CN115029397A - Preparation method of R-5, 7-difluorochroman-4-ol - Google Patents
Preparation method of R-5, 7-difluorochroman-4-ol Download PDFInfo
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- CN115029397A CN115029397A CN202210486605.0A CN202210486605A CN115029397A CN 115029397 A CN115029397 A CN 115029397A CN 202210486605 A CN202210486605 A CN 202210486605A CN 115029397 A CN115029397 A CN 115029397A
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- difluorochroman
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- ketoreductase
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- HGTYMLFMXKYIQW-SSDOTTSWSA-N (4r)-5,7-difluoro-3,4-dihydro-2h-chromen-4-ol Chemical compound FC1=CC(F)=C2[C@H](O)CCOC2=C1 HGTYMLFMXKYIQW-SSDOTTSWSA-N 0.000 title claims abstract description 32
- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- 238000006243 chemical reaction Methods 0.000 claims abstract description 72
- OJVRCPGUGZXUAP-UHFFFAOYSA-N 5,7-difluoro-2,3-dihydrochromen-4-one Chemical compound O=C1CCOC2=CC(F)=CC(F)=C21 OJVRCPGUGZXUAP-UHFFFAOYSA-N 0.000 claims abstract description 25
- 239000005515 coenzyme Substances 0.000 claims abstract description 25
- 239000000758 substrate Substances 0.000 claims abstract description 8
- 238000006722 reduction reaction Methods 0.000 claims abstract description 4
- 102000005751 Alcohol Oxidoreductases Human genes 0.000 claims abstract description 3
- 108010031132 Alcohol Oxidoreductases Proteins 0.000 claims abstract description 3
- 102000004190 Enzymes Human genes 0.000 claims description 28
- 108090000790 Enzymes Proteins 0.000 claims description 28
- 238000000034 method Methods 0.000 claims description 26
- 101001110310 Lentilactobacillus kefiri NADP-dependent (R)-specific alcohol dehydrogenase Proteins 0.000 claims description 19
- 239000007788 liquid Substances 0.000 claims description 19
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 16
- 239000008103 glucose Substances 0.000 claims description 9
- 241000894006 Bacteria Species 0.000 claims description 6
- 241000588724 Escherichia coli Species 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 4
- 101710088194 Dehydrogenase Proteins 0.000 claims description 2
- 102000009105 Short Chain Dehydrogenase-Reductases Human genes 0.000 claims description 2
- 108010048287 Short Chain Dehydrogenase-Reductases Proteins 0.000 claims description 2
- 150000001299 aldehydes Chemical class 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- XJLXINKUBYWONI-NNYOXOHSSA-O NADP(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-O 0.000 claims 1
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- 108010050375 Glucose 1-Dehydrogenase Proteins 0.000 description 10
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- XJLXINKUBYWONI-NNYOXOHSSA-N NADP zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-N 0.000 description 7
- 238000006555 catalytic reaction Methods 0.000 description 6
- 108010029645 galactitol 2-dehydrogenase Proteins 0.000 description 6
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- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical group NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 5
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- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 5
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
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- 238000002474 experimental method Methods 0.000 description 3
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- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000010791 quenching Methods 0.000 description 3
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- 239000012880 LB liquid culture medium Substances 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 238000007036 catalytic synthesis reaction Methods 0.000 description 2
- 230000002860 competitive effect Effects 0.000 description 2
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- 239000001963 growth medium Substances 0.000 description 2
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 150000002576 ketones Chemical class 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical group CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000000967 suction filtration Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- -1 (R) -5, 7-Difluorobenzochroman-4-ol Chemical compound 0.000 description 1
- WHBMMWSBFZVSSR-GSVOUGTGSA-N (R)-3-hydroxybutyric acid Chemical compound C[C@@H](O)CC(O)=O WHBMMWSBFZVSSR-GSVOUGTGSA-N 0.000 description 1
- HJSSBIMVTMYKPD-UHFFFAOYSA-N 3,5-difluorophenol Chemical compound OC1=CC(F)=CC(F)=C1 HJSSBIMVTMYKPD-UHFFFAOYSA-N 0.000 description 1
