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期刊名称:药物分析杂志
主管单位:中国科学技术协会
主办单位:中国药学会
承办:中国食品药品检定研究院
主编:金少鸿
地址:北京天坛西里2号
邮政编码:100050
电话:010-67012819,67058427
电子邮箱:ywfx@nicpbp.org.cn
国际标准刊号:ISSN 0254-1793
国内统一刊号:CN 11-2224/R
邮发代号:2-237
 

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3位硫酸化的肝素四糖与其同分异构体的结构与性质的比较分析

Structure and characteristic analysis of heparin-derived 3-O-sulfated tetrasaccharides and its isomer

作者(英文):
分类号:R917
出版年·卷·期(页码):2018,38 (12):2054-2067
DOI: 10.16155/j.0254-1793.2017.01.01
-----摘要:-------------------------------------------------------------------------------------------

目的:比较分析一种3位硫酸化的肝素四糖及其同分异构体的结构与性质。方法:以抗凝血药物肝素为原料,采用肝素酶Ⅱ对肝素进行完全酶解,完全酶解后,酶解产物通过凝胶渗透色谱将不同聚合度的寡糖片段分开,并采用强阴离子交换高效液相色谱以pH为3.5的2 mol·L-1氯化钠溶液和去离子水为流动相,采用线性洗脱,流速4 mL·min-1,检测波长232 nm,根据不同肝素寡糖的电荷强度和电荷密度对寡糖进行进一步分离纯化,获得一种对肝素酶具有抵抗作用的特殊肝素四糖和它的同分异构体。采用电喷雾傅立叶变换离子回旋共振高分辨质谱结合核磁共振波谱来确定肝素四糖的结构序列。并采用竞争型表面等离子体共振(SPR)的方法研究肝素四糖与抗凝血酶Ⅲ(AT Ⅲ)的结合作用。结果:肝素四糖的结构序列为ΔUA-GlcNAc6S-GlcUA-GlcNS3S和ΔUA-GlcNAc6S-GlcUA-GlcNS6S。通过特征肝素四糖的比较分析表明高分辨质谱在分析肝素来源结构相似修饰位点多变的寡糖结构上具有优势,并获得了肝素同分异构体不同硫酸化修饰位点核磁共振波谱的变化规律,总结了3位硫酸化肝素四糖典型的核磁特征。这对肝素四糖结构相似,但是ΔUA-GlcNAc6S-GlcUA-GlcNS3S的离子强度要略高于ΔUA-GlcNAc6S-GlcUA-GlcNS6S。然而二者均没有结合ATⅢ的能力,不具有抗凝血活性。结论:通过高分辨质谱和核磁共振波谱可以将肝素酶解产物中3位硫酸化的肝素四糖与其他四糖区别开来,肝素四糖不具有抗凝血活性。

-----英文摘要:---------------------------------------------------------------------------------------

Objective: To analyze the structures and characteristics of heparin-derived 3-O-sulfated tetrasaccharide and its isomers. Methods: After being treated with heparin lyase Ⅱ exhaustively,a Bio-Gel P2 size exclusion column and semi-preparative strong anion exchange (SAX) -HPLC were carried out to purify the products. Having been purified by Bio-Gel P2 size exclusion column based on the degree of polymerization,SAX-HPLC with an optimized gradient elution was used to obtain the individual tetrasaccharide based on the ion strength and charge density. The mobile phases were water and 2 mol·L-1 NaCl with pH at 3.5 at a flow rate of 4 mL·min-1. Continuous UV detection was performed at 232 nm. A couple of tetrasaccharides isomers were obtained. ESI-FT-ICR MS and NMR were used to elucidate the structures of the tetrasaccharides. The ability of the two tetrasaccharides binding with antithrombinⅢ(ATⅢ) was shown with competition surface plasmon resonance (SPR).Results: The structures of the two isomers were ΔUA-GlcNAc6S-GlcUA-GlcNS3S and ΔUA-GlcNAc6S-GlcUA-GlcNS6S. The results showed that the high resolution mass spectra had the advantage to determine the structure of heparin oligosaccharides. The comparative analysis of NMR spectrum revealed the variation of the two isomers with different modifications. The topic signal of 3-O-sulfo heparin was characterized. The two isomers had similar structures,but the ionic strength of ΔUA-GlcNAc6S-GlcUA-GlcNS3S was a little stronger than ΔUA-GlcNAc6S-GlcUA-GlcNS6S. The result of competition surface plasmon resonance (SPR) showed the two tetrasaccharides couldn't bind with ATⅢ. Conclusion: MS and NMR could discriminate 3-O-sulfo heparin tetrasaccharides from other similar products. Heparin tetrasaccharides couldn't bind with AT Ⅲ.

-----参考文献:---------------------------------------------------------------------------------------
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