CN116590358A - Method for preparing R-tetrahydropapaverine - Google Patents
Method for preparing R-tetrahydropapaverine Download PDFInfo
- Publication number
- CN116590358A CN116590358A CN202310046882.4A CN202310046882A CN116590358A CN 116590358 A CN116590358 A CN 116590358A CN 202310046882 A CN202310046882 A CN 202310046882A CN 116590358 A CN116590358 A CN 116590358A
- Authority
- CN
- China
- Prior art keywords
- tetrahydropapaverine
- dihydropapaverine
- imine reductase
- immobilized
- glucose dehydrogenase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 28
- YXWQTVWJNHKSCC-MRXNPFEDSA-N (R)-tetrahydropapaverine Chemical compound C1=C(OC)C(OC)=CC=C1C[C@@H]1C2=CC(OC)=C(OC)C=C2CCN1 YXWQTVWJNHKSCC-MRXNPFEDSA-N 0.000 title claims abstract description 27
- 238000006243 chemical reaction Methods 0.000 claims abstract description 28
- 102000004190 Enzymes Human genes 0.000 claims abstract description 24
- 108090000790 Enzymes Proteins 0.000 claims abstract description 24
- 108090000854 Oxidoreductases Proteins 0.000 claims abstract description 19
- 150000002466 imines Chemical class 0.000 claims abstract description 19
- UCJDFFOXXPPGLJ-UHFFFAOYSA-N 1-[(3,4-dimethoxyphenyl)methyl]-6,7-dimethoxy-3,4-dihydroisoquinoline Chemical compound C1=C(OC)C(OC)=CC=C1CC1=NCCC2=CC(OC)=C(OC)C=C12 UCJDFFOXXPPGLJ-UHFFFAOYSA-N 0.000 claims abstract description 16
- 108010050375 Glucose 1-Dehydrogenase Proteins 0.000 claims abstract description 15
- 230000000694 effects Effects 0.000 claims abstract description 8
- 239000000758 substrate Substances 0.000 claims abstract description 5
- 102000004316 Oxidoreductases Human genes 0.000 claims description 16
- 239000007853 buffer solution Substances 0.000 claims description 10
- 239000000872 buffer Substances 0.000 claims description 8
- 239000005515 coenzyme Substances 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 6
- 150000003839 salts Chemical class 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 4
- 230000009467 reduction Effects 0.000 claims description 3
- 244000063299 Bacillus subtilis Species 0.000 claims 2
- 235000014469 Bacillus subtilis Nutrition 0.000 claims 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims 1
- 102000007698 Alcohol dehydrogenase Human genes 0.000 claims 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 claims 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 claims 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims 1
- 108090000698 Formate Dehydrogenases Proteins 0.000 claims 1
- 229910019142 PO4 Inorganic materials 0.000 claims 1
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate 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-DQQFMEOOSA-N 0.000 claims 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 claims 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims 1
- 239000010452 phosphate Substances 0.000 claims 1
- 230000001172 regenerating effect Effects 0.000 claims 1
- 230000008929 regeneration Effects 0.000 claims 1
- 238000011069 regeneration method Methods 0.000 claims 1
- 239000000203 mixture Substances 0.000 abstract description 7
- 108090000623 proteins and genes Proteins 0.000 abstract description 7
- 239000003822 epoxy resin Substances 0.000 abstract description 6
- 229920000647 polyepoxide Polymers 0.000 abstract description 6
- 239000002994 raw material Substances 0.000 abstract description 6
- 108010093096 Immobilized Enzymes Proteins 0.000 abstract description 5
- 238000004132 cross linking Methods 0.000 abstract description 5
- 230000007613 environmental effect Effects 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 238000010364 biochemical engineering Methods 0.000 abstract description 2
- 230000010355 oscillation Effects 0.000 abstract description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- XXZSQOVSEBAPGS-DONVQRBFSA-L cisatracurium besylate Chemical compound [O-]S(=O)(=O)C1=CC=CC=C1.[O-]S(=O)(=O)C1=CC=CC=C1.C1=C(OC)C(OC)=CC=C1C[C@H]1[N@+](CCC(=O)OCCCCCOC(=O)CC[N@+]2(C)[C@@H](C3=CC(OC)=C(OC)C=C3CC2)CC=2C=C(OC)C(OC)=CC=2)(C)CCC2=CC(OC)=C(OC)C=C21 XXZSQOVSEBAPGS-DONVQRBFSA-L 0.000 description 8
- 229960002945 atracurium besylate Drugs 0.000 description 7
- XXZSQOVSEBAPGS-UHFFFAOYSA-L atracurium besylate Chemical compound [O-]S(=O)(=O)C1=CC=CC=C1.[O-]S(=O)(=O)C1=CC=CC=C1.C1=C(OC)C(OC)=CC=C1CC1[N+](CCC(=O)OCCCCCOC(=O)CC[N+]2(C)C(C3=CC(OC)=C(OC)C=C3CC2)CC=2C=C(OC)C(OC)=CC=2)(C)CCC2=CC(OC)=C(OC)C=C21 XXZSQOVSEBAPGS-UHFFFAOYSA-L 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- YXWQTVWJNHKSCC-UHFFFAOYSA-N 1-[(3,4-dimethoxyphenyl)methyl]-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline Chemical compound C1=C(OC)C(OC)=CC=C1CC1C2=CC(OC)=C(OC)C=C2CCN1 YXWQTVWJNHKSCC-UHFFFAOYSA-N 0.000 description 6
