WO2022127310A1 - 一种制备(s)-2-(3-吡啶)-吡咯烷的方法 - Google Patents
一种制备(s)-2-(3-吡啶)-吡咯烷的方法 Download PDFInfo
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- RWRDLPDLKQPQOW-UHFFFAOYSA-N tetrahydropyrrole Substances C1CCNC1 RWRDLPDLKQPQOW-UHFFFAOYSA-N 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title claims abstract description 24
- 150000002466 imines Chemical class 0.000 claims abstract description 49
- 238000006243 chemical reaction Methods 0.000 claims abstract description 46
- 241000894006 Bacteria Species 0.000 claims abstract description 34
- 239000000758 substrate Substances 0.000 claims abstract description 26
- 102000004190 Enzymes Human genes 0.000 claims abstract description 15
- 108090000790 Enzymes Proteins 0.000 claims abstract description 15
- 241000863423 Myxococcus fulvus Species 0.000 claims abstract description 8
- -1 2-pyridine pyrrolidine compound Chemical class 0.000 claims abstract 2
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 24
- 108090000623 proteins and genes Proteins 0.000 claims description 17
- 239000000047 product Substances 0.000 claims description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
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- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 6
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 6
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- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 4
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- 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 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 claims description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 2
- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 2
- 241001052560 Thallis Species 0.000 claims description 2
- 238000004807 desolvation Methods 0.000 claims description 2
- 230000001939 inductive effect Effects 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- 238000002390 rotary evaporation Methods 0.000 claims description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- 238000000638 solvent extraction Methods 0.000 claims description 2
- ZVJHJDDKYZXRJI-UHFFFAOYSA-N 1-Pyrroline Chemical compound C1CC=NC1 ZVJHJDDKYZXRJI-UHFFFAOYSA-N 0.000 claims 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 2
- 150000002170 ethers Chemical class 0.000 claims 1
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 abstract description 8
- 108010050375 Glucose 1-Dehydrogenase Proteins 0.000 abstract description 8
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- 102000004316 Oxidoreductases Human genes 0.000 description 35
- 210000004027 cell Anatomy 0.000 description 14
- 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 description 11
- 238000003786 synthesis reaction Methods 0.000 description 10
- 230000015572 biosynthetic process Effects 0.000 description 8
- 150000001412 amines Chemical class 0.000 description 7
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- 239000003814 drug Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 2
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 2
- 230000002210 biocatalytic effect Effects 0.000 description 2
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- 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
- 229930014626 natural product Natural products 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- NCXSNNVYILYEBC-SNVBAGLBSA-N (2r)-2-(2,5-difluorophenyl)pyrrolidine Chemical compound FC1=CC=C(F)C([C@@H]2NCCC2)=C1 NCXSNNVYILYEBC-SNVBAGLBSA-N 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000672609 Escherichia coli BL21 Species 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 102000010909 Monoamine Oxidase Human genes 0.000 description 1
- 108010062431 Monoamine oxidase Proteins 0.000 description 1
- 241000863420 Myxococcus Species 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- 208000000323 Tourette Syndrome Diseases 0.000 description 1
- 208000016620 Tourette disease Diseases 0.000 description 1
- 102000003929 Transaminases Human genes 0.000 description 1
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- 239000003905 agrochemical Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000003276 anti-hypertensive effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
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- 239000002585 base Substances 0.000 description 1
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- 230000008901 benefit Effects 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 208000012839 conversion disease Diseases 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229940083124 ganglion-blocking antiadrenergic secondary and tertiary amines Drugs 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
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- 239000008101 lactose Substances 0.000 description 1
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- 229910052751 metal Inorganic materials 0.000 description 1
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- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 150000003235 pyrrolidines Chemical class 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011946 reduction process Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
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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
- C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
- C12P17/165—Heterorings having nitrogen atoms as the only ring heteroatoms
Definitions
- the invention belongs to the field of biocatalysis, and relates to an imine reductase derived from Myxococcus as a biocatalyst and NADP (H) as a coenzyme to reduce 2-pyridine-1-pyrroline to generate (S)-2-( 3-Pyridine)-pyrrolidine method, product optical purity >98%.
- Chiral amines and their derivatives are important branches of single enantiomer drugs, and are the structural units of many pharmaceutical intermediates and agrochemicals.
- Currently, more than 70% of drugs are chiral amines and their derivatives, including neural , antihypertensive and cardiovascular and cerebrovascular drugs [Mitsukura K, Kuramoto T, Yoshida T, et al.[J].Appl Microbiol Biotechnol, 2013, 97: 8079-8086.].
- Optically pure 2-aryl(hetero)ylpyrrolidines are important building blocks commonly found in natural products, drug molecules, and synthetic intermediates. Functionalized chiral pyrrolidines have recently been shown to possess various biological activities, especially is a precursor of potentially beneficial compounds in the treatment of Parkinson's disease, Alzheimer's disease and Tourette's syndrome [Viswanath A, Joseph L, [J] ACS Comb. Sci. 2017, 19, 286-298].
