Liquid-Phase and Ultrahigh-Frequency-Acoustofluidics-Based Solid-Phase Synthesis of Biotin-Tagged 6′/3′-Sialyl-N-Acetylglucosamine by Sequential One-Pot Multienzyme System

6′/3′-Sialylated N-acetyllactosamine (6′/3′-SLN) is important for discrimination of the source (human or avian) of influenza virus strains. Biotinylated oligosaccharides have been widely used for analysis and quick detection. The development of efficient strategies to synthesize biotin-tagged 6′/3′-SLN have become necessary. Effective mixing is essential for enzymatic solid-phase oligosaccharide synthesis (SPOS). In the current study, newly developed technology ultrahigh-frequency-acoustofluidics (UHFA), which can provide a powerful source for efficient microfluidic mixing, solid-phase oligosaccharide synthesis and one-pot multienzyme (OPME) system, were used to develop a new strategy for oligosaccharide synthesis. Firstly, biotinylated N-acetylglucosamine was designed and chemically synthesized through traditional approaches. Secondly, biotinylated 6′- and 3′-sialyl-N-acetylglucosamines were prepared in solution through two sequential OPME modules in with a yield of ~95%. Thirdly, 6′-SLN was also prepared through UHFA-based enzymatic solid-phase synthesis on magnetic beads with a yield of 64.4%. The current strategy would be potentially used for synthesis of functional oligosaccharides.