Synthesis method of chiral fused ring tetrahydroisoquinoline alkaloid and analogue thereof
阅读说明：本技术 手性稠环四氢异喹啉类生物碱及其类似物的合成方法 (Synthesis method of chiral fused ring tetrahydroisoquinoline alkaloid and analogue thereof ) 是由 姚培圆 杨林松 李键煚 徐泽菲 陈曦 冯进辉 吴洽庆 朱敦明 于 2021-04-15 设计创作，主要内容包括：本发明提供了一种亚胺还原酶及其在不对称还原制备光学纯手性稠环四氢异喹啉类化合物的应用,具体地,所述亚胺还原酶是来源于Myxococcus fulvus的氧化还原酶。所述的亚胺还原酶在温和的反应条件下实现10-500mM稠环二氢异喹啉类底物的手性还原。本发明提供的亚胺还原酶底物谱宽,且对稠环二氢异喹啉具有较高的活性。(The invention provides an imine reductase and application thereof in preparing an optical pure chiral fused ring tetrahydroisoquinoline compound through asymmetric reduction, and particularly relates to the imine reductase which is oxidoreductase derived from Myxococcus fulvus. The imine reductase realizes chiral reduction of 10-500mM condensed ring dihydroisoquinoline substrate under mild reaction condition. The imine reductase substrate provided by the invention has wide spectrum and has higher activity on condensed ring dihydroisoquinoline.)
1. A novel method for synthesizing polycyclic tetrahydroisoquinoline alkaloids and analogues thereof is characterized in that substrate polycyclic tetrahydroisoquinoline alkaloids and analogues thereof are added into a certain organic cosolvent, then added into a buffer system of imine reductase IRED1 and coenzyme regeneration system recombinant bacteria, and converted to obtain chiral polycyclic tetrahydroisoquinoline alkaloids and analogues thereof.
2. The novel method for synthesizing fused ring tetrahydroisoquinoline alkaloids and analogues thereof according to claim 1, wherein the substrate fused ring dihydroisoquinoline alkaloid and analogues thereof have the following structures:
3. the novel method for synthesizing fused ring tetrahydroisoquinoline alkaloids and analogues thereof according to claim 1, wherein the amino acid sequence of said imine reductase is shown in SEQ ID No. 1.
4. The novel process for the synthesis of fused ring tetrahydroisoquinoline alkaloids and analogues thereof according to claim 1, wherein the coenzyme regeneration system comprises glucose and glucose dehydrogenase.
5. The novel method for synthesizing condensed ring tetrahydroisoquinoline alkaloid and analogues thereof according to claim 1, wherein the concentration of the condensed ring tetrahydroisoquinoline alkaloid and analogues thereof is 10-100 g/L, preferably 80 g/L.
6. The novel method for synthesizing fused ring tetrahydroisoquinoline alkaloids and analogues thereof according to claim 1, wherein the chiral product fused ring tetrahydroisoquinoline alkaloids and analogues thereof have the configuration of R.
Technical Field
The invention relates to a novel method for synthesizing condensed ring tetrahydroisoquinoline alkaloids and analogues thereof, belonging to the field of biochemical engineering. In particular to a novel method for preparing an optical pure chiral fused ring tetrahydroisoquinoline compound by enzyme catalysis asymmetric reduction.
