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Solid phase synthetic technique for thymosin alpha1

A solid-phase synthesis, thymosin technology, applied in the direction of thymopoietin, hormone peptide, peptide preparation method, etc.

Active Publication Date: 2011-06-01
苏州天马医药集团天吉生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] With comprehensive reference to the preparation methods of thymosin α1 and related synthesis reports at home and abroad, it is found that these technologies have some deficiencies, which are mainly manifested in: ①Biosynthesis technology single molecule Escherichia coli is difficult to express, and it is difficult to produce on a large scale; ②The BOC strategy uses HF for synthesis and cracking, which is violent and corrosive, and is harmful to production personnel and the environment; ③It is difficult to obtain high purity (>98 %) of Thymosin α1, or the yield is low (<5%), resulting in high production cost

Method used

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  • Solid phase synthetic technique for thymosin alpha1
  • Solid phase synthetic technique for thymosin alpha1
  • Solid phase synthetic technique for thymosin alpha1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1: Synthesis of Fmoc-Asp (Rink Amide AM Resin)-OtBu

[0052] Add 2g of Fmoc-Rink Amide AM Resin, with a substitution degree of 1.1mmol / g, into a solid-phase reactor, add 20ml of DCM to swell the resin for 30min, and remove Fmoc protection twice with 20% DBLK5+10min to obtain NH 2 -Rink Amide AM Resin, washed 4 times in DMF and 2 times in DCM.

[0053] Fmoc-Asp-OtBu0.91g, DIC0.4ml, HOBt0.327g were dissolved in 4ml of DMF, activated in ice water at low temperature for 10min, then added to the above solid-phase reactor, and reacted at room temperature for 2h. After washing twice with DMF, 6.2 ml of acetic anhydride and 5.3 ml of pyridine were added to react for 4 hours to block unreacted free amino groups on the resin. After washing twice with DMF, it was shrunk with methanol for 10+10 min to obtain Fmoc-Asp(Rink Amide AM Resin)-OtBu, and the detected substitution degree was 0.453mmol / g.

Embodiment 2

[0054] Example 2: AA 1-27 -Synthesis of Asp(resin)-OtBu

[0055] Weigh 2.2g of Fmoc-Asp(Rink Amide AM Resin)-OtBu with a degree of substitution of 0.453mmol / g, add to the reactor and swell with DCM for 0.5 hours, then use 20% DBLK (5+5)min to remove Fmoc, wash Then connect the 27th amino acid Fmoc-Glu(OtBu)-OH, dissolve 1.702g Fmoc-Glu(OtBu)-OH, 1.517gHBTU, 0.149gHOBt in 5mlDMF, add 1mlDIPEA after 10 ice baths; after low temperature activation for 10min, add the above solid In the phase reactor, react at room temperature for 1-2 hours, and the end point of the reaction is determined by the ninhydrin method. Repeat the above steps, and so on to complete the connection of amino acids from 26 to 1 to obtain AA 1-27 -Asp(resin)-OtBu. The amount of raw materials: 1.0 mmol of amino acid, 1.517 g of HBTU, 0.149 g of HOBt, and DIPE Alml.

Embodiment 3

[0056] Example 3: Ac-AA 1-27 -Synthesis of Asp(resin)-OtBu

[0057] Add 2.8ml of acetic anhydride and 2.4ml of pyridine in the reactor, and react at room temperature for 4 hours. The ninhydrin method resin is colorless and transparent, and the reaction is complete. The acetylation reagent is removed, washed twice with DMF, and shrunk with methanol (10+10 )min, Ac-AA was obtained after drying under reduced pressure for 6 hours 1-27 -Asp(resin)-OtBu 4.9 g.

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Abstract

The invention relates to a solid-phase synthesis process of a thymosin alpha 1, belonging to the polypeptide solid-phase synthesis technical field. The invention comprises the following steps: a. a Fmoc-Rink Amide AM resin or a Fmoc-Rink Amide MBHA resin is used as carrier, an H2N-Rink Amide AM resin or an H2N-Rink Amide MBHA resin is obtained after deprotection of the Fmoc; b. side chain carboxyl group of Fmoc-Asp-X is connected with resin amino by the method of solid-phase synthesis to obtain the Fmoc-Asp (resin)-X; c. the left amino acid in the sequence is synthesized in solid-phase with the Fmoc strategy; d. after the amino protection group Fmoc of N terminal amino acid is removed, the N terminal amino acid is acetylated by acetic anhydride and pyridine; e. then the acetylated N terminal amino acid is cut by a cracking agent (tri fluoroacetic acid / benzoylate sulfide / 1, 2- dithioglycol / Anisole) to obtain the thymosin alpha 1; f. crude product of the thymosin alpha 1 is prepared and separated by HPLC to obtain the pure thymosin alpha 1. The invention can significantly increase the yield of the thymosin alpha 1 and decrease the production cost, which is helpful for scale production and has better industrialization prospect.

Description

technical field [0001] The invention relates to a polypeptide whose C-terminus is Asn, in particular to thymosin α1, especially the solid-phase synthesis process of thymosin α1. Background technique [0002] Thymosin α1 is a single-component polypeptide compound present in the thymus of mammals. It is a molecule related to the development and differentiation of immune cells. It can differentiate and proliferate T lymphocytes, improve cellular immune function, and destroy the target cells it infects. , It can also activate NK cell activity and promote the production of immune-related cytokines. The activity of thymosin α1 is 10 to 1,000 times higher than that of thymopentin. It is a compound drug for the treatment of chronic hepatitis B, hepatitis C, and immunodeficiency syndrome. It also plays a greater role in the treatment of non-small cell lung cancer and malignant melanoma. Thymosin α1 was developed and marketed by Italian company Sciclone in 1997. It has been approved ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/66C07K14/47C07K1/04
CPCY02P20/55
Inventor 初虹
Owner 苏州天马医药集团天吉生物制药有限公司
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