- many chiral 2-aryl(hetero)ylpyrrolidines are natural products that can be used as chiral bases, chiral auxiliaries, and chiral ligands.
- the asymmetric synthesis methods of chiral amines mainly include chemical synthesis or biological enzyme-catalyzed synthesis.
- the chemical synthesis of chiral 2-aryl(hetero)ylpyrrolidine requires multi-step reactions, during which chiral derivatization reagents or metal catalysts are used. It can only be completed after waiting, the conditions are harsh, the pollution is serious, the optical purity is difficult to reach more than 98.0%, the yield is low, and there are many limitations in actual large-scale production [Charles H.M., Steven J.Q.[J]Journal of Medicinal Chemistry,2017,19,286- 298]. Therefore, it is particularly important to explore more green and efficient biological enzyme catalysis methods.
- the enzymes commonly used in the biological enzymatic catalysis of chiral amines mainly include transaminases [Fuchs M, Kozelewski D, et al. [J].Chem Commun, 2010, 46(30): 5500-5502], monoamine oxidase [Ghislieri D, Turner N. [J].Topics in Catalysis,2014,57(5):284-300], dehydrogenase [Abrahamson MJ,Wong JW,[J].Advanced Synthesis&Catalysis,2013,355(9):1780-1786] and sub Amine reductase [Mangassanchez J, France SP, Montgomery SL, et al. [J].
- imine reductase has the unique advantage of catalyzing the synthesis of chiral secondary and tertiary amines [Lenz M, Borlinghaus N, Weinmann L, et al.[J].World J Microbiol Biotechnol,2017,33( 11): 199], has gradually become a research hotspot in the biocatalytic synthesis of chiral amines in recent years.
- the substrate concentration in the reduction process is often not too high, otherwise the reaction conversion rate will be significantly reduced.
- the present invention provides a method for preparing (S)-2-(3-pyridine)-pyrrolidine, using imine reductase (IRED) or engineering bacteria expressing the enzyme to prepare chiral 2-pyridinepyrrolidine compounds.
- the regeneration of coenzyme is realized by using glucose dehydrogenase/glucose system.
- the specific construction method of the genetically engineered bacteria producing imine reductase described in the present invention is as follows: the MsIR1 (WP_074958336.1) gene derived from Myxococcus fulvus is codon-optimized and the corresponding sequence is fully synthesized, Nde I and EcoR I restriction sites are added to the end, and the synthesized gene is constructed into the corresponding expression vector, and then the expression vector is transformed into the recipient bacteria, that is, the genetically engineered bacteria M1 producing imine reductase are obtained respectively. ; And the genetically engineered bacteria are fermented and cultured to realize the efficient heterologous expression of imine reductase.
- the vector series used by the imine reductase-producing genetically engineered bacteria in the present invention include: pET series plasmids, pTXB1 series, pGEX series, pETduet series, and pTYB series.
- the imine reductase-producing genetically engineered bacteria in the present invention is characterized in that the host bacteria capable of efficiently expressing exogenous genes are one of the following: BL21 series, Rosetta series, Origami series, and Tuner series.
- a transformant obtained by transforming a host with a plasmid can grow and produce the imine reductase of the present invention based on known information.
- Any artificial or natural medium containing suitable carbon sources, nitrogen sources, inorganic and other nutrients can be used as long as it can satisfy the growth of the host cells and express the target protein.
- the culture method and culture conditions are not clearly limited, and can be appropriately selected according to the different culture methods and types, as long as the growth of the host can be satisfied and an imine reductase with corresponding activity can be produced.
- the imine reductase used to prepare the chiral 2-(3-pyridine)-pyrrolidine of the present invention can be the culture of the above-mentioned imine reductase genetically engineered recombinant bacteria, or the bacteria obtained by centrifuging the culture medium. Somatic cells or their processed products.
- the processed product refers to the extract obtained from the bacterial cells, the broken liquid, or the separated product obtained by separating and/or purifying the imine reductase of the extract, or the immobilized product of the immobilized extract or processed product.
- the present invention also relates to a method for converting whole cells or crude enzyme solution to synthesize chiral 2-(3-pyridine)-pyrrolidine compounds.
- S)-2-(3-pyridine)-pyrrolidine is characterized in that, comprises the steps:
- the imine reductase-producing genetically engineered bacteria are cultured on a seed medium, inoculated into a fermentation medium in a certain proportion, and after culturing for a certain period of time, an inducer IPTG or lactose or a mixture of the two is added to induce the culture for a certain period of time, and centrifugation is performed.