- LAMUXTNQCICZQX-UHFFFAOYSA-N 3-chloropropan-1-ol Chemical compound OCCCCl LAMUXTNQCICZQX-UHFFFAOYSA-N 0.000 description 1
- 241000590002 Helicobacter pylori Species 0.000 description 1
- NPYPAHLBTDXSSS-UHFFFAOYSA-N Potassium ion Chemical compound [K+] NPYPAHLBTDXSSS-UHFFFAOYSA-N 0.000 description 1
- DWAQJAXMDSEUJJ-UHFFFAOYSA-M Sodium bisulfite Chemical compound [Na+].OS([O-])=O DWAQJAXMDSEUJJ-UHFFFAOYSA-M 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 208000007107 Stomach Ulcer Diseases 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 230000001262 anti-secretory effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000009876 asymmetric hydrogenation reaction Methods 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 125000003636 chemical group Chemical group 0.000 description 1
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- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 230000008029 eradication Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 201000005917 gastric ulcer Diseases 0.000 description 1
- 208000021302 gastroesophageal reflux disease Diseases 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229940037467 helicobacter pylori Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 210000001711 oxyntic cell Anatomy 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 229920000333 poly(propyleneimine) Polymers 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 229910001414 potassium ion Inorganic materials 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 229940126409 proton pump inhibitor Drugs 0.000 description 1
- 239000000612 proton pump inhibitor Substances 0.000 description 1
- 238000004537 pulping Methods 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- UKLNMMHNWFDKNT-UHFFFAOYSA-M sodium chlorite Chemical compound [Na+].[O-]Cl=O UKLNMMHNWFDKNT-UHFFFAOYSA-M 0.000 description 1
- 229960002218 sodium chlorite Drugs 0.000 description 1
- 235000010267 sodium hydrogen sulphite Nutrition 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
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Abstract
The invention belongs to the technical field of medicines, and particularly relates to a preparation method of R-5, 7-difluorochroman-4-ol. The preparation method of the invention takes 5, 7-difluorochroman-4-one as a substrate, and asymmetric reduction reaction is carried out under the condition that ketone reductase, coenzyme and coenzyme circulating system exist to obtain R-5, 7-difluorochroman-4-ol. The enzymatic reaction has extremely high conversion rate and good chiral selectivity, only uses a small amount of environment-friendly organic solvent, has mild reaction conditions and simple process operation, is suitable for industrial production of R-5, 7-difluorochroman-4-ol, and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a preparation method of R-5, 7-difluorochroman-4-ol.
Background
The traditional drugs for the treatment of acid related diseases are Proton Pump Inhibitors (PPI). Although the therapeutic effect of PPIs is satisfactory, they still have some disadvantages, such as: short half-life in plasma, long duration of drug action, and the like, and the drug action is influenced by the amount of food ingested. The potassium ion competitive acid blocker (P-CAB) is a novel drug, and shows quick and effective antisecretory activity by competitive and reversible combination with H +/K + -ATPase in parietal cells.
Tegolrazan is a novel P-CAB, and since 7 months of 2018, tegolrazan has been approved in korea for the treatment of gastroesophageal reflux disease, gastric ulcer, and eradication of helicobacter pylori.
R-5, 7-difluorochroman-4-ol is an important intermediate for synthesizing Tegolazan, and the structural formula is as follows:
at present, R-5, 7-difluorochroman-4-ol is synthesized mainly by a chemical method, such as: patents CN101341149B, CN107849003A and CN 109320485A. The method mainly uses the chiral catalyst to achieve the effect of asymmetric hydrogenation, and has the defects of high price of the chiral catalyst, difficult product purification, non-ideal yield, environmental pollution and the like. Compared with chemical synthesis, the enzyme catalysis has the characteristics of mild reaction conditions, high stereoselectivity, high catalytic activity, simple post-treatment and the like. At present, many researches on the preparation of organic compounds by enzyme catalysis have been reported, for example, "CN 109852593A-a recombinant ketoreductase and its application in the preparation of R-3-hydroxybutyric acid and its salts" the alcohols are successfully prepared by recombinant ketoreductase.
