mixing, centrifuging at 8000rpm for 1min to obtain a supernatant phase and an organic solid phase, blowing the supernatant phase by using nitrogen, dissolving in ethanol, detecting the conversion rate and the chirality by using HPLC (high performance liquid chromatography), respectively screening 100 hydrolases from the following substrates (the 100 hydrolases are all from commercial enzymes, artificially synthesizing known sequences reported in documents or artificially mutating the sequences), detecting the conversion rate and the chirality by using HPLC, and obtaining a great amount of raw materials in most of reaction systems of the hydrolases without product generation. Table 1 shows that there was a system with product formation detected and the product e.e. value > 90%.
TABLE 1
Figure 666971DEST_PATH_IMAGE011
Figure 670699DEST_PATH_IMAGE012
Figure 456252DEST_PATH_IMAGE013
Example 2
(1) Feeding: to a 100 mL reaction flask, hydrolase Thermomyces.sp.0.5 g, 30 mL of tris-hcl buffer (300 mM, pH =9.0) was added, and stirred, and the enzyme was dissolved in tris-hcl buffer;
(2) adding a substrate: 1g of the main raw material was charged into the above reaction flask
Figure 912642DEST_PATH_IMAGE014
Stirring, system pH = 9.0;
(3) reaction: reacting the system at 30 ℃, stirring for 24 hours, and maintaining the pH of the system to 9.0 by using 1N NaOH;
(4) and (3) post-treatment: adding 20 mL of acetonitrile into the system after the reaction is finished, stirring for 30min, and then carrying outFiltering with a diatomite pad, adjusting the pH of the obtained organic phase system to 8-9 with a sodium bicarbonate solution, adding 20 mL of ethyl acetate for extraction for three times, adjusting the pH of the water phase to 2-3 with 0.5M hydrochloric acid, separating out a product, controlling the temperature of the product to be 40-45 ℃, concentrating, and filtering to obtain the product
Figure 277895DEST_PATH_IMAGE015
The purity is 99% and the e.e. value is 85.97%.
Example 3
(1) Feeding: to a 100 mL reaction flask, add hydrolase thermomyces.sp 0.05 g, 25 mL potassium phosphate buffer (100 mM, pH =7.5), stir, and dissolve the enzyme in potassium phosphate buffer; 2.5 mL of ethyl acetate was added
(2) Adding a substrate: 1g of the main raw material was added to the above reaction flask
Figure 542654DEST_PATH_IMAGE016
Stirring, and maintaining the pH of the system to 7.5 by using 1N NaOH;
(3) reaction: reacting the system at 40 ℃, stirring for 120h, and maintaining the pH of the system to 7.5 by using 1N NaOH;
(4) and (3) post-treatment: stopping reaction with 900 μ L of 50% acetonitrile in 100 μ L system, shaking thoroughly, mixing, centrifuging at 8000rpm for 1min, blowing the obtained supernatant with nitrogen, dissolving in ethanol, and detecting by HPLC that conversion rate is 45.67%, and the product is
Figure 358163DEST_PATH_IMAGE017
The e.e. value was 99.5%.
Example 4:
(1) feeding: to a 250 mL reaction flask, 0.5 g of the hydrolase, Rhizomucor miehei, 50 mL of potassium phosphate buffer (100 mM, pH =7.5) was added, stirred, and the enzyme was dissolved in the potassium phosphate buffer;
(2) adding a substrate: into the above reaction flask, 1g of the main raw material
Figure 666961DEST_PATH_IMAGE018
Stirring, and maintaining the pH of the system to 7.5 by using 1N NaOH;
(3) reaction: the system reacts at 50 ℃, is stirred and reacts for 12 hours, and 1N NaOH is used for maintaining the pH value of the system to 7.5
(4) And (3) post-treatment: stopping reaction with 900 μ L of 50% acetonitrile in 100 μ L system, shaking thoroughly, mixing, centrifuging at 8000rpm for 1min, blowing the obtained supernatant with nitrogen, dissolving in ethanol, and detecting conversion rate of 63.25% by HPLC with the residual substrate
Figure 226118DEST_PATH_IMAGE019
The e.e. value was 96.98%.
