Method for synthesizing (R)-1-tert-butoxycarbonyl-3-aminopiperidine by adopting transaminase catalyst and enzymatic way

A technology of tert-butoxycarbonyl and aminopiperidine, applied in the field of enzyme catalysis, can solve the problems of inability to obtain high optical purity, cumbersome procedures, complicated operations, etc., and achieves the effects of high specificity, broad application prospects and strong catalytic activity.

Active Publication Date: 2020-01-24
ENZYMASTER NINGBO BIO ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, the existing synthetic methods involve more reagents, and the operation is complicated and the process is cumbersome, so the cost is high, and the target product with high optical purity cannot be obtained

Method used

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  • Method for synthesizing (R)-1-tert-butoxycarbonyl-3-aminopiperidine by adopting transaminase catalyst and enzymatic way
  • Method for synthesizing (R)-1-tert-butoxycarbonyl-3-aminopiperidine by adopting transaminase catalyst and enzymatic way
  • Method for synthesizing (R)-1-tert-butoxycarbonyl-3-aminopiperidine by adopting transaminase catalyst and enzymatic way

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] According to this embodiment, the recombinant Escherichia coli expressing the transaminase catalyst is cultivated:

[0049] The synthetic transaminase catalyst gene DNA fragment was double-digested with restriction endonuclease NdeI and AvrII at 37°C for 8 h, purified by agarose gel electrophoresis, and the target fragment (SEQ ID NO. 1); then, under the action of T4 DNA ligase, the target fragment was ligated with the plasmid pACYC-Duet-B that was digested by NdeI and EcoRI overnight at 25°C to obtain a recombinant expression plasmid; the recombinant expression plasmid was transformed into In Escherichia coli competent cells, the transformation conditions are: 45°C, heat shock for 90 seconds, screen the positive recombinants on a resistance plate containing chloramphenicol, pick a single clone, culture the recombinant bacteria, and wait for the plasmid After amplification, the plasmid was extracted and retransformed into competent cells. The transformation solution was...

Embodiment 2

[0052] Transaminase Catalyst Activity and Chiral Verification

[0053] The catalytic system is as follows (total volume 5mL): 0.25g (50g / L) recombinant Escherichia coli wet cells expressing transaminase catalyst, 0.1M TEOA buffer solution (pH8), 2mM pyridoxal phosphate, 100g / L N-tert-butoxy Carbonyl-3-piperidone, 0.5M isopropylamine (adjust pH to 8 with hydrochloric acid); reaction conditions: temperature 30°C, 400rpm magnetic stirring, reaction for 24 hours. After the reaction was completed, it was inactivated with acetonitrile at a ratio of 1:1, and a sample was taken to detect the formation of the product by HPLC. It was calculated that the conversion rate was 25% after 24 hours of reaction, and 12.5 g of the product was produced. After detection, the product is the R enantiomer, and the ee value is above 99.77%.

Embodiment 3

[0055] Substrate Concentration Optimization

[0056] The catalytic system is as follows (total volume: 5 mL): 0.25 g (50 g / L) of recombinant E. coli wet cells expressing transaminase catalyst, 0.1 M TEOA buffer solution (pH8), 2 mM pyridoxal phosphate, 4-100 g / L N-tert Butoxycarbonyl-3-piperidone, 0.5M isopropylamine (adjust pH to 8 with hydrochloric acid); reaction conditions: temperature 30°C, 400rpm magnetic stirring, reaction for 24 hours.

[0057] After the reaction was finished, inactivate with acetonitrile 1:1, sample HPLC to detect the generation of the product, the results are shown in Table 2 below:

[0058] Table 2 The amount of product generation corresponding to different substrate concentrations

[0059]

[0060]

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Abstract

The invention relates to the technical field of enzyme catalysis, and specifically relates to a transaminase catalyst; and the invention further relates to a method for synthesizing (R)-1-tert-butoxycarbonyl-3-aminopiperidine by adopting an enzymatic way, as well as a production method of the (R)-1-tert-butoxycarbonyl-3-aminopiperidine. The method for synthesizing the (R)-1-tert-butoxycarbonyl-3-aminopiperidine by adopting the enzymatic way comprises a step of, in the presence of pyridoxal phosphate and the transaminase catalyst, allowing reaction of N-tert-butoxycarbonyl-3-piperidinone as a reaction substrate with an amino donor so as to produce the (R)-1-tert-butoxycarbonyl-3-aminopiperidine. The method for synthesizing the (R)-1-tert-butoxycarbonyl-3-aminopiperidine by adopting the enzymatic way utilizes relatively few reagents, and is mild in reaction conditions, so that tedious steps needed for chemical process synthesis are greatly simplified; and moreover, a target product withan ee value up to 99.77% or above can be obtained without performance of separation. Therefore, the transaminase catalyst and the process method utilizing the transaminase catalyst to synthesize the (R)-1-tert-butoxycarbonyl-3-aminopiperidine provided by the invention have broad application prospects as well as great market values.

Description

technical field [0001] The present invention relates to the technical field of enzyme catalysis, in particular to a transaminase catalyst, a method for enzymatically synthesizing (R)-1-tert-butoxycarbonyl-3-aminopiperidine, and a (R)-1- A production method of tert-butoxycarbonyl-3-aminopiperidine. Background technique [0002] (R)-1-tert-butoxycarbonyl-3-aminopiperidine (also known as R-1-BOC-3-aminopiperidine) is an important intermediate in medicine and organic synthesis, used in the production of spices, pesticides, etc. has certain applications. [0003] The synthetic method of existing R-1-BOC-3-aminopiperidine is mainly chemical synthesis method, Shen Dadong et al. Ethyl ester is used as a raw material to obtain the final product R-1-BOC-3 aminopiperidine through steps such as ammonolysis and oxidation. Shuai Xiaohua et al. (Shanghai Haobo Chemical Technology Co., Ltd., a method for preparing (R)- or (S)-3-aminopiperidine dihydrochloride, CN201510589806.3[P], 2015-1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12P17/12
CPCC12N9/1096C12P17/12C12Y206/01
Inventor 胡虎洪瑞梅刘思彤余梦娇
Owner ENZYMASTER NINGBO BIO ENG CO LTD
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