- 229960000970 cisatracurium besylate Drugs 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000001914 filtration Methods 0.000 description 5
- 238000003756 stirring Methods 0.000 description 5
- -1 1, 5-pentanediol acrylic acid diester Chemical class 0.000 description 4
- HTSGKJQDMSTCGS-UHFFFAOYSA-N 1,4-bis(4-chlorophenyl)-2-(4-methylphenyl)sulfonylbutane-1,4-dione Chemical compound C1=CC(C)=CC=C1S(=O)(=O)C(C(=O)C=1C=CC(Cl)=CC=1)CC(=O)C1=CC=C(Cl)C=C1 HTSGKJQDMSTCGS-UHFFFAOYSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- BAWFJGJZGIEFAR-NNYOXOHSSA-O NAD(+) 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](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-O 0.000 description 4
- 238000007259 addition reaction Methods 0.000 description 4
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 4
- 238000000855 fermentation Methods 0.000 description 4
- 230000004151 fermentation Effects 0.000 description 4
- 229960001031 glucose Drugs 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 229930027917 kanamycin Natural products 0.000 description 4
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 4
- 229960000318 kanamycin Drugs 0.000 description 4
- 229930182823 kanamycin A Natural products 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- 241001052560 Thallis Species 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 238000010907 mechanical stirring Methods 0.000 description 3
- CZXGXYBOQYQXQD-UHFFFAOYSA-N methyl benzenesulfonate Chemical compound COS(=O)(=O)C1=CC=CC=C1 CZXGXYBOQYQXQD-UHFFFAOYSA-N 0.000 description 3
- 238000007069 methylation reaction Methods 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 238000001308 synthesis method Methods 0.000 description 3
- 229940043375 1,5-pentanediol Drugs 0.000 description 2
- 239000004593 Epoxy Substances 0.000 description 2
- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 description 2
- BAPJBEWLBFYGME-UHFFFAOYSA-N Methyl acrylate Chemical compound COC(=O)C=C BAPJBEWLBFYGME-UHFFFAOYSA-N 0.000 description 2
- ALQSHHUCVQOPAS-UHFFFAOYSA-N Pentane-1,5-diol Chemical compound OCCCCCO ALQSHHUCVQOPAS-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 229950002863 cisatracurium besilate Drugs 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000005886 esterification reaction Methods 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 206010021118 Hypotonia Diseases 0.000 description 1
- 241000218378 Magnolia Species 0.000 description 1
- WXNXCEHXYPACJF-ZETCQYMHSA-M N-acetyl-L-leucinate Chemical compound CC(C)C[C@@H](C([O-])=O)NC(C)=O WXNXCEHXYPACJF-ZETCQYMHSA-M 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 150000001252 acrylic acid derivatives Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002999 depolarising effect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 229960001340 histamine Drugs 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 238000007327 hydrogenolysis reaction Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000036640 muscle relaxation Effects 0.000 description 1
- 239000003158 myorelaxant agent Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000005809 transesterification reaction Methods 0.000 description 1
- 230000007371 visceral function Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- C12P17/10—Nitrogen as only ring hetero atom
- C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The application discloses a method for preparing R-tetrahydropapaverine, which relates to the technical field of biochemical engineering, wherein genes containing high (R) selective imine reductase and glucose dehydrogenase are implanted into a strain, the strain is subjected to flat culture, and monoclonal is selected for gradual culture; adding the imine reductase crude enzyme solution and the glucose dehydrogenase crude enzyme solution into an activated epoxy resin carrier, and obtaining immobilized mixed enzyme through oscillation crosslinking; the immobilized enzyme mixture catalyzes dihydropapaverine to produce R-tetrahydropapaverine. The application provides a method for preparing R-tetrahydropapaverine, which creatively utilizes immobilized imine reductase with high selectivity and high activity to catalyze high-concentration dihydropapaverine to generate R-tetrahydropapaverine, and the conversion rate is more than 90% under the condition of high substrate concentration (5 percent); the ee value is more than 98.5%, and the method has high yield, mild reaction conditions and environmental protection; meanwhile, the raw materials are wide in source and low in price, and the production cost is low.
