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Solid Phase Synthesis of Thymosin α1

A technology of solid-phase synthesis and thymosin, which is applied in the field of solid-phase synthesis of thymosin α1, can solve the problems of low condensation efficiency, large influence of spatial structure, unfavorable industrial production, etc., so as to reduce the influence, improve the reaction yield, Less side effects

Active Publication Date: 2011-12-21
HARBIN PHARMA GROUP BIOLOGICAL ENG +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage is that the combination of Wang resin and Fmoc-Asn(Trt)-OH is difficult, and there is also a side reaction in which the linker is excised and connected to the peptide chain during cleavage, which will have a certain impact on the yield
Moreover, the condensation efficiency of the amino acid condensing agents TBTU-HOBt, DCC-HOBt, and DIC-HOBt in the method is not very high. For relatively long peptides such as thymosin α1, the spatial structure has a greater impact on the yield. , leading to higher costs, which is not conducive to industrial production

Method used

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  • Solid Phase Synthesis of Thymosin α1

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1: AA (1-27) -Asn(Trt) 28 -Synthesis of CTC resin

[0031]Weigh 66.7g (40mmol) of Fmoc-Asn(Trt)-CTC resin with a substitution degree of 0.60mmol / g, add it to the reactor, add 500ml DMF, turn on the nitrogen gas and mix the liquid and resin gently from bottom to top, stir and swell Stop after 30 minutes, and remove the liquid by suction filtration. Then add 500ml of 20% piperidine / DMF solution (capping solution) into the reactor, stir and react for 5 minutes under nitrogen protection, remove the liquid, and wash twice with 500ml DMF. Then add 500ml of decapping solution, stir and react for 10 minutes, then remove, wash with 500ml of DMF 8 times, and wash off the Fmoc protecting group. Weigh 68g of Fmoc-Glu(OtBu)-OH, 60.8g of HATU, dissolve in 500ml of DMF, add 26.5ml of cooled NMM under ice bath, activate for 10 minutes, add to the reactor, and react at 15℃~25℃ 2.5-3 hours, subject to ninhydrin colorimetric detection. Then the reaction liquid was removed by ...

Embodiment 2

[0032] Example 2: Ac-AA (1-27) -Asn 28 Preparation of (Trt)-CTC resin

[0033] Prepare the acetylation solution 76ml of acetic anhydride, 65ml of pyridine, and 1000ml of DMF, mix well and add to the reactor, react for 24 hours under the protection of nitrogen, and then remove the acetylation solution by suction filtration. Add 1000ml DMF to wash 6 times, 1500ml DCM to wash 3 times, 1000ml MeOH to wash 3 times, each time for 5 minutes. The resin was removed and dried under reduced pressure to obtain 242.2 g of thymosin α1 peptide resin.

Embodiment 3

[0034] Example 3: Preparation of Crude Peptides by Cleavage of Resin Peptides

[0035] (1) Prepare 2400 ml of lysis solution of trifluoroacetic acid / dimercaptoethanol / water=90:5:5 (v / v), and pre-freeze at -10°C for 12 hours.

[0036] (2) Put 242.2g of peptide resin in a round-bottomed flask, cool in an ice bath, add 2400ml of pre-frozen lysate, remove the ice bath after the addition, and stir at room temperature for 2.5 hours. Then the resin was removed by filtration, and the filtrate was added to 10 times the volume of glacial ether. Settled for 5 hours. Centrifuge and wash 8 times at 4000 rpm. The precipitate was removed and dried under reduced pressure to obtain 73.5 g of crude thymosin α1 peptide.

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Abstract

The invention relates to a solid phase synthesis process of thymosin alpha1. The process comprises the following steps of: (1) preparation of NH2-Asn(Trt)-CTC resin; (2) synthesis of AA(1-27)-Asn(Trt)28-CTC resin; (3) preparation of Ac-AA(1-27)-Asn28(Trt)-CTC resin; (4) preparation of crude thymosin alpha1 by using a cracking agent; and (5) purification of the crude thymosin alpha1.

Description

technical field [0001] The invention relates to a method for synthesizing and purifying polypeptides, in particular to a solid-phase synthesis process for thymosin α1. Background technique [0002] In 1977, Dr. Allen Goldstein, known as the "Father of the Thymus", extracted the most immunologically active component from the fifth component of thymosin: thymosin α1. Thymosin α1 is a biological response modifier (BRM), consisting of 28 amino acid residues, the amino acid sequence is as follows: [0003] N-Acetylated-L-seryl-L-aspartyl-L-alanyl-L-alanyl-L-valyl-L-aspartyl-L-threonyl-L -seryl-L-seryl-L-glutamyl-L-isoleucyl-L-threonyl-L-threonyl-L-lysyl-L-aspartyl-L -Leucyl-L-lysyl-L-glutamyl-L-lysyl-L-lysyl-L-glutamyl-L-valyl-L-valyl-L-glutamyl Aminoacyl-L-glutamyl-L-alanyl-L-glutamyl-L-asparagine. [0004] The N-terminus is acetylated and the molecular weight is 3108. It mainly exists in the thymus, and also exists in low levels in spleen, lung, kidney, brain and other tis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/575C07K1/20C07K1/06C07K1/04
Inventor 齐岩葛存慧李郑武王孝文李会成陈玉军张海涛高晶曹翊婕徐岩王丽娜
Owner HARBIN PHARMA GROUP BIOLOGICAL ENG
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