Synthesis method of D-threonine

A synthesis method and threonine technology are applied in the field of synthesis of non-natural amino acid D-threonine, and can solve the problems of only 50% utilization rate of intermediates, low yield of DL-threonine, inconvenient operation and the like, Achieve the effect of being suitable for large-scale popularization and application, low fermentation cost and easy operation

Inactive Publication Date: 2013-12-18
SHANGHAI PUYI CHEM CO LTD
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0008] The disadvantage of the above method is that in order to remove the DL-allothreonine isomers, the yield of DL-threonine obtained by repeated crystallization of the threonine copper complex is low; and hydrogen sulfide is used when decoppering Or resin, the operation is inconvenient; in addition, the obtained DL-threonine has to be further split by enzymes to obtain a single D-threonine, and the utilization rate of the intermediate is only 50%.
This method is complicated to operate, the total yield is low, and the cost is high

Method used

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  • Synthesis method of D-threonine
  • Synthesis method of D-threonine
  • Synthesis method of D-threonine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Construction of racemization of amino acid racemase and expression of L-threonine deaminase

[0056] Main reagents: restriction endonuclease and Taq DNA polymerase (Fermentas), T4 DNA ligase and alkaline phosphatase CIAP (TAKARA), KOD neo plus DNA polymerase (TOYOBO), plasmid extraction kit and mini gel recovery Kit (Axygen), Kanamycin (Shanghai Sangon Bioengineering Company). Primer synthesis, total gene synthesis and subcloning were all entrusted to Nanjing GenScript Biotechnology Co., Ltd.

[0057] Construction of amino acid racemase expression strain:

[0058] According to the reported gene sequence of the amino acid racemase alr derived from Pseudomonas putida KT2440 (NCBI accession number: NC_002947), the sequence was synthesized in the whole gene, and restriction sites NdeI and BamHI were designed at both ends, and subcloned into the vector pET24a to obtain recombinant Plasmid pET24a-alr. The constructed recombinant plasmid pET24a-alr was transforme...

Embodiment 2

[0061] Embodiment 2: the preparation of D-allothreonine

[0062] Shake flask fermentation of two enzyme expression strains: preparation medium TB (12g / L peptone, 24g / L yeast extract, 5g / L glycerol, 2.13g / L KH 2 PO 4 , 16.43g / L K 2 HPO 4 ·3H 2 o). Medium TB was divided into 500mL Erlenmeyer shake flasks with a liquid volume of 100mL, and then heated and sterilized in an autoclave at 121°C for 20min. After cooling, add kanamycin at a final concentration of 100 μg / mL to the sterilized TB medium (prepared as a high-concentration mother solution and filter it to sterilize), and then inoculate the two enzyme-expressing strains into the medium respectively, Cultivate on a shaker at 37°C and 220rpm until OD600=5-6, add 0.2mM IPTG to induce, lower the temperature to 28°C, continue to cultivate for about 24h, centrifuge the fermentation broth at 8000rpm, remove the supernatant, and collect the bacteria for later use.

[0063] Preparation of two enzyme crude enzyme liquids: the cel...

Embodiment 3

[0067] 1. Preparation of D-allothreonine methyl ester (3-2)

[0068]

[0069] Dissolve 6.0 g of D-allothreonine (3-1) in 36 mL of anhydrous methanol, cool the solution to 0°C, add 11.8 g of thionyl chloride dropwise, and react at 0°C for 48 hours, monitor the reaction by TLC After completion, concentrated to obtain 8.5 g of white solid D-allothreonine methyl ester hydrochloride (3-2), with a yield of 99.5%. 1 H NMR (400MHz,MeOD)δ4.26(d,J=6.6Hz,1H),4.09(d,J=3.5Hz,1H),3.85(s,3H),1.27(d,J=6.6Hz,3H ); MS(ESI)m / z=134(M + +1).

[0070] 2. Preparation of N-benzoyl-D-allothreonine methyl ester (3-3)

[0071]

[0072] Under nitrogen protection, dissolve 7.0 g of D-allothreonine methyl ester hydrochloride (3-2) in 35 mL of anhydrous methanol, then add 11.5 g of triethylamine, cool the system to 0°C, and slowly add benzyl Acyl chloride 5.8g, keep warm for 2h after the dropwise addition, TLC monitors that the reaction is complete, evaporate the methanol under reduced pressure, a...

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Abstract

The invention relates to a synthesis method of D-threonine (I). The method comprises the following steps: preparing D-allothreonine (V) from L-threonine (VI) serving as a raw material through racemization of amino acid racemase, action of L-threonine deaminase and purification sequentially; performing esterification on D-allothreonine (V) serving as a starting material to obtain an intermediate (IV); protecting amino by using benzoyl to obtain a compound of a formula (III); performing intramolecular cyclization on the compound of the formula (III) in the presence of sulfoxide chloride and turning over the spatial configuration of hydroxyl to obtain an oxazoline intermediate (II); performing ring opening on the intermediate (II) under the action of acid and removing a protecting group to obtain D-threonine (I). The synthesis method of D-threonine (I) is smart in design, the starting materials are cheap and readily available, the process flow is simple and practical, and a new method is provided for producing D-threonine on a large scale.

Description

technical field [0001] The invention relates to the technical field of amino acid synthesis, in particular to a synthesis method of unnatural amino acid D-threonine. Background technique [0002] D-threonine (formula (I)) is an optical isomer of the natural amino acid L-threonine, and is an important unnatural amino acid. It is mainly used in the fields of chiral drugs, chiral additives, chiral auxiliary agents, etc., and is used as a chiral source for chiral synthesis in the pharmaceutical industry. As an optically active organic acid, it plays an irreplaceable role in the asymmetric synthesis of certain chiral compounds. It is mainly used for the production of new broad-spectrum antibiotics, D-threoninol, and threonine protection in the synthesis of peptides. agent. [0003] [0004] Regarding the synthesis of D-threonine, the methods that can be found at present are very limited, and are basically in the research stage. For example, Tetrahedron: Asymmetry, 2010, (21...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07C229/22C07C227/18C12P13/04C12P41/00
CPCY02P20/55
Inventor 杨汉荣李涛陶荣盛朱傳赟李晟
Owner SHANGHAI PUYI CHEM CO LTD
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