CN114921508B - Biocatalytic preparation method of (R) -6-hydroxy chroman-3-carboxylic acid - Google Patents
Biocatalytic preparation method of (R) -6-hydroxy chroman-3-carboxylic acid Download PDFInfo
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Abstract
The invention discloses a biocatalytic preparation method of (R) -6-hydroxy chroman-3-carboxylic acid, which comprises the following steps: in a liquid reaction system, taking a compound shown in a formula I as a substrate, and carrying out asymmetric reduction reaction under the catalysis of alkene reductase in the presence of coenzyme to form a compound shown in a formula II; step (b) of separating the compound of formula II from the reaction system after the reaction of step (a); wherein the alkene reductase is selected from: alkene reductase ERED-16323 or ERED-1663. The invention also provides a reaction system. The method has the advantages of short steps, mild conditions, high conversion rate and high optical purity of the product.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a biocatalytic preparation method of (R) -6-hydroxy chroman-3-carboxylic acid.
Background
US patent US10183939B2 discloses a class of structurally novel racemic RAF inhibitors that demonstrate binding affinity for B-RAF V600E and C-RAF, which pan-RAF inhibitors are considered promising candidates to overcome the resistance mechanisms associated with clinically approved B-RAF selective drugs. Wherein, the examples describe that the racemic RAF inhibitor can be resolved by a chromatographic column to obtain two single configuration enantiomers, and the biological activity data of the two single configuration enantiomers show significant differences (such as IC of compounds 3A and 3B) 50 Values differ by a factor of about 10). Therefore, it is of practical interest to develop a process for the preparation of enantiomers of a single configuration.
The compound (R) -6-hydroxy chroman-3-carboxylic acid and derivatives thereof are key chiral intermediates for preparing the single-configuration RAF inhibitor, and the current synthetic method is to use a noble metal catalyst and a chiral ligand to perform catalytic hydrogenation reduction on the 6-hydroxy-2H-chromene-3-carboxylic acid to obtain the compound. The method has the problems of poor product stereoselectivity, poor yield, low atom economy, high price of the catalyst and the chiral ligand, harsh hydrogenation conditions, high use safety risk and the like.
Therefore, there is a need in the art to develop a novel preparation method of (R) -6-hydroxychroman-3-carboxylic acid and its derivatives.
Disclosure of Invention
The invention aims to provide a preparation method of (R) -6-hydroxy chroman-3-carboxylic acid, which has the advantages of high substrate conversion rate, high optical purity of products, mild reaction conditions, simple and convenient operation and easy industrial production.
In a first aspect of the present invention, there is provided a biocatalytic preparation method of (R) -6-hydroxychroman-3-carboxylic acid, comprising the steps of:
in a liquid reaction system, taking a compound shown in a formula I as a substrate, and carrying out asymmetric reduction reaction under catalysis of alkene reductase in the presence of coenzyme to form a compound shown in a formula II;
step (b) of separating the compound of formula II from the reaction system after the reaction of step (a);
wherein the alkene reductase is selected from: 1) Ene reductase ERED-16323 with Genbank number WP-014096091; or 2) an ene reductase ERED-1663 with Genbank accession number NC-015687.
In some embodiments, the concentration of the compound of formula I in the reaction system is from 0.1 to 500g/L.
Preferably, the concentration of the compound of formula I in the reaction system is 1g/L or more, 10g/L or more, 25g/L or more, 50g/L or more, 75g/L or more, 100g/L or more, 125g/L or more, 150g/L or more, 175g/L or more, 200g/L or more, 220g/L or more, 240g/L or more, 260g/L or more, 280g/L or more, 300g/L or more, 320g/L or more, 350g/L or more, 400g/L or more, or 450g/L or more.
Preferably, in the reaction system, the concentration of the compound shown in the formula I is 1-500g/L; more preferably, in the reaction system, the concentration of the compound shown in the formula I is 10-400g/L; most preferably, the concentration of the compound of formula I in the reaction system is 50-350g/L.
In some embodiments, the mass ratio (w/w) of the alkene reductase to the compound of formula I in the reaction system is (0.01 to 20): 1. preferably, the mass ratio (w/w) of the alkene reductase to the compound of the formula I is (0.01-10): 1. more preferably, the mass ratio (w/w) of the alkene reductase to the compound of formula I is (0.1 to 10): 1.
in some embodiments, the coenzyme is selected from the group consisting of: a reducing coenzyme, an oxidizing coenzyme, or a combination thereof.