Description
Technical Field
The application relates to the technical field of biochemical engineering, in particular to a method for synthesizing high-purity R-tetrahydropapaverine.
Background
Cis-atracurium besylate is an all-cis isomer of atracurium besylate, has great advantages compared with atracurium besylate in drug effect, including no obvious histamine release, little muscle relaxation effect, no accumulation effect, little cardiovascular reaction, little dependence on visceral functions such as liver, kidney and the like, and is a relatively ideal medium-aging non-depolarizing muscle relaxant.
Atracurium besylate was first discovered in 1976 and was used in surgery in 1982. The synthesis process mainly uses tetrahydropapaverine and 1, 5-pentanediol acrylic acid diester as raw materials, and adopts Michelia addition and methylation reaction, and finally adopts diethyl ether precipitation to obtain atracurium besylate. The synthesis process of cis-atracurium besylate is prepared by chiral resolution based on the synthesis of atracurium besylate.
The existing synthesis method of cis-atracurium besylate uses racemic tetrahydropapaverine as raw material, and R-isomer is obtained by resolution and alkalization and ionization.
In 1992, derek et al proposed a synthesis method of cis-atracurium besylate for the first time, which uses racemic tetrahydropapaverine as a raw material to react with N-acetyl-L-leucine as a resolving agent to form salt, and then re-crystallize for 3 times to obtain R-tetrahydropapaverine-N-acetyl-L-leucine salt, and alkalize to obtain R-tetrahydropapaverine; r-tetrahydropapaverine and 1, 5-pentanediol acrylic acid diester undergo Michelial addition reaction, and then undergo nitrogen methylation reaction with methyl benzenesulfonate to obtain atracurium besylate; eluting by column chromatography to obtain water-soluble oily substance, and lyophilizing to obtain cis-atracurium besilate. (HILL D A. Neuromedical blocking agents: america,5453510[ P ] 1995-09-02.)
The 1, 5-pentanediol acrylic acid diester is expensive, has high synthesis difficulty, harsh operating conditions and difficult post-treatment. In patent CN 200910068233, song Honghai and the like, R-tetrahydropapaverine and methyl acrylate are subjected to michal addition reaction to obtain a product R-3- [1- (3, 4-dimethoxybenzyl) -6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline ] -methyl acrylate, the product is subjected to transesterification reaction with 1, 5-pentanediol, the obtained intermediate and methyl benzenesulfonate are subjected to nitrogen methylation reaction to obtain atracurium besylate, and the final product is obtained through column chromatography. Avoids using 1, 5-pentanediol acrylic acid diester, has mild condition and lower cost. (CN 200910068233)
In the column chromatography process, cis-atracurium besylate has poor stability, is easy to decompose and generate byproducts, and affects the yield. Therefore, R-tetrahydropapaverine and acrylic acid or acrylic acid derivatives are subjected to Michal addition reaction to generate a compound I, then the compound I and methyl benzenesulfonate are subjected to hydrogenolysis reaction to obtain a compound II, R-type compound II is obtained through crystallization, esterification reaction is carried out on the compound II and 1, 5-pentanediol to generate an intermediate III, and esterification condensation is carried out on the compound II and R-type II to generate cis-atracurium besylate, so that byproducts are avoided, and the reaction difficulty is reduced. (US 20100168431A 1)
Although many researchers have carried out process improvement, the current process has the problem of lower total yield, which leads to no reduction of the cost of medicines, and the selection of a high-efficiency and concise synthetic route is a key for reducing the cost. Resolution of tetrahydropapaverine and isolation and purification of cis-atracurium besilate remain a research hotspot in future research. The total reaction yield can be greatly improved by changing an enzymatic reduction route in the synthesis of a key intermediate R-tetrahydropapaverine, and therefore, a method for preparing the R-tetrahydropapaverine is provided.
