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Transaminase catalyst and method for enzymatic synthesis of (R)-1-naphthylethylamine

A catalyst and transaminase technology, applied in biochemical equipment and methods, botany equipment and methods, microbial-based methods, etc., can solve the problems of low chiral resolution yield, high industrial production costs, and potential safety hazards , to achieve the effect of avoiding chemical resolution steps, easy to obtain, and stable at the end of the reaction

Active Publication Date: 2020-07-14
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, ammonia water has a strong odor and has environmental pollution problems; while high-temperature and high-pressure hydrogenation requires high equipment requirements and has potential safety hazards; the yield of chiral resolution is not high, resulting in high industrial production costs

Method used

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  • Transaminase catalyst and method for enzymatic synthesis of (R)-1-naphthylethylamine
  • Transaminase catalyst and method for enzymatic synthesis of (R)-1-naphthylethylamine
  • Transaminase catalyst and method for enzymatic synthesis of (R)-1-naphthylethylamine

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Experimental program
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Effect test

Embodiment 1

[0062] This embodiment aims to compare the impact of different solvents on the reaction conversion rate, and the specific experimental conditions include:

[0063] The total volume of the reaction system: 5ml;

[0064] 1-naphthylethanone (substrate) concentration: 100g / L;

[0065] Recombinant Escherichia coli wet cell concentration: 100g / L;

[0066] Isopropylamine concentration: 2M;

[0067] PLP concentration: 2mM;

[0068] The solvents are: ethyl acetate, isopropyl acetate, butyl acetate, isopropanol, n-butanol, methyl tert-butyl ether, toluene;

[0069] Reaction temperature: 30°C;

[0070] Reaction time: 1h, 3h, 4h, 5h, 21h, 24h;

[0071] The specific reaction results are as follows figure 1 As shown, it can be seen that when isopropanol (IPA) is used as the reaction solvent, the enzyme activity is the best.

Embodiment 2

[0073] This embodiment is intended to implement the substrate gradient experiment, and the specific experimental conditions include:

[0074] The total volume of the reaction system: 5ml;

[0075] 1-naphthylethanone (substrate) concentration: 300-916g / L (full substrate);

[0076] Recombinant Escherichia coli wet cell concentration: 100g / L;

[0077] Isopropylamine concentration: 2M;

[0078] PLP concentration: 2mM;

[0079] Isopropanol concentration: 37%;

[0080] Reaction temperature: 30°C;

[0081] Reaction time: 1h, 2h, 4h, 24.5h;

[0082] The specific reaction results are as follows figure 2 with 3 As shown, when the visible substrate concentration is 500g / L, the 24.5h substrate accumulation is the largest.

Embodiment 3

[0084] 1) In a three-necked flask, add 90 g of recombinant Escherichia coli wet cells expressing the transaminase catalyst, 450 g of 1-naphthyloethanone, place in a water bath at 30° C., and stir mechanically;

[0085] 2) Add 270ml isopropanol, 144ml isopropylamine and 9ml PLP aqueous solution while stirring;

[0086] 3) stirring and reacting at a speed of 200r / min for 48 hours;

[0087] 4) After the reaction, distill under reduced pressure at 55°C to remove isopropanol and isopropylamine;

[0088] 5) Filtrate, add 250ml of 3M hydrochloric acid aqueous solution to the filtrate, cool down and crystallize to obtain 140g of solid naphthylethylamine hydrochloride, then add sodium hydroxide to dissociate, extract with 100ml of dichloromethane, leave to separate layers, take the organic phase, and distill off the solvent to obtain The target product (R)-1-naphthylethylamine was 108g, and the ee value of the target product was >99% after detection.

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Abstract

The invention relates to the technical field of medical intermediates, specifically to a transaminase catalyst and a method for enzymatic synthesis of (R)-1-naphthylethylamine. The method comprises the following steps: adding recombinant escherichia coli wet thalli expressing the transaminase catalyst and 1-acetophenone into a reaction container, carrying out heating in a water bath, and carryingout mechanical stirring; adding an organic solvent or an aqueous solution of water, an amino donor and pyridoxal phosphate under stirring; and after a reaction is finished, carrying out post-treatmentso as to obtain the (R)-1-naphthylethylamine. The transaminase catalyst disclosed by the invention is easy to obtain and high in chiral selectivity; meanwhile, the method for enzymatic synthesis of the (R)-1-naphthylethylamine is simple in operation, mild in reaction conditions and stable in reaction completion, and can directly generate the (R)-1-naphthylethylamine with an optical purity of 99%or above, so tedious chemical resolution steps and a tedious chemical reaction route are avoided.

Description

technical field [0001] The invention relates to the technical field of pharmaceutical intermediates, in particular to a transaminase catalyst, and also to a method for enzymatically synthesizing (R)-1-naphthylethylamine. Background technique [0002] Optically pure (R)-1-naphthylethylamine is an important pharmaceutical intermediate, and its preparation method usually includes: taking naphthalene ethyl ketone as raw material, adding ammonia water to generate imine, and then hydrogenating under high temperature and high pressure conditions to obtain the eliminated The naphthylethylamine of spin, further implement chemical chiral resolution: take chiral aspartic acid or tartaric acid as chiral resolving agent, under the condition of adding solvent and heating, enantiomeric salt is formed; The difference is resolved, separated and prepared two configurations of R configuration and S configuration of naphthylethylamine. It can be seen that the resolution process of naphthalenee...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12P41/00C12P13/00C12R1/19
CPCC12N9/1096C12P13/001C12P41/006C12Y206/01
Inventor 李一军王子坤黄勇开王吉勇马克·博科拉蔡宝琴陈海滨胡虎余梦娇刘思彤
Owner ENZYMASTER NINGBO BIO ENG CO LTD
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