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Synthesis method of (2S,3R)-2-substituted aminomethyl-3-hydroxybutyrate under catalysis of enzyme

A technology of hydroxybutyrate and aminomethyl, which is applied in the field of biopharmaceuticals, can solve the problems of limited industrial application, low substrate concentration, and high production cost, and achieves the avoidance of expensive metal catalysts, excellent diastereoselectivity, and Excellent enantioselectivity effect

Active Publication Date: 2021-10-22
FUDAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Because transition metal catalysts are expensive, and the reaction needs to be carried out under high temperature and high pressure conditions, heavy metal residues are difficult to remove, resulting in high production costs and limited industrial applications
In recent years, the use of biological enzymes to catalyze asymmetric reduction has received extensive attention. However, the configuration of the product generated by the conversion is inconsistent (US Patent US4927507), the enantioselectivity is not good, and the product needs further configuration reversal (Chinese Patent CN1940079B, CN1940080B ) or the substrate concentration is low, the industrial application is limited ( Chem. Commun. 2015 ,51, 12328-12331) and so on

Method used

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  • Synthesis method of (2S,3R)-2-substituted aminomethyl-3-hydroxybutyrate under catalysis of enzyme
  • Synthesis method of (2S,3R)-2-substituted aminomethyl-3-hydroxybutyrate under catalysis of enzyme
  • Synthesis method of (2S,3R)-2-substituted aminomethyl-3-hydroxybutyrate under catalysis of enzyme

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1, construction, induced expression, and protein purification of carbonyl reductase genetically engineered bacteria

[0036] Expression host BL21(DE3) was transformed with plasmid pET28a-SsCR. Inoculate BL21(DE3)-pET28a-SsCR into a 5mL liquid LB test tube culture medium containing kanamycin resistance, place it on a shaking table at 37°C for 8 hours, and transfer the activated culture at 1% transfer volume Transferred to the liquid LB shake flask culture medium containing kanamycin resistance, cultured in the fermentation medium with constant temperature shaking for 3 hours, the culture temperature was 37° C., 200 rpm. When the cell concentration grows to OD 600 When = 0.8, add 0.1mM IPTG (final concentration), induce at 18°C ​​for 18h, collect cells by centrifugation at 8500rpm for 10min, resuspend with pH7.0, 100mM potassium phosphate buffer, break with 55% ultrasonic power for 15min, and centrifuge to obtain Cell supernatant containing SsCR.

Embodiment 2

[0037] Embodiment 2, (2 S, 3 R )-2-Benzamidomethyl-3-hydroxybutyrate (I a ) preparation (100 g / L feed).

[0038]

[0039] Add 2-benzamidomethyl-3-oxobutyrate (II a ) 1.5 g (400 mM), add 1.5 mL of toluene (10% v / v), 2.378 g auxiliary substrate glucose (2:1 equivalent ratio to substrate, 158.5 g / L), add NADP + solution to make its final concentration 0.2230g / L (0.3mM), add 3 mL of GDH cell supernatant (20% v / v), and take 6 mL of SsCR cell supernatant in Example 1 (20% v / v ), adding pH=7.0, 100 mM sodium phosphate buffer to make a final reaction volume of 15 mL, saturated with Na 2 CO 3 The pH value of the solution was adjusted between 6.9-7.1, and then placed in a constant temperature water bath at 30° C. and stirred at 600 rpm for 33 hours. After the reaction was finished, it was extracted 3 times with ethyl acetate,

[0040] The organic phases were combined, washed with saturated sodium chloride, and dried over anhydrous sodium sulfate. After concentration, the crude ...

Embodiment 3

[0042] Embodiment 3, (2 S, 3 R )-2-Benzamidomethyl-3-hydroxybutyrate (I a ) preparation (300g / L feeding).

[0043] Add 2-benzamidomethyl-3-oxobutyrate (II a ) 30 g (1200 mM), add 20 mL of toluene (10% v / v), 59.45 g of auxiliary substrate glucose (2.5:1 with substrate equivalent ratio, 3000 mM, 595 g / L), NADP + The final concentration is 0.3717 g / L (0.5mM), add 20 mL of GDH cell supernatant (20% v / v), take 60 mL of SsCR cell supernatant in Example 1 (20% v / v), add pH = 7.0, 100 mM sodium phosphate buffer to make a final reaction volume of 100 mL, saturated with Na 2 CO 3 The pH value of the solution was adjusted between 6.9-7.1, and then placed in a constant temperature water bath at 30° C. and stirred at 600 rpm for 35 hours. After the reaction was completed, it was extracted three times with ethyl acetate, and the organic phases were combined, washed with saturated sodium chloride solution, and dried over anhydrous sodium sulfate. After concentration, the crude product...

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Abstract

The invention discloses a synthesis method of (2S,3R)-2-substituted amino methyl-3-hydroxybutyrate under the catalysis of an enzyme. The method comprises the following steps: preparing engineering bacteria of carbonyl reductase; preparing a resting cell suspension of the engineering bacteria and an engineering bacteria culture containing carbonyl reductase; and then conducting mixing with a substrate 2-substituted aminomethyl-3-ketobutyrate, glucose dehydrogenase, a cosolvent, glucose and a cofactor, and carrying out asymmetric carbonyl reduction reaction to obtain (2S,3R)-2-substituted aminomethyl-3-hydroxybutyrate. The amino acid sequence of the carbonyl reductase is as shown in SEQ ID NO. 1. The biocatalyst used in the invention is convenient and easy to obtain, and expensive metal catalysts are avoided; reaction conditions are simple, complex regulation and control are not needed, enantioselectivity is excellent, diastereoselectivity is excellent, and complete conversion of a substrate up to 300 g / L can be achieved; and a new way is provided for preparing chiral drug intermediates.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, in particular to enzyme-catalyzed (2 S, 3 R )-2-substituted aminomethyl-3-hydroxybutyrate synthetic method. Background technique [0002] Carbapenem antibiotics (i.e., penem antibiotics) are a class of broad-spectrum antibiotics that are widely used, mainly including imipenem, meropenem, panipenem, etc., and the global market demand is huge. 4-AA is the abbreviation of 4-acetoxyazetidinone, which is the main raw material for the production of all carbapenem antibiotic APIs. Among them, 2-substituted aminomethyl-3-ketobutyrate (II) The asymmetry is reduced to (2 S, 3 R )-2-substituted aminomethyl-3-hydroxybutyrate (I) is a key step in the synthesis of 4-AA, and its structure is as follows: [0003] [0004] Currently (2 S, 3 R )-2-substituted aminomethyl-3-hydroxybutyrate (I) can be prepared by transition metal catalyst catalytic reduction and enzyme catalytic reduction. Beca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/00C12N1/21C12N15/53C12N9/04C12N15/70C12R1/19
CPCC12P13/001C12N9/0006C12N15/70C12Y101/01184C12P41/002C12Y101/01047
Inventor 陈芬儿陶媛黄则度程荡孟歌
Owner FUDAN UNIV
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