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Synthesis method of thymosin alpha1

A technique for synthesizing thymosin and solid-phase peptides, which is applied in the fields of peptide preparation methods, thymosin, chemical instruments and methods, etc., can solve problems such as the difficulty of synthesizing thymosin α, and achieve the effects of easy post-processing, good purity, and easy operation

Active Publication Date: 2014-07-23
HYBIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Those skilled in the art know that thymosin alpha 1 The two segments (Ser 1 -Glu 10 and Ile 11 -Asn 28 ) is easy to form a β-sheet structure, which results in the synthesis of thymosin α by the existing solid-phase peptide synthesis method 1 very difficult

Method used

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  • Synthesis method of thymosin alpha1
  • Synthesis method of thymosin alpha1
  • Synthesis method of thymosin alpha1

Examples

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preparation example Construction

[0035] The invention provides a thymosin alpha 1 The nonlinear solid-phase synthesis method comprises the following steps:

[0036] (1) Using Rink Amide resin as a carrier, synthesize Fmoc-Asp(resin)-OtBu with Fmoc-Asp-OtBu, and then follow thymosin α 1The peptide sequence of the peptide sequence, using the solid-phase peptide synthesis method on the basis of Fmoc-Asp (resin)-OtBu to couple amino acids one by one from the C-terminal to the N-terminal, in this step, the last amino acid coupled is thymosin α 1 The serine at the 9th position of the N-terminus in the peptide sequence obtains the following peptide resin A:

[0037] Boc-Ser-Glu(OtBu)-Ile-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Lys( Boc)-Glu(OtBu)-Val-Val-Glu(OtBu)-Glu(OtBu)-Ala-Glu(OtBu)-Asp(resin)-OtBu;

[0038] (2) Use Fmoc-Ser(tBu)-OH to react with the free hydroxyl group of serine at the N-terminal of the above-mentioned peptide resin A to form an ester bond. thymosin alpha 1 Th...

Embodiment 1

[0057] Embodiment 1: the degree of substitution is the synthesis of Fmoc-Asp (resin)-OtBu of 0.2mmol / g

[0058] Weigh 500 g of Rink Amide resin with a substitution degree of 0.6 mmol / g, add it to a solid-phase reaction column, wash it twice with DMF, and swell the resin with DMF for 30 minutes. The Fmoc protection was removed with DBLK (20% piperidine / DMF), followed by washing 4 times with DMF and 2 times with DCM. Weigh 51.4g Fmoc-Asp-OtBu (125mmol), 20.3g HOBt (150mmol) and dissolve in a mixed solution of DCM and DMF with a volume ratio of 1:1, add 23.4ml DIC (150mmol) under ice water bath for activation for 3min, then add solid phase reaction In the column, react at room temperature for 2 hours. Wash 3 times with DMF, add 1046.3ml of blocking solution (pyridine / acetic anhydride = 1:1, 6mol:6mol) to block for 8 hours (if the resin is not fully diffused, add DCM as a solvent). Wash 4 times with DMF, 4 times with DCM, shrink and dry with methanol to obtain Fmoc-Asp(resin)-Ot...

Embodiment 2

[0059] Embodiment 2: the degree of substitution is the synthesis of Fmoc-Asp (resin)-OtBu of 0.3mmol / g

[0060] Weigh 500 g of Rink Amide resin with a substitution degree of 0.6 mmol / g, add it to a solid-phase reaction column, wash it twice with DMF, and swell the resin with DMF for 30 minutes. The Fmoc protection was removed with DBLK (20% piperidine / DMF), followed by washing 4 times with DMF and 2 times with DCM. Weigh 77.2g Fmoc-Asp-OtBu (187.5mmol), 30.4g HOBt (225mmol) and dissolve in the mixed solution of DCM and DMF with a volume ratio of 1:1, add 35.2ml DIC (225mmol) under ice water bath for activation for 3min, then add the solid phase In the reaction column, react at room temperature for 2 hours. Wash 3 times with DMF, add 1046.3ml of blocking solution (pyridine / acetic anhydride = 1:1, 6mol:6mol) to block for 8 hours (if the resin is not fully diffused, add DCM as a solvent). Wash 4 times with DMF, 4 times with DCM, shrink and dry with methanol to obtain Fmoc-Asp(r...

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Abstract

The invention relates to a nonlinear solid phase synthesis method of thymosin alpha1. The method is characterized in that a coupling site is a side chain hydroxy group not a routine N amino end group of Ser during coupling of Ser-Ser fragments; and after a peptide chain is synthesized, ester bonds recombine under appropriate conditions to form an amide structure in order to obtain thymosin alpha1. Compared with traditional linear solid phase synthesis methods, the method provided by the invention has the advantages of maintenance of simple operation and easy post-treatment, and realization of high yield and god purity brought by reduction of the coupling difficulty due to beta-folding, alleviation of the steric hindrance, and provides a brand new idea for the large scale production of thymosin alpha1.

Description

technical field [0001] The present invention relates to a synthetic method of polypeptide, more specifically, relates to thymosin α 1 synthetic method. Background technique [0002] thymosin alpha 1 (Thymosinα 1 , hereafter referred to as Tα 1 ) is an acidic polypeptide isolated from bovine thymus preparation TF5 (Thymosin fraction5), its 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-OH [0004] Tα 1 The biological activity of bovine thymus preparation is higher than that of TF5. Specifically, Tα 1 It can promote the proliferation and maturation of T lymphocytes in the thymus, increase the secretion of IL-2, α and r interferon by lymphocytes, and enhance the expression of IL-2 receptors. In addition, Tα 1 It can enhance the number and activity of NK cells, and indirectly kill virus-infected liver cells and tumor cells through the proliferation of immune cells such...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/575C07K1/20C07K1/04
CPCC07K14/57581
Inventor 宓鹏程刘建马亚平袁建成
Owner HYBIO PHARMA
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