Glycosylation is a prevalent post-translational modification referring to the attachment of glycans or sugars to proteins. Glycosylation has significant impacts on a protein’s structure and function, and the glycans themselves can also be recognized by carbohydrate-binding proteins to enact new functions that may improve the efficacy and half-life of protein-based therapeutics. However, the mammalian glycan biosynthesis pathway produces extraordinarily diverse and heterogenous glycans, making glycan function and recognition challenging to characterize. My research focuses on the development of tools to probe glycan function, evaluate protein-glycan interactions, and leverage the known glycan specificities of carbohydrate-binding proteins for therapeutic indications and includes three major research projects. The first project was to evaluate the immunogenicity of natural N-glycans aiming to raise N-glycan specific antibodies. For the purpose, a series of N-glycan-based immunogens were prepared from five common human N-glycan structures and chemically conjugated to a bacteriophage Qβ carrier protein as an adjuvant, and the conjugates were used as immunogens for immunization in mice. Analysis of the immune response revealed that most of the antibodies elicited by all N-glycan conjugates unexpectedly targeted the conserved chitobiose core, giving cross-reactive antibodies. Importantly, terminal sialylation and linker chemistry were found to have significant effects on the titer and specificity of the elicited antibodies. This study outlines significant challenges to raising selective N-glycan-specific antibodies and provides important guidelines for the further optimization of N-glycan-based immunogens towards the development of selective N-glycan-specific monoclonal antibodies as probes for studying glycan functions. The second project was focused on application of catanionic vesicles as synthetic scaffolds for the multivalent display of N-glycans to probe protein-glycan interactions. A general platformwas developed allowing for multivalent display of various N-glycans on catanionic vesicles. It was found that the N-glycan-coated vesicles had high affinities for several plant and mammalian lectins. Furthermore, vesicles were prepared that displayed more than one glycan structure simultaneously. These well-defined vesicles were employed to recapitulate diverse glycan-coated surfaces as mimics of the mammalian glycocalyx and provided valuable insights into lectin recognition of glycan ligands in such complex environments. Indeed, for the vesicles displaying two unique N-glycan structures simultaneously, the presence of an unrelated glycan structure was found to significantly impact lectin binding to its cognate glycan ligand. Thus, N-glycan-coated catanionic vesicles have great potential as tools for characterizing complex protein-glycan interactions and elucidating glycan function. The third project explored the chemoenzymatic method developed by our lab for constructing site-specific antibody-glycan conjugates as next-generation Lysosome Targeting- Antibody Chimeras (LYTACs). These site-specific conjugates were used in evaluating optimal glycan ligands for targeted lysosomal degradation of clinically relevant protein targets. Several natural and synthetic glycan ligands containing terminal galactose or N-acetylgalactosamine (GalNAc) were attached to monoclonal antibodies and evaluated in cell-based binding and protein-degradation assays. Interesting new trends in glycan ligand binding were discovered, and natural triantennary N-glycans were reported for the first time to be effective ligands for lysosomal delivery of target proteins. Antibody conjugates containing synthetic tri-GalNAc or natural triantennary N-glycan ligands were found to significantly degrade extracellular human PCSK9, a well-validated therapeutic target for treating high cholesterol. Additional experiments indicated that targeted degradation of PCSK9 may be a promising new therapeutic strategy for lowering cholesterol, and this strategy could easily be adapted for the targeted degradation of other extracellular disease-associated proteins. In summary, these studies present methodologies for producing diverse glycoconjugates as valuable tools for elucidating glycan function and intervening in disease.