Supplemental Experimental Procedures

Chemistry & Biology 18 Supplemental Information A Possible Oligosaccharide-Conjugate Vaccine Candidate for Clostridium difficile Is Antigenic and Immunogenic Matthias A. Oberli, Marie-Lyn Hecht, Pascal Bindschädler, Alexander Adibekian, Thomas Adam, and Peter H. Seeberger Supplemental Experimental Procedures All chemicals used were reagent grade and used as supplied except where noted. Dichloromethane (CH 2 Cl 2 ), toluene and diethylether (Et 2 O) were purified by a J. C. Meyer Cycle-Tainer Solvent Delivery System. Pyridine, triethylamine, and acetonitrile were distilled over CaH 2 prior to use. Triflic anhydride (Tf 2 O) and trichloroacetonitrile (CCl 3 CN) were stirred over P 2 O 5 and distilled. Solvents for chromatography and work up procedures were distilled. Reactions were performed under an Ar-atmosphere except where noted. Analytical thin layer chromatography (TLC) was performed on Merk silica gel 60 F 254 plates (0.25mm). Compounds were visualized by UV irradiation or by dipping the plate in a cerium(iv)sulfate/ammoniummolybdate/h 2 O/H 2 SO 4 solution followed by heating. Flash column chromatography was carried out using forced flow of the indicated solvent on Fluka Kieselgel 60 (230-400 mesh). 1 H-NMR spectra were recorded on a Varian VRX-300 (300 MHz), AV-400 (400 MHz), and Brucker DRX500 (500 MHz) spectrometer, and are reported in ppm ( ) relative to the resonance of the solvent. Coupling constants (J) are reported in (Hz). 13 C-NMR spectra were obtained using a Varian VRX-300 (75 MHz), and Brucker DRX500 (125 MHz) spectrometer and are reported in ppm ( ) relative to the solvent. MALDI high-resolution mass spectra were performed by the MS-service at the Laboratory for Organic Chemistry (ETH Zürich). IR spectra were recorded on a Perkin-Elmer 1600 FTIR spectrometer (neat) or on a Perkin-Elmer Spectrum 100 Series spectrometer (CHCl 3 ); solvents and concentrations (in g/100 ml) are indicated. Recycling preparative size exclusion HPLC (LC-9101, Japan Analytical Industry Co., Ltd.); flow rate: 3.5 ml/min; solvent: CHCl 3. S1

Supporting Scheme 1. a) 1. Triethyl orthobenzoate, DMF; 2. 10-Camphorsulfonic acid; 3. NaH, BnBr, DMF; b) levulinic acid, DIPC, DMAP, CH 2 Cl 2. Ethyl 2-O-Benzoyl-4,6-di-O-benzyl-1-thio-α-D-mannopyranoside (25). A solution of ethyl 6-O-acetyl-1-thio-α-D-mannopyranoside 26 (2.0 g, 7.5 mmol) (Gemma et al. 2005) in DMF (2 ml) was treated with triethyl orthobenzoate (13.6 ml, 60 mmol) then evacuated for 5 min (1 Torr or less). 10-Camphorsulfonic acid (0.2 g) was added in one portion and the vacuum was immediately restored. After 10 min, 10-camphorsulfonic acid (0.2 g) was added again and vacuum was immediately restored again. After 5 min, TLC (DCM/Acetone 2:1) showed full conversion of the starting material into the 2,3-cyclic orthoester, additional DMF (40 ml) was added and the mixture was cooled to 0 C. Sodium hydride (1.35 g, 56.3 mmol, 60% dispersion in mineral oil) was added and stirred for 10 min. Benzyl bromide (3.1 ml, 26 mmol) was added slowly. After 5 min the cooling bath was removed and the mixture was stirred for 2 h at room temperature. The mixture was cooled to 0 C again and quenched by the addition of MeOH (10 ml) and diluted with water (300 ml). The aqueous phase was extracted with a 1:1 cyclohexane/etoac mixture (3 x 200 ml). The combined organic phases were washed with water (100 ml), brine (2 x 100 ml), dried over MgSO 4, filtered, and concentrated to afford a pale yellow oil. This oil was directly dissolved in THF (60 ml), treated with HCl (1 N in water), and stirred for 1h at room temperature. The reaction mixture was diluted with water (200 ml) and extracted with EtOAc (3 x 200 ml). The combined organic phases were washed with NaHCO 3 aq. sat. (200 ml), water (300 ml), brine (300 ml), dried over MgSO 4, filtered and concentrated. The crude product was purified by flash column chromatography (10:1 4:1 cyclohexane/etoac) to afford 25 (2.46 g, 4.8 mmol, 64%) as a colorless oil. 1 H NMR (CDCl 3, 300 MHz) 1.39 (t, J = 7.5 Hz, 3H), 2.16 (d, J = 4.8 Hz, 1H), 2.59 2.74 (m, 2H), 3.78 (d, J = 9.6 Hz, 1H), 3.96 (dd, J = 11.1, 3.6 Hz, 1H), 4.06 (t, J = 9.6 Hz, 1H), 4.17 4.23 (m, 2H), 4.54 (d, J = 12.3 Hz, 1H), 4.66 (d, J = 11.4 S2

Hz, 1H), 4.76 (d, J = 10.8 Hz, 1H), 4.79 (d, J = 11.4 Hz, 1H), 5.45 5.47 (m, 2H), 7.27 7.39 (m, 12H), 7.58 (t, J = 7.5 Hz, 1H), 8.04 (d, J = 6.9 Hz, 2H). 13 C NMR (CDCl 3, 75 MHz) 15.0, 25.7, 69.0, 71.2, 71.8, 73.5, 74.7, 74.8, 76.0, 82.4, 127.6, 127.6, 127.9, 128.0, 128.3, 128.4, 128.5, 129.8, 129.9, 133.2, 138.2, 138.3, 166.1. [ ] rt D = +41.3 (c = 1.0, CHCl 3 ). IR (CHCl 3 ): 3471, 2926, 2872, 1719, 1452, 1316, 1269, 1072 cm 1. MALDI-HRMS: m/z calcd for C 29 H 32 O 6 SNa [M + Na] + 531.1812, obsd 531.1816. Ethyl 2-O-Benzoyl-4,6-di-O-benzyl-3-O-levulinoyl-1-thio-α-D-mannopyranoside (9). To a solution of 25 (2.4 g, 4.7 mmol) in CH 2 Cl 2 (50 ml) at 0 C, DMAP (0.92 g, 7.6 mmol), DIPC (1.18 ml, 7.6 mmol) and levulinic acid (0.78 ml, 7.6 mmol) were added. The mixture was stirred for 3 h at room temperature, diluted with a mixture of cyclohexane/etoac (2:1, 100 ml), filtered over a plug of celite and concentrated. The crude product was purified by flash column chromatography on silica gel (10:1 2:1 cyclohexane/etoac) to afford 9 (2.74 g, 4.5 mmol, 96%) as a colorless oil. 1 H NMR (CDCl 3, 300 MHz) 1.30 (t, J = 7.5 Hz, 3H), 2.09 (s, 3H), 2.40 2.48 (m, 2H), 2.54 2.76 (m, 4H), 3.76 (d, J = 11.1 Hz, 1H), 3.95 (dd, J = 10.8, 3.3 Hz, 1H), 4.20 4.28 (m, 2H), 4.53 (d, J = 11.4 Hz, 2H), 4.69 (d, J = 11.4 Hz, 1H), 4.76 (d, J = 11.4 Hz, 1H), 5.39 (dd, J = 10.2, 4.2 Hz, 2H), 5.60 (s, 1H), 7.21 7.40 (m, 12H), 7.60 (t, J = 7.2 Hz, 1H), 8.06 (d, J = 7.2 Hz, 2H). 13 C NMR (CDCl 3, 75 MHz) 14.9, 25.5, 28.0, 29.7, 37.8, 68.8, 72.0, 72.1, 72.9, 73.1, 73.5, 74.8, 82.3, 127.6, 127.7, 127.8, 128.4, 128.4, 128.5, 129.8, 129.9, 133.3, 138.0, 138.3, 165.5, 171.7, 206.1. [ ] rt D = +29.5 (c = 1.0, CHCl 3 ). IR (CHCl 3 ): 2928, 2871, 1744, 1720, 1452, 1361, 1267, 1154 cm 1. MALDI-HRMS: m/z calcd for C 34 H 38 O 8 SNa [M + Na] + 629.2180, obsd 629.2169. 2-O-Benzoyl-4,6-di-O-benzyl-3-O- N-(Benzyl)benzyloxycarbonyl-5-amino-pentyl levulinoyl-α-d-mannopyranoside (10). N-(Benzyl)benzyloxycarbonyl-5-amino-pentan-1-ol (216 mg, 0.66 mmol) and thioglycoside 9 (200 mg, 0.33 mmol) were coevaporated with toluene (2 12 ml), dried in vacuo for 2 h, and S3

