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Synthetic method of polypeptide thymosin alpha1

A thymic method, solid-phase synthesis technology, applied in the preparation methods of peptides, chemical instruments and methods, peptides, etc., can solve the problems of low final yield and insufficient peptide purity.

Active Publication Date: 2011-09-28
HAINAN SHUANGCHENG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Although the solid-phase synthesis process is very mature, the final yield of amino acids of difficult-to-synthesize sequences may be very low (30%-45%), which may limit the scale-up of the process from an economic point of view, from the pharmaceutical industry The requirements for the purity of the peptide will lead to insufficient purity of the peptide

Method used

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  • Synthetic method of polypeptide thymosin alpha1
  • Synthetic method of polypeptide thymosin alpha1
  • Synthetic method of polypeptide thymosin alpha1

Examples

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preparation example Construction

[0033] In an example of the present invention, the new preparation method of the polypeptide solid-phase synthesis thymus method of the present invention includes the following steps:

[0034] In the first step, Fmoc-Asn(Trt)-OH and resin are bonded to obtain Fmoc-Asn(Trt)-resin; preferably using hydroxyl functional resin, more preferably PEG-Wang resin, the substitution rate of the PEG-Wang resin 0.2-0.8mmol / g;

[0035] In the second step, the deprotecting agent of the present invention is mixed with the Fmoc-Asn(Trt)-resin, and the Fmoc group is removed to obtain the Asn(Trt)-resin;

[0036] In the third step, Fmoc-Glu(OtBu)-OH and Asn(Trt)-resin are condensed to obtain Fmoc-Glu(OtBu)-Asn(Trt)-resin;

[0037] The fourth step, using the deprotecting agent of the present invention to remove the Fmoc group;

[0038] The 5th step, repeat above-mentioned peptide bond formation step, make peptide chain grow from C terminal to N terminal, until obtaining FmocSer(tBu)Asp(OtBu)AlaA...

Embodiment 1

[0061] Preparation of Fmoc-Asn(Trt)-PEG-King Resin

[0062] Soak 15g of PEG-King resin (substitution degree is 0.5-0.7mmol / g) in 150mlDMF for 30 minutes, drain, and 3.0 equivalents (19.33g, 32.4mmoles) of Fmoc-Asn(Trt)-OH, 4.8 equivalents (7.41 mL, 51.8mmol) of 2,6-dichlorobenzoylchloride and 8.4 equivalents (7.4mL, 90.7mmol) of DMF solution were mixed for esterification reaction for 3 hours. Then the resin was washed with DMF, and the resin was capped with 100 mL of 10% pyridine / DMF and 100 mL of 10% benzoyl chloride (benzoyl chloride) / DMF mixture for 1 hour. The resin was washed with DMF and methanol, dried in vacuum, and the degree of substitution was measured (the degree of substitution was 0.43mmol / g). See figure 1 .

[0063] Fmoc deprotection

[0064] Use 0.2MHOBt / 6%piperazine / DMF to remove Fmoc, and deprotect twice consecutively, the time is 10min and 20min respectively. Drain the deprotection solution and wash with DMF and methanol respectively. After exhaustion,...

Embodiment 2

[0080] Preparation of Fmoc-Asn(Trt)-PEG-King Resin

[0081] Soak 15g of PEG-King resin (substitution degree is 0.5-0.75mmol / g) in 150mlDMF for 30 minutes, drain, and 3.0 equivalents (19.33g, 32.4mmole) of Fmoc-Asn(Trt)-OH, 4.8 equivalents (7.41 mL, 51.8mmol) of 2,6-dichlorobenzoylchloride and 8.4 equivalents (7.4mL, 90.7mmol) of DMF solution were mixed for esterification reaction for 3 hours. Then the resin was washed with DMF, and capped with 100 mL of 10% pyridine / DMF and 100 mL of 10% benzoyl chloride / DMF mixture for 1 hour. The resin was washed with DMF and methanol, dried in vacuum, and the degree of substitution was measured.

[0082] Fmoc deprotection

[0083] Use 0.1MHOBt / 10%piperazine / DMF to remove Fmoc, and deprotect twice consecutively, the time is 10min and 20min respectively. Drain the deprotection solution and wash with DMF and methanol respectively. After exhaustion, Kaiser test was used to evaluate the removal of Fmoc.

[0084] Fmoc amino acid condensation...

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Abstract

The invention discloses a preparation method of thymosin alpha1 through solid phase polypeptide synthesis, comprising the steps of: (1) mixing Fmoc-Asn(Trt)-OH with hydroxyl functional resin, and performing esterification to the mixture so as to obtain Fmoc-Asn(Trt)- resin; (2) mixing Fmoc-Asn(Trt)- resin with a deprotection agent, thus obtaining Asn(Trt)- resin; (3) condensing Fmoc-Glu(OtBu)-OH and Asn(Trt)- resin, thus obtaining Fmoc-Glu(OtBu)-Asn(Trt)-resin; (4) according to solid phase synthesis method, repeating Fmoc removal in step (2) and condensation of amino acid and polypeptide on resin in step (3), and condensing amino acid from end C to end N in an order of Glu to Ser with polypeptide on resin, thus forming polypeptide resin as shown in formula II; and (5) separating the polypeptide on the polypeptide resin as shown in formula II and resin, thus obtaining thymosin alpha1 as shown in formula I.

Description

technical field [0001] The invention relates to the field of polypeptide solid-phase synthesis, in particular to a novel solid-phase synthesis method of the thymus method. Background technique [0002] Thymosin (also known as thymosin, thymopentin, Thymosin) is a group of physiologically active polypeptides secreted by thymus tissue. It can promote the transformation of lymphocytes, enhance the phagocytic activity of macrophages, and can be used to treat various immunodeficiency diseases. [0003] In the treatment of chronic hepatitis B with Thymosin α1 alone, studies have shown that the sustained response rate (ALT normalization + DNA negative conversion + HBeAg negative conversion) is about 37%, compared with single use of Thymosin α1 in the treatment of chronic hepatitis B Using interferon is similar, but without the side effects of interferon. [0004] The clinical research on the application of thymusfasin in the treatment of chronic hepatitis B in Taiwan, China showe...

Claims

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Application Information

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IPC IPC(8): C07K14/575C07K1/06C07K1/04A61P31/20
Inventor 袁剑琳张巍王仕银
Owner HAINAN SHUANGCHENG PHARMA
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