In some embodiments, the reducing coenzyme is selected from the group consisting of: NADH, NADPH, or a combination thereof.
In some embodiments, the oxidative coenzyme is selected from the group consisting of: NAD +, NADP +, or combinations thereof. Preferably, the oxidative coenzyme is selected from NADP +. Preferably, the oxidative coenzyme is selected from NAD +.
In some embodiments, the mass ratio (w/w) of the coenzyme to the compound of formula I is (0.0001 to 1): 1. preferably, the mass ratio (w/w) of the coenzyme to the compound of formula I is (0.0001 to 0.3): 1. more preferably, the mass ratio (w/w) of the coenzyme to the compound of formula I is (0.01 to 0.3): 1.
in some embodiments, an enzyme for coenzyme regeneration is also present in the reaction system. Preferably, the enzyme for coenzyme regeneration is selected from the group consisting of: glucose dehydrogenase, alcohol dehydrogenase, formate dehydrogenase, or a combination thereof. More preferably, the enzyme for coenzyme regeneration is selected from the group consisting of: a glucose dehydrogenase.
In some embodiments, in the reaction system, the mass ratio (w/w) of the enzyme for coenzyme regeneration to the compound of formula I is (0.01 to 10): 1. preferably, the mass ratio (w/w) of the enzyme for coenzyme regeneration to the compound of formula I is (0.01 to 6): 1. more preferably, the mass ratio (w/w) of the enzyme for coenzyme regeneration to the compound of formula I is (0.1 to 2): 1.
in some embodiments, a co-substrate is also present in the reaction system. Preferably, the co-substrate is selected from: isopropanol, glucose, ammonium formate, or a combination thereof. More preferably, the co-substrate is selected from: and (3) glucose.
In some embodiments, the mass ratio (w/w) of the co-substrate to the compound of formula I in the reaction system is (0.1-20): 1. preferably, the mass ratio (w/w) of the co-substrate to the compound of formula I is (1-10): 1. preferably, the mass ratio (w/w) of the co-substrate to the compound of formula I is (1-5): 1.
in some embodiments, the combination of the enzyme for coenzyme regeneration and the co-substrate in the reaction system is selected from the group consisting of: (1) glucose dehydrogenase with glucose; (2) alcohol dehydrogenase with isopropanol; or (3) formate dehydrogenase with ammonium formate. Preferably, the combination of the enzyme for coenzyme regeneration and the co-substrate is selected from: glucose dehydrogenase and glucose.
In some embodiments, in step (a), the reaction temperature is from 5 ℃ to 50 ℃. Preferably, the reaction temperature is 15 ℃ to 45 ℃. More preferably, the reaction temperature is 20 ℃ to 40 ℃. Most preferably, the reaction temperature is from 25 ℃ to 35 ℃.
In some embodiments, the reaction time in step (a) is 0.1 to 120 hours. Preferably, the reaction time is 0.2 to 72 hours. More preferably, the reaction time is 0.5 to 48 hours. Most preferably, the reaction time is from 1 to 24 hours.
In some embodiments, in the step (a), the pH of the reaction system is 5.5 to 9.5. Preferably, the pH is 6.0 to 8.5. More preferably, the pH is 6.5 to 8.0. Most preferably, the pH is from 6.5 to 7.5.
In some embodiments, the alkene reductase is present in the reaction system in the form of: free form, immobilized enzyme or enzyme in the form of bacterial cells.
In some embodiments, the reaction system is an aqueous system.
In some embodiments, the buffer of the reaction system is Phosphate Buffered Saline (PBS), TRIS-methylamine salt buffer (TRIS-HCl), TRIS-methylamine sulfate buffer (TRIS-H) 2 SO 4 ) Or triethanolamine salt buffer (TOEA) or a combination thereof. Preferably, the buffer of the reaction system is Phosphate Buffered Saline (PBS).
In some embodiments, the reaction system contains a solvent selected from the group consisting of: water, alcohol, or a combination thereof.
In some embodiments, the reaction system may also contain a co-solvent. Preferably, the co-solvent is selected from dimethyl sulfoxide.
