2 Results and Discussion
3.3 Automated Glycan Assembly of Arabino- and Glucuronoxylan
3.3.2 Automated Glycan Assembly of Arabinoxylan Oligosaccharides
3.3.2 Automated Glycan Assembly of Arabinoxylan
The protected tetrasaccharide was dissolved in DCM/MeOH (2:1, 3 mL) and NaOMe (0.5 M in MeOH, 0.500 mL) was added. The reaction mixture was stirred overnight and subsequently neutralized by addition of prewashed Amberlite IR-120 (H+) resin. The resin was filtered off and the solvents were removed in vacuo. The crude product was purified by reversed phase HPLC using a preparative C5 column affording the semi-protected tetrasaccharide. The product was dissolved in a mixture of EtOAc/MeOH/AcOH/H2O (4:2:2:1, 3 mL) and the resulting solution was added to a round-bottom flask containing Pd/C (10% Pd, 10.0 mg). The suspension was saturated with H2
for 30 min and stirred under a H2-atmosphere overnight. After filtration of the reaction mixture through a syringe filter (RC 0.45 m) the solvents were evaporated to provide the fully deprotected tetrasaccharide 10 (1.4 mg, 2.19 µmol, 13% over 11 steps, based on resin loading). 1H NMR (600 MHz, D2O): δ = 5.21 (s, 1H), 4.51 (d, J = 7.4 Hz, 1H), 4.39 (d, J = 7.8 Hz, 1H), 4.34 (d, J = 7.9 Hz, 1H), 4.11-4.08 (m, 1H), 4.08-4.01 (m, 3H), 3.92-3.86 (m, 2H), 3.82-3.78 (m, 1H), 3.77-3.73 (m, 2H), 3.72-3.67 (m, 1H), 3.66-3.58 (m, 3H), 3.57-3.53 (m, 1H), 3.49 (t, J = 9.2 Hz, 1H), 3.39-3.31 (m, 4H), 3.25-3.17 (m, 3H), 2.93 (t, J = 7.5 Hz, 2H), 1.65-1.55 (m, 4H), 1.40-1.34 (m, 2H) ppm. 13C NMR (151 MHz, D2O): δ = 108.5, 102.8, 101.9, 100.1, 84.5, 80.9, 79.0, 76.6, 76.4, 76.0, 75.6, 73.9, 73.7, 72.9, 72.8, 70.1, 69.2, 65.2, 62.9, 62.8, 61.3, 39.3, 28.2, 26.4, 22.1 ppm. ESI-HRMS: m/z= [M+H]+ calcd. for C25H46NO17+: 632.2760, found 632.2804.
RP-HPLC of the deprotected tetrasaccharide (ELSD trace):
Aminopentyl 2-O-[α-L-arabinofuranosyl]-β-D-xylopyranosyl-(1→4)-β-D- xylo-pyranosyl-(1→4)-β-D-xylopyranoside (11)
Linker-functionalized resin 9 (52.0 mg, 16.9 mol) was placed in the synthesizer and synthesizer modules were applied as follows:
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module A: 2 x 1.8 equiv 1c, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module E: 15 equiv. PBu3, 5% H2O in THF, 6 x 30 min, 45 °C
Module B: 2 x 1.8 equiv 2a, NIS, DCM/dioxane, 2 x 45 min, -20 to -5 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Cleavage from the resin using UV irradiation at 305 nm in a continuous flow photoreactor afforded the crude product. Purification by normal phase HPLC using a preparative YMC diol column gave the protected tetrasaccharide.
