含N-非取代葡糖胺残基肝素四糖的制备与表征

生物体内含有N-非取代葡糖胺残基（GlcNH₃⁺）结构的硫酸类肝素（HS）具有重要的生物和病理生理学功能。但这种HS在生物体内的含量较少、获得困难，而采用化学方法制备与生物体内结构相似的这种寡糖，有助于研究HS在生物体内的功能作用。本实验以高硫的肝素四糖为原料，用部分脱N位硫酸根的方法，制备了含1个和2个GlcNH₃⁺的肝素四糖，并采用液相色谱-离子阱-飞行时间质谱（LC/MS-IT-TOF）法对其进行结构检测。通过分析（EIC）-MS和MS²提取离子流图发现，含不同GlcNH₃⁺数目的肝素四糖具有不同的裂解规律，含GlcNH₃⁺数目越多，生成的碎片离子越多，这为MS方法进一步鉴定和定量测定含GlcNH₃⁺结构的寡糖奠定了基础。

Preparation and Characterization of Heparin Tetrasaccharide with N-unsubstituted Glucosamine Residues

The rare GlcNH₃⁺ residues implicated in important cellbiological and pathophysiological phenomena are also now particularly hot subjects in the pharmaceutical industry. Recently its tumour invasion and metastasis has raised interest. However, it is difficult to prepare naturally-occuring, because of their low abundance in GlcNH₃⁺ containing oligosaccharides from HS. Therefore, the ability to chemically generate a series of structurally-defined oligosaccharides containing GlcNH₃⁺ residues would greatly contribute to investigating their natural role in HS. In this study, heparin tetrasaccharides (dp4s) possessing one and two GlcNH₃⁺ residues were prepared from partially de N-sulfation of the fully sulfated dp4. Then the structure of dp4s was further detected by liquid chromatography-ion trap/time-of-flight mass spectrometry (LC/MS-IT-TOF). Two dp4s (dp4-1 and dp4-2) are obtained, in which dp4-1 with one GlcNH₃⁺ residue located in the central position, while dp4-2 with two GlcNH₃⁺ residues located in the central and reducing terminal positions.The results of extracted ion chromatogram (EIC) in LC/MS-IT-TOF system suggest that minor sulfate losses of the dp4s appear in MS ion source, but most of the samples maintain the original structure, which is helpful for determining the accurate molecular weight of heparin/HS oligosaccharides with GlcNH₃⁺ residues. MS and MS² analysis show different fragmentation patterns of dp4s with different GlcNH₃⁺ residues using the same MS parameters. ^0,2X₂ fragmentation near the non-reducing end of oligosaccharide was only detected in dp4-1. Meanwhile, ^1,5X₂, ^2,4A₄ and ^1,5A₄ fragmentations were only appeared in dp4-2. The dp4 with higher GlcNH₃⁺ residues revealed more cross-ring cleavage patterns, suggesting that higher positive charges might make the ring more fragile, presumably reflecting the markedly different conformations and chemical environments at these positions, which results in different dissociation of dp4s in MS. This provides a foundation for further structural identification and quantification of GlcNH₃⁺ oligosaccharides by mass spectrum analysis, which could lead to a greater understanding of the biological roles of GlcNH₃⁺ residues in HS/heparin.