Conception et synthèse de dérivés du ganglioside GM3 en tant que candidats vaccins anticancéreux

Glycosylation is a prevalent post-translational modification of cell-surface proteins and lipids, essential for cellular recognition, signaling, and immune modulation. In cancer, glycosylation patterns are often dysregulated, leading to the overexpression of tumor-associated carbohydrate antigens (TACAs). These aberrant structures provide targets for synthetic vaccine development, aiming to induce immune memory and improve tumor recognition. Advances in glycan synthesis have significantly enhanced the design and immunogenicity of carbohydrate-based cancer vaccines. Ganglioside GM3 (Neu5Ac-α2,3-Gal-β1,4-Glc-β1-ceramide) is widely distributed in neuronal and epithelial membranes and regulates cell growth, adhesion, apoptosis, and signaling. Notably, GM3 is overexpressed in various tumors, including melanoma, breast cancer, and lung cancer. It is also one of the nine TACAs that have been progressed into clinical trials by the FDA. In our previous studies, a series of GM3 analogues were synthesized by replacing the glycosidic units excluding sialic acid. These non-natural glycan structures demonstrated promising anti-tumor activities by in vitro assays. In this study, a series of allyl glycosides (glucose, galactose, mannose, and lactose) were synthesized. Selective protection strategies were applied to expose either the C-3 or C-6 hydroxyl groups to serve as acceptors for sialylation. The key glycosylation step employed sialyl xanthate as glycosylation donor to afford six products in good yields (41~65%). Side reactions arising from allyl electrophilic addition were minimized by careful control of promoter addition. All new compounds were purified by chromatography and fully characterized using high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) spectroscopy. Subsequently, the allyl glycosides were converted to their corresponding glycolaldehydes via ozonolysis and conjugated to carrier protein BSA and KLH. The conjugation degree was determined using MALDI-TOF-MS or specific colorimetric assays. Notably, even among structural isomers, significant differences in conjugation efficiency were observed. To further investigate this phenomenon, α- and β-glycolaldehyde glycosides of five biologically relevant monosaccharides (Man, Glc, Gal, GlcNAc, and GalNAc) were synthesized and conjugated to proteins at a fixed sugar-to-protein molar ratio. Continuous kinetic monitoring revealed that anomeric configuration markedly influences both reaction rates and final sugar loading: α-Man > β-Man; β-Gal/GalNAc > their α-counterparts; Glc and GlcNAc showed no significant anomeric differences. NMR analysis identified cyclic intermediates that reduce the effective concentration of reactive aldehyde, while DFT calculations provided complementary insight into anomer-specific electrophilic character. This work presents a systematic study on the synthesis of GM3 derivatives and the conjugation of glycolaldehyde to protein, providing valuable information for design, synthesis, and conjugation of GM3 analogues and their vaccine application.