Disclosure of Invention
(one) solving the technical problems
Aiming at the defects of the prior art, the application aims to provide an immobilized imine reductase with high selectivity and high activity for catalyzing high-concentration dihydropapaverine to generate R-tetrahydropapaverine. Under the condition of high substrate concentration (5%), the conversion rate is more than 90%; the ee value is greater than 98.5%.
(II) technical scheme
In order to achieve the above purpose, the application is realized by the following technical scheme:
a method for preparing R-tetrahydropapaverine comprises reducing dihydropapaverine (or its salt) with enzyme having imine reductase activity to obtain R-tetrahydropapaverine (or its salt)
The synthesis method comprises the following steps:
step one: genes containing high (R) selective imine reductase and glucose dehydrogenase are implanted into a strain, the strain is subjected to plate culture, and monoclonal is selected for gradual culture;
step two: adding the imine reductase crude enzyme solution and the glucose dehydrogenase crude enzyme solution into an activated epoxy resin carrier, and obtaining immobilized mixed enzyme through oscillation crosslinking;
step three: the immobilized enzyme mixture catalyzes dihydropapaverine to produce R-tetrahydropapaverine.
Preferably, in the first step, the strain culture includes the steps of:
firstly, transferring the strain into 3ml of LB culture solution containing 50 mu M kanamycin for overnight culture, transferring 1% of inoculation amount into 100ml of LB culture solution containing 50 mu M kanamycin for 4-8 hours;
then transferring into a 10L fermentation tank for culturing; when the OD of the cells reaches about 25, the temperature is reduced to 30 ℃,0.5 mM isopropyl-beta-D-thiopyran galactoside (I PTG) is added, and the culture is continued for 18 hours to induce the expression of the target protein.
Preferably, after the strain is cultured, the fermentation broth is taken out and centrifuged at high speed to collect cells to obtain 600-1000 g of wet thalli, and the wet thalli is preserved at-20 ℃ for standby.
Preferably, the high-speed centrifugation is performed at 6000rpm for 30min, and the temperature of the LB medium is maintained at 37℃and stirred at 220 rpm.
Preferably, in the second step, the preparation of the epoxy resin carrier includes the following steps:
adding 40mL 100mM PBS buffer solution into 10g epoxy carrier, stirring or oscillating for 15min at 20-25 ℃ and rotating speed of 150rpm, filtering, washing twice with 50mM BS buffer solution, and drying by suction to obtain the activated epoxy resin immobilized carrier;
wherein, ph=8.0 of PBS buffer.
Preferably, in the second step, the imine reductase crude enzyme solution and the glucose dehydrogenase crude enzyme solution are prepared by the same preparation method, and the preparation method comprises the following steps:
taking 10g of cells expressed by imine reductase or glucose dehydrogenase, re-suspending in 100ml of PBS buffer solution, uniformly mixing, homogenizing and crushing the cells under high pressure, centrifuging at 13000rpm for 60min, and filtering the supernatant by a 0.4 mu m membrane to obtain an enzyme solution;
wherein, the PBS buffer is 50mM, and the pH=8.0.
Preferably, in the second step, the oscillating crosslinking includes the following steps:
(1) Oscillating and crosslinking for 20 hours at the temperature of 25 ℃ at the rotating speed of 180rpm, and carrying out suction filtration and water washing for three times;
(2) Stirring or shaking with 50mM PBS buffer, pH=8.0, 0.5M NaCl at 150rpm for 45min;
(3) Washing with 50mM PBS buffer solution with pH=8.0 for 2 times, and suction filtering to obtain immobilized mixed enzyme.
