CN117143934A - Method for preparing (R) -tebuconazole by enzyme chemical method - Google Patents
Method for preparing (R) -tebuconazole by enzyme chemical method Download PDFInfo
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- CN117143934A CN117143934A CN202310879988.2A CN202310879988A CN117143934A CN 117143934 A CN117143934 A CN 117143934A CN 202310879988 A CN202310879988 A CN 202310879988A CN 117143934 A CN117143934 A CN 117143934A
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- tebuconazole
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- epoxy intermediate
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- PXMNMQRDXWABCY-INIZCTEOSA-N (R)-tebuconazole Chemical compound C([C@@](O)(C(C)(C)C)CN1N=CN=C1)CC1=CC=C(Cl)C=C1 PXMNMQRDXWABCY-INIZCTEOSA-N 0.000 title claims abstract description 60
- 238000000034 method Methods 0.000 title claims abstract description 39
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 21
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 21
- 239000000126 substance Substances 0.000 title claims abstract description 16
- 239000004593 Epoxy Substances 0.000 claims abstract description 67
- 241000588724 Escherichia coli Species 0.000 claims abstract description 51
- 238000006243 chemical reaction Methods 0.000 claims abstract description 40
- PXMNMQRDXWABCY-UHFFFAOYSA-N 1-(4-chlorophenyl)-4,4-dimethyl-3-(1H-1,2,4-triazol-1-ylmethyl)pentan-3-ol Chemical compound C1=NC=NN1CC(O)(C(C)(C)C)CCC1=CC=C(Cl)C=C1 PXMNMQRDXWABCY-UHFFFAOYSA-N 0.000 claims abstract description 36
- 239000005839 Tebuconazole Substances 0.000 claims abstract description 36
- 239000007787 solid Substances 0.000 claims abstract description 12
- NSPMIYGKQJPBQR-UHFFFAOYSA-N 4H-1,2,4-triazole Chemical compound C=1N=CNN=1 NSPMIYGKQJPBQR-UHFFFAOYSA-N 0.000 claims abstract description 7
- 238000007142 ring opening reaction Methods 0.000 claims abstract description 5
- 239000000203 mixture Substances 0.000 claims description 17
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 15
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 11
- 239000003960 organic solvent Substances 0.000 claims description 10
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical group [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 9
- 239000000758 substrate Substances 0.000 claims description 9
- 230000002255 enzymatic effect Effects 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 6
- 241000223252 Rhodotorula Species 0.000 claims description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 6
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 6
- 150000002009 diols Chemical group 0.000 claims description 5
- 230000007935 neutral effect Effects 0.000 claims description 5
- 239000012044 organic layer Substances 0.000 claims description 5
- 239000002585 base Substances 0.000 claims description 4
- 238000006911 enzymatic reaction Methods 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 4
- MFRIHAYPQRLWNB-UHFFFAOYSA-N sodium tert-butoxide Chemical compound [Na+].CC(C)(C)[O-] MFRIHAYPQRLWNB-UHFFFAOYSA-N 0.000 claims description 4
- 239000012295 chemical reaction liquid Substances 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 230000010355 oscillation Effects 0.000 claims description 3
- XBDQKXXYIPTUBI-UHFFFAOYSA-M Propionate Chemical compound CCC([O-])=O XBDQKXXYIPTUBI-UHFFFAOYSA-M 0.000 claims description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 2
- KXKVLQRXCPHEJC-UHFFFAOYSA-N acetic acid trimethyl ester Natural products COC(C)=O KXKVLQRXCPHEJC-UHFFFAOYSA-N 0.000 claims description 2
- 239000003513 alkali Substances 0.000 claims description 2
- 230000003287 optical effect Effects 0.000 claims description 2
- 239000012074 organic phase Substances 0.000 claims description 2
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 238000002390 rotary evaporation Methods 0.000 claims description 2
- 239000000741 silica gel Substances 0.000 claims description 2
- 229910002027 silica gel Inorganic materials 0.000 claims description 2
- 238000010025 steaming Methods 0.000 claims description 2
- 239000006228 supernatant Substances 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 238000001311 chemical methods and process Methods 0.000 claims 4
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 230000007613 environmental effect Effects 0.000 abstract description 3
- 238000010364 biochemical engineering Methods 0.000 abstract description 2
- 239000003054 catalyst Substances 0.000 abstract description 2
- 230000001404 mediated effect Effects 0.000 abstract description 2
- 239000000543 intermediate Substances 0.000 description 58
