Strategy for Isolation, Preparation, and Structural Analysis of Chondroitin Sulfate Oligosaccharides from Natural Sources
Strategy for Isolation, Preparation, and Structural Analysis of Chondroitin Sulfate Oligosaccharides from Natural Sources
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天然来源硫酸软骨素低聚糖的分离、制备和结构分析策略
DOI：
10.1021/acs.analchem.0c01410
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发表时间：
2020-09-01
影响因子：
7.4
通讯作者：
Wang, Zhongfu
中科院分区：
文献类型：
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作者：
Wei, Ming;Huang, Linjuan;Wang, Zhongfu
硫酸软骨素低聚糖(CSO)的结构，特别是其硫酸盐化模式，与许多生物途径和疾病密切相关。然而，详细的功能分析，如它们与糖链结合蛋白(GBP)的相互作用，一直滞后，可能是因为无法获得明确定义的、多样化的结构。除了对化学和酶法合成构成挑战外，这也是由于它们的不稳定性、结构复杂性和低质量谱检测灵敏度在异构体水平上的纯化和结构分析方面的挑战。本文首次采用循环制备高效液相色谱法在异构体水平上分离纯化了双功能荧光连接物2-氨基-N-2-氨基乙基苯甲酰胺(AEAB)标记的鲨鱼CS四糖组分。然后，每个异构体通过N-乙酰化、羧酰胺化、全甲基化和硅烷化试剂脱硫等多步步骤进行衍生化。用ESI-MSN在正离子模式下对每个衍生化异构体进行结构分析。共分离得到16个异构体，最小质量组分为0.007 mg，最大质量组分为17.53 mg，其中10个异构体进行了结构分析。这些CSO的制备和结构分析为进一步研究CSO构效关系奠定了基础。
The structure of chondroitin sulfate oligosaccharides (CSOs), especially their sulfation pattern, has been found to be closely related with many biological pathways and diseases. However, detailed functional analysis such as their interaction with glycan binding proteins (GBPs) has been lagging, presumably due to the unavailability of well-defined, diverse structures. Besides challenging chemical and enzymatic synthesis, this is also due to the challenges in their purification at the isomer level and structural analysis owing to their instability, structural complexity, and low mass spectrometry detection sensitivity. Herein, we first used recycling preparative HPLC to separate and purify shark CS tetrasaccharide component labeled by a bifunctional fluorescent linker 2-amino-N- 2-aminoethyObenzamide (AEAB) at the isomer level. Then, each isomer was derivatized through a multistage procedure including N-acetylation, carboxyl amidation, permethylation, and desulfation with silylating reagent. Structural analysis of each derivatized isomer was performed with ESI- MSn in positive ion mode. A total of 16 isomers of CSO-AEAB were isolated, with a minimum mass component of 0.007 mg and a maximum mass component of 17.53 mg, of which 10 isomers (>90 mu g) were structurally analyzed. This preparation and structure analysis of CSOs lay the foundation for further study of the structure-activity relationship of CSOs.