In some embodiments, the volume to mass ratio of the co-solvent to the compound of formula I is from 0.1 to 20 mL/g. Preferably, the volume-mass ratio of the cosolvent to the compound of the formula I is 1-10 mL/g. More preferably, the volume-to-mass ratio of the cosolvent to the compound of the formula I is 2-5 mL/g.
In some embodiments, in step (b), the separating comprises: heating to inactivate protein, centrifuging or filtering, extracting the filtrate with an extraction solvent, and concentrating the organic layer.
In some embodiments, in step (b), the extraction solvent is methanol.
In some embodiments, in the step (b), the e.e. value of the compound of formula II in the reaction system after the reaction is more than or equal to 99%.
In some embodiments, in step (b), ≧ 30% of the compound of formula I is converted into the compound of formula II in the reaction system after the reaction. Preferably, not less than 32% of the compound of formula I is converted into the compound of formula II. More preferably, not less than 34% of the compound of formula I is converted to the compound of formula II.
In a second aspect, the present invention provides a reaction system comprising:
(1) An aqueous solvent;
(2) A substrate which is a compound of formula I;
(3) A coenzyme;
(4) An alkene reductase; the alkene reductase is selected from: 1) The Genbank serial number of the ene reductase ERED-16323 is WP-014096091; or 2) ERED-1663 with Genbank accession number NC-015687;
(5) A co-substrate.
In some embodiments, the reaction system further comprises: (6) an enzyme for coenzyme regeneration.
In some embodiments, the concentration of the compound of formula I in the reaction system is from 0.1 to 500g/L.
Preferably, the concentration of the compound of formula I in the reaction system is 1g/L or more, 10g/L or more, 25g/L or more, 50g/L or more, 75g/L or more, 100g/L or more, 125g/L or more, 150g/L or more, 175g/L or more, 200g/L or more, 220g/L or more, 240g/L or more, 260g/L or more, 280g/L or more, 300g/L or more, 320g/L or more, 350g/L or more, 400g/L or more, or 450g/L or more.
Preferably, in the reaction system, the concentration of the compound shown in the formula I is 1-500g/L; more preferably, in the reaction system, the concentration of the compound shown in the formula I is 10-400g/L; most preferably, the concentration of the compound of formula I in the reaction system is 50-350g/L.
In some embodiments, the mass ratio (w/w) of the alkene reductase to the compound of formula I in the reaction system is (0.01 to 20): 1. preferably, the mass ratio (w/w) of the alkene reductase to the compound of the formula I is (0.01-10): 1. more preferably, the mass ratio (w/w) of the alkene reductase to the compound of formula I is (0.1 to 10): 1.
in some embodiments, the e.e. value of the compound of formula II in the reacted reaction system is 99% or more.
In some embodiments, ≧ 30% of the compound of formula I is converted into the compound of formula II in the reaction system after the reaction. Preferably, not less than 32% of the compound of formula I is converted into the compound of formula II. More preferably, not less than 34% of the compound of formula I is converted to the compound of formula II.
The reaction system provided by the invention can carry out enzymatic reaction, and can prepare (R) -6-hydroxychroman-3-carboxylic acid with high stereoselectivity, and the optical purity is that the e.e. value is more than or equal to 99%.
In a third aspect, the present invention also provides a biocatalytic preparation method of (R) -6-hydroxychroman-3-carboxylic acid, comprising: carrying out an enzymatic reaction using a reaction system according to the second aspect of the invention, thereby producing a compound of formula II:
in some embodiments, the concentration of the compound of formula I in the reaction system is from 0.1 to 500g/L.
Preferably, the concentration of the compound of formula I in the reaction system is 1g/L or more, 10g/L or more, 25g/L or more, 50g/L or more, 75g/L or more, 100g/L or more, 125g/L or more, 150g/L or more, 175g/L or more, 200g/L or more, 220g/L or more, 240g/L or more, 260g/L or more, 280g/L or more, 300g/L or more, 320g/L or more, 350g/L or more, 400g/L or more, or 450g/L or more.
Preferably, in the reaction system, the concentration of the compound shown in the formula I is 1-500g/L; more preferably, in the reaction system, the concentration of the compound shown in the formula I is 10-400g/L; most preferably, the concentration of the compound of formula I in the reaction system is 50-350g/L.