- 230000000694 effects Effects 0.000 description 24
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 21
- 230000001580 bacterial effect Effects 0.000 description 12
- 239000000725 suspension Substances 0.000 description 11
- 239000008057 potassium phosphate buffer Substances 0.000 description 8
- PXMNMQRDXWABCY-MRXNPFEDSA-N (S)-tebuconazole Chemical compound C([C@](O)(C(C)(C)C)CN1N=CN=C1)CC1=CC=C(Cl)C=C1 PXMNMQRDXWABCY-MRXNPFEDSA-N 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 7
- 238000004587 chromatography analysis Methods 0.000 description 6
- 238000004007 reversed phase HPLC Methods 0.000 description 6
- 102000005486 Epoxide hydrolase Human genes 0.000 description 5
- 108020002908 Epoxide hydrolase Proteins 0.000 description 5
- 238000004817 gas chromatography Methods 0.000 description 5
- 230000014759 maintenance of location Effects 0.000 description 5
- 239000000575 pesticide Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 150000003852 triazoles Chemical class 0.000 description 5
- 238000005481 NMR spectroscopy Methods 0.000 description 4
- 230000000844 anti-bacterial effect Effects 0.000 description 4
- 239000003899 bactericide agent Substances 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 230000003197 catalytic effect Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 239000011541 reaction mixture Substances 0.000 description 3
- -1 tebuconazole epoxide Chemical class 0.000 description 3
- 241000219194 Arabidopsis Species 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000005815 base catalysis Methods 0.000 description 2
- 238000010170 biological method Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 238000013375 chromatographic separation Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000000855 fungicidal effect Effects 0.000 description 2
- 239000000417 fungicide Substances 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 231100000053 low toxicity Toxicity 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- YNWVFADWVLCOPU-MDWZMJQESA-N (1E)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1H-1,2,4-triazol-1-yl)pent-1-en-3-ol Chemical compound C1=NC=NN1/C(C(O)C(C)(C)C)=C/C1=CC=C(Cl)C=C1 YNWVFADWVLCOPU-MDWZMJQESA-N 0.000 description 1
- OILXMJHPFNGGTO-UHFFFAOYSA-N (22E)-(24xi)-24-methylcholesta-5,22-dien-3beta-ol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)C=CC(C)C(C)C)C1(C)CC2 OILXMJHPFNGGTO-UHFFFAOYSA-N 0.000 description 1
- RQOCXCFLRBRBCS-UHFFFAOYSA-N (22E)-cholesta-5,7,22-trien-3beta-ol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CCC(C)C)CCC33)C)C3=CC=C21 RQOCXCFLRBRBCS-UHFFFAOYSA-N 0.000 description 1
- WAPNOHKVXSQRPX-SSDOTTSWSA-N (R)-1-phenylethanol Chemical compound C[C@@H](O)C1=CC=CC=C1 WAPNOHKVXSQRPX-SSDOTTSWSA-N 0.000 description 1
- OQMZNAMGEHIHNN-UHFFFAOYSA-N 7-Dehydrostigmasterol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CC(CC)C(C)C)CCC33)C)C3=CC=C21 OQMZNAMGEHIHNN-UHFFFAOYSA-N 0.000 description 1
- DNVPQKQSNYMLRS-NXVQYWJNSA-N Ergosterol Natural products CC(C)[C@@H](C)C=C[C@H](C)[C@H]1CC[C@H]2C3=CC=C4C[C@@H](O)CC[C@]4(C)[C@@H]3CC[C@]12C DNVPQKQSNYMLRS-NXVQYWJNSA-N 0.000 description 1
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 241000208822 Lactuca Species 0.000 description 1
- 235000003228 Lactuca sativa Nutrition 0.000 description 1
- XRYVAQQLDYTHCL-UHFFFAOYSA-N Marini Chemical compound O1C=2C(CC(CC=C(C)C)C(C)=C)=C(O)C=C(O)C=2C(=O)CC1C1=CC=C(O)C=C1O XRYVAQQLDYTHCL-UHFFFAOYSA-N 0.000 description 1
- 208000031888 Mycoses Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 238000013494 PH determination Methods 0.000 description 1
- 241000209504 Poaceae Species 0.000 description 1
- 102000004005 Prostaglandin-endoperoxide synthases Human genes 0.000 description 1
- 108090000459 Prostaglandin-endoperoxide synthases Proteins 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 238000011914 asymmetric synthesis Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- FBOUIAKEJMZPQG-BLXFFLACSA-N diniconazole-M Chemical compound C1=NC=NN1/C([C@H](O)C(C)(C)C)=C/C1=CC=C(Cl)C=C1Cl FBOUIAKEJMZPQG-BLXFFLACSA-N 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- DNVPQKQSNYMLRS-SOWFXMKYSA-N ergosterol Chemical compound C1[C@@H](O)CC[C@]2(C)[C@H](CC[C@]3([C@H]([C@H](C)/C=C/[C@@H](C)C(C)C)CC[C@H]33)C)C3=CC=C21 DNVPQKQSNYMLRS-SOWFXMKYSA-N 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005923 long-lasting effect Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 238000004305 normal phase HPLC Methods 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- ABBQGOCHXSPKHJ-WUKNDPDISA-N prontosil Chemical compound NC1=CC(N)=CC=C1\N=N\C1=CC=C(S(N)(=O)=O)C=C1 ABBQGOCHXSPKHJ-WUKNDPDISA-N 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000006340 racemization Effects 0.000 description 1
