Ingrid 't Hart

51 Chemoenzyma�c synthesis of DSGb5 2 5-aminopentyl αNeu5Ac-(2 → 3)-β-D-Galactopyranosyl-(1 → 3)-[ αNeu5Ac-(2 → 6)]-2- acetamido-2-deoxy-β-D-galactopyranosyl-(1 → 3)-α-D-galactopyranosyl-(1 → 4)-β-D- galactopyranosyl-(14)-β-D-glucopyra-noside (6b). ST6GalNAc5 and CIAP were added to a mixture of compound 5b (1.0 mg, 10 mM final concentration) 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 4 h and monitored by TLC (EA:MeOH:H 2 O:HOAc 3:3:3:2). Purifica�on by Bio-Gel P-2 size exclusion chromatography provided compound 6b (0.7 mg, 57%). 1 H NMR (600 MHz, D 2 O) δ 4.92 (1H, d, J = 4.0 Hz, H-1, Gal-III), 4.69 (1H, d, J = 8.5 Hz, H-1, GalNAc-IV), 4.55 – 4.49 (3H, m, H-1, Gal-II; H-1, Gal-V; H-1, Glc-I), 4.40 (1H, t, J = 6.5 Hz, H-5, Gal-III), 4.28 (1H, d, J = 2.7 Hz, H-4, Gal-III), 4.20 (1H, d, J = 3.0 Hz, H-4, GalNAc-IV), 4.11 – 4.04 (3H, m, H-2, GalNAc- IV; H-3, Gal-V, H-4, Gal-II), 4.03 – 3.53 (39H, m), 3.31 (1H, t, J = 8.5 Hz, H-2, Glc-I), 3.06 – 2.99 (2H, m, CH 2 , pentyl), 2.80 – 2.70 (2H, m, H-3eq, Neu5Ac-VI; H-3eq, Neu5Ac-VII), 2.07 – 2.00 (9H, m, 3x NHAc), 1.79 (1H, t, J = 12.1 Hz, H-3ax, Neu5Ac-VI), 1.75 – 1.63 (5H, m, 2x CH 2 , pentyl; H-3ax, Neu5Ac-VII ), 1.51 – 1.43 (2H, m, CH 2 , pentyl). ESI HRMS ( m/z ): [M + H] + calcd for C 59 H 100 N 4 O 42 , 1537.5812; found 1537.5874. Microarray Experimental procedures The synthe�c glycans (100 µM in sodium phosphate (250 mM), pH 8.5 buffer) were printed on ac�vated glass slides (Nexterion Slide H, Scho� Inc) by piezoelectric non- contact prin�ng (sciFLEXARRAYER S3, Scienion Inc) with a drop volume of ~400 pL and 1 drop per spot at 50 % rela�ve humidity. The compounds were printed as replicates of 6 with on each slide 24 subarrays (3x8). The slides were incubated overnight in a saturated NaCl chamber (providing a 75% rela�ve humidity environment), a�er which the remaining ac�vated esters were quenched with ethanolamine (50 mM) in TRIS (100 mM), pH 9.0. Slides were rinsed with DI water, dried by centrifuga�on, and stored in a desiccator at RT. Sub-arrays were incubated with bio�nylated lec�ns ( Maackia amurensis leukagglu�nin (MAL-II), Soybean agglu�nin (SBA) and Wheat Germ agglu�nin (WGA); from Vector Labs) at 10 µg/mL premixed with Streptavidin-AlexaFluor635 (5 µg/mL; ThermoFisher Scien�fic, S32364) in TSM binding buffer (20 mM Tris Cl, pH 7.4, 150 mM NaCl, 2 mM CaCl 2 , 2 mM MgCl 2, 0.05% Tween, 1% BSA) for 1 h followed by washing. Wash steps involved 4 successive washes with each 5 min soak �me with 1) TSM wash buffer (20 mM Tris Cl, pH 7.4, 150 mM NaCl, 2 mM CaCl 2 , 2 mM MgCl 2, 0.05% Tween-20); 2) TSM buffer (20 mM Tris Cl, pH 7.4, 150 mM NaCl, 2 mM CaCl 2 , 2 mMMgCl 2 ); 3) deionized H 2 O; and 4) deionized H 2 O. 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 O NH 2