Concept of Future Glycoprotein Drugs: Synthesis of a Thioglycosidically Linked α-N-Acetylgalactosamine-Carrying Cyclic Peptide as a Model of Miniature Macrophage Activating Factor

Glycoproteins are often considered as drug candidates. However, the regulation of post-translational glycan attachment remains an issue. We hypothesized that replacing the oxygen atom in the glycosidic linkage with sulfur atoms would stabilize the labile linkage against glycosidases, resulting in improved pharmacokinetics. In this study, we focused on the macrophage-activating factor (MAF) carrying O-linked N-acetylgalactosamine (GalNAc) and creating a miniature glycopeptide associated with MAF. A partial structure of MAF with a chemical mutation at three amino acid residues was designed in which threonine was replaced with cysteine (Cys), leading to a thioglycosidically linked GalNAc and a conformationally stable cyclic peptide due to the disulfide bond. GalNAc-Cys was used in solid-phase peptide synthesis, and the desired cyclic glycopeptide was synthesized. In the synthesis of GalNAc-Cys, glycosylation reactions were carried out based on the hard and soft acids and bases concept, where glycosyl trichloroacetimidate and fluoride were successfully used to couple with the thiol group in Cys. GalNAc-Cys was also evaluated as a substrate of α-GalNAc-ase and was shown to resist hydrolysis, supporting our concept. The synthesized cyclic miniature MAF induced LPS-assisted IL-12 production and resisted against α-GalNAc-ase.