- 230000036632 reaction speed Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 230000000707 stereoselective effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
- C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
- C07D249/08—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Analytical Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a method for preparing (R) -tebuconazole by an enzyme chemical method, belonging to the technical field of biochemical engineering. Firstly, splitting a racemic tebuconazole epoxy intermediate in a mild reaction system by using recombinant bacteria E.coli/Rph whole cells expressing an epoxy hydrolase as a catalyst to prepare an ee value of 92.7% and a yield of 44.0% of the (R) -tebuconazole epoxy intermediate; the (R) -tebuconazole with high enantiomeric purity (> 99%ee) is prepared by catalyzing the ring-opening reaction of the (R) -tebuconazole epoxy intermediate mediated by the 1,2, 4-triazole in solid base, thereby providing a new technology for commercialization of single enantiomer of tebuconazole. The method has the advantages of simple process, environmental friendliness, low production cost and the like, and has important application prospect.
Description
Technical Field
The invention relates to a method for preparing (R) -tebuconazole by an enzyme chemical method, belonging to the technical field of biochemical engineering.
Background
The triazole bactericide is one of the most widely used bactericides in the world at present, 84% of triazole bactericides belong to chiral pesticides, most triazole chiral bactericides exist in the form of racemates except diniconazole and uniconazole, and researches show that the single chiral pesticide has high efficacy, less dosage and three wastes, is safer to crops and environmental ecology and has lower relative cost. Therefore, more efficient and low-toxicity pesticide products with single enantiomer are developed, and the method has important significance for reducing pesticide residues, protecting the environment and maintaining the human body health.
The tebuconazole is a typical chiral triazole fungicide, effectively controls various fungal diseases on crops by inhibiting the synthesis of fungal ergosterol, has the advantages of wide sterilization spectrum, high efficiency, low toxicity, long lasting period and the like, and is widely used for seed pretreatment or foliar spray of important crops such as gramineae, fruits, vegetables and the like. A large number of researches show that the target biological activity, the ecological toxicity, the environmental enrichment and the like of the tebuconazole enantiomer have obvious stereoselectivity difference. For example, (R) -tebuconazole is more than 100 times as active as (S) -tebuconazole, but (S) -tebuconazole possesses a significant plant growth regulating effect (Stehmann C.et al. Pesticide Sci,1995, 44:183-195); (R) -tebuconazole degrades faster in lettuce than its enantiomer (Zhao Liuqing, 2021, shuoshi paper, national academy of agriculture); (R) -tebuconazole degrades faster in rabbit brain and heart sites (Zhu et al, 2007), with preferential degradation of (R) -tebuconazole in Arabidopsis, and enrichment of (S) -tebuconazole in Arabidopsis. At present, the preparation of the single enantiomer of tebuconazole mainly adopts a high performance liquid chromatography asymmetric resolution method, but the method has the defects of high cost, low yield and the like, and is difficult to realize industrialized production and large-scale application.
The asymmetric catalysis method mediated by the chiral epoxy intermediate is one of the most effective ways to obtain part of triazole fungicide optically active monomers, but the chiral epoxy intermediate with large steric hindrance group (such as tebuconazole epoxy intermediate) causes difficulty in realizing high stereoselective synthesis by chemical and biological methods, so far no report on the asymmetric synthesis of the chiral tebuconazole epoxy intermediate with large steric hindrance group by chemical or biological methods is available. Chinese patent document (application No. 201811621721.9) discloses an epoxide hydrolase derived from rhodotorula marini and uses the epoxide hydrolase in biocatalysis of ethylene oxide to prepare (R) -phenylethanol. In order to expand the application of the epoxide hydrolase, the invention is used for preparing (R) -tebuconazole, and further provides a novel green and efficient method for preparing (R) -tebuconazole by an enzyme chemical method.