However, these enzyme systems reported in the prior art do not allow their enzymatic synthesis or low conversion due to the differences in the functional groups and chiral positions in R-5, 7-difluorochroman-4-ol. There are no other suitable enzymes available to convert other readily available substrates to R-5, 7-difluorochroman-4-ols. Therefore, the enzymatic synthesis of R-5, 7-difluorochroman-4-ol remains a problem to be solved in the art.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a preparation method of R-5, 7-difluorochroman-4-ol, aiming at selecting a proper enzyme catalysis system to realize the enzymatic synthesis of the R-5, 7-difluorochroman-4-ol.
A preparation method of R-5, 7-difluorochroman-4-ol takes 5, 7-difluorochroman-4-one as a substrate, and asymmetric reduction reaction is carried out under the condition that ketone reductase, coenzyme and coenzyme circulating system exist to obtain R-5, 7-difluorochroman-4-ol;
wherein, the ketoreductase is one or the combination of two or more of short-chain dehydrogenase SDR, medium-chain dehydrogenase MDR or aldehyde ketoreductase AKR.
Preferably, the coenzyme cycle system is a GDH-glucose coenzyme cycle system or an isopropanol coenzyme cycle system.
Preferably, the coenzyme is NAD or NADP.
Preferably, the reaction is carried out in 0.01-0.4mol/L PBS buffer.
Preferably, the pH condition of the reaction is 6.5 to 7.0.
Preferably, the reaction temperature is 30-37 ℃ and the reaction time is 12-24 h.
Preferably, the concentration of the 5, 7-difluorochroman-4-one in the reaction system is 30 to 100 g/L.
Preferably, the ketoreductase is added to the reaction system during the reaction in the form of recombinant wet E.coli or liquid enzyme expressing the ketoreductase.
Preferably, the concentration of the recombinant escherichia coli wet bacteria in the reaction system is 30 g/L-100 g/L;
or the concentration of the liquid enzyme in the reaction system is 150 g/l-500 g/l.
Preferably, the concentration of the coenzyme in the reaction system is 0.1-1 g/L.
In the present invention, the "coenzyme" is a small organic molecule capable of transferring a chemical group from one enzyme to another, and is loosely bound to the enzyme and necessary for the activity of a specific enzyme to be exerted. For example, the coenzyme of the invention may be selected from NAD (nicotinamide adenine dinucleotide) or NADP (nicotinamide adenine dinucleotide phosphate). The coenzyme circulation system is a chemical reaction system generated in the coenzyme action process. For example, the coenzyme cycle system of the present invention can be selected from a GDH-glucose coenzyme cycle system or an isopropanol coenzyme cycle system. The GDH-glucose coenzyme cycle system can transfer the transfer of H atom dependent NAD (P) required for converting reaction substrate ketone into product alcohol to glucose supply, wherein GDH is glucose dehydrogenase. The "isopropanol coenzyme cycle system" can transfer the H atom-dependent NAD (P) transfer required for the conversion of the reaction substrate ketone to the product alcohol to the isopropanol supply.
The invention realizes the method for preparing R-5, 7-difluorochroman-4-ol by enzyme catalysis through the selection of enzyme and the optimization of reaction conditions, can synthesize the target product by only one step, and can achieve higher yield and chiral purity.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 is a schematic diagram of the synthesis of R-5, 7-difluorochroman-4-ol in examples 1-3 of the present invention;
FIG. 2 is an HPLC chromatogram for substrate conversion assay of example 1 of the present invention;
FIG. 3 is an HPLC chromatogram for optical purity measurement of the product of example 1 of the present invention.