- Collect the bacterial cells carry out high-pressure crushing, and transform into 2 under the reaction conditions of pH 6.0-10.0 buffer solution, 2-pyridine-1-pyrroline compound substrate 10-100 g/L, reaction temperature of 20-40 °C, and rotating speed of 200 rpm. After ⁇ 24 hours, after the reaction is complete, (S)-2-(3-pyridine)-pyrrolidine is obtained after centrifugation, alkalization, extraction and desolvation, and the yield is greater than 80%.
- the inorganic alkali used for alkalization is one or more combinations of sodium hydroxide, potassium hydroxide and sodium carbonate.
- the organic solvent used for the extraction is one or more combinations of dichloromethane, ethyl acetate and methyl tert-butyl ether.
- drying agent used for drying is anhydrous sodium sulfate or anhydrous magnesium sulfate, etc.
- the suitable medium in the reaction can be water, fermentation broth or an aqueous medium containing different buffers, and the buffer used can be one or both of phosphate, Tris hydrochloride, bicarbonate, carbonate, etc. added to the water. of the above compositions.
- the pH value of the present invention can preferably be maintained within the pH range where the imine reductase can express its activity, preferably the pH value is 6.0-10.0.
- the reaction temperature is preferably maintained within a temperature range where imine reductase can express its activity, preferably 20 to 40°C.
- the substrate concentration described in the present invention is not limited, and usually the substrate is 10-90 g/L. Considering the reaction effect, the substrate concentration is preferably greater than or equal to 50 g/L.
- substrates can be added in batches during the reaction.
- the reaction product can also be separated after the reaction is completed or the product can be continuously removed by in-situ separation.
- the present invention relates to a method for biocatalytic synthesis of (S)-2-(3-pyridine)-pyrrolidine. More specifically, the present invention provides an imine reductase derived from Myxococcus fulvus and its genetically engineered bacteria to convert The method for reducing a 2-pyridine-1-pyrroline compound to (S)-2-(3-pyridine)-pyrrolidine, the imine reductase activity derived from Myxococcus fulvus is relatively high, the reaction substrate concentration, the reaction yield The yield and the optical purity of the product are high, the operation in the reaction process is simple, the energy consumption is low, the green chemistry requirements are met, and the method can be applied to the biotransformation and preparation of industrially produced (S)-2-(3-pyridine)-pyrrolidine compounds.
