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Preparation method of sitagliptin intermediate

A sitagliptin and intermediate technology, applied in the field of enzyme engineering and biopharmaceuticals, can solve the problems of high difficulty in product separation and purification, low product yield, high production cost, etc., simplify the product separation and purification process, and increase product yield and organic solvent recovery, the effect of reducing production costs

Active Publication Date: 2021-10-08
台州酶易生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the deficiencies of the prior art, the application provides a preparation method of a sitagliptin intermediate, to improve the existing biocatalytic synthesis process for preparing the sitagliptin intermediate, which is difficult to separate and purify the product, and the product Problems such as low yield and high production cost

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  • Preparation method of sitagliptin intermediate
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  • Preparation method of sitagliptin intermediate

Examples

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preparation example Construction

[0042] The embodiment of the present application provides a preparation method of a sitagliptin intermediate, and a sitagliptin intermediate prepared by the preparation method, the preparation method comprising the following steps:

[0043] S a , Sitagliptin precursor ketone (2Z)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] shown in formula (I) ]Triazolo[4,3-a]pyrazin-7-(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-one as substrate, with in a standard Organic solvents having a boiling point of not higher than 110°C under atmospheric pressure are mixed to obtain a mixed solution; and

[0044] S b 1. Conduct transamination contact between the mixed solution and transaminase to generate an enzyme-catalyzed reaction to generate the sitagliptin intermediate represented by formula (II).

[0045] In some embodiments, step S b The method of transaminating the mixed solution with the transaminase is as follows: all the mixed solution is directly mixed with the transaminase. As an ...

Embodiment 1

[0079] Embodiment 1: provide the transaminase used for transaminase catalyzed reaction

[0080] The embodiment of the present application provides a total of five transaminases (corresponding to numbers 1 to 5), wherein the transaminases numbered 1 to 4 are obtained by one or more point mutations of the transaminase having the amino acid sequence shown in SEQ ID NO: 1 , wherein, the nucleotide sequence of the gene encoding the transaminase having the amino acid sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 2, and the specific information of the transaminases numbered 1 to 5 is shown in Table 1 below:

[0081] Table 1 Mutation mode and sequence information of transaminases No. 1 to No. 5

[0082]

[0083] 1.1. Construction of genetically engineered bacteria containing transaminase coding genes

[0084] Construct the genetically engineered bacteria containing the transaminase coding gene of No. 1 to No. 5 respectively, wherein, the steps of constructing the genetical...

Embodiment 2

[0104] Embodiment 2: select the used organic solvent of transaminase catalyzed reaction

[0105] The transaminase enzyme solutions No. 1 to No. 5 prepared in Example 1 were subjected to an organic solvent screening experiment.

[0106] 2.1. Organic solvent single agent experiment

[0107] The method flow of organic solvent single agent experiment is as follows:

[0108] S2.1.1. Take 100 μL of the single transaminase solution prepared in Example 1 (transaminase solution No. 1, transaminase solution No. 2, transaminase solution No. 3, transaminase solution No. 4, or transaminase solution No. 5) Transaminase enzyme liquid) is placed on the reaction plate, then in the transaminase enzyme liquid, add 1.5mL pH to be the stock mixture of 8.5, wherein, the stock mixture comprises the triethanolamine of 0.2mol / L, the pyridoxal phosphate (PLP) of 2mmol / L , 2mol / L isopropylamine (IPM) and sterile water to obtain a premixed system;

[0109] S2.1.2. Add 400 μL of the mixed solution to t...

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Abstract

The invention discloses a preparation method of a sitagliptin intermediate and the sitagliptin intermediate prepared by the preparation method, the preparation method comprises the step that in the presence of an organic solvent with a boiling point not higher than 110 DEG C at a standard atmospheric pressure, carrying out transamino contact between transaminase and a substrate sitagliptin precursor ketone (2Z)-4-oxo-4-[3-(trifluoromethyl)-5, 6-dihydro-[1, 2, 4]triazolo[4, 3-a]pyrazine-7-(8H)-yl]-1-(2, 4, 5-trifluorophenyl) butyl-2-one to perform enzyme catalysis reaction so as to prepare the sitagliptin intermediate (3R)-3-amino-1-[3-(trifluoromethyl)-5, 6, 7, 8-tetrahydro-1, 2, 4-triazolo [4, 3-a] pyrazine-7-yl]-4-(2, 4, 5-trifluorophenyl) butyl-1-one, a mixed solution obtained by mixing a low-boiling-point organic solvent and the substrate is added into a premixing system containing the transaminase in a fed-batch mode, ketoreductase and a coenzyme regeneration system in a specific proportion are added into the premixing system, the substrate conversion rate is increased, and the substrate conversion rate can reach 99%.

Description

technical field [0001] The application relates to the fields of enzyme engineering and biopharmaceuticals, in particular to a preparation method of a sitagliptin intermediate. Background technique [0002] Sitagliptin phosphate is a drug for the treatment of type II diabetes. Its trade name is Januvia, and its active ingredient is (3R)-3-amino-1-[3-(trifluoromethyl)- 5,6-Dihydro-1,2,4-triazolo[4,3-a]pyrazin-7(8H)-yl]-4-(2,4,5-trifluorophenyl)butane- 1-keto (Sitagliptin, Sitagliptin). Sitagliptin is a dipeptidyl peptidase-4 (DPP-4) inhibitor, which mainly controls blood sugar levels by protecting endogenous incretins and enhancing their effects, and has good safety and tolerance sex. The key step in the preparation process of sitagliptin is the synthesis of chiral amino intermediates. [0003] In the prior art, the synthetic method of the chiral amino intermediate of sitagliptin mainly contains chemical synthesis process and biocatalytic synthesis process, wherein, althou...

Claims

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Application Information

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IPC IPC(8): C12P17/18
CPCC12P17/182Y02P20/584
Inventor 周硕赖敦岳叶涛劳淑华
Owner 台州酶易生物技术有限公司
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