In some embodiments, in the method, the mass ratio (w/w) of the alkene reductase to the compound of formula I in the reaction system is (0.01 to 20): 1. preferably, the mass ratio (w/w) of the alkene reductase to the compound of the formula I is (0.01-10): 1. more preferably, the mass ratio (w/w) of the alkene reductase to the compound of the formula I is (0.1 to 10): 1.
in some embodiments, in the method, the e.e. value of the compound of formula II in the reaction system after the reaction is 99% or more.
In some embodiments, the method comprises converting ≥ 30% of the compound of formula I into the compound of formula II in the reaction system after the reaction. Preferably, not less than 32% of the compound of formula I is converted into the compound of formula II. More preferably, not less than 34% of the compound of formula I is converted to the compound of formula II.
Terminology of the technology
Alkene reductase
In the present invention, an "ene reductase" is an enzyme capable of stereoselectively catalyzing the reduction of a carbon-carbon double bond to obtain an optically active saturated compound. One typical ene reductase is ene reductase ERED-16323, genbank accession No. WP-014096091; or the ene reductase ERED-1663 with Genbank accession number NC-015687.
The above-mentioned alkene reductase may be used in the reaction system in the form of wet cells, crude enzyme solution, crude enzyme powder, pure enzyme, or the like, and is commercially available.
Coenzyme
In the present invention, "coenzyme" means a coenzyme capable of effecting electron transfer in a redox reaction.
Typically, the coenzyme of the invention is a reducing coenzyme or an oxidizing coenzyme, the reducing coenzyme being NADH or NADPH and the oxidizing coenzyme being NAD + or NADP +. Since the reducing coenzyme is expensive in terms of cost, it is preferable to select the oxidizing coenzyme NAD + or NADP +.
When selecting an oxidative coenzyme, it is necessary to select a method for realizing coenzyme regeneration, which mainly includes three methods: the method comprises the following steps of (1) glucose dehydrogenase and cosubstrate glucose, (2) alcohol dehydrogenase and cosubstrate isopropanol, and (3) formate dehydrogenase and cosubstrate ammonium formate.
In a preferred embodiment, the coenzyme is NADP +, and the coenzyme regeneration system is glucose dehydrogenase, preferably glucose dehydrogenase and glucose co-substrate. The ratio of the dosage of the NADP + to the dosage of the compound shown in the formula I is 0.01-30.0% (w/w). The buffer system is Phosphate Buffer Solution (PBS), TRIS-salt buffer solution (TRIS-HCl), TRIS-sulfate buffer solution (TRIS-H) 2 SO 4 ) Or triethanolamine salt buffer (TOEA) or a combination thereof at a concentration of 0.1 mol/L. The pH of the buffer was 5.5 to 9.5.
Cosolvent
In the present invention, a cosolvent may or may not be added to the reaction system.
As used herein, the term "co-solvent" refers to a sparingly soluble substance that forms a soluble intermolecular complex, association, double salt, or the like with an added third substance in a solvent to increase the solubility of the sparingly soluble substance in the solvent. This third material is referred to as a co-solvent.
In the present invention, the water solubility of the substrate compound is poor, and when the substrate concentration is increased, the reaction conversion rate is seriously affected. Thus, the substrate solubility can be increased by adding a co-solvent to improve the reaction conversion. Optional co-solvents are dimethyl sulfoxide, methanol, ethanol, isopropanol, acetonitrile, toluene, acetone or combinations thereof, preferably dimethyl sulfoxide.