dissolved in CH 2 Cl 2 (4 ml). The mixture was cooled to 11 C, NIS (74 mg, 0.33 mmol) and TMSOTf (6 L, 30 mol) were added. The mixture was stirred and warmed to 5 C in 1 h. The reaction mixture was quenched by the addition of pyridine (0.1 ml) and concentrated. The crude product was purified by flash column chromatography (1:0 3:1 cyclohexane/etoac) to afford 10 (258 mg, 0.30 mmol, 90%) as a colorless oil. 1 H NMR (CDCl 3, 400 MHz) 1.29 (bs, 2H), 1.53 (bs, 4H), 2.09 (s, 3H), 2.38 2.64 (m, 3H), 2.69 2.78 (m, 1H), 3.22 (bs, 1H), 3.28 (bs, 1H), 3.40 (bs, 1H), 3.67 (bs, 1H), 3.77 (d, J = 9.7 Hz, 1H), 3.90 3.93 (m, 2H), 4.18 (t, J = 9.5 Hz, 1H), 4.51 (s, 2H), 4.54 (d, J = 2.4 Hz, 1H), 4.57 (d, J = 3.2 Hz, 1H), 4.69 (d, J = 11.1 Hz, 1H), 4.77 (d, J = 12.0 Hz, 1H), 4.90 (s, 1H), 5.19 (s, 2H), 5.45 5.48 (m, 2H), 7.22 7.43 (m, 22H), 7.60 (t, J = 7.4 Hz, 1H), 8.06 (d, J = 7.1 Hz, 2H). 13 C NMR (CDCl 3, 100 MHz) 23.4, 28.0, 29.1, 29.7, 37.9, 67.1, 68.0, 68.8, 70.5, 71.6, 72.6, 73.5, 74.9, 97.6, 127.3, 127.6, 127.7, 127.8, 127.9, 128.3, 128.4, 128.5, 129.7, 129.9, 138.0, 138.4, 165.5, 171.7, 206.1. [ ] rt D = 5.9 (c = 1.0, CHCl 3 ). IR (CHCl 3 ): 2939, 1744, 1721, 1698, 1453, 1421, 1359, 1267, 1229, 1113, 1079, 1072 cm 1. MALDI-HRMS: m/z calcd for C 52 H 57 NO 11 Na [M + Na] + 894.3824, obsd 894.3839. 2-O-Benzoyl-4,6-di-O-benzyl-1-α-D- N-(Benzyl)benzyloxycarbonyl-5-amino-pentyl mannopyranoside (11). To a solution of 10 (250 mg, 287 mol) in CH 2 Cl 2 (3 ml), pyridine (0.7 ml), acetic acid (0.45 ml) and hydrazine monohydrate (28 L, 0.57 mmol) were added. The solution was stirred for 2 h at room temperature. The reaction was quenched by the addition of acetone (0.1 ml) and concentrated. The residue was coevaporated with toluene (2 12 ml) and purified by flash column chromatography on silica gel (8:1 4:1 cyclohexane/etoac) to afford 11 (223 mg, 287 mol, quant.) as a colorless oil. 1 H NMR (CDCl 3, 400 MHz) 1.31 (bs, 2H), 1.55 (bs, 4H), 2.11 (s, 1H), 3.23 (bs, 1H), 3,28 (bs, 1H), 3.40 (bs, 1H), 3.66 (bs, 1H), 3.77 3.83 (m, 2H), 3.91 (dd, J = 10.7, 3.5 Hz, 1H), 4.00 (t, J = 9.5 Hz, 1H), 4.26 (bs, 1H), 4.51 (s, 2H), 4.57 (d, J = 12.0 Hz, 1H), 4.66 (d, J = 11.1 Hz, 1H), 4.76 (d, J = 12.0 Hz, 1H), 4.82 (d, J = 11.1 Hz, 1H), 4.93 (s, 1H), 5.19 (s, 2H), 5.34 (s, 1H), 7.22 7.43 (m, 22H), 7.58 (t, J = 7.4 Hz, 1H), 8.05 (d, J = 7.1 Hz, 2H). 13 C NMR (CDCl 3, 100 MHz) 23.4, 29.1, 67.2, 67.8, 69.1, 70.6, 70.7, 71.5, 73.0, 73.5, 75.0, 97.5, 127.3, 127.6, 127.8, 127.9, 128.1, 128.5, 133.2, 136.9, 137.9, 138.2, 138.4, 166.2. [ ] rt D = 2.7 (c = 1.0, CHCl 3 ). IR (CHCl 3 ): 3031, S4

2938, 1699, 1453, 1423, 1359, 1268, 1098, 1071 cm 1. MALDI-HRMS: m/z calcd for C 47 H 51 NO 9 Na [M + Na] + 796.3456, obsd 796.3471. Phenyl 2-O-Acetyl-3,4,6-tri-O-benzyl- -D-glucopyranosyl-(1 3)-4,6-O-benzylidene-1- seleno-2-n-trichloroacetyl- -D-galactosaminopyranoside (12). Acceptor 6 (574 mg, 1.04 mmol) (Chen et al. 2005) and glycosyl phosphate 7 (594 mg, 0.86 mmol) (Ravidà et al. 2006) were coevaporated with toluene (2 10 ml), and dried in vacuo for 2 h. The mixture was dissolved in CH 2 Cl 2 (10 ml), freshly activated molecular sieves (4 Å) were added and the mixture was cooled to 30 C. TMSOTf (157 L, 0.86 mmol) was added and the mixture was stirred and warmed up to 5 C in one hour. The reaction was quenched by the addition of pyridine (0.2 ml) and concentrated. Flash column chromatography on silica gel (8:1 4:1 cyclohexane/etoac) afforded 12 (697 mg, 0.68 mmol, 78%) as a white foam. 1 H NMR (CDCl 3, 400 MHz) 1.92 (s, 3H), 3.59 3.66 (m, 2H), 3.83 (d, J = 2.6 Hz, 2H), 3.91 (t, J = 9.5 Hz, 1H), 4.03 (dd, J = 12.6, 1.6 Hz, 1H), 4.08 (s, 1H), 4.18 (dd, J = 12.5, 1.2 Hz, 1H), 4.26 (dd, J = 11.2, 3.3 Hz, 1H), 4.50 4.54 (m, 2H), 4.59 (d, J = 10.9 Hz, 1H), 4.63 4.67 (m, 3H), 4.80 4.84 (m, 3H), 5.08 (dd, J = 9.0, 8.3 Hz, 1H), 5.53 (s, 1H), 6.39 (d, J = 4.7 Hz, 1H), 7.19 7.38 (m, 22H), 7.47 7.51 (m, 4H). 13 C NMR (CDCl 3, 100 MHz) 20.9, 51.6, 65.8, 68.3, 69.2, 70.1, 72.4, 73.7, 74.3, 75.1, 75.2, 75.6, 82.7, 87.4, 92.6, 98.6, 101.0, 126.3, 127.7, 127.8, 127.9, 128.0, 128.2, 128.5, 128.5, 133.6, 137.6, 137.7, 137.7, 137.9, 161.6, 170.0. [ ] rt D = +63.6 (c = 1.0, CHCl 3 ). IR (CHCl 3 ): 3032, 2870, 1746, 1718, 1514, 1498, 1454, 1367, 1095, 1028 cm 1. MALDI-HRMS: m/z calcd for C 50 H 50 Cl 3 NO 11 SeNa [M + Na] + 1048.1506, obsd 1048.1511. Phenyl 2-O-Acetyl-3,4,6-tri-O-benzyl- -D-glucopyranosyl-(1 3)-6-O-benzyl-1-seleno-2- N-trichloroacetyl- -D-galactosaminopyranoside (13). S5