Preferably, in the third step, the formula of the immobilized enzyme mixture for catalyzing the dihydropapaverine to generate R-tetrahydropapaverine is as follows:
preferably, in the third step, the step of catalyzing the dihydropapaverine to generate R-tetrahydropapaverine by the immobilized mixed enzyme comprises the following steps:
adding coenzyme NAD+ and PBS buffer solution, glucose monohydrate, stirring uniformly, adding immobilized mixed enzyme, dissolving dihydropapaverine in DMSO, dropwise adding into the system in batches, mechanically stirring at 30 ℃ after the dropwise addition is finished, controlling the reaction pH=7.0, and detecting the conversion rate and the ee value of the product by HPLC after 20 hours of reaction.
(III) beneficial effects
The application creatively utilizes immobilized imine reductase with high selectivity and high activity to catalyze high-concentration dihydropapaverine to generate R-tetrahydropapaverine, and the conversion rate is more than 90 percent under the condition of high substrate concentration (5 percent); the ee value is greater than 98.5%. The method has high yield, mild reaction conditions and environmental protection; the method is easy for large-scale production, and simultaneously has the advantages of wide raw material source, low price, low production cost and environmental friendliness, and can be used for remarkably reducing the production cost of tetrahydropapaverine and meeting the requirements of the current green industrial production.
Detailed Description
The embodiment of the application provides a method for preparing R-tetrahydropapaverine, which creatively utilizes immobilized imine reductase with high selectivity and high activity to catalyze high-concentration dihydropapaverine to generate R-tetrahydropapaverine, and the conversion rate is more than 90% under the condition of high substrate concentration (5 percent); the ee value is greater than 98.5%. The method has high yield, mild reaction conditions and environmental protection; the method is easy for large-scale production, and simultaneously has the advantages of wide raw material source, low price, low production cost and environmental friendliness, and can be used for remarkably reducing the production cost of tetrahydropapaverine and meeting the requirements of the current green industrial production.
Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods. The experimental methods used in the examples below are conventional, unless otherwise specified
Example 1
High (R) selective imine reductase (amino acid SEQUENCE shown as SEQUENCE1, nucleotide SEQUENCE shown as SEQUENCE 2), glucose dehydrogenase (amino acid SEQUENCE shown as SEQUENCE3, nucleotide SEQUENCE shown as SEQUENCE 4), 2 genes each obtained by gene synthesis, each gene linked to vector pET28 a. The strain is subjected to plate culture, and monoclonal is selected for gradual culture. Firstly, transferring the strain into 3M L LB culture solution containing 50 mu M kanamycin (37 ℃ C., 220 rpm) for overnight culture, transferring 1% of the strain into 100M L LB culture solution containing 50 mu M kanamycin for 4-8 h, and finally transferring the strain into a 10L fermentation tank for culture; when the OD of the cells reaches about 25, cooling to 30 ℃, adding 0.5mM isopropyl-beta-D-thiopyran galactoside (I PTG), continuously culturing for 18 hours to induce the expression of target protein, taking the fermentation liquor, centrifuging at a high speed (600 rpm,30 min), collecting the cells to obtain 600-1000 g of wet thalli, and preserving at-20 ℃ for standby.
Example 2
Immobilization of enzymes
10g of epoxy carrier is added with 40mL of 100mM PBS buffer solution (pH 8.0), stirred or oscillated for 15min at the temperature of 20-25 ℃ and the rotation speed of 150rpm, filtered, washed twice with 50mM PBS buffer solution (pH 8.0) and pumped to dryness to obtain the activated epoxy resin immobilized carrier.
10g of cells expressing imine reductase or glucose dehydrogenase were resuspended in 100m l (PBS) buffer (50 mM, pH 8.0) and mixed well, the cells were broken by high pressure homogenization, centrifuged at 13000rpm at 60min, and the supernatant was filtered through a 0.4 μm membrane to obtain an enzyme solution.
Adding the imine reductase crude enzyme solution and the glucose dehydrogenase crude enzyme solution into 5g of activated epoxy resin carrier, oscillating and crosslinking for 20h at 25 ℃ and 180rpm, filtering and washing for three times, then using 50mM PBS buffer solution with pH of 8.0 and 0.5M NaC l,150rpm, stirring or oscillating for 45min, then using 50mM PBS buffer solution with pH of 8.0 to wash for 2 times, and filtering to obtain the immobilized mixed enzyme.
