Solid-phase synthesis method of artificial E selectin

A solid-phase synthesis and selectin technology, which is applied in the preparation methods of peptides, chemical instruments and methods, animal/human proteins, etc., to achieve the effects of short production cycle, low production cost and high yield

Active Publication Date: 2011-11-16
张世明
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, although the primary structure of the 23-30 residue oligopeptide of E-selectin is: H-YTHLVALQ-NH 2 ; In the prior art, the oligopept

Method used

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  • Solid-phase synthesis method of artificial E selectin
  • Solid-phase synthesis method of artificial E selectin
  • Solid-phase synthesis method of artificial E selectin

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0051] Example 1E-selectin (YTHLVAIQ-NH 2 ) Preparation

[0052] (1) Raw material preparation and identification:

[0053] Rink Amide-MBHA Resin: Product NO.: HCRAm04-1-1, Lot NO.: GRMH0706, Specification: 0.42mmol / g, 100~200mesh, 1% DVB, Manufacturer: Hecheng (Tianj ing Nankai Hecheng Sci&Tech.Co .Ltd.).

[0054] Protected amino acid raw materials were purchased from Applied Biosysterms and Gill Biochemical in the United States, and these raw materials need to be identified. The identification results are shown in Table 1. The purchased raw materials meet the requirements.

[0055] Table 1 Determination results of protected amino acid raw materials

[0056]

[0057] DMF treatment: 3A molecular sieve immersion, FDNB (2,4-nitrofluorobenzene) OD≤0.15. (The DMF used below are all processed DMF).

[0058] Hexahydropyridine treatment: re-evaporate.

[0059] (2) Specific synthesis steps:

[0060] 1) Synthetic Fmoc-Q Trt -Resin:

[0061] Fmoc removal step: Weigh 5g (0.42mmol / g) of Rink Amide-MB...

Example Embodiment

[0095] Example 2E-selectin (YTHLVAIQ-NH 2 ) Preparation

[0096] The material preparation is similar to Example 1, E-selectin (YTHLVAIQ-NH 2 The preparation of) is completed on the multiplex peptide synthesizer. The peptide reaction is carried out at 30°C for 2 to 3 hours. The final yield was 67.33%.

Example Embodiment

[0097] Example 3E-selectin (YTHLVAIQ-NH 2 ) Purification

[0098] The prepared crude peptide was subjected to column chromatography with TSK 40S (D=2.5cm, H=85cm, V=240ml), 1N HAC containing 5% acetonitrile as the eluent, flow rate 1.5ml / min, and the recovery rate was 50%. HPLC purification conditions: sample amount: 100mg, dissolved in 50% acetic acid containing 5% acetonitrile; Buffer: 1N acetic acid containing 5% acetonitrile; flow rate 1.5ml / min; detection wavelength 280nm; there are three peaks P1, P2, P3 , Collect P2, freeze-dried to obtain a soft solid. Such as figure 1 , The purity is 97%. TLC thin-plate chromatography conditions (DCM:MeOH:HAC=90:8:2) used for purification. HPLC high performance liquid chromatography, C18 column, Buffer A 0.1% TFA, Buffer B contains 0.1% TFA 100% acetonitrile, elution gradient: 0~5mins, 0% Buffer B; 5~35mins, 40%~100% Buffer B; 35~45mins, 100% Buffer B; 45~50mins, 100% buffer A.

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Abstract

The invention discloses a solid-phase synthesis method of artificial E selectin, characterized by comprising the following steps of: (1) taking RinkAmideResin protected by fluorenylmethyloxycarbonyl as a carrier, sequentially connecting Fmoc-Gln (Trt)-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-His (Trt)-OH, Fmoc-Thr (tBu)-OH and Fmoc-Tyr (otBu) according to a solid-phase polypeptide synthesis method to obtain peptide resin; (2) removing the protection of fluorenylmethyloxycarbonyl from the peptide resin, cutting off the peptide from the peptide resin by a peptide cutting reagent to obtain the artificial E selectin crude product; and (3) separating and purifying the artificial E selectin crude product by high performance liquid column chromatography to obtain the artificial E selectin. The process is relatively stable and has the advantages of easy availability of raw materials, short production cycle, low production cost, high yield and even quality.

Description

technical field [0001] The invention belongs to the technical field of pharmacy, and in particular relates to a method for solid-phase synthesis of artificial E-selectin. Background technique [0002] Adhesion molecules refer to a class of molecules that are produced by cells, exist on the surface of cells, and mediate the contact and binding between cells and cells or between cells and substrates. Adhesion molecules are mostly glycoproteins, and a few are glycolipids, which are distributed on the cell surface or in the extracellular matrix (ECM). So far, there are more than one hundred kinds of cell adhesion molecules that have been discovered. During cerebral ischemia-reperfusion, there are three groups of cell adhesion molecules involved: (1) integrin family: all integrin Both subunits contain CD11 and CD18. Representative integrins are integrin 1, integrin 2, lymphocyte function-associated antigen 1 and membrane lytic attachment complex 1. (2) Immunoglubilin family: A...

Claims

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Application Information

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IPC IPC(8): C07K14/705C07K1/06C07K1/04
Inventor 张世明
Owner 张世明
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