Protein O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is a highly dynamic and reversible monosaccharide post-translational modification in eukaryotic cells, playing critical roles in cellular regulation. To date, more than 5,000 O-GlcNAc-modified proteins have been identified, distributed in the cytoplasm, nucleus, mitochondria, and membraneless subcellular compartments. O-GlcNAcylation is catalyzed by O-GlcNAc transferase (OGT), which installs a single N-acetylglucosamine moiety onto serine and threonine residues, whereas removal of the modification is mediated by O-GlcNAcase (OGA). Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), the end product of the hexosamine biosynthetic pathway (HBP), serves as the sugar donor for this modification. This tightly coupled enzymatic cycle allows O-GlcNAcylation to act as a nutrient- and signal-responsive regulatory modification, linking metabolic state to protein function. O-GlcNAcylation regulates a broad range of fundamental cellular processes, including gene transcription, chromatin remodeling, epigenetic regulation, protein-protein interactions, and diverse intracellular signaling pathways. Dysregulation of global or protein-specific O-GlcNAcylation has been linked to the development and progression of multiple human diseases, including cancer, diabetes, and neurodegenerative disorders. With continuous advances in analytical methodologies, particularly high-resolution mass spectrometry and enrichment strategies, an increasing number of O-GlcNAc modification sites have been identified, allowing the functional roles of target proteins and their specific modification sites to be examined in detail. However, the highly dynamic nature of O-GlcNAcylation implies that chemical or genetic methods targeting OGT or OGA often lead to widespread changes in O-GlcNAc levels across thousands of proteins. Such global alterations complicate the functional interpretation of site-specific modifications and make it challenging to determine the precise role of O-GlcNAc at individual protein sites. Increasing evidence suggests that targeted modulation of specific O-GlcNAc sites on individual proteins provides improved mechanistic insight and greater translational relevance than global modulation approaches. Therefore, developing tools and strategies that allow site-specific modulation of O-GlcNAcylation on individual proteins, without altering overall O-GlcNAc levels, is critical. This review summarizes recent advances in targeted protein O-GlcNAcylation, highlights emerging chemical biology tools and strategies, and discusses their potential to reveal site-specific O-GlcNAc functions and their relevance to human disease.

Key words: O-GlcNAcylation, chemical biology, molecular tools, targeted regulation, site-specific modification