Method for synthesizing thymalfasin

A thymosin and solid-phase synthesis technology, which is applied in the preparation methods of peptides, thymosin, chemical instruments and methods, etc., can solve the problems that the yield and purity cannot reach a high level, there are many impurities, and the purification is difficult to carry out.

Active Publication Date: 2014-01-08
HYBIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the process of preparing thymus by one-by-one coupling method, the synthesis cycle is long and there are many impurities (impurities such as default peptides are easily produced in the synthesis of long peptides), and most of the impurities are similar to the target peptides, resulting in subsequent purification. Difficult to carry out, the yield and purity cannot reach a high level

Method used

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  • Method for synthesizing thymalfasin
  • Method for synthesizing thymalfasin
  • Method for synthesizing thymalfasin

Examples

Experimental program
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Effect test

Embodiment 1

[0070] Embodiment 1: the preparation of polypeptide resin 1

[0071] 1. Preparation of Fmoc-β-Asp (OtBu)-Rink amide resin

[0072] Take Rink amide resin (20g, 16mmol) with a degree of substitution sub=0.8mmol / g, add it to the reaction column, swell it with DMF for more than 30 minutes, drain the DMF, and remove it with DBLK (a DMF solution containing 20% ​​piperidine). Removal of the amino protecting group Fmoc of the resin was carried out for 5 minutes each time, twice in total. After the removal was completed, it was washed 6 times with DMF, drained and ready to be fed.

[0073] Weigh Fmoc-β-Asp (OtBu)-OH (6.6g, 16mmol), HOBt (2.6g, 19.2mmol), dissolve with DMF (40ml) and DCM (40ml), add DIC (2.4g, 19.2 mmol), activated for 5 minutes and then added to the reaction column. After 60 minutes, the reaction was completed. The resin was washed three times with DMF, and the blocking solution (pyridine (20ml) and acetic anhydride (22ml)) was added. Wash 6 times. Then add an appro...

Embodiment 2

[0076] Embodiment 2: Preparation of polypeptide fragment 1

[0077] Weigh 2-CTC resin (40g, 20mmol) with a substitution degree of 0.5mmol / g, add it to a solid-phase reaction column, wash it twice with DMF, and swell the resin with DMF for 30 minutes, then take Fmoc-Lys(Boc)- OH (28.1g, 60mmol) was dissolved in a mixed solution of DCM (30ml) and DMF (30ml) with a volume ratio of 1:1, and DIPEA (15.5g, 120mmol) was added under ice-cooling conditions, and the solution was added to the solid phase after activation for 5 minutes In the reaction column, react at room temperature for 2 hours. After the reaction, block with blocking solution (DIPEA: methanol: DCM = 1:2:17v:v) three times, each time for 3 minutes. The volume of closed liquid is calculated as 4.0ml / gram of resin. Then add an appropriate amount of methanol to wash 3 times, each time for 10 minutes, and wash 6 times with DMF. Fmoc protection was removed with DBLK, followed by 6 washes with DMF.

[0078] Dissolve Fmoc-G...

Embodiment 3

[0080] Embodiment 3: Preparation of polypeptide fragment 2

[0081] Weigh 2-CTC resin (40g, 20mmol) with a substitution degree of 0.5mmol / g, add it to a solid-phase reaction column, wash it twice with DMF, and swell the resin with DMF for 30 minutes, then take Fmoc-Ser(tBu)- OH (23.0g, 60mmol) was dissolved in a mixed solution of DCM (30ml) and DMF (30ml) with a volume ratio of 1:1, and DIPEA (15.5g, 120mmol) was added under ice-cooling conditions, and the solution was added to the solid phase after activation for 5 minutes In the reaction column, react at room temperature for 2 hours. After the reaction, use blocking solution (DIPEA: methanol: DCM = 1:2:17v:v) to block three times, each time for 3 minutes. The volume of closed liquid is calculated as 4.0ml / gram of resin. Then add an appropriate amount of methanol to wash 3 times, each time for 10 minutes, and wash 6 times with DMF. Fmoc protection was removed with DBLK, followed by 6 washes with DMF.

[0082] Dissolve Fmoc...

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Abstract

The invention relates to the field of medicine synthesis, and discloses a method for synthesizing thymalfasin. According to the method of the invention, based on the amino acid sequence from the C terminal to the N terminal of the thymalfasin peptide chain, fragments of 1-8, 9-19 and 20-28 are synthesized, and the three polypeptide fragments are coupled to obtain thymalfasin. According to the invention, a plurality of fragments are synthesized simultaneously; the synthetic period is reduced by 2 / 3; intermediates are easy to purify; the cost is low; the purity of the finished products is high; by-products are few; the product yield is high; and the method facilitates large-scale production of thymalfasin.

Description

technical field [0001] The invention relates to the field of medicine synthesis, in particular to a new method for synthesizing thymus. Background technique [0002] Thymosin α1 (also known as thymosin α1) is the main bioactive component of thymosin and an important immune regulatory substance in the body. It is a polypeptide composed of 28 amino acids acetylated at the N-terminal. Its peptide sequence is as follows: [0003] Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu -Glu-Ala-Glu-Asn-COOH [0004] Although the mechanism of action of this product in treating chronic hepatitis B and enhancing the reactivity of the immune system has not been fully elucidated, a number of in vitro tests have shown that this product promotes the maturation of T lymphocytes by stimulating peripheral blood lymphocyte mitogens, increasing antigen or filament After cleavage activation, the levels of interferon-α, interferon-γ, interleukin-2, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/575C07K1/06C07K1/04
CPCC07K14/57581Y02P20/55
Inventor 肖庆刘建马亚平袁建成
Owner HYBIO PHARMA
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