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.