Ingrid 't Hart

103 Chemoenzyma�c synthesis of heptose-ganglioside mimics 5 Experimental procedures 5-Aminopentyl αNeu5Ac-(2 → 3)-β-D-Galactopyranosyl-(1 → 3)-L- glycero -α-D- manno heptopyranoside (2). PmST1 M144D (75 µL of a 2 mg/mL solu�on) was added to compound 1 (14 mg, 30.6 µmol, 10 mM final concentra�on) in H 2 O with CMP- Neu5Ac (11 mM), MgCl 2 (20 mM) and Tris buffer (100 mM, pH = 7.7). A�er 4 h the mixture was lyophilized overnight. The residue was redissolved in H 2 O and purified by Bio-Gel P2 size exclusion column chromatography to provide compound 2 (11.5 mg, 50 %). 1 H NMR (600 MHz, D 2 O) δ 4.88 (1H, s, H-1, Hep-I), 4.58 (1H, d, J = 7.8 Hz, H-1, Gal-II), 4.13 – 4.08 (2H, m, H-2, Hep-I), 4.06 – 3.47 (22H, m), 3.00 – 2.94 (2H, m, CH 2 , pentyl), 2.75 (1H, dd, J = 12.4, 4.6 Hz, H-3eq, Neu5Ac-III), 2.01 (3H, s, CH 3 , NHAc), 1.78 (1H, t, J = 12.1 Hz, H-3ax, Neu5Ac-III), 1.71 – 1.56 (4H, m, 2 x CH 2 , pentyl), 1.49 – 1.37 (2H, m, CH 2 , pentyl). ESI HRMS ( m/z ): [M + H] + calcd for C 29 H 52 N 2 O 20 ; 749.3186 found 749.3199 5-Aminopentyl 2-acetamido-2-deoxy-β-D-galactopyranosyl-(1 → 4)-[αNeu5Ac-(2 → 3)]- β-D-Galactopyranosyl-(1 → 3)-L- glycero -α-D- manno heptopyranoside (3). CgtA (fresh lysate: 250 µL) was added to compound 2 (4.1 mg, 5.5 µmol, 10 mM final concentra�on) in H 2 O with UDP-GalNAc (11 mM), MgCl 2 (20 mM) and Tris buffer (100 mM, pH = 7.7). A�er 19 h the mixture was lyophilized overnight. The residue was redissolved in H 2 O and purified by Bio-Gel P2 size exclusion chromatography to provide compound 3 (3.5 mg, 68 %). 1 H NMR (600 MHz, D 2 O) δ 4.88 (1H, s, H-1, Hep-I), 4.71 (1H, d, J = 8.5 Hz, H-1, GalNAc-IV), 4.57 (1H, d, J = 7.9 Hz, H-1, Gal-II), 4.15 (1H, d, J = 9.9 Hz, H-3, Gal-II), 4.10 (2H, s, H-4, Gal-II; H-2, Hep-I), 4.04 (1H, t, J = 6.5 Hz, H-6, Hep-I), 3.98 – 3.93 (2H, m, H-4, Hep-I; H-3, Hep-I), 3.92 – 3.56 (19H, m), 3.54 – 3.45 (2H, m, CHH, pentyl; H-6, Neu5Ac-III), 3.41 (1H, t, J = 8.8 Hz, H-2, Gal-II), 2.98 (2H, t, J = 7.6 Hz, CH 2 , pentyl), 2.66 (1H, dd, J = 12.6, 4.4 Hz, H-3eq, Neu5Ac-III), 2.01 (6H, s, 2 x CH 3 , NHAc), 1.90 (1H, t, J = 12.0 Hz, H-3ax, Neu5Ac-III), 1.72 – 1.57 (4H, m, 2 x CH 2 , pentyl), 1.50 – 1.36 (2H, m, 2 x CH 2 , pentyl). ESI HRMS ( m/z ): [M + H] + calcd for C 37 H 65 N 3 O 25 ; 952.3980 found 952.3962. 5-Aminopentyl β-D-Galactopyranosyl-(13)-2-acetamido-2-deoxy-β-D-galacto- pyranosyl-(1 → 4)-[αNeu5Ac-(2 → 3)]-β-D-Galactopyranosyl-(1 → 3)-L- glycero -α-D- manno heptopyranoside (4). CgtB (lysate: 50 µL) was added to compound 3 (1.2 mg, 1.3 µmol, 10 mM final concentra�on) in H 2 O with UDP-Gal (11 mM), MgCl 2 (20 mM) and Tris buffer (100 mM, pH = 7.7). A�er 20 h the mixture was lyophilized overnight. The residue was redissolved in H 2 O and purified by Bio- Gel P2 size exclusion chromatography to provide compound 4 (1.3 mg, 92 %). 1 H NMR (600 MHz, D 2 O) δ 4.91 (1H, s, H-1, Hep-I), 4.79 – 4.76 (1H, m, H-1, GalNAc-IV), 4.60 (1H, OH O O HO OH O HO HO HO HO O O NH 2 O HO OH HO HO AcHN COOH OH O O O OH O HO HO HO HO O O NH 2 O HO HO HO OH NHAc HOOC O HO OH HO AcHN OH O O O OH O HO HO HO HO O O NH 2 O HO HO HO OH NHAc HOOC O HO OH O AcHN O HO OH HO HO