To a solution of 12 (614 mg, 598 mol) in CH 2 Cl 2 (6 ml) freshly activated and acid washed molecular sieves (4 Å) was added. The mixture was cooled to 78 C, triethylsilane (0.29 ml, 1.8 mmol) and trifluoromethanesulfonic acid (0.18 ml, 2.0 mmol) were added. After stirring for 3 h at 78 C the reaction was quenched by the addition of pyridine (0.2 ml) and diluted with CH 2 Cl 2 (30 ml). The solution was washed with NaHCO 3 aq. sat. (25 ml), brine (25 ml), dried over MgSO 4, filtered and concentrated. Flash column chromatography on silica (1:0 3:1 cyclohexane/etoac) afforded 13 (417 mg, 406 mol, 68%) as a white foam. 1 H NMR (CDCl 3, 400 MHz) 1.93 (s, 3H), 2.99 (s, 1H), 3.47 3.78 (m, 8H), 4.14 (s, 1H), 4.37 (t, J = 5.7 Hz, 1H), 4.41 4.50 (m, 5H), 4.60 (dd, J = 9.6, 5.5 Hz, 3H), 4.72 (dd, J = 11.2, 5.0 Hz, 2H), 4.94 (dd, J = 8.8, 7.6 Hz, 1H), 5.95 (d, J = 4.9 Hz, 1H), 6.84 (d, J = 7.8 Hz, 1H), 7.09 7.32 (m, 23H), 7.51 (d, J = 7.0 Hz, 2H). 13 C NMR (CDCl 3, 100 MHz) 21.0, 51.6, 67.8, 68.6, 69.2, 72.7, 72.8, 73.5, 73.6, 74.9, 75.0, 75.1, 82.5, 88.5, 92.5, 100.2, 127.6, 127.9, 127.9, 128.0, 128.2, 128.4, 128.4, 128.5, 128.5, 128.5, 129.3, 134.5, 137.6, 137.6, 137.8, 138.2, 161.3, 169.7. [ ] rt D = +15.2 (c = 1.0, CHCl 3 ). IR (CHCl 3 ): 2930, 2866, 1721, 1497, 1453, 1363, 1266, 1232, 1060, 1028 cm 1. MALDI-HRMS: m/z calcd for C 50 H 52 Cl 3 NO 11 SeNa [M + Na] + 1050.1663, obsd 1050.1681. Phenyl 2-O-Acetyl-3,4,6-tri-O-benzyl- -D-glucopyranosyl-(1 3)-6-O-benzyl-4-Olevulinoyl-1-seleno-2-N-trichloroacetyl- -D-galactosaminopyranoside (5). To a solution of 13 (417 mg, 0.41 mmol) in CH 2 Cl 2 (10 ml) at 0 C, DMAP (247 mg, 2.03 mmol), DIPC (0.32 ml, 2.03 mmol) and levulinic acid (0.21 ml, 2.03 mmol) were added. The mixture was stirred at room temperature over night, diluted with a mixture of cyclohexane/etoac (2:1, 100 ml), filtered over a plug of Celite and concentrated. The crude product was purified by flash column chromatography on silica gel (10:1 2:1 cyclohexane/etoac) to afford 5 (443 mg, 0.39 mmol, 94%) as a colorless foam. 1 H NMR (CDCl 3, 400 MHz) 1.99 (s, 3H), 2.17 (s, 3H), 2.53 2.94 (m, 4H), 3.46 3.63 (m, 4H), 3.67 3.77 (m, 2H), 3.81 (dd, J = 10.9, 1.9 Hz, 1H), 3.92 (dd, J = 11.0, 3.2 Hz, 1H), 4.47 4.70 (m, 9H), 4.77 (d, J = 11.0, 3.5 Hz, 2H), 4.96 (dd, J = 9.4, 7.9 Hz, 1H), 5.56 (d, J = 2.2 Hz, 1H), 5.94 (d, J = 4.9 Hz, 1H), 7.08 (d, J = 8.3 Hz, 1H), 7.16 7.40 (m, 23H), 7.54 (d, J = 6.9 Hz, 2H). 13 C NMR (CDCl 3, 100 MHz) 21.0, 28.0, 29.8, 38.0, 52.2, 68.6, 69.4, 72.1, 72.3, 72.5, 73.5, 75.1, 75.6, 77.7, 82.6, 88.6, 92.6, 99.8, 127.6, 127.6, 127.8, 127.8, 127.9, 128.0, S6

128.1, 128.4, 135.6, 137.8, 138.0, 138.0, 138.1, 161.4, 169.9, 171.5, 206.7. [ ] rt D = +95.2 (c = 1.0, CHCl 3 ). IR (CHCl 3 ): 3031, 2873, 1746, 1717, 1513, 1498, 1366, 1229, 1152, 1061, 1028 cm 1. MALDI-HRMS: m/z calcd for C 55 H 58 Cl 3 NO 13 SeNa [M + Na] + 1148.2032, obsd 1148.2014. 2-O-Acetyl-3,4,6-tri-O-benzyl- -D-glucopyranosyl-(1 3)-6-O-benzyl-4-O-levulinoyl-2-Ntrichloroacetyl- -D-galactosaminopyranoside (14). To a solution of 5 (443 mg, 0.39 mmol) in THF (6 ml), NIS (177 mg, 0.78 mmol) and HCl (1 N, 2 ml) were added. The mixture was stirred over night and diluted with CH 2 Cl 2 (40 ml), quenched by the addition of Na 2 S 2 O 3 aq. sat. (15 ml) and NaHCO 3 aq. sat. (15 ml). The phases were separated and the aqueous phase was extracted with CH 2 Cl 2 (2 x 40 ml). The combined organic phases were dried over MgSO 4, filtered, and concentrated. Flash column chromatography on silica gel (1:0 1:1 cyclohexane/etoac) afforded 14 (374 mg, 0.38 mmol, 96%) as a colorless foam. 1 H NMR (CDCl 3, 300 MHz) 1.75 (s, 1H), 1.91 (s, 3H), 2.11 (s, 3H), 2.49 2.67 (m, 3H), 2.76 2.81 (m, 1H), 3.30 3.59 (m, 6H), 3.64 (dd, J = 10.8, 1.7 Hz, 1H), 3.99 (dd, J = 10.7, 3.3 Hz, 1H), 4.13 (s, 1H), 4.17-4.31 (m, 2H), 4.32 4.47 (m, 3H), 4.49 (d, J = 1.3 Hz, 2H), 4.54 (d, J = 11.3 Hz, 1H), 4.64 (d, J = 11.4 Hz, 1H), 4.68 (d, J = 10.9 Hz, 1H), 4.78 (dd, J = 9.3, 7.8 Hz, 1H), 5.17 (t, J = 3.5 Hz, 1H), 5.37 (d, J = 2.8 Hz, 1H), 6.69 (d, J = 9.6 Hz, 1H), 7.04 7.29 (m, 20H). 13 C NMR (CDCl 3, 75 MHz) 20.9, 28.0, 29.8, 38.2, 51.3, 68.6, 68.9, 69.3, 70.3, 72.0, 72.6, 73.4, 73.7, 75.0, 75.1, 75.4, 78.0, 82.5, 91.8, 92.8, 100.8, 127.6, 127.6, 127.8, 127.8, 127.9, 128.1, 128.4, 128.4, 137.6, 137.7, 138.0, 138.1, 161.3, 169.9, 171.9, 207.1. IR (CHCl 3 ): 2872, 1747, 1717, 1454, 1364, 1231, 1179, 1151, 1064, 1028 cm 1. MALDI-HRMS: m/z calcd for C 49 H 54 Cl 3 NO 14 Na [M + Na] + 1010.2485, obsd 1010.2494. 2-O-Acetyl-3,4,6-tri-O-benzyl- -D-glucopyranosyl-(1 3)-6-O-benzyl-4-O-levulinoyl-2-Ntrichloroacetyl- -D-galactosaminopyranosyl N-Phenyltrifluoroacetimidate (15). S7

