Supporting Information Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2007
Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2006 Supporting Information for High-Throughput Substrate Specificity Studies of Sialidases by Using Chemoenzymatically Synthesized Sialoside Libraries Harshal Chokhawala, Hai Yu, and Xi Chen* General Methods 1 H NMR (300, 400 or 600 MHz) and 13 C NMR (75 or 100 MHz) spectra were recorded on a Varian Mercury-300, a Varian Inova-400, or a Varian Inova-600 spectrometer. Low and high resolution electrospray ionization (ESI) mass spectra were obtained at the Mass Spectrometry and Proteomics Facility in the Ohio State University. NMR and HRMS Data of Sialosides 4-Nitrophenyl O-(5-acetamido-3,5-dideoxy-D-glycero-a-D-galacto-2-nonulopyranosylonic acid)-(2? 3)-O-b-D-galactopyranoside (Neu5Aca2,3GalbpNP, 8) Yield, 91%; light yellow foam. 1 H NMR (400 MHz, D2O) δ 8.25 (d, 2H, J = 9.2 Hz), 7.23 (d, 2H, J = 9.2 Hz), 5.29 (d, 1H, J = 7.6 Hz), 4.22 (dd, 1H, J = 9.6 and 3.2 Hz), 4.02 (d, 1H, J = 3.2 Hz), 3.92-3.50 (m, 11H), 2.76 (dd, 1H, J = 12.4 and 4.4 Hz, H-3 eq ), 2.00 (s, 3H), 1.80 (t, 1H, J = 12.4 Hz, H-3 ax ); 13 C NMR (100 MHz, D 2 O) δ 175.33, 174.09, 161.99, 142.76, 126.37(2C), 116.75(2C), 100.18, 100.00, 75.74, 73.16, 72.32, 72.05, 69.07, 68.56, - 1 -
68.34, 67.60, 62.78, 60.95, 52.00, 39.94, 22.36. HRMS (ESI) m/z calcd for C 23 H 31 N 2 O 16 (M-Na) 591.1674, found 591.1674. 4-Nitrophenyl O-(5-acetamido-9-O-acetyl-3,5-dideoxy-D-glycero-a-D-galacto-2-nonulopyranosylonic acid)-(2? 3)-O-b-D-galactopyranoside (Neu5,9Ac2a2,3GalbpNP, 9) Yield, 87%; light yellow foam. 1 H NMR (400 MHz, D 2 O) δ 8.25 (d, 2H, J = 9.2 Hz), 7.24 (d, 2H, J = 9.2 Hz), 5.29 (d, 1H, J = 7.6 Hz), 4.37 (d, 1H, J = 11.6 Hz), 4.23 (d, 1H, J = 9.6 Hz), 4.15 (dd, 1H, J = 12.0 and 5.6 Hz), 4.08-3.50 (m,10h), 2.77 (dd, 1H, J = 12.4 and 4.4 Hz, H-3 eq ), 2.06 (s, 3H), 2.02 (s, 3H), 1.81 (t, 1H, J = 12.4 Hz, H-3 ax ); 13 C NMR (100 MHz, D 2 O) δ 175.14, 174.46, 173.96, 161.84, 142.67, 126.24 (2C), 116.56 (2C), 99.96, 99.81, 75.60, 72.83, 72.18, 69.54, 68.93, 68.51, 67.34, 65.80, 62.59, 60.80, 51.80, 39.85, 22.18, 20.35. HRMS (ESI) m/z calcd for C 25 H 33 N 2 O 17 (M-Na) 633.1779, found 633.1797. 4-Nitrophenyl O-(5-acetamido-3,5-dideoxy-9-O-lactyl-D-glycero-a-D-galacto-2-nonulopyranosylonic acid)-(2? 