By differentiating the steric hindrance of the silyl protecting groups, one-pot iterative glycosylations have been conducted to construct trisaccharides 15, 17, and 20 (Scheme 3). Structure 17 is a precursor to the core 4 structure of mucin glycans, which are catabolized by various microbial enzymes to lead to a microbial ecological network. The n-pentenyl leaving group is also compatible with the glycosylation condition, which offers the opportunity for activation at a later stage to assemble more complex oligosaccharides. The yield for glycosylated product 17 was 40%, with the lower yield being attributed to scrambling of the n-pentenyl and fluoride groups on the two monosaccharide precursors prior to coupling. According to previous research conducted in our lab, having a smaller silyl protecting group could help reduce the anomeric scrambling; 16b however, when synthesizing the desired glycosyl acceptor 16, the TMS protecting group was too labile to withstand the further reaction conditions. Therefore, a TES protecting group was installed at the C3 position instead of a TMS protecting group. Trisaccharide 20 is a precursor to LNTri II, one of the core structures of human milk oligosaccharides, which play a crucial role in breast-fed infant health. The onepot glycosylation of 18 yielded 22% of the desired product. The low yield in this case results from the relatively slow reaction of the final glycosylation with 1. After installation of the glucose substrate at the C4 axial position, the C3 position then possesses a sterically crowded environment for the final coupling. Scheme 3. One-pot iterative Glycosylation of Trisaccharides.