Ganglioside glycans are ubiquitous and complex biomolecules that are involved in a wide range of biological functions and disease processes. Variations in sialylation and sulfation render the structural complexity and diversity of ganglioside glycans, and influence protein–carbohydrate interactions. Structural and functional insights into the biological roles of these glycans are impeded due to the limited accessibility of well-defined structures. Here we report an integrated chemoenzymatic strategy for expeditious and systematic synthesis of a comprehensive 65-membered ganglioside glycan library covering all possible patterns of sulfation and sialylation. This strategy relies on the streamlined modular assembly of three common sialylated precursors by highly stereoselective iterative sialylation, modular site-specific sulfation through flexible orthogonal protecting-group manipulations and enzymatic-catalysed diversification using three sialyltransferase modules and a galactosidase module. These diverse ganglioside glycans enable exploration into their structure–function relationships using high-throughput glycan microarray technology, which reveals that different patterns of sulfation and sialylation on these glycans mediate their unique binding specificities.

神经节苷脂聚糖是普遍存在的复杂生物分子，参与广泛的生物功能和疾病过程。唾液酸化和硫酸化的变化导致神经节苷脂聚糖的结构复杂性和多样性，并影响蛋白质-碳水化合物相互作用。由于明确结构的可及性有限，对这些聚糖的生物学作用的结构和功能了解受到阻碍。在这里，我们报告了一种综合化学酶策略，用于快速、系统地合成综合的 65 元神经节苷脂聚糖库，涵盖所有可能的硫酸化和唾液酸化模式。该策略依赖于通过高度立体选择性迭代唾液酸化对三种常见唾液酸化前体进行简化的模块化组装，通过灵活的正交保护基操作进行模块化位点特异性硫酸化，以及使用三个唾液酸转移酶模块和一个半乳糖苷酶模块进行酶催化多样化。这些不同的神经节苷脂聚糖使得能够利用高通量聚糖微阵列技术探索它们的结构-功能关系，该技术揭示了这些聚糖上不同的硫酸化和唾液酸化模式介导了它们独特的结合特异性。