A solution of 14 (374 mg, 0.38 mmol) in CH 2 Cl 2 (6 ml) was cooled to 0 C, (N-phenyl)- 2,2,2-trifluoroacetimidoyl chloride (236 mg, 1.13 mmol) and Cs 2 CO 3 (247 mg, 0.76 mmol) were added and the resulting solution was stirred at room temperature over night, diluted with CH 2 Cl 2, filtered through a plug of Celite, and concentrated. Flash column chromatography on silica (1:0 3:1 cyclohexane/etoac) afforded 15 (342 mg, 0.30 mmol, 78%) as a colorless oil. 1 H NMR (CDCl 3, 400 MHz) 1.92 (s, 3H), 2.12 (s, 3H), 2.49 2.70 (m, 3H), 2.74 2.87 (m, 1H), 3.43 3.58 (m, 4H), 3.60 3.78 (m, 3H), 4.10 (dt, J = 10.8, 4.6 Hz, 2H), 4.42 4.62 (m, 9H), 4.71 (dd, J = 11.1, 6.5 Hz. 2H), 4.88 (dd, J = 9.3, 7.8 Hz, 1H), 5.58 (d, J = 2.7 Hz, 1H), 6.69 (dd, J = 12.3, 8.2 Hz, 2H), 7.03 (dd, J = 10.7, 4.2 Hz, 1H), 7.09 7.16 (m, 2H), 7.18 7.35 (m, 21H). 13 C NMR (CDCl 3, 100 MHz) 20.9, 27.9, 29.8, 38.0, 50.7, 68.3, 68.7, 68.8, 71.3, 72.7, 73.5, 73.6, 75.0, 75.6, 77.7, 82.5, 92.3, 100.1, 119.2, 124.6, 127.7, 127.8, 127.9, 128.0, 128.3, 128.4, 137.8, 137.8, 137.9, 138.0, 142.9, 161.7, 169.8, 171.5, 206.6. IR (CHCl 3 ): 2870, 1748, 1718, 1518, 1454, 1366, 1308, 1229, 1208, 1155, 1096, 1063, 1028 cm 1. MALDI-HRMS: m/z calcd for C 57 H 58 F 3 Cl 3 N 2 O 14 Na [M + Na] + 1179.2798, obsd 1179.2776. Ethyl 2,3,6-Tri-O-benzyl-4-O-fluorenylmethoxycarbonyl-1-thio-β-D-glucopyranoside (4). A solution of 16 (1.05 g, 2.1 mmol) (van Steijn et al. 1992) in pyridine (10 ml) was treated at room temperature with Fmoc-Cl (1.1 g, 4.2 mmol). The yellow suspension was stirred at room temperature for 5.5 h, and concentrated. The residue was taken up in a 1:1-mixture of cyclohexane/etoac (50 ml), washed twice with 10% aqueous citric acid solution and with brine. The organic phase was dried over MgSO 4, filtered, and concentrated. Flash column chromatography on silica gel (1:0 9:1 cyclohexane/etoac) afforded 4 (1.1 g, 1.5 mmol, 72%) as a colorless oil. 1 H NMR (CDCl 3, 300 MHz) 1.34 (t, J = 7.4 Hz, 3H), 2.68 2.84 (m, 2H), 3.49 (t, J = 9.3 Hz, 1H), 3.62 3.66 (m, 3H), 3.73 (t, J = 9.1 Hz, 1H), 4.11 (t, J = 7.2 Hz, 1H), 4.23 4.34 (m, 2H), 4.49 4.52 (m, 3H), 4.70 (d, J = 11.2 Hz, 1H), 4.72 (d, J = 10.2 Hz, 1H), 4.84 (d, J = 11.2 Hz, 1H), 4.87 (t, J = 9.4 Hz, 1H), 4.90 (d, J = 10.2 Hz, 1H), 7.18 7.41 (m, 19H), 7.50 7.57 (m, 2H), 7.74 7.76 (m, 2H). 13 C NMR (CDCl 3, 75 MHz) 15.4, 25.3, 46.8, 69.9, 70.1, 73.6, 75.6, 81.3, 83.8, 85.1, 119.9, 124.8, 124.9, 127.0, 127.4, 127.4, 127.5, 127.7, 127.7, 128.1, 128.2, 137.5, 137.7, 137.8, 141.0, 142.9, 143.0, 154.1. IR (neat): 3065, 3030, 2870, 1752, 1497, 1452, 1385, 1361, 1255, 1072, 1029, 965 cm 1. [ ] rt D = 5.4 (c = S8

1.0, CHCl 3 ). MALDI-HRMS: m/z calcd for C 44 H 44 O 7 SNa [M + Na] + 739.2700, obsd 739.2692. 2,3,6-Tri-O-benzyl-4-O-fluorenylmethoxycarbonyl-β-D-glucopyranoside (17). A solution of 4 (150 mg, 0.21 mmol) in THF (10 ml) was cooled to 0 C, and treated with NBS (130 mg, 0.73 mmol) and HCl (1 ml, 0.01 N). The mixture was stirred for 2 h at 0 C, diluted with CH 2 Cl 2 (30 ml) and quenched by the addition of Na 2 S 2 O 3 aq. sat. (10 ml) and NaHCO 3 aq. sat. (10 ml). The phases were separated and the aqueous phase was extracted with CH 2 Cl 2 (2 x 30 ml). The combined organic phases were dried over MgSO 4, filtered and concentrated. The crude product was purified by flash column chromatography on silica gel (1:0 1:1 cyclohexane/etoac) to afford 17 (98 mg, 0.146 mmol, 70%) as a colorless foam. 1 H NMR (CDCl 3, 400 MHz) 1.28, 1.59 (2 s, 1H), 3.12 (s, 1H), 3.44 3.48 (m, 1H), 3.54 3.73 (m, 3H), 4.04 (t, J = 9.4 Hz, 1H), 4.11 4.37 (m, 3H), 4.48 4.56 (m, 2H), 4.67 4.96 (m, 4H), 5.23 (s, 1H), 7.20 7.43 (m, 19H), 7.53 7.59 (m, 2H), 7.78 (d, J = 7.5 Hz, 2H). 13 C NMR (CDCl 3, 100 MHz) 23.4, 29.1, 67.2, 69.1, 70.7, 71.4, 73.1, 73.6, 74.9, 75.8, 91.3, 97.5, 127.3, 127.5, 127.8, 127.9, 128.1, 128.3, 128.4, 128.5, 129.8, 129.9, 133.2, 136.8, 137.9, 138.2, 138.4, 166.2. IR (neat): 2897, 2871, 1750, 1452, 1253, 1096, 1048, 1027 cm 1. MALDI-HRMS: m/z calcd for C 42 H 40 O 8 Na [M + Na] + 695.2615, obsd 695.2608. 2,3,6-Tri-O-benzyl-4-O-fluorenylmethoxycarbonyl-β-D-glucopyranosyl N-Phenyltrifluoroacetimidate (18). A solution of 17 (98 mg, 0.15 mmol) in CH 2 Cl 2 (2 ml) was cooled to 0 C, (N-phenyl)-2,2,2- trifluoroacetimidoyl chloride (91 mg, 0.44 mmol) and Cs 2 CO 3 (95 mg, 0.29 mmol) were added and the resulting solution was stirred for 3 h at room temperature, diluted with CH 2 Cl 2, filtered through a plug of Celite and concentrated. Flash column chromatography on silica gel (9:1 5:1 cyclohexane/etoac) afforded 18 (126 mg, 0.15 mmol, quant.) as a colorless oil. 1 H NMR (CDCl 3, 400 MHz) 3.48 3.58 (m, 2H), 3.66 (s, 1H), 3.98 4.06 (m, 2H), 4.18 4.27 (m, 2H), 4.44 (s, 2H), 4.58 4.76 (m, 4H), 4.82 4.95 (m, 1H), 6.63, 6.71 (2d, J = 7.7, 7.6 Hz, 2H), 7.12 (t, J = 7.4 Hz, 1H), 7.13 7.32 (m, 22H), 7.42 7.48 (m, 3H), 7.65 7.69 (m, 2H). 13 C S9

