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Thioester peptide synthesis method

A technology of peptide synthesis and thioester, which is applied in the field of thioester peptide synthesis, can solve the problems of high synthesis cost of thioester peptide and limit the industrial production of thioester peptide, and achieve the effects of reducing synthesis cost, saving production time, and low cost

Pending Publication Date: 2022-03-15
ANHUI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of liquid chromatography has resulted in high synthesis costs of thioester peptides and also limits the industrial production of thioester peptides

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0044] Example 1: Synthesis of thioester peptides with Phe at the C-terminus

[0045] The specific process of the preparation method for preparing thioester peptides in this embodiment includes the following steps:

[0046] (1) Preparation of polypeptide hydrazide whose C-terminus is Phe:

[0047] Preparation of 2-Cl(trt)-hydrazine resin: 5mL of DM containing 5% hydrazine hydrate (v / v) prepared in advance was added to 536mg of 2-Cl(trt)-Cl resin (loading 0.56mmol / g, 300μmol) , react at room temperature for 30 minutes, discard the reaction solution, and repeat once; add 5 mL of blocking reagent 5 mL of 20% (v / v) methanol in DMF solution, and wash the resin six times with DMF after 20 minutes of reaction.

[0048] Amino acid condensation: Weigh 697mg Fmoc-Phe-OH (6eq) and 255mg Oxyma (6eq) in a 10mL centrifuge tube, add 5.5mL of DMF and 280μL DIC (6eq) to dissolve the amino acid and add it to the solid-phase synthesis tube; in a metal bath ( After shaking at 55° C. for 40 minu...

Embodiment 2

[0057] Embodiment 2: Synthesis of C-terminal thioester peptides of Ile

[0058] The specific process of the preparation method for preparing thioester peptides in this embodiment includes the following steps:

[0059] (1) Prepare the polypeptide hydrazide whose C-terminus is Ile:

[0060] Preparation of 2-Cl(trt)-hydrazine resin: 5mL of DM containing 5% hydrazine hydrate (v / v) prepared in advance was added to 536mg of 2-Cl(trt)-Cl resin (loading 0.56mmol / g, 300μmol) , react at room temperature for 30 minutes, discard the reaction solution, and repeat once; add 5 mL of 20% (v / v) methanol DMF solution and 5 ml of blocking reagent, and wash the resin with DMF six times after reacting for 20 minutes.

[0061] Amino acid condensation: Weigh 636mg Fmoc-Ile-OH (6eq) and 255mg Oxyma (6eq) in a 10mL centrifuge tube, add 5.5mL of DMF and 280μL DIC (6eq) to dissolve the amino acid and add it to the solid-phase synthesis tube; in a metal bath ( After shaking at 55° C. for 40 minutes, th...

Embodiment 3

[0070] Example 3: Synthesis of thioester peptides with Pro at the C-terminus

[0071] The specific process of the preparation method for preparing thioester peptides in this embodiment includes the following steps:

[0072] (1) Preparation of polypeptide hydrazide whose C-terminus is Pro:

[0073] Preparation of 2-Cl(trt)-hydrazine resin: Add 5 mL of DM containing 5% hydrazine hydrate (v / v) prepared in advance to 536 mg of 2-Cl(trt)-Cl resin (loading 0.56 mmol / g, 300 μmol) , react at room temperature for 30 minutes, discard the reaction solution, and repeat once; add 5 mL of 20% (v / v) methanol DMF solution and 5 ml of blocking reagent, and wash the resin with DMF six times after reacting for 20 minutes.

[0074] Amino acid condensation: Weigh 606mg Fmoc-Pro-OH (6eq) and 255mg Oxyma (6eq) in a 10mL centrifuge tube, add 5.5mL of DMF and 280μL DIC (6eq) to dissolve the amino acid and add it to the solid-phase synthesis tube; in a metal bath ( After shaking at 55° C. for 40 minu...

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Abstract

The invention discloses a thioester peptide synthesis method, which specifically comprises the following steps: dissolving polypeptide hydrazide in a hydrochloric acid-containing mixed solvent consisting of dimethyl sulfoxide and water, and reacting with isoamyl nitrite in an ice-salt bath to generate acyl azide peptide; adding excessive methyl thioglycolate, and then adding ammonium bicarbonate prepared in advance to adjust the acidity of the solution to be neutral, so that the acyl azide peptide is converted into peptide thioester; pre-cooled trifluoroacetic acid and diethyl ether are sequentially added into a reaction system, so that thioester peptide is crystallized and separated. The thioester peptide is prepared through the low-cost, simple and rapid operation process, and the problem that high-cost high performance liquid chromatography and time-consuming freeze drying operation are needed in traditional thioester peptide synthesis is solved.

Description

technical field [0001] The invention relates to a method for synthesizing thioester peptides. The thioester peptides are prepared through low-cost, simple and rapid operation procedures. Background technique [0002] Thioester peptides are an important class of polypeptide intermediates and are widely used in the connection technology of peptide fragments. For example, in natural chemical ligation technology, thioester peptides are synthesized through chemoselective ligation with N-terminal Cys peptides to realize the synthesis of long fragment peptides. In the aminolysis of thioester peptide catalyzed by silver ions, the thioester peptide is linked to the N-terminal amino group of the polypeptide and successfully used for splicing of the polypeptide. Due to the instability to piperidine, thioester peptides cannot be directly synthesized by the Fmoc method, which has limited the popularization and application of thioester peptides for quite a long time. Recently, it was di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/30C07K1/08C07K1/06C07K1/04
CPCC07K1/303C07K1/08C07K1/06C07K1/04
Inventor 方葛敏虞飞强谢晓磊殷庆红
Owner ANHUI UNIVERSITY
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