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Method for preparing thymosin alpha 1 by liquid phase fragment condensation

A technology of thymosin and fragments, applied in the field of biochemistry, can solve the problems of time-consuming, difficult to obtain high-purity thymosin α1, and high production cost, and achieve the effects of simplifying the post-processing process, reducing the number of preparations, and reducing the cost of synthesis

Active Publication Date: 2014-03-26
NANTONG SHIMEIKANG PHARMA CHEM
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Comprehensively referring to the preparation methods of thymosin α1 and related synthesis reports at home and abroad, it is found that these technologies have some shortcomings, which are mainly manifested in: ①The whole liquid phase synthesis is time-consuming and requires good post-processing technology; ②Biosynthesis technology is difficult to scale production ; ③ It is difficult to obtain high-purity thymosin α1 by conventional solid-phase synthesis methods, and the yield is low (5-10%), resulting in high production costs

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  • Method for preparing thymosin alpha 1 by liquid phase fragment condensation
  • Method for preparing thymosin alpha 1 by liquid phase fragment condensation
  • Method for preparing thymosin alpha 1 by liquid phase fragment condensation

Examples

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Embodiment 1 2

[0035] Example 1. Preparation of thymosin α1 by 2+2 fragment method

[0036] 1. Resin preparation

[0037] 1.1 Preparation of Fmoc-Asn(Trt)-2-chloro-trityl resin: add 2-chloro-trityl chloride resin (5g, substitution value 0.8mmol / g resin, 1 eq.) to 150 mL peptide synthesis Wash the swollen resin with 60 mL of DCM for 30 minutes. The solvent was drained and a solution of Fmoc-Asn(Trt)-OH (1.2 eq.) and DIEA (2.5 eq.) in 30 mL DCM was added. The mixture was mechanically stirred under an argon atmosphere for 1 hour. Add 10 mL of chromatographic grade methanol (2ml / g resin) to block the active part on the resin for 30 minutes. Drain the solvent, wash with 3×60 mL DMF, 3×60 mL DCM, 3×60 mL MeOH, vacuum filter and dry to constant weight to obtain 6.39g Fmoc-Asn(Trt)-2-chloro-trityl resin. The amount of Fmoc in the piperidine deprotection solution was measured by ultraviolet spectrophotometry, and the loading amount of the resin was 0.39 mmol / g.

[0038] 1.2 Preparation of F...

Embodiment 2 3

[0136] Embodiment two, three-segment method synthetic thymosin α1

[0137] 1 Resin preparation

[0138] 1.1 The synthesis of Fmoc-Asn(Trt)-2-chloro-trityl resin is the same as in Example 1.

[0139] 1.2 The synthesis of Fmoc-Ser(tBu)-2-chloro-trityl resin is the same as in Example 1.

[0140] 1.3 Preparation of Fmoc-Lys(Boc)-2-chloro-trityl resin: add 2-chloro-trityl chloride resin (5 g, substitution value 0.8 mmol / g resin, 1 eq.) to 150 mL of polypeptide Synthesizer, wash swollen resin with 60 mL DCM. Drain the resin bed, since the resin substitution value must be reduced to synthesize Fmoc-AA(10-20)-OH, add Fmoc-Lys(Boc)-OH (1.2 eq.) and DIEA (2.5 eq.) in 30 mL of DCM . The mixture was mechanically stirred under an argon atmosphere for 1 hour. Add 10 mL of chromatographic methanol (2ml / g resin) to cap the active part on the resin for 30 minutes. The resin bed was drained, washed with 3×60 mLDMF, 3×60 mLDCM, 3×60 mLMeOH, vacuum filtered and dried to constant weight to o...

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Abstract

The invention provides a method for preparing thymosin alpha 1 by liquid phase fragment condensation, belonging to the technical field of biochemistry. According to the method, high capacity value (not less than 0.8mmol / g) resin is used as a starting material, firstly, a standard solid phase polypeptide synthesis (SPPS) technology is adopted for synthesizing a high-purity peptide fragments with selected structures, then, a liquid phase condensation technology is adopted for connecting the peptide fragments, and thus, a high-purity (more than 99%) target peptide is obtained. Compared with the solid phase thymosin alpha 1 synthesis technology, the method provided by the invention avoids the problem of low coupling ratio of amino acids behind the 12th position, and greatly improves yield (up to 25-30%) of the thymosin alpha 1; meanwhile, the peptide fragment formed by solid phase synthesis is free from purification, post-treatment technology is simplified, finally, the thymosin alpha 1 is purified by means of high performance liquid chromatography, preparation difficulty is reduced, preparation times is decreased, synthesis cost of the thymosin alpha 1 is lowered, and implementation of large-scale and industrialized production is facilitated.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and relates to a method for synthesizing thymosin α1, in particular to a method for preparing thymosin α1 by condensation of liquid phase fragments. Background technique [0002] Thymosin α1 (also known as Thymosin α1, Thymosin) is a thymosin with the following structure: Ac-SDAAVDTSSEITTKDLKEKKEVVEEAEN-COOH. Thymosin α1 is a single-component polypeptide compound present in the mammalian thymus. It is a polypeptide composed of 28 amino acid residues and N-terminal amino acid acetylation. It is a molecule related to the development and differentiation of immune cells. It has the ability to differentiate T lymphocytes, The role of proliferation and improving cellular immune function can destroy infected target cells, activate NK cell activity, and promote the production of immune-related cytokines. The activity of thymosin α1 is 10 to 1000 times higher than that of thymopentin. It is a compou...

Claims

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Application Information

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IPC IPC(8): C07K14/575C07K1/06C07K1/02
CPCY02P20/55C07K14/57581
Inventor 王锐常民彭雅丽薛宏祥李明生
Owner NANTONG SHIMEIKANG PHARMA CHEM
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