4.5. Acid Hydrolysis and Determination of Absolute Configurations of Monosaccharides

Acid hydrolysis of compound 2 (1.4 mg) was carried out in a solution of 2 M trifluoroacetic acid (TFA, 750 μL) in a sealed vial using a water bath at 100 ^°C for 2 h. The H₂O layer was washed with n-hexane (3 × 750 μL) and concentrated under reduced pressure. One drop of concentrated TFA and 500 μL of R-(–)-2-octanol (Sigma Aldrich) were added to the obtained mixture in the sealed vial and then heated on a glycerol bath at 130 °C for 6 h. The solution was evaporated using a Smart Evaporator (BioChromato, Japan) and the resulting products were treated with a mixture of pyridine/acetic anhydride (1:1, 400 μL) for 24 h at room temperature. The acetylated 2-octylglycosides were purified by silica gel (2 × 0.5 cm) flash chromatography in 100% MeOH and then analyzed by GC–MS using the corresponding authentic samples prepared by the same procedure (Figures S23–S27) [30]. The retention times (t_R) and EIMS spectra showed for the components of the hydrolysate corresponded to those of the authentic samples and were as follows:

D-glucose: t_R 28.05 min, m/z (%): 354 (2), 331 (97), 315 (13), 289 (4), 259 (4), 242 (17), 229 (11), 215 (9), 203 (27), 169 (98), 157 (45), 143 (100), 127 (42), 112 (67), 98 (99), 81 (42), 71 (46), 57 (54); t_R 28.51 min, m/z (%):354 (2), 331 (13), 289 (5), 271 (2), 242 (43), 229 (9), 215 (7), 200 (55), 182 (9), 169 (58), 157 (100), 140 (57), 127 (18), 115 (96), 98 (95), 81 (32), 71 (25), 57 (34); t_R 28.80 min, m/z (%): 354 (2), 331 (36), 289 (3), 271 (5), 242 (42), 229 (8), 200 (45), 182 (9), 169 (100), 157 (89), 140 (46), 127 (21), 115 (87), 98 (74), 81 (24), 71 (25), 57 (33) and t_R 29.13 min, m/z (%):354 (2), 331 (100), 315 (9), 289 (2), 259 (2), 242 (11), 227 (9), 215 (5), 203 (16), 183 (12), 169 (71), 157 (27), 143 (60), 127 (23), 115 (46), 98 (62), 81 (25), 71 (36), 57 (40);

N-acetyl-D-glucosamine: t_R 31.51 min, m/z (%): 355 (4), 330 (8), 318 (3), 288 (4), 259 (5), 241 (52), 226 (10), 210 (10), 199 (38), 181 (40), 168 (20), 156 (66), 139 (72), 126 (22), 114 (100), 96 (49), 84 (27), 72 (20), 57 (18).