Example 3
Enzymatic synthesis of R-tetrahydropapaverine
In this example, immobilized enzyme mixture was used to catalyze dihydropapaverine to form (R) -tetrahydropapaverine. The immobilized enzyme can be recycled after the reaction is finished.
(1) 1.0mg of coenzyme NAD+ and 6ml of PBS buffer (100 mM, pH=7.0) were added to 10ml of the catalytic system, 0.235g of glucose monohydrate was stirred uniformly, 1.2g of immobilized mixed enzyme (imine reductase and glucose dehydrogenase) was further added, 0.20g of dihydropapaverine was dissolved in 0.20g of DMSO, and the mixture was added dropwise to the system in portions over 30 minutes. Mechanical stirring at 30 ℃, controlling the reaction ph=7.0. After 20h of reaction, HPLC detects the conversion and the ee value of the product. The conversion rate is 95%, and the optical purity (ee) of the (R) -dihydropapaverine is more than 98.9%.
(2) 10mg of coenzyme NAD+ and 22ml of PBS buffer (100 mM, pH=7.0) are added into 100ml of a catalytic system, 5.87g of glucose monohydrate is stirred uniformly, 30g of immobilized mixed enzyme (imine reductase and glucose dehydrogenase) is added, 5.0g of dihydropapaverine is dissolved into 5g of DMSO, and the mixture is added dropwise into the system for 30 minutes. Mechanical stirring at 30 ℃, controlling the reaction ph=7.0. After 20h of reaction, HPLC detects the conversion and the ee value of the product. The conversion rate is 93%, and the optical purity (ee) of the (R) -dihydropapaverine is more than 98.5%.
(3) 10mg of coenzyme NAD+ and 22ml of PBS buffer (100 mM, pH=7.0) are added into 100ml of a catalytic system, 5.87g of glucose monohydrate is stirred uniformly, 30g of immobilized mixed enzyme (imine reductase and glucose dehydrogenase) is added, 5.0g of dihydropapaverine is dissolved into 5g of DMSO, and the mixture is added dropwise into the system for 30 minutes. Mechanical stirring at 30 ℃, controlling the reaction ph=7.0. After 20h of reaction, HPLC detects the conversion and the ee value of the product. The conversion rate is 98%, and the optical purity (ee) of the (R) -dihydropapaverine is more than 99.1%.
SEQUENCE1
MITLIGLGPMGQAMVRVLLENGHGVTVWNRTASRADGVVAAGAVRAETPADAVAASELVLLSLTDYAAMYDILGKAGETLAGKVVVNLSSDTPEKTREAAEWVKARGGQFIAGGVMVPAPLVGKEEAYVFYSGPTEVFEKHREVLALIGRADFLGEDVRLAQLFYQAQLDIFLNSLSAFMHASALVRSAGVPLEKFLPYAKDNFAMMGFYLEAAVEQIEKGDHPGDEADVIMMGASADHIVQASRDAGIDVALPEAVKSHYDRAIAAGHGRSSWTSLFEIIKADGR
SEQUENCE2
