Ingrid 't Hart

69 Synthesis of globo-series glycolipids 3 (CV) of wash 1, 10 CV of wash 2, HiLgtD was eluted in 3 CV of elu�on buffer. The eluate was concentrated using a Vivaspin 6 filter and further purified on a PBS equilibrated Superdex 200 Increase 10/300 GL column a�ached to a Shimadzu Nexera system using a flow of 0.45 ml/min collec�ng frac�ons each minute. Frac�ons containing HiLgtD were pooled, concentrated, aliquoted and stored at -20°C. Further characteriza�on of the enzyme was done by SurePage 4-12% gel, using Laemmli sample buffer and the Precision Plus Protein marker as reference. Experimental procedures (2S,3R,4E)-2-amino-3-hydroxy-octadec-4-enyl α-D-galactopyranosyl-(1->4)-β-D- galacto-pyranosyl-(1 → 4)-β-D-glucopyranoside (1a). Compound 1a (Gb3Sph) was synthesized as described before . 49 (2S,3R,4E)-2-amino-3-hydroxy-octadec-4-enyl 2-acetamido-2-deoxy-β-D- galactopyranosyl-(1 → 3)-α-D-galactopyranosyl-(1 → 4)-β-D-galactopyranosyl-(1 → 4)- β-D-glucopyranoside (2a). LgtD (100 µL, 0.5 mg) was added to a mixture of compound 1a (5.0 mg, 10 mM final concentra�on) in H 2 O with UDP-GalNAc (3.9 mg, 10 mM), MgCl 2 (20 mM), Tris buffer (100 mM, pH = 7.7). The mixture was shaken at 37 °C overnight and monitored by Maldi-TOF. Purifica�on by bench top C4 column using MeOH:H 2 O (1:0 to 0:1 v/v) as the eluent provided a mixture of Gb3Sph/Gb4Sph. The glycolipid mixture was resubmi�ed as described above. This was repeated un�l no more star�ng material was seen a�er C4 purifica�on. (compound 2a (Gb4Sph), 1.4 mg, 22 %). 1 H NMR (600 MHz, MeOD) δ 5.82 – 5.72 (1H, m, CH=C H -CH 2 ), 5.55 – 5.44 (1H, m, CHOH-C H =CH), 4.94 (1H, d, J = 3.9 Hz, H-1, Gal-III), 4.62 (1H, d, J = 8.0 Hz, H-1, GalNAc-IV), 4.44 – 4.38 (1H, m, H-1, Gal-II), 4.33 – 4.29 (1H, m, H-1, Glc-I), 4.27 (1H, t, J = 6.2 Hz, H-5, Gal-III), 4.17 (1H, d, J = 2.2 Hz, H-4, Gal-III), 4.03 (1H, t, J = 6.7 Hz, CH=CH-C H OH), 4.00 – 3.48 (22H, m), 3.44 – 3.38 (1H, m), 3.36 – 3.25 (1H, m, H-2, Glc-I), 3.01 – 2.94 (1H, m, H C-NH 2 , Sph), 2.13 – 2.05 (2H, m, CH=CH-C H 2 ), 2.01 (3H, s, CH 3 , NHAc), 1.47 – 1.24 (22H, m, 11 x CH 2 , Sph), 0.90 (3H, t, J = 7.0 Hz, CH 3 , Sph). ESI HRMS ( m/z ): [M + H] + calcd for C 44 H 80 N 2 O 22 ; 988.5203 found 989.5283. O HO OH HO HO O O OH HO OH O O OH HO HO NH 2 OH C 13 H 27 O O HO OH HO AcHN O HO OH O HO O O OH HO OH O O OH HO OH NH 2 OH C 13 H 27 O