Crude NP-HPLC (ELSD trace):
The protected tetrasaccharide was dissolved in DCM/MeOH (2:1, 3 mL) and NaOMe (0.5 M in MeOH, 0.500 mL) was added. The reaction mixture was stirred overnight and subsequently neutralized by addition of prewashed Amberlite IR-120 (H+) resin. The resin was filtered off and the solvents were removed in vacuo. The crude product was purified by reversed phase HPLC using a preparative C5 column affording the semi-protected tetrasaccharide. The product was dissolved in a mixture of EtOAc/MeOH/AcOH/H2O (4:2:2:1, 3 mL) and the resulting solution was added to a round-bottom flask containing Pd/C (10% Pd, 10.0 mg). The suspension was saturated with H2 for 30 min and stirred under a H2-atmosphere overnight. After filtration of the reaction mixture through a syringe filter (RC 0.45 m) the solvents were evaporated to provide the fully deprotected tetrasaccharide 11 (4.4 mg, 6.92 µmol, 41% over 11 steps, based on resin loading). 1H NMR (600 MHz, D2O): δ = 5.29 (s, 1H), 4.57 (d, J = 7.5 Hz, 1H), 4.48 (d, J = 7.8 Hz, 1H), 4.43 (d, J = 7.8 Hz, 1H), 4.21-4.04 (m, 4H), 4.02-3.51 (m, 12H), 3.46-3.36 (m, 3H), 3.35-3.25 (m, 3H), 3.02 (s, 2H), 1.76-1.62 (m, 4H), 1.52-1.41 (m, 2H) ppm. 13C NMR (151 MHz, D2O): δ = 111.1, 105.4, 104.3, 102.9, 87.2, 83.6, 80.7, 79.3, 79.0, 78.7, 78.3, 76.5, 76.3, 75.5, 75.3, 72.7, 71.7, 67.6, 65.6, 65.5, 63.9, 42.0, 30.8, 29.0, 24.7 ppm.
ESI-HRMS: m/z= [M+H]+calcd. for C25H46NO17: 632.2766; found: 632.2770.
RP-HPLC of the deprotected tetrasaccharide (ELSD trace):
Aminopentyl β-D-xylopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-2-O-[α-L- arabino-furanosyl]-β-D-xylopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D- xylopyranosyl-(1→4)-β-D-xylopyranoside (12)
Linker-functionalized resin 9 (104 mg, 33.8 mol) was placed in the synthesizer and synthesizer modules were applied as follows:
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module A: 2 x 1.8 equiv 1c, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module E: 15 equiv. PBu3, 5% H2O in THF, 6 x 30 min, 45 °C
Module B: 2 x 1.8 equiv 2a, NIS, DCM/dioxane, 2 x 45 min, -20 to -5 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Cleavage from the resin using UV irradiation at 305 nm in a continuous flow photoreactor afforded the crude product. Purification by normal phase HPLC using a
preparative YMC diol column gave the protected heptasaccharide.
Crude NP-HPLC (ELSD trace):
The protected heptasaccharide was dissolved in DCM/MeOH (2:1, 3 mL) and NaOMe (0.5 M in MeOH, 0.500 mL) was added. The reaction mixture was stirred overnight and subsequently neutralized by addition of prewashed Amberlite IR-120 (H+) resin. The resin was filtered off and the solvents were removed in vacuo. The crude product was purified by reversed phase HPLC using a preparative C5 column affording the semi-protected heptasaccharide. The product was dissolved in a mixture of EtOAc/MeOH/AcOH/H2O (4:2:2:1, 3 mL) and the resulting solution was added to a round-bottom flask containing Pd/C (10% Pd, 10.0 mg). The suspension was saturated with H2
for 30 min and stirred under a H2-atmosphere overnight. After filtration of the reaction mixture through a syringe filter (RC 0.45 m) the solvents were evaporated to provide the fully deprotected heptasaccharide 12 (2.6 mg, 2.48 µmol, 7% over 17 steps, based on resin loading). 1H NMR (600 MHz, D2O): δ = 5.30 (t, J = 0.9 Hz, 1H), 4.60 (d, J = 7.4 Hz, 1H), 4.52-4.46 (m, 4H), 4.43 (d, J = 7.9 Hz, 1H), 4.18 (dd, J = 2.9, 1.3 Hz, 1H), 4.17-4.07 (m, 7H), 4.01-3.95 (m, 2H), 3.92-3.61 (m, 13H), 3.60-3.54 (m, 4H), 3.49-3.36 (m, 7H), 3.35-3.25 (m, 6H), 3.01 (t, J = 7.6 Hz, 2H), 1.73-1.64 (m, 4H), 1.46 (p, J = 7.8 Hz, 2H) ppm. 13C NMR (151 MHz, D2O): δ = 111.0, 105.4, 104.4, 104.3, 104.3, 104.2, 102.6, 87.2, 83.6, 80.5, 79.3, 79.0 (2C), 78.9 (2C), 78.6, 78.2, 76.4, 76.3, 76.2 (2C), 75.6, 75.4, 75.3 (2C), 72.8, 71.8, 71.1, 67.8, 65.6, 65.5 (2C), 65.4, 63.9, 42.0, 30.8, 29.1, 25.9, 24.7, 22.6 ppm. ESI-HRMS: m/z= [M+Na]+calcd. for C40H70NO29: 1028.4034; found 1028.4050.
