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A kind of synthetic method of pregabalin intermediate

A technology of pregabalin and synthesis method, which is applied in the directions of botanical equipment and methods, biochemical equipment and methods, enzymes, etc., can solve the problems of poor chiral selection of products, low transformation efficiency and high cost, and achieves simple operation and transformation. High rate and less pollution

Active Publication Date: 2021-11-12
ENZYMASTER NINGBO BIO ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, the pregabalin intermediates synthesized by chemical methods are generally mixed rotation products of R and S configurations, which need to be resolved through cumbersome chemical processes and a large amount of organic solvents, and only half of the configuration compounds can be used for the target Purification of the product is costly and not environmentally friendly
In addition, although the existing enzymatic method can synthesize pregabalin intermediates through biological enzymes such as relatively environmentally friendly hydrolases, it has disadvantages such as low conversion efficiency and poor chiral selection of products.

Method used

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  • A kind of synthetic method of pregabalin intermediate
  • A kind of synthetic method of pregabalin intermediate
  • A kind of synthetic method of pregabalin intermediate

Examples

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Embodiment 1

[0021] This embodiment provides a hydantoinase, the nucleotide sequence of which is shown in SEQ ID NO: 1 in the Sequence Listing, and the amino acid sequence is shown in SEQ ID NO: 2 in the Sequence Listing. Concretely, the preparation method of this hydantoin enzyme comprises the following steps:

[0022] S1. Take the existing commercially available pACYCDuet vector as the expression vector.

[0023] S2. After digesting the pACYCDuet vector, insert the nucleotide sequence of hydantoinase shown in the sequence table SEQ ID NO:1 between the NdeI and AvrII restriction sites of the digested pACYCDuet vector to obtain Recombinant expression vector.

[0024] S3, the above-mentioned recombinant expression vector is transformed into BL21 (DE3) escherichia coli, obtains bacterium colony; Then, pick single bacterium colony and inoculate in the 250mL conical flask containing the LB medium of 50mL with inoculating loop, place conical flask in Shaking incubator, 30 ℃, 250rpm cultivated...

Embodiment 2

[0026] This embodiment provides a kind of synthetic method of pregabalin intermediate, and the chemical name of this pregabalin intermediate is (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid , the synthetic route of this synthetic method is as follows:

[0027]

[0028] Specifically, the synthesis method comprises the following steps:

[0029] S1. Place 0.01 g of 3-(2-methylpropyl) glutarimide and 0.25 g of the Escherichia coli wet cells containing hydantoinase obtained in Example 1 above into 5 mL of pH 7.5 The enzymatic reaction was carried out in a reactor of 50 mM aqueous phosphate buffer solution for 48 hours, and the temperature of the buffer solution in the reactor was controlled at 30° C. to obtain a reaction solution.

[0030] S2. Add 4 times the volume of acetonitrile to the above reaction solution for vibration inactivation, then centrifuge at 13000rmp for 3 minutes, and take the supernatant to obtain the reaction product containing the pregabalin intermediat...

Embodiment 3

[0032] This embodiment provides a kind of synthetic method of pregabalin intermediate, and the chemical name of this pregabalin intermediate is (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid , the synthesis method comprises the following steps:

[0033] S1. Place 0.01 g of 3-(2-methylpropyl) glutarimide and 0.2 g of E. coli wet cells containing hydantoinase obtained in Example 1 above into 5 mL of pH 6 The enzymatic reaction was carried out in a reactor of aqueous phosphate buffer solution for 48 hours, and the temperature of the buffer solution in the reactor was controlled at 20° C. to obtain a reaction solution.

[0034] S2. Add 4 times the volume of acetonitrile to the above reaction solution for vibration inactivation, then centrifuge at 12000rmp for 3 minutes, and take the supernatant to obtain the reaction product containing the pregabalin intermediate.

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Abstract

The invention is applicable to the technical field of biochemistry and provides a synthesis method of pregabalin intermediates. The synthesis method includes the following steps: carrying out an enzymatic reaction between a substrate with the general structural formula I and hydantoinase to obtain the The pregabalin intermediate; the pregabalin intermediate is an R structure and / or an S structure; the general structural formula of the pregabalin intermediate is formula II; wherein, the nucleotide sequence of hydantoinase is as follows: Any one of the lists SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5 is shown; the amino acid sequence of hydantoinase is as shown in the sequence list SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO : any one of 6. The present invention uses hydantoinase to synthesize pregabalin intermediates, and the product has extremely high chirality selectivity, simple operation, high conversion rate, environmental protection, less pollution, and is suitable for industrial production.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, in particular to a method for synthesizing a pregabalin intermediate. Background technique [0002] Pregabalin intermediate is an important raw material for the synthesis of Pregabalin. [0003] At present, there are chemical methods and enzymatic methods for the synthesis of pregabalin intermediates. Among them, the pregabalin intermediates synthesized by chemical methods are generally mixed rotation products of R and S configurations, which need to be resolved through cumbersome chemical processes and a large amount of organic solvents, and only half of the configuration compounds can be used for the target Purification of the product is costly and not environmentally friendly. In addition, although the existing enzymatic method can synthesize pregabalin intermediates through relatively environmentally friendly biological enzymes such as hydrolase, it has disadvantages such as low conver...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P13/02C12N9/86C12N15/55
CPCC12N9/86C12P13/02C12Y305/02002
Inventor 张迎新马克·博科拉蔡宝琴罗霄陈军陈海滨孙保国
Owner ENZYMASTER NINGBO BIO ENG CO LTD
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