Method for synthesizing glucagon-like peptide (GLP)-1 analogue in solid-phase mode

A GLP-1, solid-phase synthesis technology, used in peptide preparation methods, chemical instruments and methods, animal/human proteins, etc., can solve the problems of amino acid sequence connection of difficult peptides and by-products, inability to separate by-products, etc. Benefit and social benefit, increase synthesis yield, reduce the effect of His racemic impurity

Inactive Publication Date: 2013-03-20
JILIN AOTENG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the problem of missing peptides and by-products with very similar properties caused by difficult or incomplete amino acid sequence connection in the process of solid-phase synthesis of GLP-1 analogues, resulting in the inability to separate by-products during subsequent purification, the present invention proposes a solid-phase synthesis The method of GLP-1 analogs, this method adopts the method of fragment condensation and introduction of substituents at the difficult link point, which improves the yield of polypeptide synthesis

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] (1) Weigh 10g (1mmol) of the starting resin Fmoc-Rink Amide PEG MBHA Resin with a degree of substitution of 0.1mmol / g, and add it to a solid-phase reaction vessel. Add the solvent DCM to the vessel to completely immerse the starting resin in Swell in DCM solvent for 1 hour;

[0033] (2) Add 50 mL of piperidine / DMF deprotection reagent with a volume concentration of 20% to remove the Fmoc protecting group, react at a temperature of 15°C for 30 min, then wash twice with DMF and twice with methanol;

[0034] (3) Using Fmoc-Ser(tBu)-OH as the intermediate raw material, activate 3 mmol of Fmoc-Ser(tBu)-OH, DIC, and HoBt together in a DMF solvent for 5 minutes in an ice bath;

[0035] (4) Put the activated Fmoc-Ser(tBu)-OH into the container of step (2), and control the reaction temperature at 15°C;

[0036] (5) After the reaction was monitored by ninhydrin, Fmoc-Ser(tBu)-MBHA Resin was obtained by cross-washing three times with DMF and methanol, and the detection degree of ...

Embodiment 2

[0045](1) Weigh 10g (6mmol) of the starting resin Fmoc-Rink Amide PEG MBHA Resin with a degree of substitution of 0.6mmol / g, and add it to the solid-phase reaction vessel. Add the solvent DCM to the vessel to completely immerse the starting resin in Swell in DCM solvent for 2 hours;

[0046] (2) Add 100 mL of piperidine / DMF with a volume concentration of 20% to remove the Fmoc protecting group, react at 20°C for 20 min, and then wash with DMF and methanol for 3 times;

[0047] (3) Using Fmoc-Ser(tBu)-OH as the intermediate raw material, activate 12 mmol of Fmoc-Ser(tBu)-OH, DIC, and HoAt in DMF solvent for 5 minutes in an ice bath;

[0048] (4) Put the activated Fmoc-Ser(tBu)-OH into the container of step (2), and control the reaction temperature at 20°C;

[0049] (5) After the reaction was monitored by ninhydrin, Fmoc-Ser(tBu)-MBHA Resin was obtained by cross-washing with DMF and methanol three times, and the detection degree of substitution was 0.38 mmol / g.

[0050] (6) Pu...

Embodiment 3

[0058] (1) Weigh 10g (4mmol) of the starting resin Fmoc-Rink Amide PEG MBHA Resin with a degree of substitution of 0.4mmol / g, and add it to a solid-phase reaction vessel. Add the solvent DCM to the vessel to completely immerse the starting resin in Swell in DCM solvent for 0.5 hour;

[0059] (2) Add 20 mL of piperidine / DMF with a volume concentration of 20% to the container of step (1) to remove the Fmoc protecting group, react at 25°C for 30 min, then wash with DMF for 3 times, and methanol for 3 times;

[0060] (3) Using Fmoc-Ser(tBu)-OH as the intermediate raw material, activate 16 mmol of Fmoc-Ser(tBu)-OH, DIC, and HoBt together in DMF solvent for 5 minutes in an ice bath;

[0061] (4) Put the activated Fmoc-Ser(tBu)-OH into the container of step (2), and control the reaction temperature at 10°C;

[0062] (5) After the reaction was monitored by ninhydrin, it was washed 3 times with DMF and 3 times with methanol to obtain Fmoc-Ser(tBu)-MBHA Resin, and the detection degree ...

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Abstract

The invention relates to the field of polypeptide solid-phase synthesis and provides a method for synthesizing glucagon-like peptide (GLP)-1 analogue in a solid-phase mode. The method for synthesizing the GLP-1 analogue in the solid-phase mode solves the problems that in the process of synthesizing the GLP-1 analogue in the solid-phase mode, peptide deficiency products and by-products are generated because of difficult or incomplete amino acid sequence connection, and the by-products are resulted to be difficulty to separate in subsequent purification. The peptide deficiency products and by-products are quite same in properties. According to the method for synthesizing the GLP-1 analogue in the solid-phase mode, a segment convergent synthesis method and a substituent-introduced method are adopted in difficult-connection points and polypeptide synthesis yield coefficient is improved.

Description

technical field [0001] The invention relates to the field of polypeptide solid-phase synthesis, in particular to a method for solid-phase synthesis of GLP-1 (glucagon-like peptide-1) analogues. Background technique [0002] With the development of cell and molecular biology, the mysterious veil of incretin has been gradually revealed. Studies have confirmed that incretin is a gut-derived hormone in the human body. The method promotes the secretion of insulin by pancreatic β cells and reduces the secretion of glucagon by pancreatic α cells, thereby lowering blood sugar. After a normal person eats a meal, incretin starts to be secreted, which in turn promotes the secretion of insulin, so as to reduce the fluctuation of blood sugar after a meal. However, for patients with type 2 diabetes, the "incretin effect" is impaired, mainly manifested in a smaller increase in the concentration of glucagon-like peptide-1 (GLP-1) after meals than in normal people, but its The effects of p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/575C07K1/20C07K1/18C07K1/06C07K1/04
Inventor 刘秀丽
Owner JILIN AOTENG BIOTECH
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