RP-HPLC of the deprotected heptasaccharide (ELSD trace):
Aminopentyl β-D-xylopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-2-O-[β-D- xylopyra-nosyl]-β-D-xylopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D- xylopyranosyl-(1→4)-β-D-xylopyranoside (13)
Linker-functionalized resin 9 (104 mg, 33.8 mol) was placed in the synthesizer and synthesizer modules were applied as follows:
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module A: 2 x 1.8 equiv 1c, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C
Module E: 15 equiv. PBu3, 5% H2O in THF, 6 x 30 min, 45 °C
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Cleavage from the resin using UV irradiation at 305 nm in a continuous flow photoreactor afforded the crude product. Purification by normal phase HPLC using a preparative YMC diol column gave the protected heptasaccharide.
Crude NP-HPLC (ELSD trace):
The protected heptasaccharide was dissolved in DCM/MeOH (2:1, 3 mL) and NaOMe (0.5 M in MeOH, 0.500 mL) was added. The reaction mixture was stirred overnight and subsequently neutralized by addition of prewashed Amberlite IR-120 (H+) resin. The resin was filtered off and the solvents were removed in vacuo. The crude product was purified by reversed phase HPLC using a preparative C5 column affording the semi-protected heptasaccharide. The product was dissolved in a mixture of EtOAc/MeOH/AcOH/H2O (4:2:2:1, 3 mL) and the resulting solution was added to a round-bottom flask containing Pd/C (10% Pd, 10.0 mg). The suspension was saturated with H2 for 30 min and stirred under a H2-atmosphere overnight. After filtration of the reaction mixture through a syringe filter (RC 0.45 m) the solvents were evaporated to provide the fully deprotected heptasaccharide 13 (3.8 mg, 3.69 µmol, 11% over 16 steps, based on resin loading). 1H NMR (600 MHz, D2O): δ = 4.68-4.62 (m, 2H), 4.52-4.46 (m, 4H), 4.43 (d, J = 7.9 Hz, 1H), 4.17-4.06 (m, 5H), 4.01-3.95 (m, 2H), 3.92-3.74 (m, 7H), 3.72-3.51 (m, 8H), 3.50-3.24 (m, 15H), 3.05-2.98 (m, 2H), 1.73-1.64 (m, 4H), 1.50-1.43 (m, 2H) ppm. 13C NMR (151 MHz, D2O): δ = 106.3, 105.4, 104.4, 104.3 (2C), 104.2, 102.7, 82.9, 78.9 (3C), 78.8, 78.2, 78.0, 76.4, 76.3, 76.2 (3C), 75.7, 75.6, 75.4, 75.3 (2C), 72.7, 71.8, 67.8, 67.8, 65.6, 65.5 (2C), 65.0, 41.9, 30.8, 29.0, 24.7 ppm. ESI-HRMS: m/z [M+H]+ calcd. for C40H70NO29: 1028.4034; found: 1028.4048.
RP-HPLC of the deprotected heptasaccharide (ELSD trace):
Aminopentyl α-L-arabinofuranosyl-(1→2)-β-D-xylopyranosyl-(1→4)-β-D- xylo-pyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D- xylo-pyranosyl-(1→4)-β-D-xylopyranoside (14)
Linker-functionalized resin 9 (104 mg, 33.8 mol) was placed in the synthesizer and synthesizer modules were applied as follows:
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C
Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module A: 2 x 1.8 equiv 1c, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module E: 15 equiv. PBu3, 5% H2O in THF, 6 x 30 min, 45 °C
Module B: 2 x 1.8 equiv 2a, NIS, DCM/dioxane, 2 x 45 min, -20 to -5 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Cleavage from the resin using UV irradiation at 305 nm in a continuous flow photoreactor afforded the crude product. Purification by normal phase HPLC using a preparative YMC diol column gave the protected heptasaccharide.
