Ingrid 't Hart

50 Chapter 2 2 5-aminopentyl αNeu5Ac-(2 → 3)-β-D-Galactopyranosyl-(1 → 3)-2-acetamido-2-deoxy- β-D-galacto-pyranosyl-(1 → 3)-α-D-galactopyranosyl-(1 → 4)-β-D-galactopyranosyl- (1 → 4)-β-D-glucopyranose (5b). ST3Gal1 and CIAP were added to compound 4b (2.0 mg, 10 mM final concentra�on) in H 2 O with CMP-Neu5Ac (15 mM), MgCl 2 (20 mM), sodium cacodylate buffer (50 mM, pH 7.5). The mixture was shaken at 37°C for 24 h and monitored by TLC (EA:MeOH:H 2 O:HOAc 4:4:3.3:2). Purifica�on by Bio-Gel P-2 size exclusion chromatography provided compound 5b (1.8 mg, 69%). 1 H NMR (600 MHz, D 2 O) δ 4.92 (1H, d, J = 3.9 Hz, H-1, Gal-III), 4.71 (1H, d, J = 8.5 Hz, H-1, GalNAc-IV), 4.56 – 4.49 (3H, m, H-1 Gal-V; H-1, Gal-II; H-1, Glc-I), 4.39 (1H, t, J = 6.4 Hz, H-5, Gal-III), 4.26 (1H, d, J = 2.4 Hz, H-4, Gal-III), 4.19 (1H, d, J = 2.8 Hz, H-4, GalNAc-IV), 4.13 – 3.48 (34H, m), 3.36 – 3.27 (1H, m, H-2, Glc-I), 3.02 (2H, t, J = 7.5 Hz, CH 2 ), 2.76 (2H, dd, J = 12.4, 4.6 Hz, H-3eq, Neu5Ac-VI), 2.04 (6H, s, 2x NHAc), 1.79 (1H, t, J = 12.1 Hz, H-3ax, Neu5Ac-VI), 1.75 – 1.65 (4H, m, 2x CH 2 , pentyl), 1.52 – 1.43 (2H, m, CH 2 , pentyl). ESI HRMS (m/z): [M + H] + calcd for C 48 H 83 N 3 O 34 , 1246.4858; found 1246.4970. αNeu5Ac-(2 → 3)-β-D-Galactopyranosyl-(1 → 3)-[αNeu5Ac-(2 → 6)]-2-acetamido-2- deoxy-β-D-galacto-pyranosyl-(1 → 3)-α-D-galactopyranosyl-(1 → 4)-β-D-galactopyranos- yl-(1 → 4)-α/β-D-glucopyranose (6a). ST6GalNAc5 and CIAP were added to a mixture of compound 5a (4.2 mg, 10 mM final concentra�on) in H 2 O with CMP-Neu5Ac (15 mM), MgCl 2 (20 mM), sodium cacodylate buffer (50 mM, pH 7.5). The mixture was shaken at 37°C overnight and monitored by TLC (EA:MeOH:H 2 O:HOAc 3:3:3:2). More enzymes were added un�l no more star�ng material could be observed. Purifica�on by Bio-Gel P-2 size exclusion chromatography and semi-preprara�ve HILIC column (XBridge® Amide 5 µm, 4.6 mm x 250 mm column, Waters, 70% B isocra�c) provided compound 6a (2.64 mg, 50 % ). 1 H NMR (750 MHz, D 2 O) δ 5.24 (0.5H, d, J = 3.7 Hz, H-1α, Glc-I, 4.93 (1H, d, J = 3.8 Hz, H-1, Gal-III), 4.70 – 4.65 (1.5H, m, H-1β, Glc-I; H-1, GalNAc-IV), 4.54 – 4.50 (2H, m, H-1, Gal-II, H-1, Gal-V), 4.43 – 4.39 (1H, m, H-5, Gal-III), 4.28 (1H, s, H-4, Gal-III), 4.20 (1H, d, J = 3.0 Hz, H-4, GalNAc-IV), 4.10 – 4.04 (3H, m, H-2, GalNAc-IV; H-3, Gal-V), 4.02 – 3.57 (36.5H, m), 3.55 (1H, dd, J = 9.6, 8.1 Hz, H-2, Gal-V), 3.29 (0.5H, dd, J = 9.0, 8.2 Hz, H-2, Glc-I), 2.79 – 2.70 (2H, m, H-3eq, Neu5Ac-VI; H-3eq, Neu5Ac-VII), 2.07 – 2.01 (9H, m, 3x NHAc), 1.80 (1H, t, J = 12.2 Hz, H-3ax, Neu5Ac-VI), 1.66 (1H, t, J = 12.2 Hz, H-3ax, Neu5Ac-VII). ESI HRMS (m/z): [M + Na] + calcd for C 54 H 89 N 3 O 42 , 1474.481 O HO OH O AcHN O HO OH O OH O HO OH O HO O O OH HO OH O O OH HO OH O HO OH HO HO AcHN COOH O NH 2 O HO O O AcHN O HO OH O OH O HO OH O HO O O OH HO OH O O OH HO OH O HO OH HO HO AcHN COOH O HO OH HO HO AcHN COOH OH