Example 5:
(1) feeding: to a 100 mL reaction flask, 0.1g of hydrolase Bacillus sp, 10 mL of potassium phosphate buffer (100 mM, pH =7.0) was added, stirred, and the enzyme was dissolved in the potassium phosphate buffer;
(2) adding a substrate: into a 100 mL reaction flask, 1g of the main raw material
Figure 548646DEST_PATH_IMAGE020
Stirring, and maintaining the pH value of the system to 7.0 by using 0.5N NaOH;
(3) reaction: the system reacts at 25 ℃, is stirred and reacts for 24 hours, and 1N NaOH is used for maintaining the pH value of the system to 7.0
(4) And (3) post-treatment: adding 20 mL of acetonitrile into the system after the reaction is finished, stirring for 30min, filtering through a diatomite pad, adjusting the pH of the obtained organic phase system to 8-9 by using a sodium bicarbonate solution, adding 20 mL of ethyl acetate, and extracting for three times to obtain an organic phase containing the residual substrate; adjusting the pH of the water phase to 2-3 by using 0.5M hydrochloric acid, adding 20 mL ethyl acetate for extraction for three times, concentrating the organic phase obtained by the three-time extraction, and concentrating the organic phase at 40-45 ℃ to obtain a product
Figure 239784DEST_PATH_IMAGE021
The yield is 32.11% and the e.e. value is 98.69%.
Example 6:
(1) feeding: adding 0.1g of hydrolase Bacillus sp, 40 mL of an acetate-ammonium acetate buffer (100 mM, pH =6.0) into a 100 mL reaction flask, stirring, and dissolving the enzyme in the acetate-ammonium acetate buffer;
(2) adding a substrate: into the above reaction flask, 1g of the main raw material
Figure 874028DEST_PATH_IMAGE022
Stirring, and the pH value of the system is 6.0;
(3) reaction: reacting the system at 25 ℃, stirring for 24 hours, and maintaining the pH of the system to 6.0 by using 1N NaOH;
(4) and (3) post-treatment: stopping reaction with 900 μ L of 50% acetonitrile in 100 μ L system, shaking thoroughly, mixing, centrifuging at 8000rpm for 1min, blowing the obtained supernatant with nitrogen, dissolving in ethanol, and detecting conversion rate by HPLC (high performance liquid chromatography) of 37.35% to obtain product
Figure 548460DEST_PATH_IMAGE023
The e.e. value was 97.63%.
Example 7:
(1) feeding: to a 100 mL reaction flask, adding hydrolase Bacillus sp 1g, 20 mL of potassium phosphate buffer (100 mM, pH =7.5), stirring, and dissolving the enzyme in the potassium phosphate buffer;
(2) adding a substrate: into a 100 mL reaction flask, 1g of the main raw material
Figure 446534DEST_PATH_IMAGE024
Stirring, and maintaining the pH of the system to 7.5 by using 0.5N NaOH;
(3) reaction: reacting the system at 20 ℃, stirring for 8 hours, and maintaining the pH of the system to 7.5 by using 1N NaOH;
(4) and (3) post-treatment: stopping reaction with 900 μ L of 50% acetonitrile in 100 μ L system, shaking thoroughly, mixing, centrifuging at 8000rpm for 1min, blowing the obtained supernatant with nitrogen, dissolving in ethanol, and detecting conversion rate by HPLC (high performance liquid chromatography) of 33.73% to obtain product
Figure 854380DEST_PATH_IMAGE025
The e.e. value is 85.35%.