- Example 1 Obtainment of highly expressed genetically engineered bacteria
- the prepared recombinant vector is transformed into Escherichia coli BL21, Rosetta or Origami by conventional methods to construct genetically engineered bacteria in which the recombinant imine reductase exists in the bacteria in a soluble form, and screened out the genetically engineered bacteria that have been established successfully.
- the engineering bacteria with the target protein expression amount of not less than 20% are used as the engineering bacteria strains for production, and are stored in the form of glycerol bacteria or milk freeze-dried strains.
- the pH of the buffer solution is 7.0-9.0, and the conversion rate is relatively high, especially when the pH is 7.5-8.0, the conversion rate effect is very significant.
- the substrate concentration and the ratio of coenzyme NADP + in the range of 10-20:0.2 have higher conversion rate, especially when the conversion rate is 30-60:0.4-0.6, the effect of conversion rate is very significant.
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Abstract
本发明中提供了一种制备(S)-2-(3-吡啶)-吡咯烷的方法，利用亚胺还原酶(IRED)或表达该酶的工程菌制备手性2-吡啶吡咯烷化合物。利用葡萄糖脱氢酶/葡萄糖体系实现辅酶的再生。更具体的本发明提供了一种利用来源于Myxococcus fulvus的亚胺还原酶及其基因工程菌将2-吡啶-1-吡咯啉化合物还原成(S)-2-(3-吡啶)-吡咯烷的方法，所述的Myxococcus fulvus来源的亚胺还原酶活性较高，反应底物浓度、反应产率以及产物的光学纯度较高，反应过程中操作简单，能耗低，符合绿色化学要求，可应用于工业生产(S)-2-(3-吡啶)-吡咯烷化合物的生物转化制备。
Description
本发明属于生物催化领域，涉及一种利用来源于粘球菌属的亚胺还原酶为生物催化剂，以NADP(H)为辅酶，还原2-吡啶-1-吡咯啉生成(S)-2-(3-吡啶)-吡咯烷的方法，产物光学纯度>98％。
手性胺及其衍生物是单一对映体药物的重要分支，是众多医药中间体及农用化学品的结构单元，目前有超过70％的药物都是手性胺及其衍生物，包括神经类、抗高血压及心脑血管等药物[Mitsukura K,Kuramoto T,Yoshida T,et al.[J].Appl Microbiol Biotechnol,2013,97：8079-8086.]。
光学纯的2-芳(杂)基吡咯烷是重要的结构单元，常见于天然产物、药物分子以及合成中间体中，功能化的手性吡咯烷类化合物最近被证明具有多种生物活性，特别是作为治疗帕金森氏病、阿尔茨海默氏病和图雷特综合征方面的潜在的有益化合物前体[Viswanath A,Joseph L,[J]ACS Comb.Sci.2017,19,286-298]。此外，许多手性2-芳(杂)基吡咯烷是天然产物，可用作手性碱、手性助剂和手性配体。因此，近年来对光学纯的2-芳(杂)基吡咯烷衍生物的合成受到了广泛的关注[Andres,J.M,Sierra,I.H,et al.[J]European Journal of Inorganic Chemistry 2000,9,1719-1726.]。
手性胺的不对称合成方法主要包括化学法合成或生物酶催化法合成，使用化学法合成手性2-芳(杂)基吡咯烷需要多步反应，期间会用到手性衍生试剂或者金属催化剂等才能完成，条件苛刻，污染严重，光学纯度难以达到98.0％以上，收率较低，在实际大规模生产中有诸 多限制[Charles H.M.,Steven J.Q.[J]Journal of Medicinal Chemistry,2017,19,286-298]。因此，探索更加绿色高效的生物酶催化法显得尤为重要。
