GMMA Oxidation and Glycoconjugate Formation

Prior to oxidation and subsequent conjugation via reductive amination, S. typhimurium OAg-negative GMMA were concentrated at 10 mg/mL and exchanged in 100 mM acetate buffer pH 4.5. GMMA were oxidized with NaIO₄ (15 mM in the final reaction mixture) for 30 min at 25 °C, and then, the reaction was quenched with Na₂SO₃ at a final concentration of 30 mM, mixing for 15 min at RT. Reductive amination, between the previously derivatized polysaccharide with a dihydrazide linker (i.e., ADH, [36 ]) and reactive aldehydes of oxidized sugar residues of GMMA lipopolysaccharide (LPS) core, was conducted ON at RT with GAC/GMMA/NaCNBH₃ w/w/w ratio of 3:1:1. GAC-GMMA conjugate was then purified through centrifugal ultrafiltration using Amicon Ultra device with a membrane cut-off of 100 kDa (Merck, Darmstadt, Germany), and removal of unconjugated polysaccharide was verified via HPLC-SEC analysis with respect to a calibration curve of purified unconjugated polysaccharide (TSK gel G3000 PWXL column, refractive index detector). Total protein quantification in GAC-GMMA conjugate was estimated by micro-BCA analysis, while GAC amount estimation was based on Rha quantification through HPAEC-PAD analysis [37 (link),38 (link)], as GlcNAc is also present in the LPS outer core of S. typhimurium OAg-negative GMMA. Conjugate size distribution was determined by DLS [39 (link)].