Example 8:
(1) feeding: to a 100 mL reaction flask, add hydrolase thermomyces.sp 2 g, 30 mL acetic acid-sodium acetate buffer (100 mM, pH = 5.0), stir, and dissolve the enzyme in the acetic acid-sodium acetate buffer;
(2) adding a substrate: into a 100 mL reaction flask, 1g of the main raw material
Figure 179182DEST_PATH_IMAGE026
Stirring, and maintaining the pH value of the system to 5.0 by using 0.5N NaOH;
(3) reaction: reacting the system at 30 ℃, stirring for 8 hours, and maintaining the pH of the system to 5.0 by using 1N NaOH;
(4) and (3) post-treatment: stopping reaction with 900 μ L of 50% acetonitrile in 100 μ L system, shaking thoroughly, mixing, centrifuging at 8000rpm for 1min, blowing the obtained supernatant with nitrogen, dissolving in ethanol, and detecting conversion rate by HPLC to 18.52%, wherein the product is
Figure 188464DEST_PATH_IMAGE027
The e.e. value was 96.65%.
Example 9:
(1) feeding: sp 2 g of hydrolase, 30 mL of potassium phosphate buffer (100 mM, pH =7.5) was added to a 100 mL reaction flask, stirred, and the enzyme was dissolved in the potassium phosphate buffer;
(2) adding a substrate: into a 100 mL reaction flask, 1g of the main raw material
Figure 485584DEST_PATH_IMAGE026
Stirring, and maintaining the pH of the system to 7.5 by using 0.5N NaOH;
(3) reaction: reacting the system at 10 ℃, stirring for 40 h, and maintaining the pH of the system to 7.5 by using 1N NaOH;
(4) and (3) post-treatment: stopping reaction with 90 μ L of 50% acetonitrile in 100 μ L system, shaking thoroughly, mixing, centrifuging at 8000rpm for 1min, blowing the obtained supernatant with nitrogen, dissolving in ethanol, and detecting by HPLC that conversion rate is 26.79%, and the product is
Figure 813797DEST_PATH_IMAGE027
The e.e. value was 95.26%.
Example 10:
(1) feeding: to a 100 mL reaction flask, 0.2 g of hydrolase Bacillus sp, 50 mL of potassium phosphate buffer (100 mM, pH =7.5) was added, stirred, and the enzyme was dissolved in the potassium phosphate buffer;
(2) adding a substrate: to a 100 mL reaction flask, 1g of the master batch dissolved in 1.5 mL of DMSO was added
Figure 157054DEST_PATH_IMAGE028
Stirring, and maintaining the pH of the system to 7.5 by using 0.5N NaOH;
(3) reaction: reacting the system at 30 ℃, stirring for 8 hours, and maintaining the pH of the system to 7.5 by using 1N NaOH;
(4) and (3) post-treatment: stopping reaction with 900 μ L of 50% acetonitrile in 100 μ L system, shaking thoroughly, mixing, centrifuging at 8000rpm for 1min, blowing the obtained supernatant with nitrogen, dissolving in ethanol, and detecting by HPLC that conversion rate is 30.59%, and the product is
Figure 268229DEST_PATH_IMAGE029
The e.e. value is 96.57%.
Example 11:
(1) feeding: to a 100 mL reaction flask, 0.2 g of hydrolase Bacillus sp, 50 mL of potassium phosphate buffer (100 mM, pH =7.5) was added, stirred, and the enzyme was dissolved in the potassium phosphate buffer;
(2) adding a substrate: to a 100 mL reaction flask, 1g of the master batch dissolved in 1.5 mL of DMSO was added
Figure 186900DEST_PATH_IMAGE028
Stirring, and maintaining the pH of the system to 7.5 by using 0.5N NaOH;
(3) reaction: reacting the system at 50 ℃, stirring for 8 hours, and maintaining the pH of the system to 7.5 by using 1N NaOH;
(4) and (3) post-treatment: stopping reaction with 900 μ L of 50% acetonitrile in 100 μ L system, shaking thoroughly, mixing, centrifuging at 8000rpm for 1min, blowing the obtained supernatant with nitrogen, dissolving in ethanol, and detecting by HPLC that conversion rate is 40.97%, and the product is
Figure 358119DEST_PATH_IMAGE029
The e.e. value is 95.64%.
