Solvolysis of EpsA-O

Solvolysis was performed by adding TFA (1.2 mL) to 16.4 mg of EpsA-O previously sonicated as reported above. The solution was stirred at 40 °C for 1h⁷⁹. The sample was dried under a nitrogen flux and washed three times with anhydrous methanol to remove TFA. The newly formed aldehydic functions were reduced with aqueous sodium borohydride for 16 h at RT. The reduction was then stopped with acetic acid and the sample was taken to dryness by evaporation under reduced pressure. The procedure was repeated three times after adding 10% acetic acid in methanol and three times with methanol. The dried sample was dissolved in 0.15 M NaCl and subjected to middle-pressure size-exclusion chromatography on the Bioline system previously described, using a Sephacryl S-300 HR column (1 cm id x 45 cm). Elution was carried out with 0.15 M NaCl at 1 mL/min flow rate. The selection of fractions to be pooled together was obtained by applying a fitting procedure using a Gaussian function and hypothesizing two main peaks, the degraded polysaccharide, and salt. According to the fitting curves obtained, (Supplementary Fig. 9a), test tubes were pooled in three fractions, named SL1, SL2, and SL3, the latter being mainly salt. Fractions SL1 and SL2 were then desalted using a Superdex G30 prep grade column (1 cm i.d. × 82 cm) with water as eluent at 1.5 mL/min flow rate, recovered by lyophilization, and dissolved in 10 mM, pH = 7 sodium phosphate buffer, blown with N₂ to prevent degradation of QuiNAc4NAc. ¹H NMR analysis showed that fraction SL1 was less heterogeneous than SL2 and thus it was chosen for thorough NMR investigation (Supplementary Fig. 9b).