NMR (CDCl 3, 100 MHz) 45.7, 67.8, 69.1, 70.3, 72.5, 72.5, 72.6, 72.8, 73.7, 74.2, 74.4, 77.8, 79.5, 80.7, 95.8, 118.3, 119.0, 119.5, 123.4, 124.0, 124.1, 125.3, 126.2, 126.6, 126.7, 126.9, 127.0, 127.3, 128.4, 136.5, 136.8, 136.9, 140.3, 142.2, 142.2, 153.3. IR (CHCl 3 ): 2872, 1753, 1717, 1598, 1451, 1317, 1250, 1208, 1152, 1098, 1071 cm 1. MALDI-HRMS: m/z calcd for C 50 H 44 F 3 NO 8 Na [M + Na] + 866.2911, obsd 866.2911. N-(Benzyl)benzyloxycarbonyl-5-amino-pentyl 2-O-Acetyl-3,4,6-tri-O-benzyl- -Dglucopyranosyl-(1 3)-6-O-benzyl-4-O-levulinoyl-2-N-trichloroacetyl- -D-galactosaminopyranosyl-(1 3)-2-O-benzoyl-4,6-di-O-benzyl-α-D-mannopyranoside (19). Disaccharide glycosylating agent 15 (134 mg, 116 mol) and monosaccharide 11 (103 mg, 133 mol) were coevaporated with toluene (3 8 ml) and dried in vacuo for 2 h. The mixture was dissolved in CH 2 Cl 2 (4 ml) and cooled to 30 C. TMSOTf (2 L, 15 mol) was added and the mixture was stirred and warmed to 3 C in 3 h. The reaction was quenched by the addition of pyridine (two drops) and concentrated. Flash chromatography on silica gel (1:0 2:1 cyclohexane/etoac) afforded 19 (165 mg, 95 mol, 82%) as a colorless foam. 1 H NMR (CDCl 3, 400 MHz) 1.22 (bs, 2H), 1.48 (bs, 4H), 1.93 (s, 3H), 2.11 (s, 3H), 2.44 2.64 (m, 3H), 2.79 2.84 (m, 1H), 3.17 (bs, 1H), 3.23 (bs, 1H), 3.34 (bs, 1H), 3.39 3.96 (m, 13H), 4.05 (t, J = 9.5 Hz, 1H), 4.24 (d, J = 13.4 Hz, 1H), 4.37 (q, J = 11.8 Hz, 2H), 4.43 4.78 (m, 13H), 4.85 (dd, J = 14.8, 6.2 Hz, 2H), 4.93 (d, J = 8.2 Hz, 1H), 4.98 (d, J = 10.7 Hz, 1H), 5.17 (s, 2H), 5.37 (s, 1H), 5.57 (d, J = 8.4 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 7.18 7.34 (m, 42H), 7.55 (t, J = 7.4 Hz, 1H), 7.99 (d, J = 7.7 Hz, 2H). 13 C NMR (CDCl 3, 100 MHz) 20.9, 28.0, 29.7, 38.1, 55.8, 67.1, 68.1, 68.8, 69.0, 69.4, 69.7, 71.8, 72.9, 73.1, 73.4, 73.5, 73.6, 74.9, 75.0, 75.6, 78.0, 82.5, 92.3, 97.2, 97.4, 100.6, 127.5, 127.5, 127.5, 127.6, 127.7, 127.8, 128.0, 128.0, 128.2, 128.2, 128.3, 128.3, 128.4, 128.4, 128.5, 130.1, 137.9, 138.0, 138.1, 138.4, 138.4, 161.7, 166.3, 166.3, 169.6, 171.5, 171.5, 206.8. [ ] rt D = +0.7 (c = 1.0, CHCl 3 ). IR (CHCl 3 ): 3031, 2866, 1747, 1718, 1523, 1497, 1453, 1363, 1265, 1230, 1063, 1028 cm 1. MALDI-HRMS: m/z calcd for C 96 H 103 Cl 3 N 2 O 22 Na [M + Na] + 1765.5994, obsd 1765.5997. S10

N-(Benzyl)benzyloxycarbonyl-5-amino-pentyl 2-O-Acetyl-3,4,6-tri-O-benzyl- -D- glucopyranosyl-(1 3)-6-O-benzyl-2-N-trichloroacetyl- -D-galactosaminopyranosyl- (1 3)-2-O-benzoyl-4,6-di-O-benzyl-α-D-mannopyranoside (20). To a solution of 19 (165 mg, 95 mol) in CH 2 Cl 2 (1.5 ml) pyridine (0.35 ml), acetic acid (0.22 ml) and hydrazine monohydrate (18 L, 377 mol) were added. The solution was stirred for 2 h at room temperature. The reaction was quenched with acetone (0.1 ml) and concentrated. The residue was coevaporated twice with toluene and purified by flash column chromatography on silica gel (8:1 4:1 cyclohexane/etoac) to afford 20 (141 mg, 86 mol, 91%) as a colorless foam. 1 H NMR (CDCl 3, 400 MHz) 1.20 (bs, 2H), 1.44 (bs, 4H), 1.65 (s, 1H), 1.93 (s, 3H), 2.81 (s, 1H), 3.14 (bs, 1H), 3.21 (bs, 1H), 3.30 (bs, 1H), 3.37 3.83 (m, 13H), 4.30 (t, J = 9.5 Hz, 1H), 4.13 (s, 1H), 4.28 4.75 (m, 15H), 4.85 (s, 1H), 4.93 5.03 (m, 3H), 5.15 (s, 2H), 5.39 (s, 1H), 7.06 (d, J = 7.7 Hz, 1H), 7.11 7.36 (m, 42H), 7.52 (t, J = 7.5 Hz, 1H), 7.97 (d, J = 7.1 Hz, 2H). 13 C NMR (CDCl 3, 100 MHz) 21.1, 23.2, 29.0, 55.8, 67.2, 67.5, 68.6, 69.1, 70.3, 71.4, 72.7, 73.4, 73.4, 73.5, 73.9, 74.8, 74.9, 75.0, 82.7, 92.3, 97.5, 98.0, 100.8, 127.4, 127.5, 127.5, 127.6, 127.8, 127.9, 127.9, 128.0, 128.1, 128.2, 128.3, 128.3, 128.4, 128.4, 128.5, 129.7, 130.1, 133.3, 137.8, 138.0, 138.3, 138.5, 138.8, 161.9, 166.5, 169.6. [ ] rt D = +5.1 (c = 1.0, CHCl 3 ). IR (CHCl 3 ): 3031, 2869, 1718, 1709, 1496, 1453, 1364, 1262, 1231, 1066, 1028 cm 1. MALDI-HRMS: m/z calcd for C 91 H 97 Cl 3 N 2 O 20 Na [M + Na] + 1667.5596, obsd 1667.5616. N-(Benzyl)benzyloxycarbonyl-5-amino-pentyl 2,3,6-Tri-O-benzyl-4-O-fluorenylmethoxycarbonyl- -D-glucopyranosyl-(1 4)-[2-O-acetyl-3,4,6-tri-O-benzyl- -D-glucopyranosyl-(1 3)]-6-O-benzyl-2-N-trichloroacetyl- -D-galactosaminopyranosyl-(1 3)-2- O-benzoyl-4,6-di-O-benzyl-α-D-mannopyranoside (21). S11