Example 12:
(1) feeding: to a 100 mL reaction flask, 0.2 g of hydrolase Bacillus sp, 50 mL of potassium phosphate buffer (100 mM, pH =7.5) was added, stirred, and the enzyme was dissolved in the potassium phosphate buffer;
(2) adding a substrate: to a 100 mL reaction flask, 1g of the master batch dissolved in 1.5 mL of DMSO was added
Figure 985409DEST_PATH_IMAGE028
Stirring, and maintaining the pH of the system to 7.5 by using 0.5N NaOH;
(3) reaction: reacting the system at 20 ℃, stirring for 8 hours, and maintaining the pH of the system to 7.5 by using 1N NaOH;
(4) and (3) post-treatment: stopping reaction with 900 μ L of 50% acetonitrile in 100 μ L system, shaking thoroughly, mixing, centrifuging at 8000rpm for 1min, blowing the obtained supernatant with nitrogen, dissolving in ethanol, and detecting conversion rate by HPLC (high performance liquid chromatography) of 26.43% to obtain product
Figure 41221DEST_PATH_IMAGE029
The e.e. value is 95.88%.
Example 13:
(1) feeding: to a 100 mL reaction flask, 0.2 g of hydrolase Bacillus sp, 30 mL of potassium phosphate buffer (100 mM, pH =7.5) was added, stirred, and the enzyme was dissolved in the potassium phosphate buffer;
(2) adding a substrate: into a 100 mL reaction flask, 1g of the main raw material
Figure 171988DEST_PATH_IMAGE030
Stirring, and maintaining the pH of the system to 7.5 by using 0.5N NaOH;
(3) reaction: reacting the system at 40 ℃, stirring for 8 hours, and maintaining the pH of the system to 7.5 by using 1N NaOH;
(4) and (3) post-treatment: stopping reaction of 100 mu L system with 900 mu L of 50% acetonitrile, shaking thoroughly, mixing uniformly, centrifuging at 8000rpm for 1min, blowing the obtained supernatant with nitrogen, dissolving in ethanol, and determining the conversion rate by HPLC to be 35.45% and the product to be e.e. 96.79%.
Example 14:
(1) feeding: to a 100 mL reaction flask, add hydrolase Thermomyces.sp.0.5 g, 50 mL Tris-Cl buffer (100 mM, pH =7.5), stir, enzyme dissolved in Tris-Cl buffer;
(2) adding a substrate: into a 150 mL reaction flask, 1g of the main raw material was added
Figure 215905DEST_PATH_IMAGE030
Stirring, and maintaining the pH of the system to 7.5 by using 0.5N NaOH;
(3) reaction: reacting the system at 30 ℃, stirring for 16 h, and maintaining the pH of the system to 7.5 by using 1N NaOH;
(4) sampling: stopping reaction with 900 μ L of 50% acetonitrile in 100 μ L system, shaking thoroughly, mixing, centrifuging at 8000rpm for 1min, blowing the obtained supernatant with nitrogen, dissolving in ethanol to obtain the final product
Figure 268175DEST_PATH_IMAGE031
The results are shown in the following table.
In addition, 1g of the main starting material was added dissolved in the cosolvent in table 2 at the time of substrate addition, and the corresponding conversion and product e.e. values were tested and reported in table 2.
