Project Summary/Abstract The proposed project aims to address the critical need for a comprehensive library of structurally defined sulfated O-GalNAc glycans to advance our understanding of their biological functions in human health and diseases. Despite their widespread occurrence and potential importance in health and disease, our understanding of the precise functions of these glycans remains limited, primarily due to the lack of access to a comprehensive library of well-defined synthetic sulfated O-GalNAc glycans and associated toolkits for functional glycomics. This proposal aims to synthesize sulfated O-GalNAc glycans using Sulfated Modular Synthesis / Enzymatic Assembly (SMSEA) approach. The SMSEA method stands out as it offers a cost-effective convergent modular synthesis backed by well-established methodologies. Subsequently, glycosyltransferases are employed to expand the core, utilizing diverse biosynthesis pathways. This results in the creation of complex, larger, and highly diverse sulfated O-GalNAc glycans. The use of Fmoc-protected threonine as a terminal linker enables synthetic glycans for versatile research applications, including glycoconjugates and glycopeptides. By synthesizing 38 unique sulfated O-GalNAc glycans, including specific types such as the 6-sulfo sialyl Lewis X structure, this library will act as an invaluable toolkit for functional glycomics. Furthermore, the project also innovates in glycan microarray technology, developing coating chemistry tailored to the multivalent presentation of sulfated O-GalNAc glycans on the array surface. This paves the way for the debut of a first-ever sulfated O-GalNAc glycan microarray, establishing another invaluable toolkit for functional glycomics. This project holds significant promise to advance our understanding of the glycobiology of sulfated O-GalNAc glycans.