Disclosure of Invention
According to the invention, recombinant bacteria E.coli/Rph whole cells (epoxide hydrolase RpEH1 from marine rhodotorula in Chinese patent document with application number 201811621721.9) derived from rhodotorula palustris are used as a biocatalyst, racemic tebuconazole is asymmetrically split to prepare a high-optical pure (R) -tebuconazole epoxy intermediate, and then the (R) -tebuconazole is prepared through solid base catalysis of ring-opening reaction of 1,2, 4-triazole on the (R) -tebuconazole epoxy intermediate, so that compared with an expensive chiral chromatographic separation method, the preparation process is simpler, the environment is more friendly, the production cost is lower, and the like, and the preparation method has a wide application prospect.
The technical scheme adopted for realizing the purpose of the invention is as follows: a method for preparing (R) -tebuconazole by using an enzymatic chemical method, comprising the steps of:
(1) Enzymatic preparation of (R) -tebuconazole epoxy intermediate: adding a racemic tebuconazole epoxy intermediate into recombinant bacterium genetic engineering bacteria E.coli/Rph containing recombinant rhodotorula palustris epoxy hydrolase, carrying out constant-temperature oscillation reaction at a reaction temperature, adding an organic solvent extractant after the reaction is finished, fully and uniformly oscillating, collecting supernatant, and carrying out rotary evaporation to obtain a mixture of the (R) -tebuconazole epoxy intermediate prepared by an enzymatic method and corresponding (S) -vicinal diol;
(2) Preparing (R) -tebuconazole by catalyzing ring opening of (R) -tebuconazole epoxy intermediate by a chemical method: adding 1,2, 4-triazole and solid alkali into an organic solvent, adding a mixture of an (R) -tebuconazole epoxy intermediate prepared by an enzymatic method and corresponding (S) -o-diol, heating for reaction, cooling the reaction liquid to room temperature after the reaction is finished, washing an organic layer with water until the solution is neutral, removing water from the organic phase, filtering and steaming the organic layer in a rotary manner, and purifying the obtained crude residue through a silica gel chromatographic column to obtain white solid (R) -tebuconazole with high optical purity.
In step 1 of the invention, the concentration of recombinant bacteria genetically engineered bacteria E.coli/Rpoh in the reaction system is 5-200 mg/mL.
Furthermore, in the step 1 of the invention, the dosage ratio of the racemic tebuconazole epoxy intermediate substrate to the recombinant bacterium genetically engineered bacterium E.coli/Rph is 2:1-10:1 (mM: mg/mL).
Further, in step 1 of the present invention, the pH of the reaction system is 5.5 to 9.0, preferably 6.5 to 8.5, more preferably 7.0 to 8.0.
Further, in step 1 of the present invention, the reaction temperature is 20 to 50 ℃, preferably 20 to 40 ℃, more preferably 30 to 35 ℃.
Further, in step 1 of the present invention, the concentration of the racemic tebuconazole epoxy intermediate in the reaction system is 20 to 200mM.
Further, in step 1 of the present invention, the organic solvent extractant is preferably any one or a mixture of several of ethyl acetate, methyl acetate and methylene chloride.
Further, in step 2 of the present invention, the organic solvent is preferably any one or more of n-butanol, toluene, and methylene chloride.
Further, in step 2 of the present invention, the volume amount of the organic solvent is such that the molar concentration of the (R) -tebuconazole epoxy intermediate is 100 to 200mM.
Further, in step 2 of the present invention, the solid base is preferably any one or more of potassium hydroxide, sodium hydroxide, potassium carbonate or sodium tert-butoxide.
Further, in step 2 of the present invention, the molar ratio of 1,2, 4-triazole to solid base (R) -tebuconazole epoxy intermediate is 4-1:1:1.
Further, in step 2 of the present invention, the reaction temperature is 110 to 140 ℃.
Further, in step 2 of the present invention, the reaction time is 4 to 24 hours.
The invention also claims: the method is applied to the fields of chiral pesticides, chiral medicines and the like.
The invention has the beneficial effects that: the invention uses recombinant bacteria E.coli/Rph whole cells from rhodotorula palustris epoxide hydrolase as a catalyst to asymmetrically split racemic tebuconazole epoxide intermediates, successfully obtains (R) -tebuconazole epoxide intermediates, and prepares high-purity (R) -tebuconazole enantiomers through solid base catalysis of ring-opening reaction of 1,2, 4-triazole on the (R) -tebuconazole epoxide intermediates. The method disclosed by the invention provides a novel method for preparing (R) -tebuconazole by using a green and effective enzyme chemical method, and has the advantages of simpler preparation process, more environment-friendly performance, lower production cost and the like compared with an expensive chiral chromatographic separation method, and has a wide application prospect.