Trisaccharide acceptor 20 (121 mg, 74 mol) and monosaccharide building block 18 (124 mg, 147 mol) were coevaporated with toluene (3 8 ml) and dried in vacuo. The mixture was dissolved in CH 2 Cl 2 (1 ml) and Et 2 O (4 ml) and cooled to 45 C. TMSOTf (3 L, 15 mol) was added and the mixture was stirred and warmed to 5 C over 150 min. The reaction was quenched by the addition of two drops of pyridine. Flash chromatography on silica gel (1:0 0:1 cyclohexane/etoac) afforded 182 mg of a pale yellow foam. This foam was purified using recycling preparative size exclusion HPLC to yield 21 (141 mg, 61 mol, 83%) as a colorless foam. 1 H NMR (CDCl 3, 400 MHz) 1.22 (bs, 2H), 1.48 (bs, 4H), 1.91 (s, 3H), 3.16 (bs, 1H), 3.24 (bs, 1H), 3.29 3.42 (m, 2H), 3.47 3.87 (m, 16H), 3.98 (t, J = 9.6 Hz, 1H), 4.06 4.33 (m, 8H), 4.39 4.64 (m, 18H), 4.69 4.77 (m, 2H), 4.89 4.97 (m, 2H), 5.01 5.19 (m, 5H), 5.37 (s, 1H), 7.01 7.38 (m, 62H), 7.46 (t, J = 7.4 Hz, 1H), 7.56 7.62 (m, 2H), 7.75 (d, J = 7.5 Hz, 2H), 8.01 (d, J = 7.1 Hz, 2H). 13 C NMR (CDCl 3, 100 MHz) 21.0, 23.2, 29.0, 46.7, 56.2, 67.2, 67.9, 68.2, 68.8, 68.8, 69.1, 69.9, 70.6, 71.7, 72.4, 73.1, 73.1, 73.3, 73.4, 73.5, 73.6, 74.2, 74.7, 74.9, 74.9, 75.0, 75.2, 75.3, 76.0, 79.5, 79.9, 82.8, 92.3, 97.2, 98.1, 98.2, 101.4, 119.9, 125.4, 127.1, 127.2, 127.2, 127.2, 127.4, 127.4, 127.4, 127.5, 127.5, 127.5, 127.6, 127.7, 127.7, 127.8, 127.8, 127.9, 127.9, 128.0, 128.0, 128.1, 128.3, 128.3, 128.4, 128.4, 128.4, 128.5, 133.3, 136.9, 137.9, 137.9, 138.0, 138.1, 138.2, 138.3, 138.5, 138.6, 138.8, 141.2, 143.6, 143.6, 154.5, 162.0, 166.1, 169.2. [ ] rt D = +21.6 (c = 1.0, CHCl 3 ). IR (CHCl 3 ): 2866, 1749, 1721, 1453, 1364, 1258, 1230, 1095, 1067 cm 1. MALDI-HRMS: m/z calcd for C 133 H 135 Cl 3 N 2 O 27 Na [M + Na] + 2321.8232, obsd 2321.8245. N-(Benzyl)benzyloxycarbonyl-5-amino-pentyl 2,3,6-Tri-O-benzyl-α-D-glucopyranosyl- (1 4)-[2-O-acetyl-3,4,6-tri-O-benzyl- -D-glucopyranosyl-(1 3)]-6-O-benzyl-2-Ntrichloroacetyl- -D-galactosaminopyranosyl-(1 3)-2-O-benzoyl-4,6-di-O-benzyl-α-Dmannopyranoside (22). Triethylamine (1 ml) was added to a solution of 21 (140 mg, 61 mol) in CH 2 Cl 2 (4 ml). The mixture was stirred over night at room temperature, diluted with toluene (10 ml) and concentrated. Flash chromatography on silica gel (1:0 1:1 cyclohexane/etoac) afforded 22 (108 mg, 52 mol, 85%) as a colorless foam. 1 H NMR (CDCl 3, 400 MHz) 1.19 (bs, 2H), S12

1.45 (bs, 4H), 1.94 (s, 3H), 2.95 (bs, 1H), 3.13 (bs, 1H), 3.20 (bs, 1H), 3.24 3.34 (m, 2H), 3.38 3.89 (m, 17H), 4.02 (t, J = 9.4 Hz, 1H), 4.15 4.72 (m, 22H), 4.78 (dt, J = 10.7, 7.1 Hz, 2H), 4.87 (s, 1H), 4.94 (dd, J = 9.5, 5.3 Hz, 1H), 4.98 (d, J = 8.1, 1H), 5.03 (d, J = 3.3 Hz, 1H), 5.14 (d, J = 1.9 Hz, 2H), 5.38 (s, 1H), 7.02 7.35 (m, 57H), 7.41 (t, J = 7.5 Hz, 1H), 7.95 (d, J = 7.2 Hz, 2H). 13 C NMR (CDCl 3, 100 MHz) 21.1, 23.2, 23.3, 29.0, 41.8, 56.6, 62.5, 67.2, 67.8, 67.9, 68.5, 69.1, 69.4, 70.4, 70.8, 71.7, 72.2, 73.0, 73.2, 73.3, 73.6, 73.7, 74.2, 74.6, 74.8, 74.9, 75.0, 75.1, 75.3, 78.0, 79.3, 81.7, 82.3, 92.3, 97.2, 97.4, 98.3, 101.2, 127.2, 127.2, 127.2, 127.2, 127.3, 127.4, 127.5, 127.5, 127.6, 127.6, 127.6, 127.7, 127.8, 127.8, 127.9, 128.1, 128.2, 128.3, 128.3, 128.4, 128.4, 128.5, 129.7, 130.0, 133.4, 137.8, 137.9, 138.0, 138.1, 138.5, 138.7, 138.8, 138.9, 139.3, 161.9, 166.4, 170.2. [ ] rt D = +10.3 (c = 1.0, CHCl 3 ). IR (CHCl 3 ): 2868, 1721, 1453, 1364, 1258, 1232, 1096 cm 1. MALDI-HRMS: m/z calcd for C 118 H 125 Cl 3 N 2 O 25 Na [M + Na] + 2099.7545, obsd 2099.7597. N-(Benzyl)benzyloxycarbonyl-5-amino-pentyl 2-O-Acetyl-3,4,6-tri-O-benzyl- -Dglucopyranosyl-(1 3)-6-O-benzyl-4-O-levulinoyl-2-N-trichloroacetyl- -D-galactosaminopyranosyl-(1 4)2,3,6-tri-O-benzyl-α-D-glucopyranosyl-(1 4)-[2-O-acetyl-3,4,6- tri-o-benzyl- -D-glucopyranosyl-(1 3)]-6-O-benzyl-2-N-trichloroacetyl- -D-galactosaminopyranosyl-(1 3)-2-O-benzoyl-4,6-di-O-benzyl-α-D-mannopyranoside (23). Disaccharide glycosylating agent 15 (120 mg, 104 mol) and tetrasaccharide acceptor 22 (108 mg, 52 mol) were coevaporated with toluene (3 8 ml) and dried in vacuo for 2 h. The mixture was dissolved in CH 2 Cl 2 (8 ml), freshly activated and acid washed molecular sieves (4 Å) were added and the mixture was cooled to 30 C. TMSOTf (2 L, 15 mol) was added and the mixture was stirred and warmed to 0 C over 210 minutes. The reaction was quenched by the addition of two drops of pyridine and concentrated. Flash chromatography on silica gel (1:0 2:1 cyclohexane/etoac) afforded a colorless oil (120 mg) that was purified using recycling preparative size exclusion HPLC to yield 23 (99 mg, 32 mol, 63%) as a colorless foam. 1 H NMR (CDCl 3, 400 MHz) 1.22 (bs, 2H), 1.47 (bs, 4H), 1.87 (s, 3H), 1.96 (s, 3H), 2.16 (s, 3H), 2.50 2.72 (m, 3H), 2.80 2.93 (m, 1H), 3.12 3.82 (m, 29H), 3.95 4.10 (m, 5H), S13

