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Baeyer-Villiger monooxygenase and application thereof in synthesis of briracetam

A monooxygenase, briracetam technology, applied in the application, oxidoreductase, enzyme and other directions, can solve the problems of explosion risk, difficult to meet medicinal requirements, harsh reaction conditions, etc.

Pending Publication Date: 2022-05-13
成都栩哲医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The above synthetic method has the following disadvantages: (1) the steps are cumbersome; using oxazolidinone as the chiral inducing group, its price is higher, and it will go through assembly and removal steps, and the economic cost is higher; (2) the second step Reaction conditions at -70°C are required, and the reaction conditions are relatively harsh, making it difficult to achieve large-scale production; (3) the third step uses hydrogen peroxide, which has a risk of explosion as a peroxide; (4) the fourth step uses dimethyl sulfide borane, There is a risk of explosion and a pungent smell, which poses safety and environmental risks
[0011] The above-mentioned synthetic method has the following disadvantages: (1) the steps are cumbersome, and the linear steps are as long as 9 steps before the final product brivaracetam can be obtained; (2) the key intermediate is prepared by enzymatic catalysis in a key step, and the yield is only 42%. The enantiomeric excess percentage is only 97.7%. (3) The final product Brivaracetam obtained from this isomer has an enantiomeric excess percentage of only 95.9%, which is difficult to meet the pharmaceutical requirements

Method used

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  • Baeyer-Villiger monooxygenase and application thereof in synthesis of briracetam
  • Baeyer-Villiger monooxygenase and application thereof in synthesis of briracetam
  • Baeyer-Villiger monooxygenase and application thereof in synthesis of briracetam

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1: the preparation of 3-propyl cyclobutanone (compound 1)

[0063] The process route is as follows:

[0064]

[0065] Dissolve trichloroacetyl chloride (2.24mL, 20mmol) and phosphorus oxychloride (1.02mL, 11.0mmol) in ether (10mL), then slowly drop this solution into a solution containing 1-pentene (1.09mL, 10mmol) , diethyl ether (20mL) and zinc copper reagent (Zinc-Copper couple, CAS#: 53801-63-1, 1.96g, 30.0mmol) in a flask. Heated to 40°C and stirred for 2 hours, then naturally cooled to room temperature and stirred for 8 hours. Afterwards, the solution was filtered with diatomaceous earth, and 80 mL of n-hexane was added to the filtrate to precipitate zinc chloride salt. The clear solution was obtained by filtration, washed successively with water, saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and evaporated to remove the solvent. The light yellow oily substance ...

Embodiment 2

[0067] Embodiment 2: the preparation of Baeyer-Villiger monooxygenase

[0068] 1.1 Acquisition of enzyme gene

[0069] Select CHMO from Acinetobacter sp. (CHMO Acineto ), for rational enzyme design and site-directed mutagenesis. According to the above monooxygenase sequence retrieved by NCBI, the wild-type CHMO from Acinetobacter sp. monooxygenase gene (CHMO Acineto Gene).

[0070] 2.2 Construction and transformation of enzyme gene

[0071] CHMO synthesized in step 1.1 Acineto The gene was inserted between the restriction enzyme sites Nde I and BamH I in the multiple cloning site region of the pEt-22b (+) plasmid to obtain wild-type CHMO Acineto gene plasmid. Primers were designed for each mutation site of the protein according to the amino acid sequence shown in sequence 1-5, and wild-type CHMO Acineto The plasmid of the gene is used as a template, and polymerase chain reaction (PCR) is used to perform site-directed mutation to obtain the plasmids of genes encoding Bae...

Embodiment 3

[0076] Example 3: Preparation of (R)-4-propyl-dihydrofuran-2-one (compound 2)

[0077] The process route is as follows:

[0078]

[0079] Add 100 mL of the whole cell buffer solution obtained in Example 2 and 100 mmol of cyclic ketone substrate into a 500 mL Erlenmeyer flask, react for 8 hours, break the cells with an ultrasonic cell disruptor, centrifuge, extract with ethyl acetate (100 mL*3), and combine In the organic phase, the extract was rotary evaporated to remove the solvent, and ethyl acetate containing 0.2 mg / mL dodecane was dissolved again. The extract was detected by gas chromatography for product yield and enantiomeric excess value. The results are shown in Table 1.

[0080] Table 1

[0081] Enzyme type Conversion rates(%) Product ee value (%) L143A / L244A (SEQ ID No: 1) 98.9 99.7 L143V / F277V (SEQ ID No: 2) 99.2 99.8 L244A / F432I (SEQ ID No: 3) 99.1 99.9 L143V / L244A / F432I (SEQ ID No: 4) 99.5 99.9 L244G / F277V / F432L (S...

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Abstract

The invention relates to a Baeyer-Villiger monooxygenase and an application of the Baeyer-Villiger monooxygenase in synthesis of briracetam. The Baeyer-Villiger monooxygenase is preferably selected from any one of polypeptides with an amino acid sequence as shown in SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4 or SEQ ID No: 5. Under the catalysis of the Baeyer-Villiger monooxygenase disclosed by the invention, (R)-4-propyl-dihydrofuran-2-ketone can be synthesized economically, efficiently and environmentally friendly, and on the basis, a Briracetam bulk drug with high optical purity can be prepared through a one-step reaction. The route for preparing the briracetam provided by the invention avoids chiral resolution and tedious separation and purification used in the traditional process, is suitable for industrial production, and has a remarkable cost advantage.

Description

technical field [0001] The invention belongs to the technical field of raw material drug synthesis, and in particular relates to a Baeyer-Villiger monooxygenase and its application in the synthesis of brivaracetam. Background technique [0002] Brivaracetam is a third-generation antiepileptic drug, a new type of high-affinity ligand for synaptic vesicle protein 2A (SV2A), and has a certain inhibitory effect on voltage-dependent sodium ion channels. In 2016, Briracetam was approved by the FDA for the treatment of epileptic seizures, and it has a good effect on generalized epileptic seizures. [0003] Patent CN1882535A discloses a chemical synthesis preparation method of brivaracetam, the synthesis route is as follows: [0004] [0005] In the above method, after the brivaracetam racemate is synthesized, the optical isomers are purified and separated by using a chromatographic column, so as to obtain optically pure brivaracetam. However, the above synthesis method needs t...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/70C12P17/10
CPCC12N9/0069C12N15/70C12P17/10C12N2800/101
Inventor 金帅江王科杨立开杨仁明王梦馨汪建刚罗晓勇
Owner 成都栩哲医药科技有限公司
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