Crude NP-HPLC (ELSD trace):
The protected heptasaccharide was dissolved in DCM/MeOH (2:1, 3 mL) and NaOMe (0.5 M in MeOH, 0.500 mL) was added. The reaction mixture was stirred overnight and subsequently neutralized by addition of prewashed Amberlite IR-120 (H+) resin. The resin was filtered off and the solvents were removed in vacuo. The crude product was purified by reversed phase HPLC using a preparative C5 column affording the semi-protected heptasaccharide. The product was dissolved in a mixture of EtOAc/MeOH/AcOH/H2O (4:2:2:1, 3 mL) and the resulting solution was added to a round-bottom flask containing Pd/C (10% Pd, 10.0 mg). The suspension was saturated with H2 for 30 min and stirred under a H2-atmosphere overnight. After filtration of the reaction mixture through a syringe filter (RC 0.45 m) the solvents were evaporated to provide the fully deprotected heptasaccharide 14 (1.65 mg, 1.61 µmol, 5% over 17 steps, based on resin loading). 1H NMR (600 MHz, D2O): 5.22 (s, 1H), 4.49 (d, J = 7.6 Hz, 1H), 4.42 (d, J = 7.7 Hz, 4H), 4.35 (d, J = 7.8 Hz, 1H), 4.12 – 3.99 (m, 6H), 3.92 (dd, J = 11.7, 5.4 Hz, 1H), 3.88 (dd, J = 5.8, 2.9 Hz, 1H), 3.84 – 3.79 (m, 1H), 3.78 – 3.68 (m, 6H), 3.67 – 3.57 (m, 4H), 3.53 – 3.45 (m, 6H), 3.38 – 3.29 (m, 6H), 3.27 – 3.19 (m, 6H), 2.94 (t, J = 7.6 Hz, 2H), 1.61 (dp, J = 21.2, 7.2, 6.8 Hz, 4H), 1.38 (p, J = 7.9 Hz, 2H) ppm. 13C NMR (151 MHz, D2O): δ = 108.4, 102.7, 101.6, 101.6, 100.2, 84.5, 80.9, 78.1, 76.6, 76.3, 76.2, 76.0, 75.6, 73.8, 73.7, 73.6, 72.9, 72.6, 70.1, 69.0, 64.9, 63.0, 62.9, 62.8, 61.2, 39.3, 28.1, 26.3 ppm. ESI-HRMS: m/z [M+H]+ calcd. for C40H70NO29: 1028.4034; found: 1028.4022.
RP-HPLC of the deprotected heptasaccharide (ELSD trace):
Aminopentyl β-D-xylopyranosyl-(1→4)-2-O-[α-L-arabinofuranosyl]-3-O-[α-L-arabino-furanosyl] β-D-xylopyranosyl-(1→4)-β-D-xylopyranoside (15)
Linker-functionalized resin 9 (104 mg, 33.8 mol) was placed in the synthesizer and synthesizer modules were applied as follows:
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module A: 2 x 1.8 equiv 1d, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module E: 15 equiv. PBu3, 5% H2O in THF, 6 x 30 min, 45 °C
Module B: 2 x 1.8 equiv 2a, NIS, DCM/dioxane, 2 x 45 min, -20 to -5 °C Module D: DDQ, DCE/MeOH/H2O (64:16:1), 7 x 30 min, 40 °C
Module B: 2 x 1.8 equiv 2a, NIS, DCM/dioxane, 2 x 45 min, -20 to -5 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Cleavage from the resin using UV irradiation at 305 nm in a continuous flow photoreactor afforded the crude product. Purification by normal phase HPLC using a preparative YMC diol column gave the protected pentasaccharide.
