Divergent Enzymatic Assembly of a Comprehensive 64-Membered IgG N-Glycan Library for Functional Glycomics.

N-Glycosylation, a main post-translational modification of Immunoglobulin G (IgG), plays a significant role in modulating the immune functions of IgG. However, the precise function elucidation of IgG N-glycosylation remains impeded due to the obstacles in obtaining comprehensive and well-defined N-glycans. Here, an easy-to-implement divergent approach is described to synthesize a 64-membered IgG N-glycan library covering all possible biantennary and bisected N-glycans by reprogramming biosynthetic assembly lines based on the inherent branch selectivity and substrate specificity of enzymes. The unique binding specificities of 64 N-glycans with different proteins are deciphered by glycan microarray technology. This unprecedented collection of synthetic IgG N-glycans can serve as standards for N-glycan structure identification in complex biological samples and the microarray data enrich N-glycan glycomics to facilitate biomedical applications.