Detailed Description
The reagents and materials used in the following examples and experimental examples are commercially available unless otherwise specified.
EXAMPLE 1 catalytic Synthesis of (R) -5, 7-Difluorobenzochroman-4-ol by ketoreductase (wet cell), preparation of wet cell
Preparation of wet cells expressing ketoreductase SDR: inoculating an escherichia coli strain expressing ketoreductase SDR into 50ml of LB liquid culture medium, carrying out shake culture at 37 ℃ for 16h, transferring 8ml of cultured bacterium liquid into 400ml of TB liquid culture medium after the culture is finished, detecting the OD value to be 0.6-0.8 after the culture is carried out for 2-3h, adding isopropyl-beta-D-thiogalactoside with the final concentration of 0.1mM to induce protein expression, and carrying out shake culture at 28 ℃ for 18 h. The cells were collected by centrifugation at 8000 rpm. .
Preparation of Wet cells expressing glucose dehydrogenase GDH: inoculating an Escherichia coli strain expressing GDH into 50ml of LB liquid culture medium, carrying out shake culture at 37 ℃ for 16h, transferring 8ml of cultured bacterium liquid into 400ml of TB liquid culture medium after the culture is finished, carrying out culture for 2-3h, detecting the OD value to be 0.6-0.8, adding isopropyl-beta-D-thiogalactoside with the final concentration of 0.1mM to induce protein expression, and carrying out shake culture at 28 ℃ for 18 h. The wet cells were collected by centrifugation at 8000 rpm.
Second, detection method
The detection method of the conversion rate of the 5, 7-difluorochroman-4-one comprises the following steps:
HPLC analytical method: analytical column Welch
C18, wherein the mobile phase is acetonitrile and water respectively, the flow rate is 1ml/min, the column temperature is 30 ℃, the detection wavelength is 220nm, the analysis time is 15min, the retention time of R-5, 7-difluorochroman-4-ol is 9.118min, and the retention time of 5, 7-difluorochroman-4-one is 11.319 min.
The method for detecting the optical purity of the R-5, 7-difluorochroman-4-ol comprises the following steps:
HPLC analytical method: analytical column
And AD-H, wherein the mobile phase is n-hexane and isopropanol respectively, the flow rate is 1.2ml/min, the column temperature is 25 ℃, the detection wavelength is 220nm, the analysis time is 30min, the retention time of R-5, 7-difluorochroman-4-ol is 12.126min, the retention time of S-5, 7-difluorochroman-4-ol is 10.862min, and the retention time of 5, 7-difluorochroman-4-one is 9.398 min.
Preparation of tris, 5, 7-difluorochroman-4-ones
A25L glass reactor was charged with acetonitrile (10L), and 3, 5-difluorophenol (1Kg, 7.69mol), potassium carbonate (2.66Kg, 19.23mol), potassium iodide (0.26Kg, 1.54mol) and 3-chloro-1-propanol (1.09Kg, 11.54mol) were added. Then, heating the reaction system to 80-85 ℃ under stirring, carrying out reflux reaction for 22-24h, and cooling and filtering after the reaction is finished. Pumping acetonitrile (4.5L) and water (10L) into another 50L kettle, adding Tempo (0.156Kg, 1.0mol), sodium chlorite (80%, 1.74Kg, 15.38mol), sodium hypochlorite (7.5%, 0.154Kg, 0.154mol), potassium dihydrogen phosphate (0.44Kg, 3.23mol) and disodium hydrogen phosphate (0.56Kg, 3.23mol), stirring for dissolving, cooling to 0-10 deg.C, slowly adding the above filtered reaction solution dropwise, keeping the internal temperature at 0-15 deg.C, heating to 20-25 deg.C, and stirring for 30-60 min. After the reaction was completed, the reaction mixture was quenched by dropwise addition of a solution of sodium bisulfite (0.96kg, 9.23mol) in water (4L). And (3) evaporating under reduced pressure to remove the acetonitrile solvent, cooling to room temperature, stirring, crystallizing, filtering, washing with water and drying. Adding concentrated sulfuric acid (2.8L) and the dried solid into a 5L four-mouth reaction bottle, stirring and heating to 50-55 ℃, reacting for 2-3h, ending, adding water (20L) into another 50L kettle, stirring and cooling to 0-10 ℃, then transferring the reaction liquid into a dropping funnel, slowly dripping into the water, keeping the internal temperature not more than 15 ℃, heating to room temperature after dripping, stirring and crystallizing for 1-2h, filtering, washing with water, washing with alkali, and drying to obtain the white-like solid 5, 7-difluorochroman-4-one (1.17Kg, 83%).