手性胺的生物酶催化法常用的酶主要包括转氨酶[Fuchs M,Kozelewski D,et al.[J].Chem Commun,2010,46(30)：5500-5502]、单胺氧化酶[Ghislieri D,TurnerN.[J].Topics in Catalysis,2014,57(5)：284-300]、脱氢酶[Abrahamson MJ,Wong JW,[J].Advanced Synthesis&Catalysis,2013,355(9)：1780-1786]和亚胺还原酶[Mangassanchez J,France SP,Montgomery SL,et al.[J].Current Opinion in Chemical Biology,2016,37：19-25.]等。相比其他几种酶，亚胺还原酶具有催化合成手性仲胺和叔胺的独特优势[Lenz M,Borlinghaus N,Weinmann L,et al.[J].World J Microbiol Biotechnol,2017,33(11)：199]，近几年来逐渐成为生物催化合成手性胺的研究热点。2018年，黄冈等人利用重组亚胺还原酶催化合成了R构型的吡咯烷，(R)-2-(2,5-二氟苯基)吡咯烷或其盐[CN201811582029.X]。
但是由于胺类底物独特的性质，导致在还原过程中底物浓度往往不能过高，否则反应转化率会明显降低，为了提高底物浓度，需要增加昂贵辅酶NAD(P)H的用量，生产成本较高。因此寻找更高活性的亚胺还原酶通成为了高效还原亚胺底物2-吡啶-1-吡咯啉的关键因素。
发明内容：
本发明中提供了一种制备(S)-2-(3-吡啶)-吡咯烷的方法，利用亚胺还原酶(IRED)或表达该酶的工程菌制备手性2-吡啶吡咯烷化合物。利用葡萄糖脱氢酶/葡萄糖体系实现辅酶的再生。
本发明中所述的产亚胺还原酶的基因工程菌，具体的构建方法是：对来源于Myxococcus fulvus的MsIR1(WP_074958336.1)基因进行密码子优化后全合成相对应序列，并在基因两端加上Nde I和EcoR I酶切位点，并将合成的基因构建到相应表达载体中，然后表达载体转化入受体菌，即分别得到所述的产亚胺还原酶的基因工程菌M1；并对基因工程菌进行发酵培养， 实现了亚胺还原酶的高效异源表达。
本发明中所述产亚胺还原酶的基因工程菌所用到的载体系列包括：pET系列质粒、pTXB1系列、pGEX系列、pETduet系列、pTYB系列。
本发明中所述的产亚胺还原酶的基因工程菌，其特征在于所述的能高效表达外源基因的宿主菌为下列之一：BL21系列、Rosetta系列、Origami系列、Tuner系列。
在本发明中，由质粒转化宿主得到的转化体可以基于已知信息生长并产生本发明所述的亚胺还原酶。任何一种人工的或天然的含有合适碳源、氮源、无机和其他营养物质的介质，只要能满足宿主菌体的生长并且能够表达出目的蛋白均可使用。培养方法和培养条件没有明确的限制，可以根据培养方法和类型等的不同而进行适当的选择，只要能满足宿主生长并能产生相应活性的亚胺还原酶即可。
用于制备本发明的手性2-(3-吡啶)-吡咯烷的亚胺还原酶可以是上述亚胺还原酶基因工程重组菌的培养物，也可以是通过将培养基离心后得到的菌体细胞或其加工制品。其中加工制品指的是菌体得到的提取物、破碎液、或者对提取物亚胺还原酶进行的分离和/或纯化得到的分离产品，或者通过固定化提取物或者加工制品的固定化制品。
本发明还涉及全细胞或粗酶液转化合成手性2-(3-吡啶)-吡咯烷化合物的方法，所述方法为：通过亚胺还原酶催化2-吡啶-1-吡咯啉化合物得到(S)-2-(3-吡啶)-吡咯烷，其特征在于，包括如下步骤：
(a)将亚胺还原酶基因工程菌在发酵培养基中扩增培养，诱导产生目的蛋白后，离心收集菌体；
(b)将收集的重组菌体细胞或对重组菌体破菌得到的粗酶液加入到缓冲液中，并加入2-吡啶-1-吡咯啉进行反应；
(c)反应完全后从反应体系中收集上层清液，并使用无机碱调节pH值；
(d)使用有机溶剂萃取，单次或合并多次的有机相；
(e)使用干燥剂干燥有机相，过滤，旋蒸回收溶剂得到目标产物。
本发明将所述产亚胺还原酶的基因工程菌经种子培养基培养，按一定比例接种到发酵培养基，培养一定时间后，加入诱导剂IPTG或乳糖或二者混合物诱导培养一定时间，离心收集菌体，进行高压破碎，在pH值为6.0～10.0的缓冲液，2-吡啶-1-吡咯啉化合物底物10～100g/L，反应温度20～40℃，转速200rpm反应条件下转化2～24小时，反应完全后经离心、碱化、萃取、脱溶后得到(S)-2-(3-吡啶)-吡咯烷，收率大于80％。
进一步地，所述碱化用的无机碱为氢氧化钠、氢氧化钾、碳酸钠中一种或两种以上组合物。
进一步地，所述萃取用的有机溶剂为二氯甲烷、乙酸乙酯、甲基叔丁基醚中一种或两种以上组合物。
进一步地，所述干燥用的干燥剂为无水硫酸钠或无水硫酸镁等。
反应中适用的介质可以是水、发酵液或含有不同缓冲液的水介质，其所用的缓冲液可以是水中加入磷酸盐、Tris盐酸盐、碳酸氢盐、碳酸盐等中一种或两种以上组合物。
本发明所述的pH值优选能够保持在亚胺还原酶可以表达其活性的pH范围内，优选pH值为6.0～10.0。反应温度优选保持在亚胺还原酶可以表达其活性的温度范围内，优选20～40℃。
本发明所述的底物浓度并没有限制，通常底物为10～90g/L，考虑到反应效果，底物浓度优选大于等于50g/L。同时为了提高生产效率，可以在反应时批次添加底物。反应产物也可以在反应结束后分离或通过原位分离的方法不断的将产物移走。
本发明涉及一种生物催化合成(S)-2-(3-吡啶)-吡咯烷的方法，更具体的本发明提供了一种利用来源于Myxococcus fulvus的亚胺还原酶及其基因工程菌将2-吡啶-1-吡咯啉化合物还原成(S)-2-(3-吡啶)-吡咯烷的方法，所述的Myxococcus fulvus来源的亚胺还原酶活性较高，反应底物浓度、反应产率以及产物的光学纯度较高，反应过程中操作简单，能耗低，符合绿色化学要求，可应用于工业生产(S)-2-(3-吡啶)-吡咯烷化合物的生物转化制备。