TABLE 2
Figure 924415DEST_PATH_IMAGE032
Figure 847372DEST_PATH_IMAGE033
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (15)

1. Chiral resolution of isobutyrate compoundThe method is characterized in that the isobutyrate compound is racemic isobutyrate compound and has the following structural general formula
Figure 900997DEST_PATH_IMAGE001
Wherein R is1Selected from: any one of substituted or unsubstituted pyridine, substituted or unsubstituted pyrazole, substituted or unsubstituted pyrimidone, substituted or unsubstituted 4-hydroxyquinoline, substituted or unsubstituted quinazoline and substituted or unsubstituted quinazolinone, R2Is selected from C1~C7Substituted or unsubstituted alkyl of, C6~C10The substituted or unsubstituted aryl group of (a), the chiral resolution process comprising:
step S1, carrying out hydrolysis treatment on the racemic isobutyrate compound by using a hydrolase to obtain a hydrolyzed system, wherein the hydrolase is derived from Thermomyces sp, Rhizomucor miehei and Bacillus sp;
step S2, separating the isobutyrate ester compound which is not hydrolyzed from the acid in the hydrolyzed system.
2. The chiral resolution process of claim 1, wherein R is1Is selected from
Figure 349296DEST_PATH_IMAGE002
、
Figure 863454DEST_PATH_IMAGE003
、
Figure 665188DEST_PATH_IMAGE004
、
Figure 10719DEST_PATH_IMAGE005
、
Figure 239706DEST_PATH_IMAGE006
、
Figure 303477DEST_PATH_IMAGE007
、
Figure 908902DEST_PATH_IMAGE008
And
Figure 108939DEST_PATH_IMAGE009
wherein R is3Is H, methyl, halogen or alkoxy, said R2Is any one of ethyl, butyl, propyl, isobutyl, phenyl and benzyl.
3. The chiral resolution method of claim 2, wherein said alkoxy is selected from any one of methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy and tert-butoxy.
4. The chiral resolution method according to claim 1, wherein the amount of the hydrolase used is 0.005-2.0 g/g of racemic isobutyrate compound.
5. The chiral resolution method according to claim 4, wherein the amount of the hydrolase used is 0.005-0.50 g/g of racemic isobutyrate compound.
6. The chiral separation method according to any one of claims 1 to 5, wherein the step S1 comprises:
mixing an isobutyrate compound, a cosolvent, a hydrolase and a buffer solution to form a hydrolysis reaction system;
and carrying out heat preservation treatment on the hydrolysis reaction system to carry out hydrolysis reaction to obtain a hydrolyzed system.
7. The chiral resolution method according to claim 6, wherein the buffer solution is selected from phosphate buffer solution, acetic acid-acetate buffer solution, tris-hydrochloric acid buffer solution.
8. The chiral resolution method according to claim 6, wherein the pH value of the buffer solution is 5.0-9.0.
9. The chiral resolution method of claim 6, wherein the cosolvent is selected from any one or more of ethyl acetate, dimethyl sulfoxide, polyethylene glycol, methanol, ethanol, acetonitrile, methyl tert-butyl ether, n-heptane, n-hexane, dichloromethane, tetrahydrofuran and n-butanol.
10. The chiral resolution method according to claim 9, wherein the temperature of the hydrolysis reaction is 10-50 ℃.
11. The chiral resolution method according to claim 10, wherein the temperature of the hydrolysis reaction is 20-40 ℃.
12. The chiral resolution method according to claim 11, wherein the temperature of the hydrolysis reaction is 25-35 ℃.
13. The chiral resolution method according to claim 6, wherein the hydrolysis reaction time is 8-120 h.
14. The chiral resolution method according to claim 13, wherein the hydrolysis reaction time is 8-24 h.
15. The chiral separation method according to claim 1, wherein the step S2 comprises:
extracting and separating the hydrolyzed system to obtain a first organic phase containing the isobutyrate compounds and a first water phase containing the hydrolysis products;
adjusting the pH value of the first aqueous phase to be below 3, extracting the first aqueous phase, carrying out phase separation to obtain a second organic phase and a second aqueous phase, and concentrating the second organic phase to obtain the hydrolysate.
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