CN113755552B - Synthesis method of chiral fused ring tetrahydroisoquinoline alkaloid and analogue thereof - Google Patents
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Abstract
The invention provides an imine reductase and application thereof in preparing an optical pure chiral fused ring tetrahydroisoquinoline compound by asymmetric reduction, and particularly provides the imine reductase which is an 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.
Description
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 nucleus have biological activities such as anti-tumor, anti-pathogenic microorganism, anti-inflammation 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 post tetrahydroisoquinoline Alkaloids or antibiotics [ J ]. Natural Product Reports,2015,32 (2): 328-347, johnson J L, nair D S, pilai S M, et al.Dissymetric factory Can Provide Useful diestereomer discrimination: 6154-6164, liu D Y, jiang L.Bioactivity diversity and functional mechanism of tetrahydroisoquinoline alloys [ J ]. Acta pharmaceutical Sinica,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 catalyst affinity hydrolysis of Ketone [ J ]. Journal of the American Chemical Society,2015 136 (10): 1-4039, lamberton R P, franJ M, et al, total Synthesis of the colloidal gold nanoparticles of microorganisms R T, P., 11. J.) (III, J.), 3-epi-Jorumycin, and 3-epi-Renieramycin G [ J ] Journal of the American Chemical Society,2005,127 (36): 12684-12690). 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 analogues thereof is mainly divided into chiral resolution, chiral induction synthesis, metal-catalyzed asymmetric synthesis (Luk L Y P, bunn S, liscombe D K, et al, mechanical students on Norcoclear kinase Synthesis of Benzylisoquinoline Alkaloid Biosynthesis: an Enzymatic Picture-Spectrer 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): 6843-6847). 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 (19): 3693-3712). Alternatively, some researchers have studied enzymatic synthesis of tetrahydroisoquinoline alkaloids and their analogues. 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 synthesized tetrahydroisoquinoline parent nucleus only has 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 tetrahydroisoquinolines and analogs using imine reductases, but they worked equally well to synthesize compounds having two amino groupsA cyclic tetrahydroisoquinoline nucleus.
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 in a buffer solution, adding 10-500mM fused ring dihydroisoquinoline substrate (dissolved in 10% DMSO), 10-50g/L of wet cells, and adding 4-50U/mL GDH,30-1000mM glucose, 0.5mg/mL NADP + Reacting at 25 ℃ and 200rpm for 10-24h. 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 removal of the organic solvent.
Drawings
FIGS. 1-12: 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 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 OD 600 About =0.6-0.8, adding IPTG with final concentration of 0.1mmol/L, inducing at 25 deg.C for 12h, and centrifuging at 6000rpm to collect the thallus.
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
1mM of 8, 9-dimethoxy-2, 3,5, 6-tetrahydro-1H-pyrrolo [2,1-a ] was added]Isoquinoline-4-ammonium chloride (dissolved in 10% DMSO), 30mM glucose, 4U/mL GDH,0.5mg/mL NADP + 2.50g IRED1 gene engineering bacteria, the buffer solution is 100mL of phosphate buffer solution with pH7.5, and the reaction is carried out for 10 hours 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 87.5%, the yield was 68.1%, and the product configuration was (R).
Example 3: preparation of optically pure (R) -9, 10-dimethoxy-2, 3,4,6,7, 11b-hexahydro-1H-pyrido [2,1-a ] isoquinoline-5-ammonium chloride by IRED1 catalysis of 9, 10-dimethoxy-1, 2,3,4, 7-hexahydropyrido [2,1-a ] isoquinoline-5-ammonium chloride
The reaction solution was mixed with 30mM 9,10-dimethoxy-1, 2,3,4,6, 7-hexahydropyrido [2,1-a ]]Isoquinoline-5-ammonium chloride (dissolved in 10% DMSO), 90mM glucose, 12U/mL GDH,0.5mg/mL NADP + 2.50g IRED1 gene engineering bacteria, the buffer solution is 100mL of phosphate buffer solution with pH7.5, and the reaction is carried out for 10 hours 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 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, 11b-hexahydro- [1,3] dioxolane [4,5-g ] pyrrolo [2,1-a ] isoquinoline
100mM of 2,3,5, 6-tetrahydro-1H- [1,3]Dioxolane [4,5-g]Pyrrolo [2,1-a]Isoquinoline-4-ammonium chloride (dissolved in 10% DMF), 300mM glucose, 20U/mL GDH,0.5mg/mL NADP + 4.5g of IRED1 gene engineering bacteria, wherein the buffer solution is 100mL of phosphate buffer solution with the pH value of 7.5, and the reaction is carried out for 10 hours 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 96.5%, the yield was 85.1%, and the product configuration was (R).