Background
Tetrahydroisoquinoline alkaloids are widely distributed in nature and are important Chiral amine compounds, and alkaloids having tetrahydroisoquinoline as a structural parent nucleus have biological activities such as anti-tumor, anti-pathogenic microorganism, anti-inflammatory and central nervous system regulation (Le V H, Inai M, Williams R M, et al. Ecteinascidins.A. review of the chemistry, biology and clinical utility of pore biochemical Analysis or biological chemistry [ J ]. Natural Product Reports,2015,32(2): 328. 347; Johnson J L, Nair D S, Pilai S M, et al. diagnosis and use diagnosis: biological Analysis: Molecular Analysis of Molecular Analysis [ 614, Molecular Analysis of biological chemistry J.54; Molecular Analysis of biological chemistry J.12. III, Molecular Analysis of biological chemistry, Molecular Analysis of biological Analysis [ III, Molecular Analysis of biological chemistry, biological Analysis of biological chemistry [ J.4, biological Analysis of biological chemistry, biological Analysis of, 2010,45(1):9-16) certain tetrahydroisoquinoline alkaloids such as berberine hydrochloride, tetrahydropalmatine, etc. have been widely used clinically (Li Y, Yu S, Wu X, et al. Iron Catalyzed asymmetry of Hydrogenation of Ketone [ J ] Journal of the American Chemical Society,2014,136(10): 4031-4039; larson R T, Pemberton R P, Franke J M, et al.Total Synthesis of the Gallimima alkalides Himandrane and GB17 use biomedical Diels-Alder Reactions of Double Diene condensers [ J ] Journal of the American Chemical Society,2015,137(34): 11197) 11204; lane J W, Chen Y, Williams R M. asymmetry Total Synthesis of (-) -Journal, (-) -Renilamycin G, 3-epi-Journal, and 3-epi-Renilamycin G [ J ]. Journal of the American Chemical Society,2005,127(36):12684 and 12690). Because of their special structures and various biological activities, tetrahydroisoquinoline alkaloids and their analogues are receiving more and more attention.
The existing methods for synthesizing tetrahydroisoquinoline and analogues thereof mainly comprise two major types, namely chemical methods and enzymatic synthesis. The chemical synthesis of tetrahydroisoquinoline and its analogues is mainly divided into chiral resolution, chiral induction synthesis, and metal-catalyzed asymmetric synthesis (Luk L Y P, Bunn S, Liscombe D K, et alsis:An Enzymatic Pictet-Spengler Reaction[J].Biochemistry,2007,46(35):10153-10161；Benedetti S,Bucciarelli S,Canestrari F,et al.Platelet's Fatty Acids and Differential Diagnosis of Major Depression and Bipolar Disorder through the Use of an Unsupervised Competitive-Learning Network Algorithm(SOM)[J].Open Journal of Depression,2014,03(2):52-73；Lee S C,Choi S Y,Chung Y K,et al.Preparation of pilot library with tetrahydro-β-carboline alkaloid core skeleton using tandem intramolecular Pictet–Spengler cyclization[J]Tetrahedron Letters,2006,47(38): 6843-. However, most literature reports that the existing chemical synthesis methods for tetrahydroisoquinoline Alkaloids have problems of complicated Reaction steps, harsh Reaction conditions, need to use expensive transition metal catalysts, and yet to improve product stereoselectivity, which affects the application of the existing chemical synthesis methods on an industrial scale (Rao R N, Maiti B, Chanda K. application of Picture-Generator Reaction to Industrial-Based Alkaloids control trap-beta-carboline Scanfold in Combinatorial Chemistry [ J].Acs Combinatorial Science,2017,19(4):199-228；Marti C,Carreira E.Construction of Spiro[pyrrolidine-3,3′-oxindoles]Recent Applications to the Synthesis of Oxindole Alkaloids[J].Ruropean Journal of Organic Chemistry,2003,2003(12):2209-2219；Chrzanowska M,Rozwadowska M D.Asymmetric Synthesis of Isoquinoline Alkaloids[J].Chemical Reviews,2004,104(7):3341-3370；-Xavier Felpin,Lebreton J.Recent Advances in the Total Synthesis of Piperidine and Pyrrolidine Natural Alkaloids with Ring-Closing Metathesis as a Key Step(p 3693-3712)[J]European Journal of Organic Chemistry,2003 (2003), (19): 3693-. Alternatively, some researchers have studied enzymatic synthesis of tetrahydroisoquinoline alkaloids and analogues thereof. The currently reported method for synthesizing tetrahydroisoquinoline alkaloids by biocatalysis mainly utilizes NCS enzyme to catalyze the Pictee-Spengler reaction, namely the intermolecular cyclization reaction between beta-arylethylamine and aldehyde or ketone, but most of the methods are used for catalyzing the Pictee-Spengler reactionSynthesized are tetrahydroisoquinoline parent nuclei with only two rings. The Helen c. hailes group asymmetrically synthesized tricyclic neferine and its analogs using NCS enzyme in 2018, but the selectivity of this method remained to be improved. The Turner group achieved biocatalytic synthesis of (R) -Harmicine using monoamine oxidase, but this approach required the use of large amounts of reducing agents. Qu et al asymmetrically synthesized tetrahydroisoquinoline and the like using imine reductase, but they worked equally well to synthesize only a tetrahydroisoquinoline nucleus having two rings.