Crude NP-HPLC (ELSD trace):
The protected pentasaccharide was dissolved in DCM/MeOH (2:1, 3 mL) and NaOMe (0.5 M in MeOH, 1.00 mL) was added. The reaction mixture was stirred overnight and subsequently neutralized by addition of prewashed Amberlite IR-120 (H+) resin. The resin was filtered off and the solvents were removed in vacuo. The crude product was purified by reversed phase HPLC using a preparative C5 column affording the semi-protected pentasaccharide. The product was dissolved in a mixture of EtOAc/MeOH/AcOH/H2O (4:2:2:1, 3 mL) and the resulting solution was added to a round-bottom flask containing Pd/C (10% Pd, 10.0 mg). The suspension was saturated with H2 for 30 min and stirred under a H2-atmosphere overnight. After filtration of the reaction mixture through a syringe filter (RC 0.45 m) the solvents were evaporated. After purification by preparative HPLC using a Hypercarb column fully deprotected pentasaccharide 15 (1.1 mg, 1.45 µmol, 4% over 13 steps, based on resin loading) was obtained. 1H NMR (700 MHz, D2O): δ = 5.14 (s, 1H), 5.09 (s, 1H), 4.50 (d, J = 7.2 Hz, 1H), 4.29 (dd, J = 17.5, 7.9 Hz, 2H), 4.18 (d, J = 4.7 Hz, 1H), 4.06-3.95 (m, 5H), 3.86-3.39 (m, 16H), 3.29 (t, J = 9.5 Hz, 3H), 3.13 (q, J = 11.0, 9.4 Hz, 3H), 2.84 (q, J = 7.1 Hz, 1H), 1.58-1.46 (m, 4H), 1.37-1.26 (m, 2H) ppm. 13C NMR (176 MHz, D2O): δ = 111.3, 110.7, 105.4, 104.0, 103.9, 102.5, 87.0, 83.8, 83.6, 83.3, 81.2, 80.1, 79.8, 79.2, 78.4, 78.2, 76.5, 76.3, 75.6, 72.7, 71.8, 67.7, 65.5, 65.1, 63.8, 63.7, 41.9, 30.8, 29.0, 24.7 ppm. ESI-HRMS:
m/z= [M+H]+calcd. for C30H54NO21+: 764.3188; found 764.3169.
RP-HPLC (ELSD trace) of the deprotected pentasaccharide:
Aminopentyl β-D-xylopyranosyl-(1→4)-3-O-[2-O-[β-D-xylopyranosyl]-α-L- arabino-furanosyl]-β-D-xylopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranoside (16)
Linker-functionalized resin 9 (104 mg, 33.8 mol) was placed in the synthesizer and synthesizer modules were applied as follows:
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module A: 2 x 1.8 equiv 1b, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module F: Ac2O, pyridine, 30 min
Module D: DDQ, DCE/MeOH/H2O (64:16:1), 7 x 30 min, 40 °C
Module B: 2 x 1.8 equiv 2a, NIS, DCM/dioxane, 2 x 45 min, -20 to -5 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Cleavage from the resin using UV irradiation at 305 nm in a continuous flow photoreactor afforded the crude product. Purification by normal phase HPLC using a preparative YMC diol column gave the protected hexasaccharide.
Crude NP-HPLC (ELSD trace):
The protected Hexasaccharide was dissolved in DCM/MeOH (2:1, 3 mL) and NaOMe (0.5 M in MeOH, 1.00 mL) was added. The reaction mixture was stirred overnight and subsequently neutralized by addition of prewashed Amberlite IR-120 (H+) resin. The resin was filtered off and the solvents were removed in vacuo. The crude product was purified by reversed phase HPLC using a preparative C5 column affording the semi-protected hexasaccharide. The product was dissolved in a mixture of EtOAc/MeOH/AcOH/H2O (4:2:2:1, 3 mL) and the resulting solution was added to a round-bottom flask containing Pd/C (10% Pd, 10.0 mg). The suspension was saturated with H2
for 30 min and stirred under a H2-atmosphere overnight. After filtration of the reaction mixture through a syringe filter (RC 0.45 m) the solvents were evaporated to provide the fully deprotected hexasaccharide 16 (15.5 mg, 17.3 µmol, 17% over 17 steps, based on resin loading). 1H NMR (600 MHz, D2O): δ = 5.56 (s, 1H), 4.57 (d, J = 7.9 Hz, 1H), 4.52 (d, J = 7.7 Hz, 1H), 4.49 (d, J = 7.7 Hz, 1H), 4.45 (d, J = 7.8 Hz, 1H), 4.43 (d, J = 7.9 Hz, 1H), 4.31 (td, J = 5.5, 3.6 Hz, 1H), 4.29 – 4.26 (m, 1H), 4.16-4.05 (m, 4H), 3.98 (dd, J = 11.6, 5.5 Hz, 1H), 3.95-3.53 (m, 13H), 3.50-3.21 (m, 12H), 3.02 (t, J = 7.6 Hz, 2H), 1.75-1.62 (m, 4H), 1.50-1.40 (m, 2H) ppm. 13C NMR (151 MHz, D2O): δ = 109.0, 105.4, 105.2, 104.3, 104.2, 104.0, 91.3, 86.6, 80.1, 79.0, 78.9, 78.3, 78.2, 78.1, 76.5, 76.2, 76.2, 75.8, 75.6, 75.6, 75.4, 75.3, 72.7, 71.8, 71.7, 67.9, 67.7, 65.6, 65.5, 65.4, 63.5, 41.9, 30.8, 29.0, 24.7 ppm. ESI-HRMS: m/z [M+H]+ calcd. for C35H62NO25: 896.3611; found: 896.3641.