Drawings
FIG. 1 effect of pH on recombinant E.coli/Rpeh enzyme activity and stability;
FIG. 2 effect of temperature on recombinant E.coli/Rpeh enzyme activity and stability;
FIG. 3 reaction progress of recombinant E.coli/Rph resolution of racemic tebuconazole epoxy intermediate;
FIG. 4 effect of different concentrations of recombinant E.coli/Rpoh on resolution of racemic tebuconazole epoxy intermediate;
FIG. 5 effect of recombinant E.coli/Rph on resolution of different substrate concentrations of racemic tebuconazole epoxy intermediate;
FIG. 6 (R) -tebuconazole 1 H NMR nuclear magnetic pattern.
Detailed Description
The present invention is not limited to the following embodiments, and those skilled in the art can implement the present invention in various other embodiments according to the present invention, or simply change or modify the design structure and thought of the present invention, which fall within the protection scope of the present invention. It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
Chiral gas chromatography conditions: 7820B Agilent gas chromatograph, flame ionization detector, sample inlet and detection port temperature of 250deg.C, temperature programmed from 100deg.C to 220deg.C at 5deg.C/min, isothermal for 2min; chiral gas chromatography column CYCLOSIL-B (30 m. Times.0.25 mm. Times.0.25 μm). The retention times of the (S) -and (R) -tebuconazole epoxy intermediates were 20.835 and 20.920min, respectively.
Reversed phase HPLC chromatography conditions: 1260 Infinicity II Agilent high performance liquid chromatograph, prontoSIL C18 column (150×4.6 mm), detection column temperature 30 ℃, mobile phase flow rate 0.8mL/min, mobile phase methanol: water=90:10, UV detector monitoring at 220nm, tebuconazole epoxy intermediate and corresponding vicinal diol retention times 3.115 and 5.162min, respectively.
Normal phase HPLC chromatographic conditions: 1260 Infinicity II Agilent high performance liquid chromatograph using chiral Chiralcel OD-H column (250 mm. Times.4.6 mm), detection column temperature of 30deg.C, mobile phase of n-hexane/isopropanol (90:10, v/v) at flow rate of 0.6mL/min, UV detector at 220nm, retention times of (S) -and (R) -tebuconazole epoxy intermediates of 6.9min and 7.5min, respectively; the retention time of the (S) -vicinal diol intermediate was 11.4min; the retention times of (S) -and (R) -tebuconazole were 33.2min and 36.9min, respectively.
The method for measuring the specific activity of the recombinant bacteria E.coli/Rpeh comprises the following steps: to a 1.5mL EP tube were added 50. Mu.L of 50mg/mL E.coli/Rpeh bacterial suspension (final concentration 5mg/mL wet cell) and 400. Mu.L of potassium phosphate buffer (100 mM, pH 7.0), and the mixture was preheated at 25℃for 5min; after adding 50. Mu.L of 200mM of the intermediate of the racemic tebuconazole epoxy (final concentration: 20 mM), and reacting for 10 minutes, 100. Mu.L of the intermediate was added to 900. Mu.L of methanol and mixed well, and the mixture was passed through an organic film of 0.22. Mu.m, and subjected to reversed-phase HPLC chromatography. Definition of enzyme activity unit: under this measurement condition, the amount of wet cells required to consume 1. Mu. Mol of tebuconazole epoxy intermediate per minute was defined as 1 cyclooxygenase activity unit (U). The gene sequence of the recombinant strain E.coli/Rph in the following examples is a genetically engineered strain shown as SEQ ID NO.2 in Chinese patent document with application number 201811621721.9.
EXAMPLE 1 optimal pH and pH stability of recombinant E.coli/Rph catalyzed racemic tebuconazole epoxy intermediate
Determination of optimum pH: 50 mu L of 200mM substrate tebuconazole epoxy intermediate (final concentration 5mg/mL wet thallus) is added with 450 mu L of potassium phosphate buffer solution with different pH values (100 mM, pH 5.5-9.0) and preheated at 25 ℃ for 5min; adding 50 mu L of E.coli/Rph bacterial suspension incubated at different pH values, reacting for 10min, adding 100 mu L of the E.coli/Rph bacterial suspension into 900 mu L of methanol, uniformly mixing, passing through an organic film of 0.22 mu m, performing reversed phase HPLC chromatographic analysis, and determining the specific activity of recombinant bacteria E.coli/Rph.
Determination of pH stability: taking 100 mu L of 50mg/mL E.coli/Rpeh bacterial suspension, and incubating in potassium phosphate buffer solutions with different pH values (20 mM, pH value is 5.5-9.0) for 1h; immediately pipetting 50. Mu.L of E.coli/Rph bacterial suspension incubated at different pH values, adding 450. Mu.L of potassium phosphate buffer (100 mM, pH 7.0), and pre-heating at 25℃for 5min; after adding 50. Mu.L of 200mM substrate tebuconazole epoxy intermediate (final concentration 5mg/mL wet cell) and reacting for 10min, 100. Mu.L of the mixture is added into 900. Mu.L of methanol and mixed uniformly, and the mixture is subjected to reversed phase HPLC chromatographic analysis through an organic film of 0.22 μm to determine the residual specific activity of recombinant bacteria E.coli/Rpeh, and the specific activity of non-recombinant bacteria E.coli/Rpeh is defined as 100% and calculated as relative enzyme activity.
The result shows that the optimal reaction pH of the recombinant bacterium E.coli/Rph is 7.5, the high catalytic activity is maintained under the neutral pH environment (pH is 6.5-8.5), the relative enzyme activity is more than 80%, and the enzyme activity is rapidly reduced when the pH is less than 6.0. The recombinant bacterium E.coli/Rph has high pH stability at pH 7.0-8.0, and the residual relative enzyme activity can be more than 95%. The above results indicate that: rpEH has high catalytic activity and stability in the neutral pH range (FIG. 1).
EXAMPLE 2 optimal temperature and temperature stability of recombinant E.coli/Rph catalyzed racemic Tebuconazole epoxy intermediate
Determination of optimal reaction stability: into a 1.5mL EP tube were added 50. Mu.L of 200mM of the racemic tebuconazole epoxy intermediate (final concentration 20 mM) and 450. Mu.L of potassium phosphate buffer (100 mM, pH 7.5), and the mixture was preheated at 20 to 50℃for 5 minutes; adding 50 mu L of E.coli/Rph bacterial suspension (final concentration of 5mg/mL wet bacterial), reacting for 10min at different temperatures of 20-50 ℃, taking 100 mu L of the mixture, adding into 900 mu L of methanol, uniformly mixing, passing through an organic film of 0.22 mu m, and carrying out reversed phase HPLC chromatographic analysis to determine the initial reaction speed of the recombinant bacteria E.coli/Rph.
Temperature stability determination: taking 100 mu L of 50mg/mL E.coli/Rph bacterial suspension, incubating for 1h in a water bath at the temperature of 20-50 ℃, and cooling in an ice bath; to a 1.5mL EP tube were added 50. Mu.L of E.coli/Rpeh bacterial suspension incubated at different temperatures (to a final concentration of 5mg/mL wet cells) and 450. Mu.L of potassium phosphate buffer (100 mM, pH 7.5), and pre-warmed at 25℃for 5min; after adding 50. Mu.L of 200mM of the intermediate of the racemization tebuconazole epoxy (with a final concentration of 20 mM), reacting for 10min, adding 100. Mu.L of the intermediate into 900. Mu.L of methanol, uniformly mixing, passing through a 0.22 μm organic film, performing reversed phase HPLC chromatographic analysis, measuring the residual specific activity of the recombinant bacteria E.coli/Rpeh after incubation treatment, and defining the specific activity of the untreated E.coli/Rpeh bacterial suspension as 100 percent and calculating the relative enzyme activity.
Studies have shown that: the optimal reaction temperature of the recombinant bacteria E.coli/Rph is 30 ℃, the recombinant bacteria E.coli/Rph has higher catalytic activity within the range of 25-35 ℃, the relative enzyme activity is more than 80%, and the specific activity of the E.coli/Rph bacterial suspension is improved by 84% compared with that of the initial wet bacterial suspension under the conditions of the optimal temperature of 30 ℃ and the optimal pH of 7.5. The recombinant bacterium E.coli/Rph has higher thermal stability at the temperature lower than 30 ℃ and the residual relative enzyme activity is more than 98%. Therefore, 30℃was chosen as the optimal reaction temperature in the subsequent catalytic reaction (FIG. 2).
