The rare N-unsubstituted glucosamine (GlcNH 3
+ ) residues in heparan sulfate (HS) have important biological and pathophysiological roles. Therefore, the ability to chemically generate a series of oligosaccharides, which have a similar structure to the naturally-occurring, GlcNH 3
+ - containing oligosaccharides from HS, would greatly contribute to investigating their natural role in HS. In this study, a hexasaccharide library that possess GlcNH 3
+ residues were prepared from the chemical modification of the fully sulfated dp6. Chemical reaction conditions were optimized to generate different pattern of GlcNH 3
+ - containing oligosaccharides, then the structure of the library was detected by high-performance liquid chromatography-ion trap/time-of-flight mass spectrometry (LC/MS-ITTOF) analysis. EIC/MS and MS 2 analysis showed different fragmentation patterns of dp6s with different GlcNH 3
+ residues. This provides a foundation for further identification and quantification of GlcNH 3
+ - oligosaccharides by mass spectrum analysis.
The O -sulfation, including 2- O - and 6- O -sulfation, in heparan sulfate (HS) have important biological and pathophysiological roles. Therefore, the ability to chemically generate a series of oligosaccharides , which have a similar structure to the naturally-occurring, 2- O - and 6- O -sulfating oligosaccharides from HS, would greatly contribute to investigating their natural role in HS. In this study, a heparin oligosaccharide library, including dp2, dp4 and dp6, were prepared from the chemical modification of the fully sulfated dp2, dp4 and dp6. Chemical reaction conditions were optimized to generate different patterns of 2- O - and 6- O -sulfated oligosaccharides, then the disaccharide composition and structure of the library was detected by high-performance liquid chromatography-ion trap/time-of-flight mass spectrometry (LC-IT-TOF/MS) analysis. This provides a foundation for further structural and functional studies of O -sulfated groups in HS.
Carbohydrates are among the most essential biomolecules for human survival, and their synthesis has garnered considerable research attention. Enzymatic synthesis provides significant advantages by addressing the challenges in controlling stereoselectivity and regioselectivity. However, broader application is hindered by labor-intensive purification procedures and substantial product loss during repeated vessel transfers. The implementation of solid-phase strategies in enzymatic carbohydrate synthesis reduces material transfer losses and streamlines downstream purification. Nevertheless, integrating enzymatic reactions with solid-phase systems presents significant compatibility issues, such as steric hindrance and restricted substrate accessibility, which diminish enzymatic efficiency and reduce productivity. To address these limitations, we developed a universal and efficient platform termed SWITCH (support-based workflow integrated temperature-sensitive enzymatic carbohydrate synthesis). This platform utilizes a temperature-responsive polymer as the support, where substrates are immobilized via click chemistry. This design enables glycan synthesis under homogeneous aqueous conditions, enhancing enzymatic accessibility and catalytic activity. Upon heating, the polymer undergoes a phase transition and precipitates from solution, enabling product isolation by filtration without organic solvents. On this platform, by integrating diverse enzyme modules to construct distinct glycosylation pathways, we successfully built a library of structurally defined glycans, including human milk oligosaccharides and glycosaminoglycans. This platform streamlines precise glycan synthesis and establishes a foundation for advancing glycoscience and biopharmaceutical applications.
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