Stereoisomers
In the present invention, stereoisomers refer to isomers produced by different arrangement of atoms in a molecule, and can be divided into cis-trans isomers and enantiomers, and also can be divided into two broad categories, enantiomers and diastereomers. In a chemical or enzymatic reaction, one stereoisomer forms preferentially over the other, referred to as stereoselectivity. Stereoselectivity can be partial, where the formation of one stereoisomer favors the other, or complete, where only one stereoisomer is formed. When a stereoisomer is an enantiomer, stereoselectivity refers to enantioselectivity, i.e., the fraction (usually reported as a percentage) of one enantiomer in the sum of the two enantiomers. It (typically as a percentage) is optionally reported in the art as the enantiomeric excess (e.e.) calculated therefrom according to the formula: [ major enantiomer-minor enantiomer ]/[ major enantiomer + minor enantiomer ]. Where the stereoisomers are diastereomers, stereoselectivity refers to diastereomeric selectivity, i.e., the fraction of one diastereomer in a mixture of two diastereomers (usually reported as a percentage), usually optionally reported as diastereomeric excess (d.e.). Enantiomeric excess and diastereomeric excess are types of stereoisomeric excess. In the present invention, substantially stereoisomerically pure, enzymes are capable of converting a substrate to the corresponding product with at least about 95%, 96%, 97%, 98% or 99% stereoisomeric excess; preferably, at least about 98% stereoisomeric excess; more preferably, at least about 99% stereoisomeric excess.
Biocatalytic preparation method
The invention provides a method for preparing a compound of a formula II by catalytic reduction of a compound of a formula I from a commercially available alkene reductase (ERED-16323 or ERED-1663). The reaction formula is shown as follows:
one specific implementation process of the preparation method is as follows: fully dissolving a substrate in a cosolvent such as dimethyl sulfoxide, adding the cosolvent into a phosphate buffer solution, uniformly stirring, adding wet thalli, crude enzyme liquid, crude enzyme powder or pure enzyme of an alkene reductase, adding coenzyme NADP +, glucose dehydrogenase and cosubstrate glucose, and maintaining the temperature at 5-50 ℃ for reaction for 0.1-120 hours. After the reaction is finished, adding methanol into the reaction solution, centrifuging and/or filtering by diatomite, taking supernatant, detecting and separating the chiral compound by a super-junction chromatography, and concentrating the collected solution to obtain the product.
The present inventors have conducted extensive and intensive studies and, as a result of extensive screening experiments, have unexpectedly developed for the first time a process for the biological production of (R) -6-hydroxychroman-3-carboxylic acid, in which an alkene reductase has high stereoselectivity for the product and specificity for the substrate.
The main advantages of the present invention include:
1) Compared with the prior art, the alkene reductase screened by the application and the preparation method thereof are used for preparing the compound shown in the formula II, and the compound has the advantages of high yield and low cost.
2) The alkene reductase screened by the application has high stereoselectivity to the product of the compound shown in the formula II, the optical purity of the product is high, and the e.e. value reaches more than 99%.
3) The alkene reductases screened by the present application are specific for the substrate compound of formula I.
4) The alkene reductase screened by the method can be used for preparing the compound shown in the formula II from the compound shown in the formula I through one-step reaction, the reaction steps are short, the reaction conditions are mild, only centrifugal filtration is needed for post-treatment, the operation is simple and convenient, and the industrial production is easy.
Drawings
FIG. 1 is a racemic chiral spectrum of a compound of formula II, wherein the retention time of S configuration is 5.826 min and the retention time of R configuration is 6.486 min; in the figure, the abscissa represents time, and the ordinate represents absorbance.
FIG. 2 is a chiral spectrum of a compound of formula II obtained by the catalysis of the alkenylreductase of the present application, showing that the e.e. value of R configuration is more than 99%; in the figure, the abscissa represents time, and the ordinate represents absorbance.
Detailed Description
The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Experimental procedures without specific conditions noted in the following examples, generally following conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: cold Spring Harbor laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight. Unless otherwise specified, the test materials of the present invention are commercially available reagents and are commercially available.
Example 1: screening for ene reductases
The reaction formula is as follows:
reaction operation: to a 10 mL reaction flask, 5 mL of a phosphate buffer solution having a volume concentration of 0.2M and a pH of 7.0, 1 mg of NADP + and 50 mg of alkene reductase (commercially available), 5 mg of glucose dehydrogenase and 20 mg of glucose were added, and the temperature was adjusted to 27 ℃.5 mg of Compound I dissolved in 25. Mu.L of dimethyl sulfoxide was charged into a reaction flask, vigorously stirred, and reacted for 18 hours. After the reaction, 100. Mu.L of the reaction solution was taken, 900. Mu.L of methanol was added, and after centrifugation, separation by SFC detection (supercritical fluid chromatography). When the wavelength is 210 nm, the compound II is detected to have the maximum ultraviolet absorption peak, the maximum absorption wavelength is 293.7 nm, and the peak-off time is 6.501 min, refer to FIG. 2. The reaction results are shown in table 1.
