Complex mammalian glycans regulate diverse physiological and pathological processes by mediating interactions with glycan-binding proteins (GBPs). However, the structural complexity, diversity, and limited availability of well-defined glycans have restricted systematic studies of glycan structure-function relationships. This dissertation focuses on the development of chemoenzymatic strategies to access structurally defined complex mammalian glycans and their application in functional glycomics to elucidate GBP recognition. Chemoenzymatic synthesis has become the most powerful tool to access complex glycans, however, it still suffers from limited enzymes tools and methods to achieve site- and branch-specific glycan extensions. Chapter 1 focuses on site-selective glycan synthesis, describing an orthogonal-group-controlled strategy for site-selective I-branching of poly-N-acetyllactosamine (poly-LacNAc) chains. Poly-LacNAc serves as a common scaffold on mammalian glycoconjugates, and its β1-6 branching forms I-antigens associated with physiological and pathological processes including cancer progression. Because GCNT2-mediated I-branching lacks intrinsic site selectivity, three orthogonal capping groups were introduced onto internal galactose (Gal) residues and selectively removed to expose defined branching sites. This strategy enabled rapid access to a diverse library of linear and site-selectively branched i/I-antigens from a single precursor. Glycan microarray analysis revealed unique binding preferences of lectins, anti-i/I antibodies, and galectins toward specific I-branching patterns. Chapter 2 identifies novel enzyme activity of human ST6GalNAc enzymes, and presents an enzymatic synthesis strategy of disialyllacto-N-tetraose (DSLNT) and related human milk oligosaccharides (HMOs) containing the atypical Neu5Acα2-6GlcNAc linkage. By evaluating human ST6GalNAc enzymes, ST6GalNAc6 was identified as the most efficient and broadly tolerant sialyltransferase for constructing this atypital sialoside motif. Integration of ST6GalNAc6 with a set of specific enzymatic modules enabled the modular synthesis of DSLNT and structurally related Neu5Acα2-6GlcNAc-containing HMOs. These defined glycans were used to generate microarrays and probe their interactions with Siglecs. The results revealed broad but distinct recognition of the atypical Neu5Acα2-6GlcNAc motif by Siglecs, suggesting potential receptor-ligand relationships relevant to HMO-mediated immunomodulation and neonatal intestinal protection. Chapter 3 explores the broad substrate specificity of human sulfotransferase CHST1 for the efficient synthesis of sulfated O-glycans and gangliosides. By combining CHST1-mediated sulfation with glycosyltransferase- and sialyltransferase-catalyzed elongation, a panel of structurally defined sulfated glycans was constructed. These compounds provided valuable probes to investigate how sulfation patterns, glycan class, and terminal epitope regulate recognition by GBPs.