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Preparation method for thymosin [alpha]1 and analogues thereof

An analog, thymosin technology, applied in the field of solid-phase synthesis of thymosin α1 and its analogs, can solve the problems of inability to use industrialized production, low purity, low solid-phase synthesis yield, etc. Low yield, the effect of increasing yield and purity

Inactive Publication Date: 2013-08-14
CHANGCHUN BEYEL PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Comprehensively referring to domestic and foreign patents on the preparation of thymosin α1 and related synthesis reports, it is found that it mainly has the disadvantages of low solid-phase synthesis yield and low purity in technology, so it cannot be used in industrial production

Method used

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  • Preparation method for thymosin [alpha]1 and analogues thereof
  • Preparation method for thymosin [alpha]1 and analogues thereof
  • Preparation method for thymosin [alpha]1 and analogues thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] The preparation of embodiment 1 Thymosin α1 thick peptide

[0071] 1. Preparation of Fmoc-Asp (Tricyclic amide linker resin)-OtBu

[0072] Fmoc-Tricyclic amide linker resin 8.772g (Bachem) with a substitution degree of 0.57mmol / g was added to the solid phase reactor, 100ml of DCM was added to swell the resin for 60 minutes, and washed twice with 20% DBLK (5 minutes + 25 min) to remove the Fmoc protection to obtain a Tricyclic amide linker resin with an N-terminal amino group, which was washed twice with DMF, twice with DCM, and twice with DMF.

[0073] Carry out coupling reaction: Fmoc-Asp-OtBu 8.230g, HOBt 3.04g, dissolved in 40ml (DMF / DCM=3 / 1) solution, ice bath for 10-20 minutes to reduce the temperature to about -5 °C, and then to 6.25ml (DIC / DCM=1 / 1) solution was slowly added dropwise to the solution, and activated at low temperature (minus 5-10°C) for 30 minutes, then the solution was added to the above reactor and reacted at room temperature for 1.5 hours. Th...

Embodiment 2

[0123] Synthesis of Example 2 Thymosin α1 Analogs

[0124] Tricyclic amide linker resin resin was applied to the solid-phase synthesis of thymosin α1 analogs, the sequence is as follows

[0125] Ac-Ser-β-Asp-Ala-Ala-Val-β-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val- Val-Glu-Glu-Ala-Glu-Asn-OH

[0126] 1. Preparation of Fmoc-Asp(Tricyclic amide linker resin)-OtBu

[0127] Add 8.772g of Fmoc-Tricyclic amide linker resin, with a substitution degree of 0.57mmol / g, into the solid-phase reactor, add DCM 100ml to swell the resin for 60 minutes, and use 20% DBLK (5+25)min twice to remove the Fmoc protection , get N 2 H-Tricyclic amide linker resin, wash 2 times with DMF, 2 times with DCM, 2 times with DMF.

[0128] Dissolve 8.230g of Fmoc-Asp-OtBu and 3.04g of HOBt in 40ml (DMF / DCM=3 / 1), put it in an ice bath for 10-20 minutes to lower the temperature to about -5°C, and then slowly add it dropwise into the solution 6.25ml (DIC / DCM=1 / 1) solution was added...

Embodiment 3

[0147] Synthesis of Example 3 Thymosin α1 Analogs

[0148] Tricyclic amide linker resin was applied in solid-phase synthesis of thymosin α1 analogs (thymosin α1 N-terminal unacetylated), the sequence is as follows:

[0149] H-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

[0150] 1. Preparation of Fmoc-Asp (Tricyclic amide linker resin)-OtBu

[0151] Fmoc-Tricyclic amide linker resin 8.772g, with a substitution degree of 0.57mmol / g, was added to the solid phase reactor, 100ml of DCM was added to swell the resin for 60 minutes, and the Fmoc protection was removed twice with 20% DBLK (5+25)min , get N 2 H-Tricyclic amide linker resin, washed 2 times with DMF, 2 times with DCM, and 2 times with DMF.

[0152] Dissolve Fmoc-Asp-OtBu 8.230g, HOBt 3.04g in 40ml (DMF / DCM=3 / 1), ice bath for 10-20 minutes to reduce the temperature to about -5°C, then slowly add dropwise to the solution 6.25ml (DIC / DCM=1 / 1) solu...

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Abstract

The invention discloses a solid phase synthesis and purification process for thymosin [alpha]1 and analogues thereof, and belongs to the technical field of peptide synthesis. The process comprises the following steps of 1) taking a tricyclic amide linker resin as a carrier; 2) connecting side chain carboxyl groups of Fmoc-Asp-Y to amino groups of the resin after the resin is subjected to deprotection of Fmoc; 3) synthesizing residual amino groups in order in a solid phase by using a Fmoc strategy; 4) removing Fmoc protection groups from N-terminal amino protection groups, acetylating the N-terminal amino protection groups by using a DMF solution of Ac2O and DIPEA; 5) cutting by using a lysis reagent to obtain a crude peptide and precipitating the crude peptide with ether; and 6) separating the crude peptide by HPLC to obtain a pure product.

Description

technical field [0001] The invention belongs to the technical field of solid-phase synthesis and purification of polypeptides, and specifically relates to a solid-phase synthesis process of Tricyclic amide linker resin resin and polypeptides whose C-terminal is Asn, especially thymosin α1 and its analogs. Background technique [0002] Thymosin alpha1 (Thymosin alpha1, Tα1) is isolated and purified from thymosin fraction 5 (TF5), an acidic polypeptide consisting of 28 amino acid residues, free of methionine, cysteine ​​and aromatic amino acids, relatively The molecular mass is 3108, the pI value is 4.2, the N-terminal acetylation has no significant effect on the activity, and its sequence is 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 [0003] Thymosin α1 is a small molecule polypeptide with strong immune function secreted by the thymus. In in vitro and in vivo experiments, thymosin α1 affects the sec...

Claims

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Application Information

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IPC IPC(8): C07K14/575C07K1/06C07K1/04
CPCY02P20/55
Inventor 王丽凤殷玉和李纪龙代丽萍
Owner CHANGCHUN BEYEL PHARMA
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