TABLE 1
As can be seen from the reaction results of the tests A1 to A5, different alkene reductases have obvious difference in the reaction effect of the substrate compound shown in the formula I.
Example 2: specificity of ene reductases for substrates
The reaction formula is as follows:
reaction operation: the same reaction procedure as in example 1 was followed using different substrates, using the alkene reductases ERED-16323 or ERED-1663. The reaction results are shown in table 2.
TABLE 2
As shown by the comparison between the tests B1-B4 and the comparative examples A1-A2, the ene reductase ERED-16323 or ERED-1663 has obvious difference in reaction effect on different R4 substituted substrates.
Example 3: amplification of the biocatalytic preparation of (R) -6-hydroxychroman-3-carboxylic acid (compound of formula II)
To a 2L reaction flask, 1L of a phosphate buffer solution having a volume concentration of 0.2M and a pH of 7.0, 1g of NADP + and 10g of an ene reductase (ERED-16323, genbank No. WP-014096091), 1g of glucose dehydrogenase and 4 g of glucose were added, and the temperature was adjusted to 27 ℃. 1g of Compound I dissolved in 5 mL of dimethyl sulfoxide was charged into a reaction flask, vigorously stirred, and reacted for 18 hours. After the reaction is finished, after post-treatment and SFC detection, the conversion rate is 35.5 percent, and the e.e. > 99 percent.
In summary, the above embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A biocatalytic preparation method of (R) -6-hydroxy chroman-3-carboxylic acid is characterized by comprising the following steps:
in a liquid reaction system, taking a compound shown in a formula I as a substrate, and carrying out asymmetric reduction reaction under catalysis of alkene reductase in the presence of coenzyme to form a compound shown in a formula II;
step (b) of separating the compound of formula (II) from the reaction system after the reaction of step (a);
wherein the alkene reductase is selected from: 1) The Genbank serial number of the ene reductase ERED-16323 is WP-014096091; or 2) ERED-1663 with Genbank accession number NC-015687.
2. The method of claim 1, wherein the concentration of the compound of formula I is from 0.1 to 500g/L.
3. The method of claim 1, wherein the mass ratio of the alkene reductase to the compound of formula I is (0.01 to 20): 1.
4. the method of claim 1, wherein the coenzyme is selected from the group consisting of: a reducing coenzyme, an oxidizing coenzyme, or a combination thereof; the reducing coenzyme is selected from the group consisting of: NADH, NADPH, or a combination thereof; the oxidative coenzyme is selected from the group consisting of: NAD +, NADP +, or combinations thereof.
5. The method according to claim 1, wherein the mass ratio of the coenzyme to the compound of formula I is (0.0001 to 1): 1.
6. the method according to claim 1, wherein in the reaction system, an enzyme for coenzyme regeneration; the enzyme for coenzyme regeneration is selected from: glucose dehydrogenase, alcohol dehydrogenase, formate dehydrogenase, or a combination thereof.
7. The method of claim 1, wherein in the reaction system, a co-substrate is also present; the co-substrate is selected from: isopropanol, glucose, ammonium formate, or a combination thereof.
8. The method of claim 1, wherein in the step (a), the reaction temperature is 5 ℃ to 50 ℃, the reaction time is 0.1 to 120 hours, and the pH of the reaction system is 5.5 to 9.5.
9. A reaction system, characterized in that the reaction system comprises:
(1) An aqueous solvent;
(2) A substrate which is a compound of formula I;
(3) A coenzyme;
(4) An alkene reductase; the alkene reductase is selected from: 1) Ene reductase ERED-16323 with Genbank number WP-014096091; or 2) ERED-1663 with Genbank accession number NC-015687;
(5) A co-substrate.
10. A biocatalytic preparation of (R) -6-hydroxychroman-3-carboxylic acid, comprising: performing an enzymatic reaction using the reaction system of claim 9, thereby producing a compound of formula II:
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| WO2022023450A1 (en) * | 2020-07-28 | 2022-02-03 | Jazz Pharmaceuticals Ireland Limited | Chiral synthesis of fused bicyclic raf inhibitors |
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