Modular Enzymatic Construction of Glycan Libraries via SWITCH: A Solid-Phase Platform with Temperature-Controlled Phase Transition.

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.