Preparation method of tetra, R-5, 7-difluorochroman-4-ol
The flow of the preparation method is shown in figure 1.
29.3g of glucose was weighed, dissolved in 650ml of pure water with stirring, and 20g of 5, 7-difluorochroman-4-one, 20g of wet cell expressing ketoreductase SDR and 3.3g of wet cell expressing glucose dehydrogenase GDH were added thereto, and after sufficiently and uniformly stirred, 0.02g of NADP was added thereto, the reaction was carried out at 30 ℃ with pH 7.0 being controlled by an alkali solution (5M NaOH solution), and the reaction was carried out for 24 hours, and the reaction was quenched with anhydrous ethanol for sampling, and the conversion rate was measured by the above-mentioned HPLC analysis method, as shown in FIG. 2, the conversion rate of 5, 7-difluorochroman-4-one was 99.5%.
After the reaction is finished, heating the system to 70 ℃, preserving heat for 30min for sterilization, after the sterilization is finished, cooling the system to 5-10 ℃, adding 20g of diatomite, filtering, collecting a filter cake, extracting the filter cake for 2-3 times by using 60ml of absolute ethyl alcohol, and evaporating the solvent to dryness to obtain 19.4g of (R) -5, 7-difluorochroman-4-ol crude product, wherein the yield is as follows: 96% and the optical purity was measured by the HPLC analysis method described above, and as shown in FIG. 3, the ee value was 99.4%.
Adding the crude product obtained in the previous step into 60ml of pure water, maintaining the temperature of the system at 30 ℃, stirring for 5h, cooling to 0-5 ℃, performing suction filtration, washing a filter cake with a small amount of pure water, collecting the filter cake, drying in a 45 ℃ forced air oven for 24h, and obtaining 19.08g of (R) -5, 7-difluorochroman-4-ol pure product, wherein the yield is as follows: 94.4%, optical purity according to the HPLC analysis method described above, ee value 99.9%. The pure water is adopted for pulping, so that the chiral value of the product can be improved to 99.9 percent, and the loss of the product is less.
EXAMPLE 2 catalytic Synthesis of (R) -5, 7-Difluorobenzoman-4-ol by ketoreductase (liquid enzyme)
Preparation of liquid enzyme
SDR liquid enzyme: wet cells expressing ketoreductase SDR prepared in example 1 were mixed and spun with 0.1M PBS buffer (pH 7.0), homogenized and disrupted, and the supernatant was collected by centrifugation to obtain ketoreductase liquid enzyme, wherein the weight ratio of added PBS to wet cells was 4: 1.
glucose dehydrogenase GDH liquid enzyme: the wet GDH cells prepared in example 1 were mixed and spun with 0.1M PBS buffer (pH 7.0), homogenized and disrupted, and the supernatant was collected by centrifugation to obtain a liquid GDH enzyme, wherein the weight ratio of added PBS to wet cells was 4: 1.
second, detection method
The method for measuring the conversion of 5, 7-difluorochroman-4-one and the method for measuring the optical purity of R-5, 7-difluorochroman-4-ol are the same as in example 1.
Preparation of tris, 5, 7-difluorochroman-4-ones
Same as in example 1.