Disclosure of Invention
The invention provides a novel method for asymmetric synthesis of chiral fused ring tetrahydroisoquinoline compounds under catalysis of imine reductase, which has the remarkable characteristics of simple process route, mild reaction conditions, high yield, less pollution and the like.
The amino acid sequence of the imine reductase is IRED1(SEQ ID NO: 1), and the imine reductase has the capacity of converting a condensed ring dihydroisoquinoline compound into a condensed ring tetrahydroisoquinoline compound. The imine reductase is derived from Myxococcus fulvus.
The carrier used by the genetic engineering bacteria for producing the imine reductase is pET series plasmids.
The imine reductase for preparing the optically pure tetrahydroisoquinoline compound is a somatic cell obtained by centrifuging a culture medium or a processed product thereof. Wherein the processed product refers to extract obtained from thallus, broken solution or separated product obtained by separating and/or purifying imine reductase extracted from thallus. The most used in the present invention is bacterial cells obtained by centrifuging a culture medium.
The invention also relates to a method for generating corresponding chiral condensed ring tetrahydroisoquinoline compounds by converting condensed ring dihydroisoquinoline substrates with different substituents under the catalysis of whole cells, which comprises the following steps:
and (3) respectively culturing the genetically engineered bacteria of the imine reductase for a certain time, adding an inducer IPTG (isopropyl-beta-D-thiogalactoside) for culturing for a certain time, and centrifugally collecting bacteria. Resuspending the cells with a buffer solution, adding 10-500mM fused ring dihydroisoquinoline baseThe resulting mixture (dissolved in 10% DMSO), 10-50g/L wet cells, and 4-50U/mL GDH, 30-1000mM glucose, 0.5mg/mL NADP+Reacting at 25 ℃ and 200rpm for 10-24 h. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the product was purified by silica gel column chromatography after removing the organic solvent.
Drawings
FIGS. 1 to 12: nuclear magnetic hydrogen spectrum of product
Detailed Description
The following examples are further illustrated for the purpose of better understanding the present invention, but are not to be construed as limiting the invention.
Example 1: construction and culture of genetically engineered bacteria
The specific construction and culture method of the genetic engineering bacteria for producing the imine reductase comprises the following steps: an amino acid sequence of Myxococcus fulvus-derived SEQ ID NO: 1, carrying out gene synthesis, constructing into pET series vectors, and carrying out heterologous expression in host bacteria escherichia coli. The strain preserved at-80 ℃ was thawed, streaked on a plate, and cultured overnight in a 37 ℃ incubator. Selecting single colony on the plate, inoculating into 20mL LB culture medium containing corresponding antibiotic, culturing for about 12h to obtain seed solution, inoculating into 700mL LB culture medium containing corresponding antibiotic according to 1% of inoculum size, and culturing on a shaker at 37 deg.C and 200rpm to OD600When the concentration was about 0.6 to 0.8, IPTG was added to the mixture at a final concentration of 0.1mmol/L to induce the cells at 25 ℃ for 12 hours, and the cells were collected by centrifugation at 6000 rpm.
