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Application of imine reductase and mutant thereof in synthesis of (S)-1-aryl-1, 2, 3, 4-tetrahydroisoquinoline

A reductase and mutant technology, applied in the field of enzyme engineering, can solve the problems of unavailable catalysts, large steric hindrance of phenyl substituents, complex and harsh operating conditions, etc., achieve high product conversion rate and optical purity, and simplify post-processing steps , the effect of improving the quality of the product

Active Publication Date: 2017-11-24
WUHAN UNIV
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AI Technical Summary

Problems solved by technology

Since the kinetic resolution of monoamine oxidase requires the use of expensive and highly toxic borane, and artificial imine reductase requires the use of expensive iridium catalysts with low conversion and e.e., these enzyme-catalyzed methods have major defects. not suitable for actual production
Correspondingly, it is more advantageous to directly use imine reductase (IRED) to reduce the imine substrate 1a to synthesize (S)-1b; No corresponding IRED was found that can catalyze the reduction of compound 1a
Until recently, Zheng et al. reported the first imine reductase SnIRED from the microorganism Stackebrandtia nassauensis (Org. Lett. 2017, 19, 3151-3154), which can directly reduce the imine compound 1a to obtain (S)-1b( following formula C); but because its catalytic conversion rate and the e.e. value of product only have 81% and 51%, still can't satisfy actual production requirement
It can be seen that although enzyme-catalyzed synthesis of 1-phenyl-1,2,3,4-tetrahydroisoquinoline compounds has begun to attract people's attention, from the current reports, either the optical purity of the product is not high, or the operation is complicated The conditions are harsh, or the catalyst is not easy to obtain, and the application in actual industrial production is greatly limited

Method used

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  • Application of imine reductase and mutant thereof in synthesis of (S)-1-aryl-1, 2, 3, 4-tetrahydroisoquinoline
  • Application of imine reductase and mutant thereof in synthesis of (S)-1-aryl-1, 2, 3, 4-tetrahydroisoquinoline
  • Application of imine reductase and mutant thereof in synthesis of (S)-1-aryl-1, 2, 3, 4-tetrahydroisoquinoline

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Effect test

Embodiment 1

[0045] Embodiment 1 Obtaining of imine reductase of the present invention

[0046] (1) Acquisition of the gene encoding imine reductase

[0047] The three imine reductases (IR45, IR96 and IR99) with catalytic ability obtained in the present invention are obtained by screening the imine reductase enzyme library to catalyze the reaction of compounds 1a-4a (the following formula). The imine reductase enzyme library is the 100 genes that may encode imine reductase constructed by the applicant in the early stage. The DNA sequences of these 100 genes are optimized according to the codon preference of Escherichia coli and sent to the company for synthesis. The codon-optimized genes were respectively added with NdeI (CATATG) restriction site at the 5' end and HindⅢ (TTCACT) restriction site at the 3' end, and cloned into pET28a plasmid to obtain recombinant plasmids pET28a-IR45, pET28a- IR96, pET28a-IR99.

[0048]

[0049] The amino acid sequences of imine reductases IR45, IR97 a...

Embodiment 2

[0056] The synthesis of embodiment 2 imine substrates

[0057] (1) Synthesis of 1-m-methylphenyl-3,4-dihydroisoquinoline

[0058] The synthetic route is as follows:

[0059]

[0060] Under the protection of nitrogen, add 2.5mL of phenethylamine (20mmol, 1.0equiv) and 60mL of anhydrous dichloromethane into the previously dried 250mL round bottom flask, cool down to 0°C, and slowly add 4.2mL of triethylamine (30mmol , 1.5equiv) and 2.9mL (22mmol, 1.1equiv) of 3-methylbenzoyl chloride, reacted at 0°C for 30 minutes after the dropwise addition, and then rose to room temperature for 24 hours. After the reaction was completed, the solvent was removed by rotary evaporation, and 50mL was added 1M HCl, extracted three times with ethyl acetate, 50 mL each time, combined the ethyl acetate phases, washed once with water, washed once with saturated brine and dried with anhydrous magnesium sulfate for 1 hour, and the solvent was removed by rotary evaporation to obtain the crude product ...

Embodiment 3

[0072] Synthesis of embodiment 3 imine reduction product racemate

[0073] (1) Synthesis of 1-phenyl-1,2,3,4-tetrahydroisoquinoline

[0074] Synthesized by the following route:

[0075]

[0076] The specific operation is as follows: under the condition of nitrogen protection, in a 10 mL pre-dried round bottom flask, add 0.5 mmol 1-phenyl-3,4-dihydroisoquinoline (1a), add 5 mL of anhydrous methanol, and cool down to 0°C, add 0.6mmol NaBH 4 , after stirring at 0°C for 30 minutes, warm to room temperature and stir for 2 hours, add 20 mL of water to quench the reaction, extract three times with ethyl acetate, 20 mL each time, combine the ethyl acetate phases and dry with anhydrous magnesium sulfate for 30 minutes, spin dry to remove The solvent racemate 1-phenyl-1,2,3,4-tetrahydroisoquinoline (rac-1b) was a white solid with a yield of 96%.

[0077] Compound rac-1b NMR / MS data ( 1 H and 13 C spectrum see figure 2 , 3 ): 1 H NMR (400MHz, Chloroform-d) δ7.38–7.28(m,5H),7....

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Abstract

The invention discloses application of imine reductase and a mutant thereof in synthesis of (S)-1-aryl-1, 2, 3, 4-tetrahydroisoquinoline, and belongs to the field of enzyme engineering. The imine reductase provided by the invention is one of the following enzymes: IR45, IR96 and IR99 as shown in SEQ ID NO. 1-3, having corresponding coding nucleotides preferably as shown in SEQ ID NO. 1-3; the mutant of the imine reductase is one of mutants IR45-W191A and IR45-W191L of IR45 and mutant IR99-L173Aof IR99. The imine reductase or the mutant thereof can catalyze a compound of formula I to synthesize a compound of formula II, wherein R in the formula I and the formula II is hydrogen, methoxy or halogen; the catalysis efficiency is high; the obtained target product has high optical purity, simple post-processing and good environmental-protection and safety.

Description

technical field [0001] The invention relates to the field of enzyme engineering, in particular to the application of an imine reductase and a mutant thereof in synthesizing (S)-1-aryl-1,2,3,4-tetrahydroisoquinoline. Background technique [0002] Solifenacin (Solifenacin, following formula A), trade name Vesicare, is a kind of urinary system antispasmodic drug developed by Japan Yamanouchi Pharmaceutical Company. It acts selectively on M 3 Receptors are widely used clinically to treat overactive bladder syndrome. Following its first launch in 2004, Sophinaxin was approved by China's SFDA in 2009 and launched in China. Due to its outstanding efficacy and safety, Solifenacin was welcomed by patients all over the world once it was launched, and occupied a large market. In 2014 alone, the sales in the US market reached 994 million US dollars. [0003] [0004] Structurally, solifenacin consists of two chiral fragments, namely (S)-1-phenyl-1,2,3,4-tetrahydroisoquinoline (S-1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12P17/12
CPCC12N9/0004C12P17/12
Inventor 瞿旭东朱进妹
Owner WUHAN UNIV
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