ATGATCACCCTGATTGGCCTGGGTCCGATGGGTCAGGCTATGGTTCGCGTGCTGCTGGAAAATGGCCACGGTGTTACCGTTTGGAATCGCACCGCCAGTCGCGCTGATGGTGTGGTGGCAGCAGGTGCAGTTCGCGCAGAAACCCCTGCAGATGCCGTTGCTGCCAGCGAACTGGTTCTGCTGAGTCTGACCGATTATGCAGCAATGTATGATATTCTGGGCAAAGCCGGCGAAACCCTGGCAGGTAAAGTGGTTGTTAATCTGAGTAGCGATACCCCGGAAAAAACCCGCGAAGCAGCAGAATGGGTTAAAGCCCGTGGCGGTCAGTTTATTGCCGGTGGTGTTATGGTGCCGGCACCGTTAGTGGGCAAAGAAGAAGCCTATGTGTTTTATAGTGGCCCGACCGAAGTGTTTGAAAAACATCGTGAAGTGCTGGCACTGATTGGCCGCGCAGATTTTCTGGGTGAAGATGTGCGCCTGGCCCAGTTATTTTATCAGGCACAGCTGGATATTTTCCTGAATAGTCTGAGTGCATTCATGCATGCAAGTGCACTGGTGCGTAGCGCCGGTGTTCCTTTAGAAAAATTTCTGCCGTATGCCAAAGACAATTTCGCCATGATGGGCTTTTATCTGGAAGCAGCAGTGGAACAGATTGAAAAAGGCGATCATCCGGGTGACGAAGCAGATGTGATTATGATGGGTGCCAGCGCAGATCATATTGTTCAGGCAAGCCGTGATGCCGGTATTGATGTTGCACTGCCGGAAGCCGTGAAAAGCCATTATGATCGTGCAATTGCCGCCGGTCATGGCCGTAGTAGTTGGACAAGTCTGTTTGAAATTATCAAGGCAGACGGCCGTTAA。
SEQUENCE3
MYPDLKGKVVAITGAASGLGKAMAIRFGKEQAKVVINYYSNKQDPNEVKEEVIKAGGEAVVVQGDVTKEEDVKNIVQTAIKEFGTLDIMINNAGLENPVPSHEMPLKDWDKVIGTNLTGAFLGSREAIKYFVENDIKGNVINMSSVHEVIPWPLFVHYAASKGGIKLMTETLALEYAPKGIRVNNIGPGAINTPINAEKFADPKQKADVESMIPMGYIGEPEEIAAVAAWLASKEASYVTGITLFADGGMTQYPSFQAGRG
SEQUENCE4
ATGTATCCGGATTTAAAAGGAAAAGTCGTCGCTATTACAGGAGCTGCTTCAGGGCTCGGAAAGGCG
ATGGCCATTCGCTTCGGCAAGGAGCAGGCAAAAGTGGTTATCAACTATTATAGTAATAAACAAGAT
CCGAACGAGGTAAAAGAAGAGGTCATCAAGGCGGGCGGTGAAGCTGTTGTCGTCCAAGGAGATGTC
ACGAAAGAGGAAGATGTAAAAAATATCGTGCAAACGGCAATTAAGGAGTTCGGCACACTCGATATT
ATGATTAATAATGCCGGTCTTGAAAATCCTGTGCCATCTCACGAAATGCCGCTCAAGGATTGGGAT
AAAGTCATCGGCACGAACTTAACGGGTGCCTTTTTAGGAAGCCGTGAAGCGATTAAATATTTCGTA
GAAAACGATATTAAGGGAAATGTCATTAACATGTCCAGTGTGCACGAAGTGATTCCTTGGCCGTTA
TTTGTCCACTATGCGGCAAGTAAAGGCGGGATAAAGCTGATGACAGAAACATTAGCGTTGGAATAC
GCGCCGAAGGGCATTCGCGTCAATAATATTGGGCCAGGTGCGATCAACACGCCAATCAATGCTGAA
AAATTCGCTGACCCTAAACAGAAAGCTGATGTAGAAAGCATGATTCCAATGGGATATATCGGCGAA
CCGGAGGAGATCGCCGCAGTAGCAGCCTGGCTTGCTTCGAAGGAAGCCAGCTACGTCACAGGCATC
ACGTTATTCGCGGACGGCGGTATGACACAATATCCTTCATTCCAGGCAGGCCGCGGTTAA
Finally, it should be noted that: it is apparent that the above examples are only illustrative of the present application and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present application.
Claims (9)
1. A process for the preparation of R-tetrahydropapaverine, characterized in that: preparation of R-tetrahydropapaverine (or salts thereof) by reduction of dihydropapaverine (or salts thereof) with an enzyme having imine reductase activity
2. The method according to claim 1, characterized in that: the coenzyme NADH or NADPH regenerating system is added in the reaction.
3. The method according to claim 1, characterized in that: wherein the amino acid sequence of the imine reductase is the sequence shown in SEQ ID NO. 1.
4. The method according to claim 2, characterized in that: the coenzyme regeneration system comprises an enzyme selected from the group consisting of: glucose dehydrogenase, alcohol dehydrogenase or formate dehydrogenase.
5. The method according to claim 4, wherein: the glucose dehydrogenase is derived from bacillus subtilis (Bacillus subtilis).
