Reaction Steps
Neu5Acα2-3Galβ1-9Kdn was prepared first by following the general one-pot two-enzyme α2-3-sialylation system described above from Galβ1-9Kdn (101 mg, 0.22 mmol) in Tris-HCl buffer (100 mM, pH 8.5) containing Neu5Ac (109 mg, 0.33 mmol), CTP (188 mg, 0.33 mmol), MgCl2 (20 mM), NmCSS (0.8 mg), and PmST1 (0.04 mg). The reaction was incubated at 37° C. for 3 h with shaking (120 rpm). The crude product was purified by Bio-Gel P-2 gel filtration to afford the sialoside product Neu5Acα2-3Galβ1-9Kdn (148 mg, 89%). 1H NMR (600 MHz, D2O): δ 4.33 (d, 1H, J=7.8 Hz, H′-1), 4.00 (dd, 1H, J=1.8 and 10.8 Hz), 3.93 (dd, 1H, J=3.6 and 11.2 Hz), 3.82-3.65 (m, 9H), 3.60-3.39 (m, 9H), 2.58 (dd, 1H, J=4.2 and 12.6 Hz, H-3 eq″), 2.01 (dd, 1H, J=4.8 and 13.2 Hz, H-3 eq), 1.85 (s, 3H, CH3), 1.62 (t, 1H, J=12.0 Hz, H-3ax), 1.60 (t, 1H, J=12.0 Hz, H-3ax″). 13C NMR (75 MHz, D2O): δ 176.29, 175.17, 173.95, 103.28, 99.93, 96.35, 75.87, 75.07, 73.02, 71.93, 71.90, 71.86, 71.61, 70.44, 69.64, 69.50, 69.15, 68.48, 68.25, 67.97, 67.67, 62.75, 61.13, 59.50, 51.84, 39.82, 39.14, 22.22. HRMS (ESI) calcd. for C26H41NNaO22 (MNa), 742.2018, found 742.2001.
