肝素衍生六糖的分离与表征。通过高效液相色谱法分析及通过核磁共振氢谱解析结构。

Isolation and characterization of hexasaccharides derived from heparin. Analysis by HPLC and elucidation of structure by 1H NMR.

作者信息

Larnkjaer A, Hansen S H, Ostergaard P B

机构信息

Biopharmaceutical Division, Heparin Research Laboratory, Gentofte, Denmark.

出版信息

Carbohydr Res. 1995 Jan 3;266(1):37-52. doi: 10.1016/0008-6215(94)00247-d.

DOI:10.1016/0008-6215(94)00247-d

PMID:7697649

Abstract

Four hexasaccharides representing major structural sequences of heparin were isolated and characterized after degradation of heparin by heparinase. The structures were determined from two-dimensional 1H NMR spectroscopy including TOCSY (total correlated spectroscopy), COSY (correlated spectroscopy), and ROESY (rotating frame nuclear Overhauser enhancement spectroscopy) methods, providing new data on hexasaccharides. One of the hexasaccharides, the last eluting component from anion exchange chromatography, was derived from the tri-sulfated repeating disaccharide, alpha-L-idopyranosyluronic acid 2-sulfate-(1-->4)-2-amino-2-deoxy-D-glucopyranose 6,N-disulfate, and having the structure delta UAp2S-(1)-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L- IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L- IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S. The second hexasaccharide contained a nonsulfated D-glucuronic acid unit instead of the L-iduronic acid adjacent to the reducing end, and having the structure delta UAp2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L- IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-beta-D- GlcAp-(1-->4)-alpha-D-GlcNp2S6S. The last two hexasaccharides were obtained in lower yield and they have not been isolated and characterized before. The structure of the third saccharide corresponded to a trimer of the repeating disaccharide except for the lack of a 6-O-sulfate group at the reducing end glucosamine residue; deltaUAp2S-(1-->4)-alpha-D-Glcnp2S6S-(1-->4)-alpha-L- IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L-IdoAp2S -(1-->4)-alpha- D-GlcNp2S. The fourth and last hexasaccharide were less sulfated and the following structure was established delta UAp2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L- Idop2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L- IdoAp-(1-->4)-alpha-D-GlcNpAc6S. Analysis of the ROESY spectra revealed conformational difference of the glucosidic linkage alpha-L-IdoAp-(1-->4)-alpha-D-GlcNp between the hexasaccharides and longer heparin chains.

摘要

通过肝素酶降解肝素后，分离并鉴定了四种代表肝素主要结构序列的六糖。这些结构通过二维¹H NMR光谱法确定，包括全相关谱（TOCSY）、相关谱（COSY）和旋转框架核Overhauser增强谱（ROESY）方法，提供了关于六糖的新数据。其中一种六糖是阴离子交换色谱中最后洗脱的组分，它衍生自三硫酸化重复二糖，即2-硫酸-α-L-艾杜吡喃糖醛酸-(1→4)-2-氨基-2-脱氧-D-葡萄糖吡喃糖6,N-二硫酸酯，其结构为ΔUAp2S-(1→4)-α-D-GlcNp2S6S-(1→4)-α-L-IdoAp2S-(1→4)-α-D-GlcNp2S6S-(1→4)-α-L-IdoAp2S-(1→4)-α-D-GlcNp2S6S。第二种六糖在还原端相邻位置含有一个非硫酸化的D-葡萄糖醛酸单元而非L-艾杜糖醛酸，其结构为ΔUAp2S-(1→4)-α-D-GlcNp2S6S-(1→)-α-L-IdoAp2S-(1→4)-α-D-GlcNp2S6S-(1→4)-β-D-GlcAp-(1→4)-α-D-GlcNp2S6S。最后两种六糖的产率较低，之前未被分离和鉴定。第三种糖的结构对应于重复二糖的三聚体，但还原端葡糖胺残基缺少6-O-硫酸基团；ΔUAp2S-(1→4)-α-D-Glcnp2S6S-(1→4)-α-L-IdoAp2S-(1→4)-α-D-GlcNp2S6S-(1→4)-α-L-IdoAp2S-(1→4)-α-D-GlcNp2S。第四种也是最后一种六糖的硫酸化程度较低，确定的结构为ΔUAp2S-(1→4)-α-D-GlcNp2S6S-(1→4)-α-L-Idop2S-(1→4)-α-D-GlcNp2S6S-(1→4)-α-L-IdoAp-(1→4)-α-D-GlcNpAc6S。对ROESY光谱的分析揭示了六糖和更长肝素链之间α-L-IdoAp-(1→4)-α-D-GlcNp糖苷键的构象差异。