Development of Fluorinated Substrate-Based Inhibitors and Prodrugs for Fucosyltransferases and Sialyltransferases

Glycosylation, the enzymatic attachment of sugars to proteins and lipids, is a fundamental post-translational modification that regulates numerous biological processes, including cell signaling, immune responses, and tumor progression. This process is mediated by glycosyltransferases, among which fucosyltransferases (FUTs) and sialyltransferases (STs) play key roles in shaping cell-surface glycan structures. Dysregulated fucosylation and sialylation are associated with a variety of diseases, yet the development of selective and cell-compatible inhibitors for these enzymes remains challenging due to complex biosynthetic pathways, feedback regulation, and limitations of existing inhibitors. This thesis focuses on the design, synthesis, and biological evaluation of fluorinated nucleotide-sugar analogues and their corresponding prodrugs as chemical tools to modulate FUT and ST activity. A central objective is to overcome limitations of commonly used inhibitors, such as slow enzymatic turnover and unwanted incorporation into glycans, while achieving precise control over cellular glycosylation. An improved fucosyltransferase inhibitor, GDP-2,2-di-F-Fuc, and its cell-permeable prodrugs were developed to address these challenges. Unlike previously used mono-fluorinated analogues, this compound exhibits strong competitive inhibition without detectable enzymatic transfer. In cellular systems, its prodrug effectively reduces intracellular GDP-fucose levels and enables dose-dependent suppression of cell-surface fucosylation, providing controlled modulation of glycan structures. To further explore enzyme selectivity, a series of C-6-modified GDP-2-F-Fuc analogues were synthesized to investigate structure–activity relationships among human FUT isoforms. Several compounds displayed enhanced potency and selectivity in enzymatic assays, offering insights into molecular features governing FUT recognition, although cellular activity was limited for most derivatives. In addition, this work investigates selective inhibition of sialyltransferases through the design of 9-modified CMP-3-fluoro-sialic acid analogues. Enzymatic studies revealed that specific C-9 modifications can bias inhibition toward particular ST isoforms, demonstrating a rational strategy for achieving isoform selectivity. However, limited metabolic conversion in cells constrained their cellular efficacy. Overall, this thesis provides new chemical tools and mechanistic insights for modulating fucosylation and sialylation. While not all compounds exhibit strong cellular activity, the results establish design principles for developing selective, non-transferable, and cell-compatible glycosyltransferase inhibitors, contributing to the broader understanding of glycosylation control in chemical biology and disease-related research.