Example 2: preparation of optically pure (R) -8, 9-dimethoxy-1, 2,3,5,6,10 b-hexahydropyrrolo [2,1-a ] isoquinoline-4-ammonium chloride by IRED1 catalysis of 8, 9-dimethoxy-2, 3,5, 6-tetrahydro-1H-pyrrolo [2,1-a ] isoquinoline-4-ammonium chloride
10mM of 8, 9-dimethoxy-2, 3,5, 6-tetrahydro-1H-pyrrolo [2,1-a ] is added]Isoquinoline-4-ammonium chloride (dissolved in 10% DMSO), 30mM glucose, 4U/mL GDH, 0.5mg/mL NADP+2.50g of IRED1 genetically engineered bacteria, wherein the buffer solution is 100mL of phosphate buffer solution with pH7.5, and the reaction is carried out at 25 ℃ and 220rpm for 10 h. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the completion of the extraction was confirmed by thin layer chromatography. Rotating the organic solventAnd (4) evaporating to remove to obtain a crude product, detecting the substrate conversion rate and the product yield by using a gas phase, and detecting the ee value of the product by using a liquid phase HPLC (high performance liquid chromatography) to be 99%. The conversion was 87.5%, the yield was 68.1%, and the product configuration was (R).
Example 3: IRED1 catalysis of 9, 10-dimethoxy-1, 2,3,4,6, 7-hexahydropyrido [2,1-a ] isoquinoline-5-ammonium chloride to prepare optically pure (R) -9, 10-dimethoxy-2, 3,4,6,7,11 b-hexahydro-1H-pyrido [2,1-a ] isoquinoline
30mM of 9, 10-dimethoxy-1, 2,3,4,6, 7-hexahydropyrido [2,1-a ] was added]Isoquinoline-5-ammonium chloride (dissolved in 10% DMSO), 90mM glucose, 12U/mL GDH, 0.5mg/mL NADP+2.50g of IRED1 genetically engineered bacteria, wherein the buffer solution is 100mL of phosphate buffer solution with pH7.5, and the reaction is carried out at 25 ℃ and 220rpm for 10 h. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the completion of the extraction was confirmed by thin layer chromatography. And (3) removing the organic solvent by rotary evaporation to obtain a crude product, detecting the substrate conversion rate and the product yield by using a gas phase, and detecting the ee value of the product by using liquid phase HPLC (high performance liquid chromatography) to be 99%. The conversion was 83.3%, the yield was 74.1%, and the product configuration was (R).
Example 4: IRED1 catalysis of 2,3,5, 6-tetrahydro-1H- [1,3] dioxolane [4,5-g ] pyrrolo [2,1-a ] isoquinoline-4-ammonium chloride to prepare optically pure (R) -1,2,3,5,6,11 b-hexahydro- [1,3] dioxolane [4,5-g ] pyrrolo [2,1-a ] isoquinoline
Mixing 100mM 2,3,5, 6-tetrahydro-1H- [1, 3%]Dioxolane [4,5-g]Pyrrolo [2,1-a]Isoquinoline-4-ammonium chloride (in 10% DMF), 300mM glucose, 20U/mL GDH, 0.5mg/mL NADP+4.5g of IRED1 genetically engineered bacteria, wherein the buffer solution is 100mL of phosphate buffer solution with pH7.5, and the reaction is carried out at 25 ℃ and 220rpm for 10 h. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the completion of the extraction was confirmed by thin layer chromatography. And (3) removing the organic solvent by rotary evaporation to obtain a crude product, detecting the substrate conversion rate and the product yield by using a gas phase, and detecting the ee value of the product by using liquid phase HPLC (high performance liquid chromatography) to be 99%. The conversion was 96.5%, the yield was 85.1%, and the product configuration was (R).
Example 5: IRED1 catalysis of 1,2,3,4,6, 7-hexahydro- [1,3] dioxolane [4,5-g ] pyrido [2,1-a ] isoquinoline-5-ammonium chloride to prepare optically pure (R) -2,3,4,6,7,12 b-hexahydro-1H- [1,3] dioxolane [4,5-g ] pyrrolo [2,1-a ] isoquinoline-5-chloride
200mM of 1,2,3,4,6, 7-hexahydro- [1,3]]Dioxolane [4,5-g]Pyrido [2,1-a ]]Isoquinoline-5-ammonium chloride (in 10% DMF), 600mM glucose, 40U/mL GDH, 0.5mg/mL NADP+12g of IRED1 genetically engineered bacteria, wherein the buffer solution is 100mL of phosphate buffer solution with pH7.5, and the reaction is carried out at 25 ℃ and 220rpm for 18 h. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the completion of the extraction was confirmed by thin layer chromatography. And (3) removing the organic solvent by rotary evaporation to obtain a crude product, detecting the substrate conversion rate and the product yield by using a gas phase, and detecting the ee value of the product by using liquid phase HPLC (high performance liquid chromatography) to be 99%. The conversion was 88.1%, the yield was 72.2%, and the product configuration was (R).
Example 6: IRED1 catalysis of 1,2,3,5,6, 11-hexahydroindolizino [8,7-b ] indole-4-ammonium chloride to prepare optically pure (R) -2,3,5,6,11,11 b-hexahydro-1H-indolizino [8,7-b ] indole
500mM of 1,2,3,5,6, 11-hexahydroindolizino [8,7-b ] is added]Indole-4-ammonium chloride (dissolved in 10% DMSO), 1M glucose, 50U/mL GDH, 0.5mg/mL NADP+20g of IRED2 gene engineering bacteria, wherein the buffer solution is 100mL of phosphate buffer solution with pH7.5, and the reaction is carried out for 16h at 25 ℃ and 220 rpm. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the completion of the extraction was confirmed by thin layer chromatography. And (3) removing the organic solvent by rotary evaporation to obtain a crude product, detecting the substrate conversion rate and the product yield by using a gas phase, and detecting the ee value of the product by using liquid phase HPLC (high performance liquid chromatography) to be 99%. The conversion was 81.7%, the yield was 67.5%, and the product configuration was (R).
Example 7: preparation of optically pure (R) -1,2,3,4,6,7,12,12 b-octahydroindolo [2,3-a ] quinolizine by IRED1 catalysis of 2,3,4,6,7, 12-hexahydro-1H-indolo [2,3-a ] quinolizine-5-ammonium chloride
200mM of 2,3,4,6,7, 12-hexahydro-1H-indolo [2,3-a ] are added]Quinazine-5-ammonium chloride (in 10% DMSO), 600mM glucose, 30U/mL GDH, 0.5mg/mL NADP+10g of IRED1 genetically engineered bacteria, wherein the buffer solution is 100mL of phosphate buffer solution with pH7.5, and the reaction is carried out at 25 ℃ and 220rpm for 10 h. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the completion of the extraction was confirmed by thin layer chromatography. Removing organic solvent by rotary evaporation to obtain crude product, and detecting substrate conversion rate and product by gas phaseThe yield of the product is 99 percent according to the ee value of the product detected by liquid phase HPLC. The conversion was 97.1%, the yield was 59.3%, and the product configuration was (R).
Example 8: IRED1 catalysis of 8-methoxy-1, 2,3,5,6, 11-hexahydroindolizino [8,7-b ] indole-4-ammonium chloride to prepare optically pure (R) -8-methoxy-2, 3,5,6,11,11 b-hexahydro-1H-indolizino [8,7-b ] indole
200mM of 8-methoxy-1, 2,3,5,6, 11-hexahydroindolizino [8,7-b ] are added]Indole-4-ammonium chloride (dissolved in 10% DMSO), 600mM glucose, 20U/mL GDH, 0.5mg/mL NADP+11g of IRED1 genetically engineered bacteria, wherein the buffer solution is 100mL of phosphate buffer solution with pH7.5, and the reaction is carried out at 25 ℃ and 220rpm for 10 h. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the completion of the extraction was confirmed by thin layer chromatography. And (3) removing the organic solvent by rotary evaporation to obtain a crude product, detecting the substrate conversion rate and the product yield by using a gas phase, and detecting the ee value of the product by using liquid phase HPLC (high performance liquid chromatography) to be 99%. The conversion was 79.7%, the yield was 56.1% and the product configuration was (R).
Example 9: preparation of optically pure (R) -9-methoxy-1, 2,3,4,6,7,12,12 b-octahydroindolo [2,3-a ] quinolizine by catalyzing 9-methoxy-2, 3,4,6,7, 12-hexahydro-1H-indolo [2,3-a ] quinolizine-5-ammonium chloride with IRED1
Adding 300mM of 9-methoxy-2, 3,4,6,7, 12-hexahydro-1H-indolo [2,3-a ]]Quinazine-5-ammonium chloride (in 10% DMSO), 700mM glucose, 4U/mL GDH, 0.5mg/mL NADP+12g of IRED1 genetically engineered bacteria, pH7.5 phosphate buffer IRED1, 25 ℃, 220rpm reaction for 16 h. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the completion of the extraction was confirmed by thin layer chromatography. And (3) removing the organic solvent by rotary evaporation to obtain a crude product, detecting the substrate conversion rate and the product yield by using a gas phase, and detecting the ee value of the product by using liquid phase HPLC (high performance liquid chromatography) to be 99%. The conversion was 75.2%, the yield was 46.9%, and the product configuration was (R).
Example 10: IRED1 catalysis of 8-chloro-1, 2,3,5,6, 11-hexahydroindolizino [8,7-b ] indole-4-ammonium chloride to prepare optically pure (R) -8-chloro-2, 3,5,6,11,11 b-hexahydro-1H-indolizino [8,7-b ] indole
150mM of 8-chloro-1, 2,3,5,6, 11-hexahydroindolizino [8,7-b ] is added]Indole-4-ammonium chloride (dissolved in 10% DMSO)) 350mM glucose, 10U/mL GDH, 0.5mg/mL NADP+7.50g of IRED1 genetically engineered bacteria, wherein the buffer solution is phosphate buffer solution with pH7.5, and the reaction is carried out at 25 ℃ and 220rpm for 10 h. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the completion of the extraction was confirmed by thin layer chromatography. And (3) removing the organic solvent by rotary evaporation to obtain a crude product, detecting the substrate conversion rate and the product yield by using a gas phase, and detecting the ee value of the product by using liquid phase HPLC (high performance liquid chromatography) to be 99%. The conversion was 88.5%, the yield was 66.3%, and the product configuration was (R).
Example 11: preparation of optically pure (R) -9-chloro-1, 2,3,4,6,7,12 b-octahydroindolo [2,3-a ] quinolizine by IRED1 catalysis of 9-chloro-2, 3,4,6,7, 12-hexahydro-1H-indolo [2,3-a ] quinolizine-5-ammonium chloride
Mixing 100mM of 9-chloro-2, 3,4,6,7, 12-hexahydro-1H-indolo [2,3-a ]]Quinazine-5-ammonium chloride (dissolved in 10% DMSO), 300mM glucose, 20U/mL GDH, 0.5mg/mL NADP+7.5g of IRED1 genetically engineered bacteria, wherein the buffer solution is phosphate buffer solution with pH7.5, and the reaction is carried out at 25 ℃ and 220rpm for 10 h. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the completion of the extraction was confirmed by thin layer chromatography. And (3) removing the organic solvent by rotary evaporation to obtain a crude product, detecting the substrate conversion rate and the product yield by using a gas phase, and detecting the ee value of the product by using liquid phase HPLC (high performance liquid chromatography) to be 99%. The conversion was 81.6%, the yield was 55.7%, and the product configuration was (R).
Example 12: IRED2 catalysis of 8-methyl-1, 2,3,5,6, 11-hexahydroindoxazino [8,7-b ] indole-4-ammonium chloride to prepare optically pure (R) -8-methyl-2, 3,5,6,11,11 b-hexahydro-1H-indoxazino [8,7-b ] indole
250mM of 8-methyl-1, 2,3,5,6, 11-hexahydroindolizino [8,7-b ] is added]Indole-4-ammonium chloride (dissolved in 10% DMSO), 750mM glucose, 30U/mL GDH, 0.5mg/mL NADP+10g of IRED2 genetically engineered bacteria, the buffer solution is phosphate buffer solution with pH7.5, and the reaction is carried out for 20h at 25 ℃ and 220 rpm. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the completion of the extraction was confirmed by thin layer chromatography. And (3) removing the organic solvent by rotary evaporation to obtain a crude product, detecting the substrate conversion rate and the product yield by using a gas phase, and detecting the ee value of the product by using liquid phase HPLC (high performance liquid chromatography) to be 99%. The conversion was 78.4%, the yield was 63.1%, and the product configuration was (R).