EXAMPLE 3 resolution of 20mM Ractozol epoxy intermediate by recombinant E.coli/Rph different reaction temperatures
In a 2mL reaction system, containing 20mM of racemic tebuconazole epoxy intermediate and 200 mu L of proper 100mg/mL E.coli/Rph recombinant bacteria (final concentration 10 mg/mL) and 1.6mL of potassium phosphate buffer (100 mM, pH 7.5), and respectively carrying out oscillation reaction for 3-6 h at 10 ℃,20 ℃,25 ℃ and 30 ℃, adding 100 mu L of the mixture into 800 mu L of ethyl acetate for extraction and extinction at fixed time, and drying the mixture through an organic film of 0.22 mu m for chiral gas chromatography analysis.
The results show that: the recombinant bacterium E.coli/Rph catalyzes the preferential hydrolysis of the (S) -tebuconazole epoxy intermediate to (S) -vicinal diol, retaining the (R) -tebuconazole epoxy intermediate. Conversion times at 10 ℃,20 ℃,25 ℃ and 30 ℃ are 6h, 5h, 3h and 3h respectively; the enantiomeric excess ee values of the (R) -tebuconazole epoxy intermediate were 49.5%, 74.4%, 86.6% and 92.7%, respectively; the yields of the (R) -tebuconazole epoxy intermediate were 30.3%, 20.0%, 41.5% and 44.0%, respectively. Thus, recombinant E.coli/Rph resolved 20mM racemic tebuconazole epoxy intermediate at 30℃to prepare (R) -tebuconazole epoxy intermediate with ee value and yield of up to 92.7% and 44.0% (FIG. 3).
EXAMPLE 4 Effect of different concentrations of recombinant bacteria E.coli/Rpoh on resolution of racemic tebuconazole epoxy intermediate
In a 2mL reaction system containing 200mM of the racemic tebuconazole epoxy intermediate and a proper amount of potassium phosphate buffer (100 mM, pH 7.5), under the reaction conditions of enzyme concentrations of 50, 100, 150 and 200mg/mL recombinant bacteria E.coli/Rpeh at 30 ℃, the reaction is carried out for 6 hours under shaking, 20 mu L of the mixture is absorbed and added into 800 mu L of ethyl acetate for extraction, 10 mu L of the mixture is simultaneously added into 800 mu L of methanol for extraction, and the mixture passes through an organic film of 0.22 mu m for chiral gas chromatography and HPLC analysis.
The results show that: at 50 (4:1), 100 (2:1), 150 (4:3) and 200 (1:1) mg/mL recombinant E.coli/Rpoh catalyzed racemic tebuconazole epoxy intermediate, the enantiomeric excess ee values of the (R) -tebuconazole epoxy intermediate were 86%, 89%, 84% and 82%, respectively; the yields of the (R) -tebuconazole epoxy intermediate were 43.8%, 46.5%, 46.0% and 46.3%, respectively. Therefore, the recombinant bacterium E.coli/Rph is resolved into 200mM of racemic tebuconazole epoxy intermediate under the condition of different engineering bacterium concentrations, and the optimal ratio of the racemic tebuconazole epoxy intermediate substrate to the recombinant bacterium genetic engineering bacterium E.coli/Rph wet thalli is 2:1 (mM: mg/mL) (FIG. 4).
EXAMPLE 5 Effect of recombinant E.coli/Rpeh resolution of different substrate concentrations of Ractozol epoxy intermediate
In a 2mL reaction system, the concentration of E.coli/Rpeh recombinant bacteria is 200mg/mL, the concentrations of the racemic tebuconazole epoxy intermediates are 100mM, 200mM, 300 mM, 400 mM and 500mM respectively, the reaction is carried out for 12h under the reaction condition of 30 ℃, a proper amount of reaction liquid is absorbed and added into 1mL of ethyl acetate for extraction and sterilization, and an organic film with the thickness of 0.22 mu m is dried for chiral gas chromatography analysis.
The results show that: recombinant bacteria E.coli/Rph catalyze the conversion of tebuconazole epoxy intermediates (100, 200, 300, 400 and 500mM, respectively) at 30℃for 12h, with the (R) -tebuconazole epoxy intermediates remaining with enantiomeric excess ee values of 90.0%, 88.9%, 79.6%, 74.5% and 61.6%, respectively. Thus, recombinant E.coli/Rph resolved the high concentration of the racemic tebuconazole epoxy intermediate at 30℃and the substrate concentration at 100mM gave the (R) -tebuconazole epoxy intermediate with the optimal ee for enantiomeric excess (FIG. 5).