6. The method according to claim 4, wherein: the glucose dehydrogenase has an amino acid sequence shown in SEQ ID NO. 3.
7. The method according to claim 1, wherein the buffer solution with a pH value of 6.0-10.0 has a substrate concentration of 1-100 g/L of R-dihydropapaverine (or salt thereof) and is converted for 2-24 hours at a reaction temperature of 20-40 ℃.
8. The method of claim 7, wherein the buffer is one or more of phosphate, tris hydrochloride, bicarbonate, and carbonate.
9. The method according to claim 1, characterized in that: the formula of the immobilized mixed enzyme for catalyzing dihydropapaverine to generate R-tetrahydropapaverine is as follows:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310046882.4A CN116590358A (en) | 2023-01-31 | 2023-01-31 | Method for preparing R-tetrahydropapaverine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310046882.4A CN116590358A (en) | 2023-01-31 | 2023-01-31 | Method for preparing R-tetrahydropapaverine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116590358A true CN116590358A (en) | 2023-08-15 |
Family
ID=87588638
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310046882.4A Pending CN116590358A (en) | 2023-01-31 | 2023-01-31 | Method for preparing R-tetrahydropapaverine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116590358A (en) |
-
2023
- 2023-01-31 CN CN202310046882.4A patent/CN116590358A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112368262B (en) | Method for preparing pregabalin intermediate (R) -3- (carbamoylmethyl) -5-methylhexanoic acid | |
| CN106916857A (en) | A kind of method for producing L glufosinate-ammoniums | |
| Chibata | Industrial application of immobilized enzyme systems | |
| CN104928340A (en) | Process for enzymatic synthesis of cefprozil | |
| EP3255147B1 (en) | Immobilized cell and preparation method thereof | |
| CN109182410B (en) | Enzymatic preparation method of (S) -N-Boc-3-hydroxypiperidine | |
| WO2022127310A1 (en) | Method for preparing (s)-2-(3-pyridine)-pyrrolidine | |
| Zhao et al. | Efficient production of diltiazem chiral intermediate using immobilized lipase from Serratia marcescens | |
| CN114807265A (en) | Synthetic method of S-nicotine | |
| CN103966275A (en) | Method for preparing highly pure L-tertiary leucine through biological process | |
| CN102373246B (en) | Method for preparing L-homophenylalanine | |
| CN116590358A (en) | Method for preparing R-tetrahydropapaverine | |
| CN114940983A (en) | Eutectic solvent based on polyalcohol hydrogen bond donor and method for improving catalytic activity of omega-transaminase by using eutectic solvent | |
| CN112175892B (en) | Engineering bacterium for co-expressing L-threonine aldolase and PLP synthase and application thereof | |
| CN104830940A (en) | An enzymatic synthesis process of Amoxicillin | |
| WO2014146242A1 (en) | Enzymatic preparation method for oxidized coenzyme ii | |
| CN111662937B (en) | Method for preparing chloramphenicol | |
| CN101104862B (en) | Method for synthesizing D-arylglycine by using heterogeneous enzyme to catalytically hydrolyzing 5-arylhydantoin | |
| CN111635917A (en) | Preparation method of beta-nicotinamide ribodinucleotide | |
| CN105420307A (en) | Method for preparing (S)-N-t-butyloxycarboryl-3-hydroxypiperidine | |
| CN106520857B (en) | Method for synthesizing aztreonam by enzyme method | |
| EP3676374A1 (en) | Highly efficient enzymatic process to produce (r)-3-quinuclidinol | |
| CN110982800B (en) | Sulfonyl transferase, application and mediated aziridine ring biosynthesis method thereof | |
| CN112359028B (en) | Biosynthesis method of tomoxetine intermediate and carbonyl reductase | |
| CN106636292B (en) | Preparation method of (1R,4S) - (-) -2-azabicyclo [2.2.1] hept-5-ene-3-ketone |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information |
Country or region after: China Address after: 201206, 3rd to 4th floors, Building 3, No. 55 Jindian Road, China (Shanghai) Pilot Free Trade Zone, Pudong New Area, Shanghai Applicant after: Shanghai Yusong Biotechnology Co.,Ltd. Address before: Building 3, Building 3-4, No. 55 Jindian Road, China (Shanghai) Pilot Free Trade Zone, Pudong New Area, Shanghai, 200000 Applicant before: Shanghai Yusong Pharmaceutical Technology Co.,Ltd. Country or region before: China |
|
| CB02 | Change of applicant information |