Efficient Syntheses of Complex-Type N-Glycans using Microflow Systems and Convergent Synthetic Routes

Abstract

N-Glycans are glycans attached to asparagine residues in proteins via post-translational modification reactions. They have diverse structures, based on which they control protein functions. We chemically synthesized N-glycans to elucidate their molecular bases to exert functions. In this study, we synthesized a core fucose-containing dodecasaccharide and a bisecting GlcNAc-containing octasaccharide. Our N-glycan syntheses were characterized by practical preparation of fragments using microflow reactions and efficient construction of the N-glycan backbone through convergent synthetic routes. First, α-sialylation, β-mannosylation, and N-glycosylation were examined in a microflow system. While these reactions require strict control of reaction conditions, desired glycosylated products could be reproducibly obtained in large quantities using a microflow system. Furthermore, our convergent strategy of assembling synthetic fragments into desired oligosaccharide backbone structures allowed the successful syntheses of N-glycans in short steps. The key in this strategy was how to achieve a satisfactory level of efficiency in glycosylation between less reactive large fragments. We showed that amide groups (NHAc) form intermolecular hydrogen bonds to reduce the reactivity and found that the glycosylation reactivity could be markedly improved by protecting them as imides (NAc₂). A high yield of the desired product could be achieved using an ether solvent for coordination stabilization of the intermediate cation even in otherwise poorly reactive glycosylation. In addition, we successfully improved stereoselectivity by carefully reviewing protection patterns.