RP-HPLC (ELSD trace) of the deprotected hexasaccharide:
Aminopentyl -L-arabinofuranosyl-(1→3)--L-arabinofuranosyl-(1→4)-β-D- xylo-pyranosyl-(1→4)-3-O-[3-O-[a-L-arabinofuranosyl]-α-L-arabinofuranosyl]-β-D- xylo-pyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranoside (17)
Linker-functionalized resin 9 (104 mg, 33.8 mol) was placed in the synthesizer and synthesizer modules were applied as follows:
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module A: 2 x 1.8 equiv 1b, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module A: 2 x 1.8 equiv 1a, TMSOTf, DCM, 2 x 35 min, -35 °C to -15 °C Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module D: DDQ, DCE/MeOH/H2O (64:16:1), 7 x 30 min, 40 °C
Module B: 2 x 1.8 equiv 2c, NIS, DCM/dioxane, 2 x 45 min, -20 to -5 °C
Module C: 20% NEt3 in DMF, 4 x 5 min, r.t.
Module B: 2 x 1.8 equiv 2a, NIS, DCM/dioxane, 2 x 45 min, -20 to -5 °C
Cleavage from the resin using UV irradiation at 305 nm in a continuous flow photoreactor afforded the crude product. Purification by normal phase HPLC using a preparative YMC diol column gave the protected octasaccharide.
Crude NP-HPLC (ELSD trace):
The protected octasaccharide was dissolved in DCM/MeOH (2:1, 3 mL) and NaOMe (0.5 M in MeOH, 1.00 mL) was added. The reaction mixture was stirred overnight and subsequently neutralized by addition of prewashed Amberlite IR-120 (H+) resin. The resin was filtered off and the solvents were removed in vacuo. The crude product was purified by reversed phase HPLC using a preparative C5 column affording the semi-protected octasaccharide. The product was dissolved in a mixture of EtOAc/MeOH/AcOH/H2O (4:2:2:1, 3 mL) and the resulting solution was added to a round-bottom flask containing Pd/C (10% Pd, 10.0 mg). The suspension was saturated with H2 for 30 min and stirred under a H2-atmosphere overnight. After filtration of the reaction mixture through a syringe filter (RC 0.45 m) the solvents were evaporated to provide the fully deprotected octasaccharide 17 (2.4 mg, 2.07 µmol, 8% over 15 steps, based on resin loading). 1H NMR (600 MHz, D2O): = 5.17 (s, 1H), 5.02 (s, 2H), 5.01 (s, 1H), 4.99 (s, 1H), 4.38 (d, J = 7.7 Hz, 1H), 4.34 (d, J = 7.7 Hz, 1H), 4.31 (d, J = 7.7 Hz, 1H), 4.28 (d, J
= 7.2 Hz, 4H), 4.20 – 4.18 (m, 1H), 4.13 – 4.08 (m, 2H), 4.03 – 3.92 (m, 7H), 3.91 – 3.87 (m, 4H), 3.82 – 3.79 (m, 2H), 3.76 – 3.52 (m, 20H), 3.42 (t, J = 9.2 Hz, 2H), 3.38 (t, J = 9.2 Hz, 1H), 3.32 – 3.21 (m, 5H), 3.20 – 3.10 (m, 5H), 2.87 (t, J = 7.6 Hz, 2H), 1.58 – 1.49 (m, 4H), 1.31 (p, J = 7.8 Hz, 2H) ppm. 13C NMR (151 MHz, D2O): = 110.8, 109.8, 109.6, 108.3, 105.4, 104.2, 104.2, 103.7, 86.7, 86.3, 85.6, 85.2, 84.9, 84.7, 84.0, 83.7, 82.7, 82.2, 81.2, 79.1, 78.9, 76.6, 76.4, 76.3, 76.2, 75.8, 75.5, 75.3, 72.7, 65.5, 65.5, 65.2, 63.8, 63.7, 63.5, 63.1, 41.9, 30.8, 29.0, 24.7 ppm. ESI-HRMS: m/z [M+H]+ calcd. for C45H78NO33+: 1160.4462; found: 1160.4506.
RP-HPLC (ELSD trace) of the deprotected octasaccharide: