Ingrid 't Hart

68 Chapter 3 3 – 2.06 (6H, m, 2 x CH 3 , OAc), 2.04 – 1.95 (11H, m, 3 x CH 3 , OAc; CH=CH-C H 2 ), 1.38 – 1.18 (22H, m, 11 x CH 2 ), 0.97 (9H, s, 3 x CH 3 , t -Bu), 0.93 (9H, s, 3 x CH 3 , t -Bu), 0.88 (3H, t, J = 7.0 Hz, CH 3 , Sph). 13 C NMR (151 MHz, CDCl 3 ) δ 170.4 (C=O, Ac), 170.4 (C=O, Ac), 170.3 (C=O, Ac), 170.1 (C=O, Ac), 170.0 (C=O, Ac), 169.4 (C=O, Ac), 165.1 (C=O, Bz), 153.8 (C=O, Troc), 139.8, 138.9 (CH= C H-CH 2 ), 138.5, 138.5, 138.3, 138.2, 138.0, 138.0, 133.2, 130.0, 129.8, 129.7, 128.6, 128.5, 128.4, 128.4, 128.4, 128.3, 128.3, 128.3, 127.8, 127.8, 127.8, 127.6, 127.6, 127.5, 127.3, 122.7 (CHOBz- C H=CH), 103.4 (C-1, Glc-I), 102.6 (C-1, Gal-II), 102.4 (C-1, GalNAc-IV), 100.9 (C-1 Gal-V), 100.0 (C-1, Gal-III), 95.2 (CCl 3 , Troc), 81.5 (C-2, Glc-I), 81.1 (C-3, Gal-II), 81.0 (C-3, Gal-III), 80.7 (C-3, Gal-I), 79.5 (C-3, GalNAc-IV), 76.2 (C-4, Gal- II), 75.2, 75.0, 75.0 (C-2, Gal-II), 74.9 ( C HOBz), 74.2, 74.0 (C-4, Gal-III), 73.7, 73.5, 73.1, 73.0 (C-2, Gal-III), 72.6 (C-4, Glc-I), 72.0, 70.8 (C-5, Gal-V), 70.60, 70.5 (C-3, Gal-V), 68.6 (C-2, Gal-V), 68.4, 68.3 (C-4, GalNAc-IV), 68.1, 67.5, 67.2, 67.1, 66.8 (C-4, Gal-V), 64.0 (C HN 3 ), 62.3, 61.2, 54.0 (C-2, GalNAc-IV), 32.4 (CH=CH= C H 2 ), 31.9, 29.7, 29.6, 29.4, 29.4, 29.2, 28.7, 27.5 (3x CH 3 , t -Bu), 27.3 (3x CH 3 , t -Bu), 23.3, 22.7, 20.8 (CH 3 , OAc), 20.8 (CH 3 , OAc), 20.8 (CH 3 , OAc), 20.8 (CH 3 , OAc), 20.7 (CH 3 , OAc), 20.6 (CH 3 , OAc), 14.2 (CH 3 , Sph). ESI HRMS ( m/z ): [M + NH 4 ] + calcd for C 127 H 161 Cl 3 N 4 O 35 Si; 2453.0114 found 2453.0146. Chemoenzyma�c synthesis Expression of LgtD from Haemophilus influenzae Materials . The full-length codon-op�mized gene encoding for HiLgtD (ENA: AAC23227) was synthesized by Genscript and subcloned into pET15b+ (NdeI, BamHI). Bl21(DE3) (C2527H) cells were purchased from New England Biolabs (NEB). Ampicillin (sodium salt) (14417) was ordered through Cayman Chemicals. Imidazole (56750), TRIS-HCl (T5941), NaCl (S9888), lysozyme (62971) and triton X-100 (T8787) came from Sigma-Aldrich. 2xYT medium (BP97432) and 2xYT Agar (BP2466) were ordered from Fisher BioReagents. Isopropyl-beta-D-thiogalactopyranoside (IPTG) (R0393) as well as GelCode Blue Stain Reagent (24592) was ordered from Thermo Scien�fic. The Ni-NTA resin (17-5318-01) and the Superdex 200 Increase 10/300 GL column (28990944) were ordered from GE Healthcare. Sartorius provided Vivaspin 6, 10000 MWCO (VS0602) spinfilters. For SDS- PAGE, SurePAGE, Bis-Tris, 10x8, 4-12%, 12 wells (M00653) from GenScript were used. Laemmli sample buffer 2x (161-0737) and Precision Plus Protein Dual Color Standards (161-0374) were ordered from Bio-Rad Laboratories. A standard buffer consis�ng of 50 mM TRIS-HCl and 250 mM NaCl pH 8 was made. Imidazole was added to the standard buffer at concentra�ons of 20 mM, 50 mM or 250 mM to make wash 1, wash 2 and elu�on buffer (re-adjusted to pH 8 if needed) respec�vely. To wash 1, lysozyme (1mg/ mL) and triton X-100 (0.1%) was added to make the lysis buffer. Protocol. BL21(DE3) cells were transformed with each of the individual vectors, a colony was picked from the 2xYT agar plate with ampicillin (100 μg/mL) and expanded to a cell culture volume of 500 mL ampicillin (100 μg/mL) containing 2xYT medium at 37°C. The cells were induced at OD 600 = 0.6 with IPTG (final concentra�on was 1 mM) and cultured overnight at 20°C. Then, cells were pelleted at 3000 xg , resuspended in lysis buffer (5% of culture volume), incubated at 37°C for 1 h and sonicated for 30’ on ice. Clear supernatant, by removal of cell debris by centrifuga�on at 10000 xg , was loaded onto a gravity flow 4 mL Ni-NTA column in case of HiLgtD. Once washed with 10 column volumes