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Preparation method of semaglutide

A semaglutide and preparation technology, applied in the field of semaglutide preparation, can solve the problems of reducing product yield, increasing production cost, resin polycondensation, etc.

Pending Publication Date: 2021-12-17
NINGBO KUNPENG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is easy to cause polycondensation of the resin during the synthesis process, which greatly reduces the coupling efficiency, and is easy to produce racemic impurities, especially the racemization of the last amino acid His, which greatly reduces the yield of the product and increases the production cost.

Method used

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  • Preparation method of semaglutide
  • Preparation method of semaglutide
  • Preparation method of semaglutide

Examples

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preparation example Construction

[0161] The preparation of semaglutide is to use genetic recombination technology to obtain the semaglutide precursor whose 17th or 18th position is Boc-protected lysine, and then connect the semaglutide side chain tBuO-Ste-Glu (AEEA-AEEA- OSu)-OtBu, thereby obtaining semaglutide.

[0162] Preparation of semaglutide

[0163] The present invention provides two synthetic routes of semaglutide, which are respectively as follows, the Fmoc complex modified compound 2 is prepared from the Boc-semaglutide precursor (compound 1), and the compound 2 is obtained after de-Boc protection 3. Compound 3 was reacted with activated semaglutide side chain tBuO-Ste-Glu(AEEA-AEEA-OSu)-OtBu to obtain compound 4, and then compound 5 was obtained by removing the Fmoc reaction, and the side chain removed the tBu protecting group , and finally semaglutide compound 6 was obtained.

[0164]

[0165]

[0166] Specifically, the present invention provides a method for preparing semaglutide, the met...

Embodiment 1

[0178] Embodiment 1 Construction of semaglutide expression strain

[0179] The construction of the semaglutide expression plasmid refers to the description in the example in the patent application number 201910210102.9. The DNA fragment of fusion protein FP1-TEV-EK-GLP-1 (18) or FP2-TEV-EK-GLP-1 (17) was cloned into the expression vector plasmid pBAD / His A (purchased from NTCC Company, Kanamyces The downstream NcoI-XhoI site of the araBAD promoter of protein resistance) was used to obtain the plasmid pBAD-FP1-TEV-EK-GLP-1 (18) or pBAD-FP2-TEV-EK-GLP-1 (17). Plasmid map such as figure 1 , as shown in 2.

[0180] Fusion protein 1 and fusion protein 2 were constructed based on the semaglutide precursor with 2-5 amino acids missing from the N-terminus shown in SEQ ID NO:1 or SEQ ID NO:2

[0181] The amino acid sequence of fusion protein 1 is shown in SEQ ID NO:4:

[0182] MVSKGEELFTGV KLTLKFICTTYVQERTISFKDTYKTRAEVKFEGD ENLYFQGDDDDKEGTFTSDVSSYLEGQAA K EFIAWLVRGRG

[0183]Th...

Embodiment 2B

[0197] Embodiment 2 Expression of Boc-semaglutide precursor

[0198] Two kinds of recombinant Escherichia coli seed solutions were respectively inoculated into the fermentation medium according to the inoculum amount of 5%, cultured in batches at 37°C and pH 7.0 until the pH rose to 7.05, and the carbon and nitrogen sources were fed separately. The carbon and nitrogen source flow-feeding method is used. After feeding, 7.5M ammonia water is automatically added, and the pH is controlled at 7.0-7.2. Cultivate for 4-6 hours, add L-arabinose to induce, and continue to induce for 14±2h. Two fermentation broths containing the semaglutide precursor fusion protein were obtained.

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Abstract

The invention provides a semaglutide derivative and a preparation method thereof. Specifically, according to the method disclosed by the invention, the semaglutide is prepared by utilizing semaglutide fusion protein containing green fluorescent protein folding units while performing Fmoc modification on a Boc modified semaglutide main chain and side chain addition on the semaglutide through orthogonal protection. The invention also provides the semaglutide main chain modified by Fmoc and Boc and a fusion protein containing the semaglutide main chain involved in the preparation method.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a preparation method of semaglutide. Background technique [0002] Diabetes is a major disease that threatens human health worldwide. In China, with the change of people's lifestyle and the acceleration of the aging process, the prevalence of diabetes is rising rapidly. Acute and chronic complications of diabetes, especially chronic complications that accumulate in multiple organs, cause disability and high mortality, seriously affect the physical and mental health of patients, and bring heavy burdens to individuals, families and society. [0003] Semaglutide is a hypoglycemic drug developed by Novo Nordisk. This product can significantly reduce the level of glycosylated hemoglobin (HbA1c) and reduce weight in patients with type 2 diabetes, while greatly reducing the risk of hypoglycemia. Semeglutide is obtained by modifying and transforming GLP-1(7-37). Compared with Liraglutide, S...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K14/605C12P21/06A61K38/26A61P3/10
CPCC07K14/605C12P21/06A61P3/10C07K2319/60C07K2319/50C07K2319/02A61K38/00Y02P20/55
Inventor 陈卫刘慧玲骆莉
Owner NINGBO KUNPENG BIOTECH CO LTD
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