Ingrid 't Hart

49 Chemoenzyma�c synthesis of DSGb5 2 recombined via BP clonase reac�on into the pDONR221 vector and the DNA sequences were confirmed. The pDONR221 clone was then recombined via LR clonase reac�on into a custom Gateway adapted version of the pGEn2 mammalian expression vector 14, 39, 41 to assemble a recombinant coding region comprised of a 25 amino acid NH 2 -terminal signal sequence from the T. cruzi lysosomal α-mannosidase 42 followed by an 8xHis tag, 17 amino acid AviTag, 43 “superfolder” GFP, 44 the nine amino acid sequence encoded by a�B1 recombina�on site, followed by the TEV protease cleavage site and the respec�ve glycosyltransferase cataly�c domain coding region. Suspension culture HEK293 cells (Freestyle 293-F cells, Life Technologies, Grand Island, NY) were transfected as previously described 14, 39 and the culture supernatant was subjected to Ni-NTA superflow chromatography (Qiagen, Valencia, CA). Enzyme prepara�ons eluted with 300 mM imidazole were concentrated to ~1 mg mL -1 using an ultrafiltra�on pressure cell membrane (Millipore, Billerica, MA) with a 10 kDa molecular weight cutoff. Table S2. Enzyme expression details. 14 Enzyme Amino Acid Residues Uniprot ID ST3GAL1 52 - 340 Q11201 ST6GALNAC5 50 - 336 Q9BVH7 ST6GALNAC6 31 - 333 Q969X2 Experimental procedures for enzyma�c synthesis αNeu5Ac-(2 → 3)-β-D-Galactopyranosyl-(1 → 3)-2-acetamido-2-deoxy-β-D- galactopyranosyl-(1 → 3)-α-D-galactopyranosyl-(1 → 4)-β-D-galactopyranosyl-(14)-α/ β-D-glucopyranose (5a). ST3Gal1 and CIAP were added to compound 4a (6.5 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 94 h andmonitored by TLC (EA:MeOH:H 2 O:HOAc 4:3:2:1). 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 5a (4.57 mg, 53 %). 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 = 3.9 Hz, H-1, Gal-III), 4.70 (1H, d, J = 8.5 Hz, H-1, GalNAc- IV), 4.68 (0.5H, d, J = 8.0 Hz, H-1β, Glc-I), 4.55 – 4.51 (2H, m, H-1, Gal-V; H-1, Gal-II), 4.41 – 4.37 (1H, m, H-5, Gal-III), 4.26 (1H, s, H-4, Gal-III), 4.19 (1H, d, J = 3.0 Hz, H-4, GalNAc-IV), 4.08 (2H, dd, J = 9.9, 3.2 Hz, H-2, GalNAc-IV; H-3, Gal-V), 4.05 (1H, d, J = 2.9 Hz, H-4, Gal-II), 4.01 – 3.57 (29.5H, m), 3.55 (1H, dd, H-2, Gal-V), 3.29 (0.5H, t, J = 8.6 Hz, H-2, Glc-I), 2.76 (1H, 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). ESI HRMS (m/z): [M + Na] + calcd for C 43 H 72 N 2 O 34 , 1183.3864; found 1183.3861. 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 OH