Ingrid 't Hart

70 Chapter 3 3 (2S,3R,4E)-2-amino-3-hydroxy-octadec-4-enyl β-D-galactopyranosyl-(1 → 3)-2- acetamido-2-deoxy-β-D-galactopyranosyl-(1 → 3)-α-D-galactopyranosyl-(1 → 4)-β-D- galactopyranosyl-(1 → 4)-β-D-glucopyranoside (3a). LgtD (50 µL, 0.25 mg) was added to a mixture of compound 2a (0.9 mg, 10 mM final concentra�on) in H 2 O with UDP-Gal (0.6 mg, 11 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 Gb4Sph/Gb5Sph. 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 3a (Gb5Sph), 0.92 mg, 88 %). 1 H NMR (600 MHz, MeOD) δ 5.83 – 5.73 (1H, m, CH= CH -CH 2 ), 5.49 (1H, dd, J = 15.1, 8.0 Hz, CHOH-C H =CH), 4.93 (1H, d, J = 4.0 Hz, H-1, Gal-III), 4.71 (1H, d, J = 8.4 Hz, H-1, GalNAc-IV), 4.41 (1H, s, H-1, Gal-II), 4.34 (1H, d, J = 7.8 Hz, H-1, Gal-V), 4.32 (1H, d, J = 7.7 Hz, H-1, Glc-I), 4.30 – 4.23 (1H, m), 4.17 (1H, s), 4.12 – 4.02 (2H, m), 3.99 (1H, s), 3.96 – 3.16 (29H, m), 2.10 (2H, dd, J = 14.1, 6.8 Hz, CH=CH-C H 2 ), 1.98 (3H, s, CH 3 , NHAc), 1.47 – 1.19 (22H, m, 11 x CH 2 ), 0.90 (3H, t, J = 6.9 Hz, CH 3 , Sph). ESI HRMS ( m/z ): [M + H] + calcd for C 50 H 90 N 2 O 27 ; 1150.5731 found 1151.5823. General procedure for ceramide synthesis Glycosylsphingosine (1 eq, 10mM), was dissolved in DMF and palmi�c acid (3 eq), EDC·HCl (3 eq), Et 3 N (5-10% v/v) and HOBt (3 eq) were added from freshly prepared stock solu�ons in DMF. The reac�on mixture was shaken in an 1.5mL eppendorf tube at room temperature overnight. The reac�on was monitored by MALDI-TOF and a�er overnight shaking, H 2 O was added and the mixture was lyophilized. The crude residue was purified on a pre-equilibrated C4 or C18 cartridge. (2S,3R,4E)-3-hydroxy-2-(hexadecanamido)octadec-4-enyl (α-D-galactopyranosyl)- (1 → 4)-(β-D-galactopyranosyl)-(1 → 4)-β-D-glucopyranoside (1b). Compound 1b was synthesized from compound 1a according to the general procedure for ceramide synthesis. The crude was purified by a 1mL C18 cartridge using H 2 O:CH 3 CN (1:0 to 2:8 v/v) as the eluent. The organic solvent was removed under N 2 flow and the residual water was removed by lyophiliza�on. (0.03mg, 3%) 1 H NMR (600 MHz, MeOD) δ 5.72 – 5.63 (1H, m, CH=C H -CH 2 ), 5.44 (1H, dd, J = 15.2, 7.7 Hz, CHOH-C H =CH), 4.94 (1H, d, J = 3.7 Hz, H-1, Gal-III), 4.40 (1H, d, J = 6.7 Hz, H-1, Gal-II), 4.29 (1H, d, J = 7.8 Hz, H-1, Glc-I), 4.25 (1H, t, J = 6.6 Hz), 4.17 (1H, dd, J = 10.1, 4.6 Hz), 4.05 (1H, t, J = 8.2 Hz, HOC H -CH=CH), 3.97 – 3.22 (22H, m), 2.24 – 2.11 (2H, m, J = 16.4, 14.4, 8.2 Hz, CH 2 , Cer), 2.06 – 1.97 (2H, m, CH 2 , Cer), 1.66 – 1.50 (2H, m, CH 2 , Cer), 1.42 – 1.21 (46, m, J = 13.8 Hz, 23 x CH 2 , Cer), 0.89 (6H, t, J = 6.9 Hz, 2x CH 3 , Cer). ESI HRMS ( m/z ): [M + Na] + calcd for C 52 H 97 NO 18 ; 1046.6598 found 1046.6606. O HO OH O AcHN O HO OH HO OH O HO OH O HO O O OH HO OH O O OH HO OH NH 2 OH C 13 H 27 O O HO OH HO HO O O OH HO OH O O OH HO HO HN OH C 13 H 27 O O C 15 H 31