EXAMPLE 6 chemical conversion of (R) -Tebuconazole epoxy intermediate preparation of (R) -Tebuconazole
1,2, 4-triazole (37.7 mg,0.55 mmol) and KOH (10 mg,0.18 mmol) were added to a solution of n-butanol (10 mL), the reaction mixture was heated to 110℃for 2 hours, then the (R) -tebuconazole epoxy intermediate (50 mg,0.18 mmol) prepared as described above was added, the reaction mixture was heated to 133℃and stirred for 4 hours, the reaction mixture was cooled to room temperature, and the organic layer was washed with water (10 mL. Times.3) until the solution became neutral, and then dried over anhydrous MgSO 4 Drying, filtering and concentrating in vacuum. The crude residue is purified by silica gel chromatography column to obtain white solid (R) -tebuconazole 1 H NMR nuclear magnetic resonance spectroscopy (FIG. 6). (R) -tebuconazole:>99％ee； 1 H NMR(400MHz,CDCl 3 ,ppm)δ8.14(s,1H),7.88(s,1H),7.09(d,J＝8.0Hz,2H),6.86(d,J＝8.0Hz,2H),4.26(s,2H),3.36(s,1H),2.40–2.33(m,1H),1.73–1.59(m,3H),0.94(s,9H)。
while the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (8)
1. A method for preparing (R) -tebuconazole by an enzyme chemical method, which is characterized by comprising the following steps:
(1) Enzymatic preparation of (R) -tebuconazole epoxy intermediate: adding a racemic tebuconazole epoxy intermediate into recombinant bacteria E.coli/Rph containing expressed recombinant rhodotorula palustris epoxy hydrolase, performing constant-temperature oscillation reaction at a reaction temperature, adding an organic solvent extractant after the reaction is finished, sufficiently and uniformly oscillating and mixing, collecting supernatant, and performing rotary evaporation to obtain a mixture of the (R) -tebuconazole epoxy intermediate prepared by an enzymatic method and corresponding (S) -vicinal diol;
(2) Preparing (R) -tebuconazole by catalyzing ring opening of (R) -tebuconazole epoxy intermediate by a chemical method: adding 1,2, 4-triazole and solid alkali into an organic solvent, adding a mixture of an (R) -tebuconazole epoxy intermediate prepared by an enzymatic method and corresponding (S) -o-diol, heating for reaction, cooling the reaction liquid to room temperature after the reaction is finished, washing an organic layer with water until the solution is neutral, removing water from the organic phase, filtering and steaming the organic layer in a rotary manner, and purifying the obtained crude residue through a silica gel chromatographic column to obtain white solid (R) -tebuconazole with high optical purity.
2. The method for preparing (R) -tebuconazole by using the enzyme chemical method according to claim 1, wherein the dosage ratio of the racemic tebuconazole epoxy intermediate substrate to the recombinant E.coli/Rph wet cell in the reaction system of the step (1) is 2:1-10:1 (mM: mg/mL).
3. The method for preparing (R) -tebuconazole by using the enzyme chemical method according to claim 1, wherein the concentration of recombinant bacteria E.coli/Rph in the reaction system of the step (1) is 5-200 mg/mL, and the concentration of the substrate of the racemic tebuconazole epoxy intermediate is 20-200 mM.
4. The method for preparing (R) -tebuconazole by using the enzymatic chemical process according to claim 1, wherein the pH of the reaction system in the step (1) is 6.5-9.0, and the reaction temperature is 20-50 ℃.
5. The method for preparing (R) -tebuconazole by using the enzymatic chemical process according to claim 1, wherein the pH of the reaction system in the step (1) is 7.0-8.0, and the reaction temperature is 30-35 ℃.
6. The method for preparing (R) -tebuconazole by using the enzymatic chemical process according to claim 1, wherein the organic solvent extractant in the step (1) is any one or a mixture of more of ethyl acetate, methyl acetate and methylene dichloride.
7. The method for preparing (R) -tebuconazole by using the enzymatic chemical process according to claim 1, wherein the organic solvent in the step (2) is any one or more of n-butanol, toluene and methylene chloride.
8. The method for preparing (R) -tebuconazole by using the enzymatic chemical method according to claim 1, wherein the volume amount of the organic solvent in the step (2) is that the molar concentration of the (R) -tebuconazole epoxy intermediate is 100-200 mM, the solid base is potassium hydroxide, sodium hydroxide, potassium carbonate or sodium tert-butoxide, the molar ratio of the (R) -tebuconazole epoxy intermediate to the solid base is 4-1:1:1, the reaction temperature is 110-140 ℃, and the reaction time is 4-24 h.
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