Preparation method of tetra, R-5, 7-difluorochroman-4-ol
Weighing 58.65g of glucose, dissolving in 120ml of pure water under stirring, adding 40g of 5, 7-difluorochroman-4-one, 210g of SDR liquid enzyme (liquid enzyme converted wet bacteria content is 9.5 percent) and 70g of glucose dehydrogenase GDH (liquid enzyme converted wet bacteria content is 9.5 percent), fully stirring uniformly, adding 0.04g of NADP, maintaining the system temperature at 30 ℃, controlling the pH to be 7.0 by alkali liquor, reacting for 12 hours, sampling, quenching the reaction by absolute ethyl alcohol, detecting the conversion rate according to the HPLC analysis method, wherein the conversion rate of the 5, 7-difluorochroman-4-one is 99.9 percent.
After the reaction is finished, heating the system to 70 ℃, preserving heat for 30min to denature protein, after the heat preservation is finished, cooling the system to 5-10 ℃, adding 10g of diatomite, filtering, collecting filter cakes, extracting the filter cakes for 2-3 times by 120ml of absolute ethyl alcohol, and evaporating the solvent to dryness to obtain 45g of (R) -5, 7-difluorochroman-4-ol crude product, wherein the yield is as follows: 96% and the optical purity according to the HPLC analysis method described above, ee value 99.4%.
Adding the crude product obtained in the previous step into 120ml of pure water, maintaining the temperature at 30 ℃, stirring for 5h, cooling to 0-5 ℃, performing suction filtration, washing a filter cake with a small amount of pure water, collecting the filter cake, drying in a 45 ℃ forced air oven for 24h to obtain 38.3g of (R) -5, 7-difluorochroman-4-ol pure product, wherein the yield is as follows: 94.8%, optical purity according to the HPLC analysis method described above, ee value 99.9%.
EXAMPLE 3 Synthesis of (R) -5, 7-Difluorobenzoman-4-ol by Isopropanol Recycling System
Preparation of wet thallus
Same as in example 1.
Second, detection method
The method for measuring the conversion of 5, 7-difluorochroman-4-one and the method for measuring the optical purity of R-5, 7-difluorochroman-4-ol are the same as in example 1.
Preparation of tris, 5, 7-difluorochroman-4-ones
Same as in example 1.
Fourthly, preparation method
40g of ketoreductase SDR wet cells were weighed out, mixed uniformly in 320ml of 0.1M phosphate buffer solution having a pH of 7.0, and then 40g of 5, 7-difluorochroman-4-one, 80ml of isopropanol and 0.04g of NADP were added, stirred at 30 ℃ for 24 hours, and then a sample was taken and quenched with absolute ethanol, and the conversion was determined according to the analytical method described in example 2, and the conversion of 5, 7-difluorochroman-4-one was 99.5% and the ee value was 99.4%.
The technical effects of the present invention will be further described below by experiments. Experimental conditions not specifically described in the experimental examples were set with reference to the first to third sections in examples 1 and 2.
Experimental example 1 optimization of reaction temperature
Three sets of parallel experiments were carried out, and 5ml of SDR liquid enzyme and 1.5ml of GDH liquid enzyme were taken, respectively, added to 3.5ml of purified water, and 1.5g of glucose, 1.5g of 5, 7-difluorochroman-4-one, and 0.001g of NADP were added with stirring, and the reaction system was stirred at 25 to 35 ℃ for 24 hours with the pH of 7.0 controlled.
After the reaction is finished, sampling and quenching with absolute ethyl alcohol, and detecting the conversion rate of the 5, 7-difluoro chroman-4-one by HPLC (detection method).
TABLE 1 conversion of substrate under different reaction temperature conditions
| Temperature of | Conversion rate of 24h |
| 25℃ | 68％ |
| 30℃ | 98.5％ |
| 35℃ | 42.3％ |
As can be seen from the results of the measurement in table 1, the conversion rate obtained under the reaction condition at 30 ℃ is significantly higher than that obtained at other temperatures, and thus 30 ℃ is the optimum reaction temperature.
