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Method for preparing swine O-type foot-and-mouth disease synthetic peptide antigen 2700 by solid-phase fragment method

A technology for foot-and-mouth disease and synthetic peptides, which is applied to peptide preparation methods, chemical instruments and methods, and peptides, and can solve problems such as incomplete core sequences, pollution, and unreachable yields.

Active Publication Date: 2017-05-17
SHANGHAI SHEN LIAN BIOMEDICAL CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although a lot of research has been done on most of the possible side reactions and their mechanisms in the past few years, the yield of a single-step cycle still cannot reach more than 99.5%, and the remaining 0.5% of each step accumulates as by-products of polluting products.
Therefore, the target peptide is easily contaminated by a series of compounds with similar structures and chemical properties, such as diastereoisomers formed by epimerization, wrong sequences and incomplete core sequences, etc.

Method used

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  • Method for preparing swine O-type foot-and-mouth disease synthetic peptide antigen 2700 by solid-phase fragment method
  • Method for preparing swine O-type foot-and-mouth disease synthetic peptide antigen 2700 by solid-phase fragment method
  • Method for preparing swine O-type foot-and-mouth disease synthetic peptide antigen 2700 by solid-phase fragment method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] This embodiment relates to the preparation of SEQ ID No: 3 modified by 9-fluorenylmethoxycarbonyl at the amino end, comprising the following steps:

[0069] (1) Preparation of Fmoc-Pro-2-chlororityl resin

[0070] Weigh 100g of 2-chlororityl resin (100-200 mesh, 1.1mmol / g, 110mmol) into the EST-50 polypeptide synthesizer, swell with 1L of DCM for 30min, filter to dryness, add 1L to dissolve 37.1g of Fmoc-Pro- OH (110mmol, 1.0eq) and 14.2g of DIEA (18.7ml, 110mmol) in DCM were reacted at 20-25°C for 60min. After filtering to dryness, add 1L of MeOH / DIEA (9:1) solution to carry out blocking reaction for 30min. After filtering, the resin was washed several times with NMP, MeOH, and NMP in turn, and then drained to obtain Fmoc-Pro-2-chlorotrityl resin. Take a small amount of resin and wash it several times with MeOH to measure the loading by removing Fmoc method, and the measured loading is 0.70mmol / g.

[0071] (2) Preparation of Fmoc-Leu-Pro-2-chlorotrityl resin

[0072...

Embodiment 2

[0078] This embodiment relates to the preparation of SEQ ID No: 4 modified by 9-fluorenylmethoxycarbonyl at the amino end, comprising the following steps:

[0079] (1) Preparation of Fmoc-Gly-2-chlororityl resin

[0080] Weigh 100g of 2-chlororityl resin (100-200 mesh, 1.1mmol / g, 110mmol) into the EST-50 peptide synthesizer, swell with 1L of DCM for 30min, filter to dryness, add 1L to dissolve 32.7g of Fmoc-Gly- OH (110mmol) and 14.2g of DIEA (18.7ml, 110mmol) in DCM were reacted at 20-25°C for 60min. After filtering to dryness, add 1L of MeOH / DIEA (9:1) solution for capping reaction for 30min. After filtering, the resin was washed several times with NMP, MeOH, and NMP in sequence, and then drained to obtain Fmoc-Gly-2-chlorotrityl resin. Take a small amount of resin and wash it several times with MeOH to measure the loading by removing Fmoc method, and the measured loading is 0.65mmol / g.

[0081] (2) Preparation of Fmoc-Arg(Pbf)-Gly-2-chlorotrityl resin

[0082] Add 1L of ...

Embodiment 3

[0088] This embodiment relates to the preparation of SEQ ID No: 5 modified by 9-fluorenylmethoxycarbonyl at the amino end, comprising the following steps:

[0089] (1) Preparation of Fmoc-Gly-2-chlororityl resin

[0090] Weigh 100g of 2-chlororityl resin (100-200 mesh, 1.1mmol / g, 110mmol) into the EST-50 polypeptide synthesizer, swell with 1L of DCM for 30min, filter to dryness, add 1L to dissolve 49.1g of Fmoc-Gly- OH (165mmol) and 14.2g of DIEA (21.3ml, 165mmol) in DCM were reacted at 20-25°C for 60min. After filtering to dryness, add 1L of MeOH / DIEA (9:1) solution for capping reaction for 30min. After filtering, the resin was washed several times with NMP, MeOH, and NMP in sequence, and then drained to obtain Fmoc-Gly-2-chlorotrityl resin. Take a small amount of resin and wash it several times with MeOH to measure the loading by removing Fmoc method, and the measured loading is 0.80mmol / g.

[0091] (2) Preparation of Fmoc-Asn(Trt)-Gly-2-chlorotrityl resin

[0092] Add 1L...

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Abstract

The invention provides a method for preparing a synthetic peptide antigen 2700 of a swine O-type foot and mouth disease through a solid-phase fragment process. The method comprises the steps of with resin as a starting raw material, sequentially connecting 9-fluorenylmethoxycarbonyl protective amino acid to prepare a protected peptide fragment, meanwhile sequentially removing a Fmoc group, carrying out linker reaction by using a condensing agent, and cutting off the protected peptide fragment by using dilute acid or weak acid; connecting the fragment and 4-(4'-dimethoxy-9-fluorenylmethoxyaminomethyl)-phenyloxymethyl resin step by step, and then, connecting the fragment and a T helper cell epitope; removing a protecting group and the resin by using concentrated acid or strong acid to obtain a crude peptide of the synthetic peptide antigen 2700; and purifying by using ion exchange resin and a tangential flow film packaging system, and removing small molecules and salt through a concentration system to obtain the synthetic peptide antigen 2700. The method provided by the invention has the characteristics of stable process, short production period, convenience in obtaining raw and auxiliary materials, few wastewater, waste gas and waste residues, low production cost, high yield and the like.

Description

technical field [0001] The invention relates to a method for preparing swine O-type foot-and-mouth disease synthetic peptide antigen 2700 by a solid-phase fragment method. Background technique [0002] The structural formula of pig type O foot-and-mouth disease synthetic peptide antigen 2700 is as follows: [0003] [0004] Pig foot-and-mouth disease is an acute, febrile, highly contagious infectious disease caused by foot-and-mouth disease virus. In recent years, foot-and-mouth disease has broken out in a large area in Jiangsu, Sichuan, the three northeastern provinces and Inner Mongolia, causing huge economic losses. Currently, immunization remains the main means of controlling the disease. The traditional inactivated vaccine is still the leading method for the prevention and treatment of foot-and-mouth disease at home and abroad, but it is easy to cause side effects such as allergies, and there are also problems in biosafety. Therefore, a lot of research has been do...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/09C07K1/36C07K1/34C07K1/18C07K1/06C07K1/04
CPCY02P20/55
Inventor 张震姬明放
Owner SHANGHAI SHEN LIAN BIOMEDICAL CORP
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