Example 5: preparation of optically pure (R) -2,3,4,6,7,1 b-hexahydro-1H- [1,3] dioxolane [4,5-g ] pyrrolo [2,1-a ] isoquinoline-5-ammonium chloride by IRED1 catalysis of 1,2,3,4,6, 7-hexahydro- [1,3] dioxolane [4,5-g ] pyrrolo [2,1-a ] isoquinoline
Example 6: preparation of optically pure (R) -2,3,5,6,11, 11b-hexahydro-1H-indolizino [8,7-b ] indole-4-ammonium chloride by IRED1 catalysis of 1,2,3,5,6, 11-hexahydro-indolizino [8,7-b ] indole-4-ammonium chloride
Example 7: preparation of optically pure (R) -1,2,3,4,6,7,12, 12b-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
Example 8: preparation of optically pure (R) -8-methoxy-2, 3,5,6,11, 11b-hexahydro-1H-indolizino [8,7-b ] indole-4-ammonium chloride by IRED1 catalysis of 8-methoxy-1, 2,3,5,6, 11-hexahydroindolizino [8,7-b ] indole-4-ammonium chloride
200mM 8-methoxy-1, 2,3,5,6, 11-hexahydroindoxazino [8,7-b ] is added]Indole-4-ammonium chloride10% DMSO), 600mM glucose, 20U/mL GDH,0.5mg/mL NADP + 11g of IRED1 gene engineering bacteria, the buffer solution is 100mL of phosphate buffer solution with pH7.5, and the reaction is carried out for 10h 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 to be 99% by using liquid phase HPLC. 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, 12b-octahydroindolo [2,3-a ] quinolizine-5-ammonium chloride by IRED1 catalysis of 9-methoxy-2, 3,4,6,7, 12-hexahydro-1H-indolo [2,3-a ] quinolizine-5-ammonium chloride
300mM of 9-methoxy-2, 3,4,6,7, 12-hexahydro-1H-indolo [2,3-a ]]Quinolizine-5-ammonium chloride (dissolved in 10% DMSO), 700mM glucose, 4U/mL GDH,0.5mg/mL NADP + 12g IRED1 gene engineering bacteria, pH7.5 phosphate buffer IRED1, 25 ℃, 220rpm reaction for 16h. 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 to be 99% by using liquid phase HPLC. The conversion was 75.2%, the yield was 46.9%, and the product configuration was (R).
Example 10: preparation of optically pure (R) -8-chloro-2, 3,5,6,11, 11b-hexahydro-1H-indolizino [8,7-b ] indole-4-ammonium chloride by IRED1 catalysis of 8-chloro-1, 2,3,5,6, 11-hexahydroindolizino [8,7-b ] indole-4-ammonium chloride
150mM 8-chloro-1, 2,3,5,6, 11-hexahydroindolizino [8,7-b ]]Indole-4-ammonium chloride (dissolved in 10% DMSO), 350mM glucose, 10U/mL GDH,0.5mg/mL NADP + 7.50g IRED1 gene engineering bacteria, the buffer solution is phosphate buffer solution with pH7.5, and the reaction is carried out for 10h 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 rate is 88.5 percent, and the yield is 66.3 percentThe compound has the structure of (R).
Example 11: preparation of optically pure (R) -9-chloro-1, 2,3,4,6,7,12, 12b-octahydroindolo [2,3-a ] quinolizine-5-ammonium chloride by IRED1 catalysis of 9-chloro-2, 3,4,6,7, 12-hexahydro-1H-indolo [2,3-a ] quinolizine-5-ammonium chloride
100mM 9-chloro-2, 3,4,6,7, 12-hexahydro-1H-indolo [2,3-a ] was added]Quinolizine-5-ammonium chloride (dissolved in 10% DMSO), 300mM glucose, 20U/mL GDH,0.5mg/mL NADP + 7.5g of IRED1 gene engineering bacteria, wherein the buffer solution is phosphate buffer solution with pH7.5, and the reaction is carried out for 10 hours 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 to be 99% by using liquid phase HPLC. The conversion was 81.6%, the yield was 55.7%, and the product configuration was (R).