以下通过具体的实施例来进一步说明，其目的在于更好的理解发明内容，但是这些实施例不构成对本发明的限制。
实施例1：高表达基因工程菌的获得
全基因合成由通用生物系统(安徽)有限公司完成。
根据细菌(Myxococcus fulvus)的亚胺还原酶MsIR1(WP_074958336.1),并对其进行密码子优化，以期使基因能够在大肠杆菌表达宿主中进行表达。并在基因两端加上Nde I和EcoR I酶切位点，构建到pET-28a(+)载体中，得到基因工程菌M1。
将制备得到的重组载体用常规方法转化入大肠杆菌BL21、Rosetta或Origami以构建重组亚胺还原酶以可溶形式存在于菌体内的基因工程菌，筛选出组建成功的基因工程菌，其中以大肠杆菌BL21为宿主菌的重组菌目的蛋白表达相对较好。以目的蛋白表达量不低于20％的工程菌，作为生产用工程菌菌种，并以甘油菌或牛奶冻干菌种形式保存。
实施例2基因工程菌的培养和粗酶液的制备
挑取平板上单菌落接种至5ml含相应抗生素的发酵培养基中，培养15h左右作为种子液，按照1％的接种量接种至含600ml的发酵培养基中，在37℃，200rpm的摇床上培养至OD
600＝0.6～0.8左右，加入终浓度为0.1mM的IPTG进行诱导10h以上，以8000rpm离心培养液收集菌体，进行高压破碎，得到亚胺还原酶的粗酶液。
实施例3利用亚胺还原酶的全细胞催化合成(S)-2-(3-吡啶)-吡咯烷
10ml磷酸缓冲液(pH7.5)，30mg/ml菌体，2eq葡萄糖,0.2mg/ml NADP
+,10mg GDH粉末，底物浓度50mg/ml，28℃反应，TLC点板判断反应进程。12小时后，加氢氧化钠饱和溶液调节pH值至10以上，离心除去变性的蛋白，上清液用二氯甲烷萃取，干燥，旋干收集产品，HPLC检测。
| 序号 | pH | 转化率 | ee |
| 1 | 6.0 | 26.1 | 99.7 |
| 2 | 6.5 | 48.9 | 99.8 |
| 3 | 7.0 | 84.6 | 99.7 |
| 4 | 7.5 | 98.7 | 99.8 |
| 5 | 8.0 | 89.1 | 99.8 |
| 6 | 8.5 | 80.3 | 99.7 |
| 7 | 9.0 | 72.6 | 99.7 |
| 8 | 9.5 | 55.8 | 99.7 |
根据上表可知，缓冲液pH7.0-9.0时具有较高的转化率，尤其pH7.5-8.0时转化率效果非常显著。
实施例4利用亚胺还原酶的全细胞催化合成(S)-2-(3-吡啶)-吡咯烷
10ml磷酸缓冲液(pH 7.5)，60mg/ml菌体，2eq葡萄糖,0-0.8mg/ml NADP
+,10mg GDH粉末，底物浓度10-90mg/ml，28℃反应，TLC点板判断反应进程。24小时后，加氢氧化钠饱和溶液调节pH值至10以上，离心除去变性的蛋白，上清液用二氯甲烷萃取，干燥，旋干收集产品，HPLC检测。
| 序号 | 底物浓度 | 辅酶NADP + | 转化率 | ee |
| 1 | 10 | 0 | 45.4 | 99.7 |
| 2 | 10 | 0.2 | 99.8 | 99.8 |
| 3 | 20 | 0.2 | 99.8 | 99.7 |
| 4 | 30 | 0.2 | 79.7 | 99.8 |
| 5 | 30 | 0.4 | 99.7 | 99.8 |
| 6 | 40 | 0.4 | 99.5 | 99.7 |
| 序号 | 底物浓度 | 辅酶NADP + | 转化率 | ee |
| 7 | 50 | 0.4 | 89.6 | 99.7 |
| 8 | 50 | 0.6 | 99.4 | 99.7 |
| 9 | 60 | 0.6 | 98.5 | 99.7 |
| 10 | 70 | 0.8 | 96.3 | 99.7 |
| 11 | 80 | 0.8 | 94.9 | 99.7 |
| 12 | 90 | 0.9 | 60.4 | 99.7 |
根据上表可知，底物浓度和辅酶NADP
+比例范围为10-20：0.2时具有较高的转化率，尤其30-60：0.4-0.6时转化率效果非常显著。
实施例5利用亚胺还原酶的粗酶液催化合成(S)-2-(3-吡啶)-吡咯烷
10ml磷酸缓冲液(pH 7.5)，60mg/ml亚胺还原酶菌体破菌粗酶液，2eq葡萄糖,0-0.8mg/ml NADP
+,10mg GDH粉末，底物浓度10-90mg/ml，30℃反应，TLC点板判断反应进程。24小时后，加氢氧化钠饱和溶液调节pH值至10以上，离心除去变性的蛋白，上清液用二氯甲烷萃取，干燥，旋干收集产品，HPLC检测。
| 序号 | 底物浓度 | 辅酶NADP + | 转化率 | ee |
| 1 | 10 | 0 | 21.6 | 99.7 |
| 2 | 10 | 0.2 | 99.2 | 99.8 |
| 3 | 20 | 0.2 | 97.8 | 99.7 |
| 4 | 30 | 0.3 | 94.5 | 99.8 |
| 5 | 30 | 0.4 | 99.3 | 99.8 |
| 6 | 40 | 0.4 | 99.2 | 99.7 |
| 7 | 50 | 0.4 | 83.4 | 99.7 |
| 8 | 50 | 0.6 | 89.4 | 99.7 |
| 9 | 60 | 0.6 | 88.5 | 99.7 |
| 10 | 70 | 0.8 | 76.3 | 99.7 |
| 11 | 80 | 0.8 | 64.9 | 99.7 |
