Synthesis of Biotinylated Sialylated Glycans

Biotinylated glycans were prepared as previously described^{21 (link)}. Briefly, the poly-LacNAc chains were obtained by the enzymatic extension of glycan cores with H. pylori β1,3-GlcNAcT and recombinant N. meningitidis β4galT-galE fusion. Subsequent sialylation of the poly-LacNAc chains was achieved by using either recombinant human ST6GAL1 or rat ST3GAL3. The resultant sialosides were treated with NHS-LCLC-biotin (#21343, Thermo scientific) in the presence of DIPEA (#496219, Sigma-Aldrich) to provide the desired biotinylated linear or N-linked sialosides. Synthesized compounds were confirmed by high-resolution mass spectrometer (HRMS) as follows: 6SLN1-N, ESI TOF-HRMS m/z calculated for C110H183N12O68S, [M + 3H]³⁺: 931.0328, found 931.0348; 6SLN2-N, ESI TOF-HRMS m/z calculated for C138H229N14O88S, [M + 3H]³⁺: 1174.4543, found 1174.4567; 6SLN3-N, ESI TOF-HRMS m/z calculated for C166H276N16O108S, [M + 3H]³⁺: 1418.2117, found 1418.2285; 3SLN3-L, ESI TOF-HRMS m/z calculated for C77H131N9O43S, [M + 2H]²⁺: 1901.8061, found 1901.7952.