Method for preparing 3-phenyl-L-serine or derivative thereof and ethyl ester of 3-phenyl-L-serine

A technology for serine and its derivatives, which is applied in the field of preparation of chiral 3-phenyl-L-serine or its derivatives and its ethyl ester, which can solve problems such as low conversion rate, lack of industrialization conditions, poor stereoselectivity, etc. question

Active Publication Date: 2020-07-07
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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  • Method for preparing 3-phenyl-L-serine or derivative thereof and ethyl ester of 3-phenyl-L-serine
  • Method for preparing 3-phenyl-L-serine or derivative thereof and ethyl ester of 3-phenyl-L-serine
  • Method for preparing 3-phenyl-L-serine or derivative thereof and ethyl ester of 3-phenyl-L-serine

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 Pivot Biotechnology Co., Ltd., amino acid sequence shown in SEQ ID NO.12), and LTTA sp01 (amino acid sequence shown in SEQ ID NO.13 shown), LTTA sp02 (amino acid sequence as shown in SEQ ID NO.14) and LTTA sp03 (amino acid sequence as shown in SEQ ID NO.15) gene sequences can be obtained in the NCBI database, by means of gene synthesis and molecular cloning Construct pET30a expression plasmid containing threonine transaldolase gene. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) cells to obtain recombinant bacteria.

Embodiment 2

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

[0114] Inoculate the recombinant bacteria in Example 1 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 culture overnight at 37°C. 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, add IPTG with a final concentration of 0.4mM, and induce expression overnight at 30°C. Cells were collected by centrifugation and resuspended in 20mM pH 7.0 phosphate buffer 4 times the wet weight of the cells, sonicated to break the cells, centrifuged, and the supernatant was taken for reaction or frozen at -20°C.

Embodiment 3

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

[0116] In the 1mL reaction system, add the final concentration of 5g / L p-thiamphenicol benzaldehyde, 6.4g / L L-threonine, 1g / L magnesium chloride, 0.005g / L pyridoxal phosphate, 100mM phosphate buffer (pH7. 5) Heat to 30°C and stir evenly with magnetic force, add 20 μl of the L-threonine transaldolase obtained in Example 2 (YH1058 (Suzhou Pivot Biotechnology Co., Ltd.), LTTA sp01, LTTA sp02 and LTTA sp03) enzyme solution, start stirring reaction, after 16 hours, sampling HPLC detects, and test result is 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 LTTA sp0...

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Abstract

The invention relates to a method for preparing 3-phenyl-L-serine or a derivative thereof. The preparation method comprises the following steps of: a, obtaining 3-phenyl-L-serine or a derivative thereof according to a reaction general formula as shown in a formula I, in which R is selected from a hydrogen group, an alkyl group, an alkoxy group, an alkyl sulfonyl group, an alkyl sulfinyl group, analkyl sulfenyl group, a sulfonic group, a sulfinyl group, a sulfydryl group, a nitro group and halogen; n is 0, 1, 2 or 3; wherein the transaldolase is L-threonine transaldolase,; and acetaldehyde reductase is added in the step a.

Description

technical field [0001] The invention belongs to the technical field of biological pharmacy and biochemical industry, and in particular relates to a preparation method of chiral 3-phenyl-L-serine or its derivatives and its ethyl ester. Background technique [0002] 3-phenyl-L-serine and its derivatives, the general structure of which is as follows: [0003] [0004] 3-Phenyl-L-serine and its derivatives are very important intermediates in organic synthesis, which are widely used in drug synthesis. For example: (2S,3R)-p-thymphenylphenylserine is a key intermediate for the synthesis of veterinary drugs florfenicol and thiamphenicol, and (2S,3R)-p-nitrophenylserine can be used as a compound for the antibacterial drug chloramphenicol key intermediates. In addition, 3-phenyl-L-serine and derivatives thereof can also prepare chiral aziridines (David Tanner, ChiralAziridines-Their Synthesis and Use in Stereoselective Transformations, Angew.Chem., Int.Ed. Engl., 33,599 (1994))...

Claims

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Application Information

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Patent Type & Authority Applications(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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