实施例6利用亚胺还原酶的全细胞催化合成(S)-2-(3-吡啶)-吡咯烷
1l磷酸缓冲液(pH 7.5)，40mg/ml亚胺还原酶菌体，2eq葡萄糖,0.4mg/ml NADP
+,100mg GDH粉末，分批补加底物，底物终浓度50mg/ml，28℃反应，TLC点板判断反应进程。反应完全后，加氢氧化钠饱和溶液调节pH值至10以上，离心除去变性的蛋白，上清液用二氯甲烷萃取，干燥，旋干收集产品。收率96％，纯度98％，e.e.值99.8％。
实施例7利用亚胺还原酶的全细胞催化合成(S)-2-(3-吡啶)-吡咯烷
1l磷酸缓冲液(pH 7.5)，40mg/ml亚胺还原酶菌体，2eq葡萄糖,0.5mg/ml NADP
+,100mg GDH粉末，分批补加底物，底物终浓度70mg/ml，30℃反应，TLC点板判断反应进程。反应完全后，加氢氧化钠饱和溶液调节pH值至10以上，离心除去变性的蛋白，上清液用二氯甲烷萃取，干燥，旋干收集产品。收率93％，纯度98％，e.e.值99.6％。
实施例8利用亚胺还原酶的粗酶液催化合成(S)-2-(3-吡啶)-吡咯烷
1l磷酸缓冲液(pH 7.5)，60mg/ml亚胺还原酶菌体破菌粗酶液，2eq葡萄糖,0.5mg/ml NADP
+,100mg GDH粉末，分批补加底物，底物终浓度50mg/ml，30℃反应，TLC点板判断反应进程。反应完全后，加氢氧化钠饱和溶液调节pH值至10以上，离心除去变性的蛋白，上清液用二氯甲烷萃取，干燥，旋干收集产品。收率94％，纯度98％，e.e.值99.7％。
以上所述仅为本发明的较佳实施例而已，是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
Claims (14)
- 一种制备(S)-2-(3-吡啶)-吡咯烷的方法，通过亚胺还原酶催化2-吡啶-1-吡咯啉化合物得到(S)-2-(3-吡啶)-吡咯烷，其特征在于，包括如下步骤：(a)将亚胺还原酶基因工程菌在发酵培养基中扩增培养，诱导产生目的蛋白后，离心收集菌体；(b)将收集的重组菌体细胞或对重组菌体破菌得到的粗酶液加入到缓冲液中，并加入2-吡啶-1-吡咯啉进行反应；(c)反应完全后从反应体系中收集上层清液，并使用无机碱调节pH值；(d)使用有机溶剂萃取，合并有机相；(e)使用干燥剂干燥有机相，过滤，旋蒸回收溶剂得到目标产物。
- 如权利要求1所述的制备(S)-2-(3-吡啶)-吡咯烷的方法，其特征在于，所述的亚胺还原酶来源于Myxococcus fulvus，其氨基酸序列为SEQ ID.NO 1。
- 如权利要求2所述的制备(S)-2-(3-吡啶)-吡咯烷的方法，其特征在于，所述亚胺还原酶的基因工程菌所用到的载体系列为pET系列质粒、pTXB1系列、pGEX系列、pETduet系列或pTYB系列。
- 如权利要求3所述的制备(S)-2-(3-吡啶)-吡咯烷的方法，其特征在于，所述的亚胺还原酶的基因工程菌的宿主菌为BL21系列、Rosetta系列、Origami系列或Tuner系列。
- 如权利要求1所述的制备(S)-2-(3-吡啶)-吡咯烷的方法，其特征在于，所述步骤(b)在pH值为6.0～10.0的缓冲液，2-吡啶-1-吡咯啉底物浓度为10～100g/L，反应温度20～40℃，转速200rpm反应条件下转化2～24小时。
- 如权利要求1所述的制备(S)-2-(3-吡啶)-吡咯烷的方法，其特征在于，所述步骤(c)反应完全后经离心、碱化、萃取、脱溶后得到(S)-2-(3-吡啶)-吡咯烷，收率大于80％。
- 如权利要求5所述的制备(S)-2-(3-吡啶)-吡咯烷的方法，其特征在于，所述2-吡啶-1-吡咯啉底物浓度为70g/L。
- 如权利要求5所述的制备(S)-2-(3-吡啶)-吡咯烷的方法，其特征在于，所述缓冲液为磷酸盐、Tris盐酸盐、碳酸氢盐、碳酸盐中一种或两种以上组合物。
- 如权利要求6所述的制备(S)-2-(3-吡啶)-吡咯烷的方法，其特征在于，所述碱化用的无机碱为氢氧化钠、氢氧化钾、碳酸钠中一种或两种以上组合物。
- 如权利要求6所述的制备(S)-2-(3-吡啶)-吡咯烷的方法，其特征在于，所述萃取用的 有机溶剂为二氯甲烷、乙酸乙酯、甲基叔丁基醚中一种或两种以上组合物。
- 如权利要求1所述的制备(S)-2-(3-吡啶)-吡咯烷的方法，其特征在于，所述干燥用的干燥剂为无水硫酸钠或无水硫酸镁。
- 一种制备(S)-2-(3-吡啶)-吡咯烷的亚胺还原酶，其特征在于，所述亚胺还原酶来源于Myxococcus fulvus，其氨基酸序列为SEQ ID.NO 1。
- 如权利要求12所述的制备(S)-2-(3-吡啶)-吡咯烷的亚胺还原酶，其特征在于，所述亚胺还原酶的基因工程菌所用到的载体系列为pET系列质粒、pTXB1系列、pGEX系列、pETduet系列或pTYB系列。
- 如权利要求13所述的制备(S)-2-(3-吡啶)-吡咯烷的亚胺还原酶，其特征在于，所述的亚胺还原酶的基因工程菌的宿主菌为BL21系列、Rosetta系列、Origami系列或Tuner系列。
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| CN115404250A (zh) * | 2021-05-29 | 2022-11-29 | 重庆博腾制药科技股份有限公司 | 一种利用还原方式制备(s)-尼古丁的方法 |
| CN114836490A (zh) * | 2022-04-29 | 2022-08-02 | 上海健康医学院 | 一种亚胺还原酶在催化合成手性2-芳基吡咯烷中的应用 |
