The activated OAg was designated OAg-oxNaIO4. For conjugation to CRM197, OAg-oxNaIO4 was added to CRM197 in NaH2PO4 100 mM pH 7.2 to give a final concentration of 10 and 5 mg/mL, respectively. NaBH3CN was added immediately after (OAg-oxNaIO4:NaBH3CN = 1:1 w/w),

and the reaction mixture stirred overnight at 37 °C. After this time, NaBH4 (OAg-oxNaIO4:NaBH4 = 1:1 w/w) was added and the mixture was stirred at 37 °C for 2 h. The conjugate was designated OAg-oxNaIO4-CRM197. OAg-oxTEMPO-CRM197: random activation of the OAg chain with TEMPO and conjugation to CRM197. OAg (3 mg/mL, corresponding to [CH2OH] of 7.69 mM) and NaHCO3 (molar ratio NaHCO3/CH2OH = 30), were added to a stirred solution of TEMPO (molar ratio TEMPO/CH2OH = 0.05) in DMF. The reaction was cooled Selleck PD98059 to 0 °C and TCC (molar ratio TCC/CH2OH = 1.6) was added. The activated sugar was recovered from the reaction mixture by precipitation with EtOH (85 v/v% in the final mixture) after 2 h of stirring at 0 °C. The pellet was washed twice with 100% EtOH (1.5 volumes with respect to the reaction mixture volume) and lyophilized. The activated OAg was designated OAg-oxTEMPO2h. The same procedure was used for the synthesis of OAg-oxTEMPO12h, increasing the reaction time to 12 h. OAg-oxTEMPO2 h

and OAg-oxTEMPO12h were conjugated to CRM197, using the same conditions for OAg-oxNaIO4. The two corresponding conjugates were designated Z-VAD-FMK supplier OAg-oxTEMPO2h-CRM197 and OAg-oxTEMPO12h-CRM197, respectively. OAg-ADH-SIDEA-CRM197: selective

activation of the terminal KDO with ADH, followed by reaction with SIDEA and conjugation to CRM197. The synthesis of this conjugate was performed as previously Florfenicol described [28] and detailed in SI. OAg-NH2-SIDEA-CRM197: selective activation of the terminal KDO with NH4OAc, followed by reaction with SIDEA and conjugation to CRM197. OAg was solubilized in 500 mM NH4OAc pH 7.0 at a concentration of 40 mg/mL. NaBH3CN was added immediately (NaBH3CN:OAg = 2:5 w/w). The solution was mixed at 30 °C for 5 days. The reaction mixture was desalted on a G-25 column and the OAg-NH2 was dried. The following steps of conjugation were performed as for OAg-ADH-SIDEA-CRM197 and the resulting conjugate was designed OAg-NH2-SIDEA-CRM197. All conjugates were purified by hydrophobic interaction chromatography (HIC) on a Phenyl HP column [GE Healthcare], loading 500 μg of protein for mL of resin in 50 mM NaH2PO4 3 M NaCl pH 7.2. The purified conjugate was eluted in water and the collected fractions were dialyzed against 10 mM NaH2PO4 pH 7.2. Total saccharide was quantified by phenol sulfuric assay [29], protein content by micro BCA (using BSA as standard and following manufacturer’s instructions [Thermo Scientifics]) and the ratio of saccharide to protein calculated. OAg-CRM197 conjugates profiles were compared with free CRM197 by HPLC-SEC and SDS-PAGE (see SI).