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A method for preparing 3-phenyl-l-serine or derivatives thereof and ethyl ester thereof

A technology for serine and derivatives, which is applied in the field of preparation of chiral 3-phenyl-L-serine or its derivatives and ethyl esters thereof, can solve the problems of poor stereoselectivity, low conversion rate, difficulty in product separation and purification, etc.

Active Publication Date: 2022-06-17
SUZHOU LEAD BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, this method still has defects such as low conversion rate, poor stereoselectivity, and difficult separation and purification of products, and does not have the conditions for industrialization

Method used

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  • A method for preparing 3-phenyl-l-serine or derivatives thereof and ethyl ester thereof
  • A method for preparing 3-phenyl-l-serine or derivatives thereof and ethyl ester thereof
  • A method for preparing 3-phenyl-l-serine or derivatives thereof and ethyl ester thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Example 1: Gene cloning and expression vector construction

[0112] Four wild-type L-threonine transaldolases, namely: YH1058 (Suzhou Pilot Biotechnology Co., Ltd., amino acid sequence as shown in SEQ ID NO.12), and LTTA sp01 (amino acid sequence as shown in SEQ ID NO.13) The gene sequences of LTTA sp02 (amino acid sequence shown in SEQ ID NO.14) and LTTA sp03 (amino acid sequence shown in SEQ ID NO.15) can be obtained in the NCBI database, and can be obtained through gene synthesis and molecular cloning. The pET30a expression plasmid containing the threonine transaldolase gene was constructed. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) cells to obtain recombinant bacteria.

Embodiment 2

[0113] Example 2: Expression of recombinant L-threonine transaldolase

[0114] The recombinant bacteria in Example 1 were inoculated into LB medium (10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, pH 7.0) containing 50 μg / mL kanamycin, and cultured at 37°C overnight. The overnight culture was transferred to TB medium (peptone 12g / L, yeast extract 24g / L, glycerol 4mL / L, potassium dihydrogen phosphate 2.31g / L, dipotassium hydrogen phosphate 12.54g / L), and cultured at 37°C until OD 600 To 0.6-0.8, IPTG was added at a final concentration of 0.4 mM, and expression was induced at 30°C overnight. After the cells were collected by centrifugation, they were resuspended in 20 mM phosphate buffer, pH 7.0, 4 times the wet weight of the cells. The cells were disrupted by sonication, centrifuged, and the supernatant was taken for reaction or frozen at -20°C.

Embodiment 3

[0115] Example 3: Testing of wild-type L-threonine transaldolase activity at low substrate concentrations - no acetaldehyde removal system

[0116] The final concentration of 5g / L p-methylsulfonylbenzaldehyde, 6.4g / L L-threonine, 1g / L magnesium chloride, 0.005g / L pyridoxal phosphate, 100mM phosphate buffer (pH7. 5) Heating to 30°C with magnetic stirring, adding 20 μl of L-threonine transaldolase (YH1058 (Suzhou Pilot Biotechnology Co., Ltd.) obtained in Example 2 at a concentration of 30 g / L, as well as LTTA sp01, LTTA sp02 and LTTA sp03) enzyme solution, start stirring reaction, 16 hours later sampling HPLC detection, the detection results are shown in Table 5.

[0117] Table 5 Wild-type L-threonine transaldolase activity detection results

[0118] Numbering serial number Conversion rate(%) Enantiomeric excess (ee%) a

Diastereomeric Ratio (DR) b

YH1058 SEQ ID NO. 12 93.6 98.6 94.7:5.3 LTTA sp01 SEQ ID NO. 13 91.5 98.5 94.2:5.8 ...

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Abstract

The present invention relates to the method for preparing 3-phenyl-L-serine or its derivatives, which comprises the following steps: a. the step of obtaining 3-phenyl-L-serine or its derivatives according to the general reaction formula shown in formula I , wherein, R is selected from: hydrogen, alkyl, alkoxy, alkylsulfonyl, alkylsulfinyl, alkylthio, sulfonic acid, sulfinic acid, mercapto, nitro and halogen; n It is 0, 1, 2 or 3; the transaldolase is L-threonine transaldolase; wherein, acetaldehyde reductase is added in the step a.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals and biochemical industries, and in particular relates to a preparation method of chiral 3-phenyl-L-serine or a derivative thereof and its ethyl ester. Background technique [0002] 3-Phenyl-L-serine and its derivatives have the following general structural formula: [0003] [0004] 3-Phenyl-L-serine and its derivatives are important intermediates in organic synthesis, which are widely used in drug synthesis. For example: (2S,3R)-p-methylsulfonyl phenylserine is a key intermediate in the synthesis of veterinary drugs florfenicol and thiamphenicol, and (2S,3R)-p-nitrophenylserine can be used as the antibacterial drug chloramphenicol. key intermediates. In addition, 3-phenyl-L-serine and its derivatives can also be prepared by simple transformation to chiral aziridines (David Tanner, Chiral Aziridines-Their Synthesis and Use in Stereoselective Transformations, Angew. Chem., Int. Ed. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07C317/48C07C315/04C12P13/04
CPCC07C315/04C12P13/04C07B2200/07C07C317/48
Inventor 谢新开黄晓飞徐伟
Owner SUZHOU LEAD BIOTECH CO LTD
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