| CN116217544B (zh) * | 2023-05-08 | 2023-08-29 | 济南悟通生物科技有限公司 | 一种(s)-降烟碱的合成方法 |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110287494A1 (en) * | 2008-09-01 | 2011-11-24 | Daicel Chemical Industries Ltd | Process for production of optically active amine derivative |
| CN102633773A (zh) * | 2011-02-14 | 2012-08-15 | 迪维斯实验室有限公司 | 一种(r,s)-烟碱的制备方法 |
| CN107406411A (zh) * | 2014-10-22 | 2017-11-28 | 下代实验室有限责任公司 | (r,s)‑烟碱的制备方法 |
| WO2020098978A1 (en) * | 2018-11-16 | 2020-05-22 | Zanoprima Lifesciences Limited | Process for the preparation of (s)-nicotin from myosmine |
| CN111511726A (zh) * | 2017-12-22 | 2020-08-07 | 斯福瑞股份有限公司 | 在醇化物碱存在下通过烟酸乙酯与n-乙烯基吡咯烷酮反应制备外消旋烟碱及后续处理步骤 |
| CN112795603A (zh) * | 2020-12-14 | 2021-05-14 | 山东金城医药化工有限公司 | 一种制备(s)-2-(3-吡啶)-吡咯烷的方法 |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101672423B1 (ko) * | 2005-02-28 | 2016-11-03 | 22엔디 센츄리 리미티드, 엘엘씨 | 식물의 니코틴 알칼로이드 수준의 감소 |
| WO2013170050A1 (en) * | 2012-05-11 | 2013-11-14 | Codexis, Inc. | Engineered imine reductases and methods for the reductive amination of ketone and amine compounds |
| US10913962B2 (en) * | 2018-11-16 | 2021-02-09 | Zanoprima Lifesciences Limited | Process of making (S)-nicotine |
| CN109593802B (zh) * | 2018-12-24 | 2021-10-01 | 上海健康医学院 | 一种(r)-2-(2,5-二氟苯基)吡咯烷或其盐的制备方法 |
| CN111057729B (zh) * | 2019-06-03 | 2023-08-29 | 弈柯莱生物科技(上海)股份有限公司 | 一种(r)-2-(2,5-二氟苯基)吡咯烷及其制备方法和应用 |
| CN110564788B (zh) * | 2019-09-30 | 2021-05-04 | 江南大学 | 一种利用亚胺还原酶生产麻黄碱的方法 |
| CN112852771B (zh) * | 2020-04-17 | 2022-04-08 | 中国科学院天津工业生物技术研究所 | 酶催化合成手性氨基醇化合物 |
-
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110287494A1 (en) * | 2008-09-01 | 2011-11-24 | Daicel Chemical Industries Ltd | Process for production of optically active amine derivative |
| CN102633773A (zh) * | 2011-02-14 | 2012-08-15 | 迪维斯实验室有限公司 | 一种(r,s)-烟碱的制备方法 |
| CN107406411A (zh) * | 2014-10-22 | 2017-11-28 | 下代实验室有限责任公司 | (r,s)‑烟碱的制备方法 |
| CN111511726A (zh) * | 2017-12-22 | 2020-08-07 | 斯福瑞股份有限公司 | 在醇化物碱存在下通过烟酸乙酯与n-乙烯基吡咯烷酮反应制备外消旋烟碱及后续处理步骤 |
| WO2020098978A1 (en) * | 2018-11-16 | 2020-05-22 | Zanoprima Lifesciences Limited | Process for the preparation of (s)-nicotin from myosmine |
| CN112795603A (zh) * | 2020-12-14 | 2021-05-14 | 山东金城医药化工有限公司 | 一种制备(s)-2-(3-吡啶)-吡咯烷的方法 |
Non-Patent Citations (1)
| Title |
|---|
| DATABASE Protein 27 September 2021 (2021-09-27), ANONYMOUS: "NAD(P)-binding domain-containing protein [Myxococcus fulvus]", XP055944423, retrieved from Genbank Database accession no. WP_074958336 * |
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