4.19 5.05 (m, 38H), 5.17 (s, 2H), 5.29 (s, 1H), 5.53 (d, J = 3.4 Hz, 1H), 7.04 (d, J = 7.5 Hz, 1H), 7.10 7.37 (m, 76H), 7.48 (t, J = 7.4 Hz, 1H), 8.02 (d, J = 7.1 Hz, 2H). 13 C NMR (CDCl 3, 100 MHz) 20.5, 21.0, 22.8, 27.7, 28.6, 29.4, 37.9, 51.8, 55.7, 55.8, 62.1, 66.8, 67.4, 67.9, 68.1, 68.3, 68.6, 68.8, 69.2, 70.0, 70.5, 71.3, 72.0, 72.5, 72.6, 72.7, 72.8, 72.9, 73.0, 73.2, 73.2, 74.0, 74.4, 74.5, 74.6, 74.6, 74.8, 75.3, 75.3, 75.8, 77.7, 79.3, 79.4, 82.2, 82.5, 91.9, 92.2, 96.7, 98.0, 98.1, 98.8, 99.6, 100.4, 101.0, 127.0, 127.0, 127.0, 127.1, 127.2, 127.3, 127.4, 127.4, 127.4, 127.5, 127.5, 127.6, 127.7, 127.8, 127.8, 127.9, 128.0, 128.0, 128.1, 128.1, 128.2, 128.4, 129.7, 132.8, 136.5, 137.4, 137.5, 137.6, 137.8, 138.0, 138.1, 138.2, 138.3, 138.7, 139.1, 161.0, 161.6, 165.8, 169.2, 169.2, 171.2, 206.4. [ ] rt D = +15.0 (c = 0.5, CHCl 3 ). IR (CHCl 3 ): 3031, 2926, 2866, 1747, 1721, 1497, 1454, 1363, 1231, 1063, 1028 cm 1. MALDI-HRMS: m/z calcd for C 167 H 177 Cl 6 N 3 O 38 Na [M + Na] + 3069.0053, obsd 3069.0127. N-(Benzyl)benzyloxycarbonyl-5-amino-pentyl 2-O-Acetyl-3,4,6-tri-O-benzyl- -Dglucopyranosyl-(1 3)-6-O-benzyl-4-O-levulinoyl-2-N-acetyl- -D-galacto-saminopyranosyl-(1 4)2,3,6-tri-O-benzyl-α-D-glucopyranosyl-(1 4)-[2-O-acetyl-3,4,6-tri-Obenzyl- -D-glucopyranosyl-(1 3)]-6-O-benzyl-2-N-acetyl- -D-galactosamino-pyranosyl- (1 3)-2-O-benzoyl-4,6-di-O-benzyl-α-D-mannopyranoside (27). A solution of 23 (99 mg, 32 mol) in toluene (3 ml) was degased by a stream of Argon for 20 minutes. Tributyltin hydrate (175 L, 0.65 mmol) and AIBN (107 mg, 0.65 mmol) were added and stirred at 90 C for 4 h. The reaction mixture was directly loaded on to a sephadex column (LH-20, MeOH/DCM 1:1) to afford 27 (63 mg, 22 mol, 68%) as a colorless oil. 1 H NMR (CDCl 3, 400 MHz) 1.26 (bs, 2H), 1.51 (bs, 4H), 1.68 (s, 3H), 1.78 (s, 3H), 1.83 (s, 3H), 1.90 (s, 3H), 2.08 (s, 3H), 2.46 2.64 (m, 3H), 2.73 (dt, J = 17.8, 7.5 Hz, 1H), 3.09 3.89 (m, 30H), 3.95 4.08 (m, 2H), 4.09 4.26 (m, 35H), 4.95 (d, J = 3.8 Hz, 1H), 4.98 (d, J = 2.3 Hz, 1H), 5.01 (d, J = 3.3 Hz, 1H), 5.18 (s, 2H), 5.34 (s, 1H), 5.48 (d, J = 3.5 Hz, 1H), 5.69 (d, J = 7.3 Hz, 1H), 6.37 (s, 1H), 7.08 7.37 (m, 77H), 7.54 (t, J = 7.7 Hz, 1H), 8.02 (d, J = 7.2 Hz, 2H). 13 C NMR (CDCl 3, 100 MHz) 20.4, 20.7, 22.9, 23.1, 23.7, 28.0, 29.3, 38.1, 62.1, S14

66.8, 67.5, 67.5, 68.1, 68.4, 68.8, 69.5, 69.9, 70.3, 71.3, 71.8, 72.5, 72.6, 72.9, 73.0, 73.0, 73.2, 73.8, 74.0, 74.2, 74.5, 74.6, 74.8, 75.0, 75.1, 78.2, 78.6, 79.5, 82.6, 97.0, 97.8, 98.6, 98.9, 199.8, 101.3, 126.8, 126.9, 126.9, 127.0, 127.1, 127.1, 127.2, 127.2, 127.3, 127.4, 127.4, 127.5, 127.5, 127.5, 127.5, 127.6, 127.7, 127.8, 127.8, 127.9, 127.9, 128.0, 128.0, 128.1, 128.1, 128.2, 128.2, 129.3, 133.1, 136.5, 136.5, 137.3, 137.6, 137.7, 137.8, 137.8, 138.1, 138.2, 138.3, 138.4, 138.4, 138.5, 138.5, 139.4, 165.9, 168.9, 169.3, 170.4, 171.1, 171.2, 206.3. [ ] rt D = +11.5 (c = 0.5, CHCl 3 ). IR (CHCl 3 ): 3031, 2921, 2867, 1745, 1718, 1697, 1670, 1497, 1453, 1364, 1233, 1060 cm 1. MALDI-HRMS: m/z calcd for C 167 H 183 N 3 O 38 Na [M + Na] + 2861.2372, obsd 2861.2259. N-(Benzyl)benzyloxycarbonyl-5-amino-pentyl 3,4,6-Tri-O-benzyl- -D-glucopyranosyl- (1 3)-6-O-benzyl-2-N-acetyl- -D-galactosaminopyranosyl-(1 4)2,3,6-tri-O-benzyl-β-Dglucopyranosyl-(1 4)-[3,4,6-tri-O-benzyl- -D-glucopyranosyl-(1 3)]-6-O-benzyl-2-Nacetyl- -D-galactosaminopyranosyl-(1 3)-4,6-di-O-benzyl-α-D-mannopyranoside (28). To a solution of 27 (36 mg, 13 mol) in THF (2 ml) and MeOH (2 ml), Potassium hydroxide (0.2 ml, aq. 3N) was added. The reaction was stirred at room temperature over night, neutralized by the addition of Amberlite acidic resin IR-120, filtered and concentrated. Purification using recycling preparative size exclusion HPLC (CHCl 3 ) yielded 28 (28 mg, 11 mol, 86%) as a colorless solid. 1 H NMR (CDCl 3, 400 MHz) 1.25 (bs, 2H), 1.48 (bs, 2H), 1.53 (bs, 2H), 1.87 (s, 6H), 3,17 (bs, 1H), 3.23 (bs, 1H), 3.27 5.06 (m, 71H), 5.01 5.17 (m, 3H), 7.08 7.48 (m, 75H). 13 C NMR (CDCl 3, 100 MHz) 21.2, 23.6, 23.7, 29.0, 67.2, 68.8, 68.9, 72.9, 73.1, 73.2, 73.4, 73.4, 73.5, 73.6, 73.7, 74.1, 74.6, 75.0, 75.0, 75.1, 75.1, 75.2, 75.6, 79.3, 80.2, 80.5, 80.7, 84.3, 84.4, 84.5, 84.8, 98.9, 99.2, 100.0, 102.8, 104.1, 105.6, 127.1, 127.2, 127.4, 127.5, 127.5, 127.6, 127.7, 127.9, 127.9, 128.0, 128.1, 128.3, 128.3, 128.3, 128.4, 128.4, 128.4, 128.5, 128.6, 128.6, 136.8, 137.2, 137.8, 137.9, 138.0, 138.1, 138.1, 138.2, 138.2, 138.3, 138.4, 138.4, 138.4, 138.5, 138.5, 138.6, 138.7, 138.7 138.8, 138.9, 139.3, 172.5, 173.3, 176.2. [ ] rt D = +33.8 (c = 0.5, CHCl 3 ). IR (CHCl 3 ): 3030, 2868, S15