Example 13: preparation of optically pure (R) -9-methyl-1, 2,3,4,6,7,12,12 b-octahydroindolo [2,3-a ] quinolizine by catalyzing 9-methyl-2, 3,4,6,7, 12-hexahydro-1H-indolo [2,3-a ] quinolizine-5-ammonium chloride with IRED1
10mM of 9-methyl-2, 3,4,6,7, 12-hexahydro-1H-indolo [2,3-a ]]Quinazine-5-ammonium chloride (dissolved in 10% DMSO), 30mM glucose, 4U/mL GDH, 0.5mg/mL NADP+2.5g of IRED1 genetically engineered bacteria, wherein the buffer solution is phosphate buffer solution with pH7.5, and the reaction is carried out at 25 ℃ and 220rpm for 10 h. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the completion of the extraction was confirmed by thin layer chromatography. And (3) removing the organic solvent by rotary evaporation to obtain a crude product, detecting the substrate conversion rate and the product yield by using a gas phase, and detecting the ee value of the product by using liquid phase HPLC (high performance liquid chromatography) to be 99%. The conversion was 86.1%, the yield was 68.5%, and the product configuration was (R).
Sequence listing
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> synthetic method of chiral condensed ring tetrahydroisoquinoline alkaloid and analogue thereof
<130> amino acid sequence
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 291
<212> PRT
<213> Myxococcus fulvus
<400> 1
Met Lys Pro His Ile Ser Ile Leu Gly Ala Gly Arg Met Gly Ser Ala
1 5 10 15
Leu Val Lys Ala Phe Leu Gln Asn Glu Tyr Thr Thr Thr Val Trp Asn
20 25 30
Arg Thr Arg Ala Arg Cys Glu Pro Leu Ala Ala Ala Gly Ala Arg Ile
35 40 45
Ala Asp Ser Val Arg Asp Ala Val Gln Thr Ala Ser Val Val Ile Val
50 55 60
Asn Val Asn Asp Tyr Asp Thr Ser Asp Ala Leu Leu Arg Gln Asp Glu
65 70 75 80
Val Thr Gln Glu Leu Arg Gly Lys Val Leu Val Gln Leu Thr Ser Gly
85 90 95
Ser Pro Lys Leu Ala Arg Glu Gln Ala Thr Trp Ala Arg Arg His Gly
100 105 110
Ile Asp Tyr Leu Asp Gly Ala Ile Met Ala Thr Pro Asp Leu Ile Gly
115 120 125
Arg Pro Asp Cys Thr Leu Leu Tyr Ala Gly Pro Lys Ala Leu Tyr Asp
130 135 140
Lys His Gln Ala Val Leu Ala Ala Leu Gly Gly Asn Thr Gln His Val
145 150 155 160
Ser Glu Asp Glu Gly His Ala Ser Ala Leu Asp Ser Ala Ile Leu Phe
165 170 175
Gln Leu Trp Gly Ser Leu Phe Ser Gly Leu Gln Ala Ala Ala Ile Cys
180 185 190
Arg Ala Glu Gly Ile Ala Leu Asp Ala Leu Gly Pro His Leu Glu Ala
195 200 205
Val Ala Ala Met Ile Gln Phe Ser Met Lys Asp Leu Leu Gln Arg Ile
210 215 220
Gln Lys Glu Gln Phe Gly Ala Asp Thr Glu Ser Pro Ala Thr Leu Asp
225 230 235 240
Thr His Asn Val Ala Phe Gln His Leu Leu His Leu Cys Glu Glu Arg
245 250 255
Asn Ile His Arg Ala Leu Pro Glu Ala Met Asp Ala Leu Ile Gln Thr
260 265 270
Ala Arg Lys Ala Gly His Gly Gln Asp Asp Phe Ser Val Leu Ala Arg
275 280 285
Phe Leu Arg
290