Preparation method of glucagon

A technology of glucagon and resin, which is applied in the field of polypeptide drug preparation, can solve the problems of low product purity, increased connection difficulty, high price, etc., and achieves the effects of reasonable reaction process, reduced side reactions, and fewer side reactions.

Pending Publication Date: 2020-04-17
CHINESE PEPTIDE CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as a special amino acid, the pseudo-proline protected dipeptide is more expensive than ordinary amino acids, which is not conducive to cost control in peptide synthesis and is not conducive to large-scale production
[0004] Moreover, in the connection of several amino acids at the end of the N-terminus, due to the increase in the difficulty of connection, the solid-phase synthesis process of each amino acid will increase side reactions, and the closer to the end, the more side reactions, the side reactions in each step will increase. Step by step accumulation, such a result, resulting in low purity of the final product
From another point of view, the difficulty of linking the terminal amino acids is also very high, and the closer to the terminal, the higher the difficulty. The prior art represented by this patent does not solve these problems

Method used

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  • Preparation method of glucagon
  • Preparation method of glucagon

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Present embodiment adopts following technical scheme:

[0021] Step 1) Connect 5-29 fragments one by one according to the glucagon sequence by solid phase synthesis to obtain H-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu) -Ser(tBu)-Lys(Boc)-Tyr(tBu)-Leu-Asp(OtBu)-Ser(tBu)-Arg(Pbf)-Arg(Pbf)-Ala-Gln(Trt)-Asp(OtBu)- Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met-Asn(Trt)-Thr(tBu)-Wang resin.

[0022] Specifically, the method for synthesizing the 5-29 fragment peptide resin in step 1) of the present invention is as follows: Fmoc-Thr(tBu)-OH is coupled with Wang resin to obtain Fmoc-Thr(tBu)-Wang resin. Wherein, the coupling reagent is 2,6-dichlorobenzoyl chloride and pyridine.

[0023] More specifically, Fmoc-Thr(tBu)-OH and Wang resin were mixed and dissolved in DCM, and pyridine was added to react with 2,6-dichlorobenzoyl chloride.

[0024] Preferably, the molar ratio of Wang resin to Fmoc-Thr(tBu)-OH, 2,6-dichlorobenzoyl chloride and pyridine is 1:1.2:2.4:4.8.

[0025] Prefe...

example 1

[0043] Example 1: the preparation of the Fmoc-Thr(tBu)-Wang resin that the degree of substitution is 0.23mmol / g

[0044] Weigh 50.00g of blank Wang resin with a substitution degree of 0.79mmol / g, Fmoc-Thr(tBu)-OH 9.41g, mix and add DCM to dissolve, then add 7.65mL of 4 equivalents of pyridine to amino acids, and finally slowly add 2,6 -Dichlorobenzoyl chloride 6.78mL, stirred and reacted for 3h, transferred to the reaction column, pumped off the reaction solution, washed 3 times with DMF, 1 time with methanol, 3 times with DMF and 3 times with methanol, and dried the solvent After vacuum drying, Fmoc-Thr(tBu)-Wang resin was obtained, and the detected substitution degree was 0.23 mmol / g.

example 2

[0045] Example 2: the preparation of Fmoc-Thr(tBu)-Wang resin with a degree of substitution of 0.41mmol / g

[0046] Weigh 50.00g of blank Wang resin with a substitution degree of 0.79mmol / g, Fmoc-Thr(tBu)-OH 18.83g, mix and add DCM to dissolve, then add 15.3mL of 4 equivalents of pyridine to amino acids, and finally slowly add 2,6 -Dichlorobenzoyl chloride 13.56mL, after stirring for 3 hours, transfer to the reaction column, remove the reaction liquid, wash 3 times with DMF, wash 1 time with methanol, wash 3 times with DMF, wash 3 times with methanol, and drain the solvent After vacuum drying, Fmoc-Thr(tBu)-Wang resin was obtained, and the detected substitution degree was 0.41 mmol / g.

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Abstract

The invention relates to a preparation method for glucagon by a solid-phase fragment method. The method comprises the following steps: connecting 5-29 fragments one by one by utilizing a solid-phase synthesis method according to a sequence; and then connecting the tetrapeptide fragment Fmoc-His(Trt)-Ser(tBu)-Gln(Trt)-Gly-OH to 5-29 fragment peptide resin, carrying out acidolysis to remove a protecting group and cut peptide to obtain a glucagon crude product, and carrying out purification and freeze-drying to obtain a glucagon pure product. The method has the advantages of easiness in synthesis, low impurity content and cost saving.

Description

technical field [0001] The invention relates to the field of polypeptide drug preparation, in particular to a method for preparing glucagon by a solid-phase fragment method. Background technique [0002] Glucagon is a hormone secreted by pancreatic islet α-cells. It is a straight-chain polypeptide composed of 29 amino acids, also known as glucagon or anti-insulin or insulin B, which is opposed to insulin. , plays the role of increasing blood sugar, in 1953, glucagon was separated and precipitated to obtain crystallization. Its chemical structure is H-His 1 -Ser 2 -Gln 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Ser 11 -Lys 12 -Tyr 13 -Leu 14 -Asp 15 -Ser 16 -Arg 17 -Arg 18 -Ala 19 -Gln 20 -Asp 21 -Phe 22 -Val 23 -Gln 24 -Trp 25 -Leu 26 -Met 27 -Asn 28 -Thr 29 -OH, the molecular weight is 3485 Daltons. [0003] Glucagon is a relatively difficult 29-peptide to prepare, especially in the linkage of a few amino acids at the end of the N-ter...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/605C07K1/04C07K1/06
CPCC07K14/605Y02P20/55
Inventor 刘宝生马佳颀李湘徐琪
Owner CHINESE PEPTIDE CO
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