1699, 1667, 1496, 1453, 1363, 1093, 1057, 1027 cm 1. MALDI-HRMS: m/z calcd for C 151 H 169 N 3 O 33 Na [M + Na] + 2575.1531, obsd 2575.1464. 5-Amino-pentyl -D-Glucopyranosyl-(1 3)-2-N-acetyl- -D-galactosaminopyranosyl- (1 4)-α-D-glucopyranosyl-(1 4)-[ -D-glucopyranosyl-(1 3)]-2-N-acetyl- -D-galactosaminopyranosyl-(1 3)-α-D-mannopyranoside (2). A mixture of 28 (19 mg, 7.4 mol), palladium on activated charcoal (19 mg), acetic acid (50 L), THF (2 ml), MeOH (2 ml), and water (2 ml) was degassed by a stream of Argon for 30 minutes. Under stirring a balloon with H 2 was applied for 48 h. After 12, 24 and 36 h, additional water (2 ml) was added. The mixture was filtered over a plug of Celite and concentrated. Purification on a sephadex column (G-25, EtOH 5% in water) afforded 10.3 mg of a colorless solid. This solid was submitted to reverse phase HPLC (C-18 column, acetonitrile 1% 10% in water) and yielded after lyophilization 2 (8.2 mg, 7.1 mol, 95%) as a colorless fluffy solid. 1 H NMR (D 2 O, 500 MHz) 1.41 1.55 (m, 2H), 1.61 1.77 (m, 4H), 2.05 (s, 3H), 2.09 (s, 3H), 3.02 (t, J = 7.6 Hz, 2H), 3.10 (dd, J = 9.2, 8.0 Hz, 1H), 3.32 (dd, J = 9.1, 8.0 Hz, 1H), 3.38 3.45 (m, 4H), 3.48 (t, J = 9.1 Hz, 2H), 3.57 (dd, J = 10.2, 3.6 Hz, 1H), 3.59 (t, J = 5.0 Hz, 1H), 3.63 4.08 (m, 25 H), 4.22 (d, J = 3.1 Hz, 1H), 4.27 4.34 (m, 2H), 4.45 (d, J = 7.9 Hz, 1H), 4.53 (d, J = 7.9 Hz, 1H), 4.64 (d, J = 8.5 Hz, 1H), 4.77 (s, 1H), 4.89 (d, J = 1.7 Hz, 1H), 4.99 (d, J = 3.7 Hz, 1H). 13 C NMR (D 2 O, 125 MHz) 22.3, 22.4, 26.5, 28.0, 39.4, 51.5, 52.3, 59.4, 60.2, 60.5, 60.7, 60.9, 61.0, 65.1, 67.5, 67.8, 69.4, 69.7, 69.8, 71.3, 71.4, 72.8, 72.8, 73.1, 74.7, 75.0, 75.4, 75.5, 75.6, 75.7, 75.8, 78.5, 78.7, 78.7, 79.9, 98.7, 99.5, 99.5, 101.4, 104.4, 105.0, 174.8, 175.0. MALDI-HRMS: m/z calcd for C 45 H 79 N 3 O 31 H [M + H] + 1158.4770, obsd 1158.4778. 3-(3,4-Dioxo-2-ethoxycyclobut-1-enylamino)pentyl -D-Glucopyranosyl-(1 3)-2-Nacetyl- -D-galactosaminopyranosyl-(1 4)- -D-glucopyranosyl-(1 4)-[ -Dglucopyranosyl-(1 3)]-2-N-acetyl- -D-galactos-aminopyranosyl-(1 3)-α-Dmannopyranoside (24). S16

Diethyl squarate (0.5 L, 3.5 mol) was added to a solution of 2 (0.5 mg, 0.4 mol) in EtOH (0.3 ml) and phosphate buffer (0.3 ml, 50 mm, ph 7.2) and shaken for 16 h at room temperature. Most of the ethanol was removed by a stream of N 2. The mixture was purified by a HPLC superdex size exclusion column (95:5 H 2 O, EtOH) to afford 24 (0.2 mg, 1.6 mol, 36%) of a cololess solid. ESI-HRMS: m/z calcd for C 51 H 84 N 3 O 34 [M + H] + 1282.4931, obsd 1282.4921. 2 O OH AcHN OH O OH O O O OH AcHN O NH 2 OH S17

2 O OH AcHN OH O OH O O O OH AcHN O NH 2 OH S18

OH O OH AcHN OH O OH O O O OH AcHN O NH 2 2 S19

OH O OH AcHN OH O OH O O O OH AcHN O 24 H N OEt S20

Comparison Isolated Polysaccharide PS-II and Hexasaccharide 2 Comparing the NMR spectra of our hexasaccharide hapten 2 with a recently published very closely related hexasaccharid (Danieli et. al 2011), the spectra match enough to confirm the structure. Comparing hapten 2 with the natural polysaccharide PS-II, differs slightly. Specifically, the signal of the anomeric region of the mannose at the reducing end is shifted, where the isolated polysaccharide PS-II is tethered via a phosphate to the next repeating unit. All other diagnostic anomeric signals match very well. Comparison of diagnostic NMRsignals: Isolated polysaccharide PS-II: ( 1 H NMR (D 2 O, 400 MHz), ( 13 C NMR (D 2 O, 100 MHz))): -Man (A) (5.44 (H-1), 97.0 (C- 1); 4.07 (H-2), 69.2 (C-2)), -Glc (B) (4.99 (H-1), 99.6 (C-1)), -GalNAc (C) (4.76 (H-1), 100.7 (C-1)), -GalNAc (D) (4.64 (H-1), 102.3 (C-1)), -Glc (E) (4.53, (H-1), 105.5 (C-1)), -Glc (F) (4.45 (H-1), 106.0 (C-1)). Hexassaccharide 2: 1 H NMR (D 2 O, 500 MHz): 4.99 (d, J = 3.7 Hz, H(1 A )), 4.22 (d, J = 3.1 Hz, H(2 A )), 4.89 (d, J = 1.7 Hz, H-(1 B )), 4.77 (s, H-(1 C )), 4.64 (d, J = 8.5 Hz, H-(1 D )), 4.53 (d, J = 7.9 Hz, H-(1 E )), 4.45 (d, J = 7.9 Hz, (H-1 F )). 13 C NMR (D 2 O, 125 MHz): 105.0, 104.4, 101.4, 99.5, 99.5, 98.7. S21

Literature Chen, C.-T., Weng, S.-S., Kao, J.-Q., Lin, C.-C., and Jan, M.-D. (2005). Stripping off Water at Ambient Temperature: Direct Atom-Efficient Acetal Formation between Aldehydes and Diols Catalyzed by Water-Tolerant and Recoverable Vanadyl Triflate. Org. Lett. 7, 3343-3346. Danieli E., Lay L., Proietti D., Berti F., Constantino P., and Adamo R. (2011). First Synthesis of C. difficile PS-II Cell Wall Polysaccharide Repeating Unit. Org. Lett. 12, DOI: 10.1021/ol11026188. Gemma, E., Lahmann, M., and Oscarson, S. (2005). Synthesis of the tetrasaccharide -D- Glcp(1 3)- -D-Manp-(1 2)- -D-Manp-(1 2)- -D-Manp recognized by Calreticulin/Calnexin. Carbohydr. Res. 340, 2558-2562. Ravidà, A., Liu, X., Kovac, L., and Seeberger, P. H. (2006). Synthesis of Glycosyl Phosphates from 1,2-Orthoesters and Application to in Situ Glycosylation Reactions. Org. Lett. 8, 1815-1818. Van Steijn, A. M. P., Kamerling, J. P., and Vliegenhart, F. G. (1992). Synthesis of trisaccharide methyl glycoside related to fragments of the capsular polysaccharide of Streptococcus pneumoniae type 18C. Carbohydr. Res. 225, 229-245. S22