Experimental example 2 optimization of reaction pH
Four sets of parallel experiments were performed, taking 5ml SDR liquid enzyme and 1.5ml GDH liquid enzyme, respectively. Adding 3.5ml of pure water, adding 1.5g of glucose, 1g of 5, 7-difluorochroman-4-one and 0.001g of NADPD under stirring, controlling the reaction temperature to be 30 ℃, and respectively stirring and reacting for 24 hours under the condition of different pH values of 5.0-8.0.
After the reaction is finished, sampling and quenching with absolute ethyl alcohol, and detecting the conversion rate of the 5, 7-difluoro chroman-4-one by HPLC (detection method).
TABLE 2 conversion at different pH
| PH | Conversion rate of 24h |
| 5.0-5.5 | 43％ |
| 5.5-6.0 | 64％ |
| 6.5-7.0 | 98.5％ |
| 7.5-8.0 | 5.7％ |
As can be seen from the test results in table 2, the conversion rate obtained under the reaction conditions of pH 6.5 to 7.0 is significantly better than that obtained under other pH ranges, and thus, pH 6.5 to 7.0 is the optimum reaction pH condition.
As can be seen from the above examples, the present application selects an appropriate ketoreductase and prefers the reaction conditions. The method realizes the reduction of 5, 7-difluorochroman-4-one into (R) -5, 7-difluorochroman-4-ol by enzyme catalysis, has extremely high conversion rate of enzyme catalysis reaction, good chiral selectivity, mild reaction conditions and simple process operation, only uses a small amount of environment-friendly organic solvent, is suitable for industrial production of the R-5, 7-difluorochroman-4-ol, and has good application prospect.
Claims (10)
1. A preparation method of R-5, 7-difluorochroman-4-ol is characterized by comprising the following steps: the method takes 5, 7-difluorochroman-4-one as a substrate, and performs asymmetric reduction reaction in the presence of ketone reductase, coenzyme and coenzyme circulating system to obtain R-5, 7-difluorochroman-4-ol;
wherein, the ketoreductase is one or the combination of two or more of short-chain dehydrogenase SDR, medium-chain dehydrogenase MDR or aldehyde ketoreductase AKR.
2. The method of claim 1, wherein: the coenzyme circulating system is a GDH-glucose coenzyme circulating system or an isopropanol coenzyme circulating system.
3. The method of claim 1, wherein: the coenzyme is NAD or NADP.
4. The method of claim 1, wherein: the reaction is carried out in 0.01-0.4mol/L PBS buffer.
5. The method of claim 1, wherein: the pH condition of the reaction is 6.5-7.0.
6. The method of claim 1, wherein: the reaction temperature is 30-37 ℃, and the reaction time is 12-24 h.
7. The method of claim 1, wherein: the concentration of the 5, 7-difluoro chroman-4-one in the reaction system is 30-100 g/L.
8. The method of claim 1, wherein: during the reaction process, the ketoreductase is added into the reaction system in the form of recombinant escherichia coli wet bacteria or liquid enzyme for expressing the ketoreductase.
9. The method of claim 8, wherein: the concentration of the recombinant escherichia coli wet thallus in a reaction system is 30-100 g/L;
or the concentration of the liquid enzyme in the reaction system is 150 g/l-500 g/l.
10. The method of claim 1, wherein: the concentration of the coenzyme in the reaction system is 0.1-1 g/L.
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| Title |
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| 薛群，等: "荧光假单胞菌短链脱氢酶的克隆、表达及酶学性质分析", 生物工程学报, vol. 27, no. 9, 25 September 2011 (2011-09-25), pages 1317 - 1325 * |
| 陈敏，等: "重组短链脱氢酶 LcSDR 催化(R)-1-苯基乙醇不对称合成的工艺开发", 化学反应工程与工艺, vol. 32, no. 4, 31 August 2016 (2016-08-31), pages 320 - 325 * |
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