Example 12: preparation of optically pure (R) -8-methyl-2, 3,5,6,11, 11b-hexahydro-1H-indolizino [8,7-b ] indole-4-ammonium chloride by IRED2 catalysis of 8-methyl-1, 2,3,5,6, 11-hexahydroindolizino [8,7-b ] indole-4-ammonium chloride
250mM 8- methyl 1,2,3,5,6, 11-hexahydroindolizino [8,7-b ]]Indole-4-ammonium chloride (dissolved in 10% DMSO), 750mM glucose, 30U/mL GDH,0.5mg/mL NADP + 10g of IRED2 genetically engineered bacteria, wherein 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 to be 99% by using liquid phase HPLC. 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, 12b-octahydroindolo [2,3-a ] quinolizine-5-ammonium chloride by IRED1 catalysis of 9-methyl-2, 3,4,6,7, 12-hexahydro-1H-indolo [2,3-a ] quinolizine
1mM of 9-methyl-2, 3,4,6,7, 12-hexahydro-1H-indolo [2,3-a ] was reacted with]Quinolizine-5-ammonium chloride (dissolved in 10% DMSO), 30mM glucose, 4U/mL GDH,0.5mg/mL NADP + 2.5g of IRED1 gene engineering bacteria, and the buffer solution is phosphate buffer with pH7.5Washing, reacting at 25 deg.C and 220rpm for 10h. 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 to be 99% by using liquid phase HPLC. 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
Claims (4)
1. A method for synthesizing polycyclic tetrahydroisoquinoline alkaloid and analogues thereof is characterized in that substrate polycyclic tetrahydroisoquinoline alkaloid and analogues thereof are added into a certain organic cosolvent, then an imine reductase IRED1 and a buffer system of coenzyme regeneration system recombinant bacteria are added, and chiral polycyclic tetrahydroisoquinoline alkaloid and analogues thereof are obtained after conversion;
the structure of the substrate condensed ring dihydroisoquinoline alkaloid and the analogue thereof is as follows, and the configuration of the chiral product condensed ring tetrahydroisoquinoline alkaloid and the analogue thereof is R;
the amino acid sequence of the imine reductase is shown as SEQ ID NO. 1.
2. The method of claim 1, wherein the coenzyme regeneration system comprises glucose and glucose dehydrogenase.
3. The method for synthesizing the condensed ring tetrahydroisoquinoline alkaloid and the analogue thereof according to claim 1, wherein the concentration of the condensed ring tetrahydroisoquinoline alkaloid and the analogue thereof is 10 to 100g/L.
4. The method for synthesizing the condensed ring tetrahydroisoquinoline alkaloid and the analogue thereof according to claim 1, wherein the concentration of the condensed ring tetrahydroisoquinoline alkaloid and the analogue thereof is 80g/L.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628841A (en) * | 2018-06-25 | 2019-12-31 | 中国科学院天津工业生物技术研究所 | Novel method for synthesizing key intermediate of dextromethorphan through enzyme catalysis asymmetry |
| CN110643650A (en) * | 2019-09-20 | 2020-01-03 | 复旦大学 | Preparation method of (S) -1-benzyl-1, 2,3,4,5,6,7, 8-octahydroisoquinoline compound |
| CN111254181A (en) * | 2018-11-30 | 2020-06-09 | 苏州同力生物医药有限公司 | Method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid by chemoenzymatic method |
| CN111254170A (en) * | 2018-11-30 | 2020-06-09 | 浙江大学 | Method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid by multi-enzyme coupling |
-
2021
- 2021-04-15 CN CN202110408471.6A patent/CN113755552B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628841A (en) * | 2018-06-25 | 2019-12-31 | 中国科学院天津工业生物技术研究所 | Novel method for synthesizing key intermediate of dextromethorphan through enzyme catalysis asymmetry |
| CN111254181A (en) * | 2018-11-30 | 2020-06-09 | 苏州同力生物医药有限公司 | Method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid by chemoenzymatic method |
| CN111254170A (en) * | 2018-11-30 | 2020-06-09 | 浙江大学 | Method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid by multi-enzyme coupling |
| CN110643650A (en) * | 2019-09-20 | 2020-01-03 | 复旦大学 | Preparation method of (S) -1-benzyl-1, 2,3,4,5,6,7, 8-octahydroisoquinoline compound |
Non-Patent Citations (2)
| Title |
|---|
| Hosoyama, A.等.6-phosphogluconate dehydrogenase[Myxococcus fulvus].Genbank Database.2019,ORIGIN部分. * |
| 杨璐.生物催化合成6,7-双甲氧四氢异喹啉类生物碱的研究.中国优秀硕士学位论文全文数据库 工程科技1辑.2020,(第02期),摘要，正文第34页第2.2.6节，第43-44页第3.3节. * |
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