3)-O-b-D-galactopyranoside (Neu5Ac9Lta2,3GalbpNP, 10) Yield, 75%; light yellow foam. 1 H NMR (600 MHz, D 2 O) δ 8.25 (d, 2H, J = 9.0 Hz), 7.24 (d, 2H, J = 9.0 Hz), 5.28 (d, 1H, J = 7.8 Hz), 4.41-4.36 (m, 2H), 4.31 (dd, 1H, J = 12.0 and 5.4 Hz), 4.23 (dd, 1H, J = 9.6 and 3.0 Hz), 4.10-4.07 (m, 1H), 4.03 (d, 1H, J = 3.0 Hz), 3.92-3.52 (m, 8H), 2.77 (dd, 1H, J = 12.6 and 4.8 Hz, H-3 eq ), 2.01 (s, 3H), 1.81 (t, 1H, J = 12.6 Hz, H-3 ax ), 1.35 (d, 3H, J = 7.2 Hz); 13 C NMR (150 MHz, D 2 O) δ 176.32, 175.09, 173.91, 161.85, 142.67, 126.26(2C), 116.56(2C), 100.02, 100.01, 75.66, 72.80, 69.55, 68.41, 68.07, 67.37, 66.74, 66.09, 62.61, 60.82, 59.45, 51.83, 39.88, 22.18, 19.35. HRMS (ESI) m/z calcd for C 26 H 35 N 2 O 18 (M-Na) 663.1885, found 663.1904. 4-Nitrophenyl O-(3,5-dideoxy-5-glycolylamido-D-glycero-a-D-galacto-2-nonulopyranosylonic acid)-(2? 3)-O-b-D-galactopyranoside (Neu5Gca2,3GalbpNP, 11) Yield, 76%; light yellow foam. 1 H NMR (400 MHz, D 2 O) δ 8.24 (d, 2H, J = 9.2 Hz), 7.23 (d, 2H, J = 9.2 Hz), 5.28 (d, 1H, J = 7.6 Hz), 4.23 (dd, 1H, J = 9.6 and 2.4 Hz), 4.09 (s, 2H), 4.03 (d, 1H, J = 3.2 Hz), 3.93-3.55 (m, 11H), 2.78 (dd, 1H, J = 12.4 and 4.4 Hz, H-3 eq ), 1.82 (t, 1H, J = 12.4 Hz, H-3ax ); 13 C NMR (100 MHz, D2O) δ 175.92, 174.00, 161.84, 142.66, 126.23 (2C), 116.56 (2C), 100.00, 99.78, 75.61 (2C), 72.75, 72.00, 68.94, 68.23 (2C), - 2 -
67.39, 62.67, 61.11, 60.80, 51.50, 39.91. HRMS (ESI) m/z calcd for C 23 H 31 N 2 O 17 (M-Na) 607.1623, found 607.1626. 4-Nitrophenyl O-(3-deoxy-D-glycero-a-D-galacto-2-nonulopyranosylonic acid)- (2? 3)-O-b-D-galactopyranose (KDNa2,3GalbpNP, 12). Yield, 75%; white foam. 1 H NMR (400 MHz, D2O) δ 8.25 (d, 2H, J = 9.6 Hz), 7.23 (d, 2H, J = 9.6 Hz), 5.28 (d, 1H, J = 8.0 Hz), 4.20 (dd, 1H, J = 9.6 and 2.4 Hz), 4.02 (d, 1H, J = 2.8 Hz), 3.92-3.82 (m, 5H), 3.74 (d, 2H, J = 6.0 Hz), 3.66-3.51 (m, 4H), 2.72 (dd, 1H, J = 12.0 and 4.8 Hz, H-3 ax ), 1.76 (t, 1H, J = 12.0 Hz, H-3 eq ); 13 C NMR (100 MHz, D 2 O) δ 174.1, 161.8, 142.7, 126.3 (2C), 116.6 (2C), 100.0, 99.8, 75.6, 75.6, 74.0, 72.3, 70.4, 69.9, 68.9, 67.9, 67.3, 62.8, 60.8, 39.5; HRMS (ESI) m/z calcd for C 21 H 29 NO 16 Na (M+H) 574.1384, found 574.1406. 4-Nitrophenyl O-(5-acetamido-3,5-dideoxy-D-glycero-a-D-galacto-2-nonulopyranosylonic acid)-(2? 6)-O-b-D-galactopyranoside (Neu5Aca2,6GalbpNP, 13) Yield, 88%; light yellow foam. 1 H NMR (300 MHz, D 2 O) δ 8.27 (d, 2H, J = 7.5 Hz), 7.24 (d, 2H, J = 7.5 Hz), 5.17 (d, 1H, J = 7.8 Hz), 4.00-3.50 (m, 13H), 2.74 (dd, 1H, J = 12.0 and 3.9 Hz, H-3 eq ), 1.99 (s, 3H), 1.80 (t, 1H, J = 12.0 Hz, H-3 ax ); 13 C NMR (75 MHz, D 2 O) δ 175.16, 173.69, 162.01, 142.69, 126.28 (2C), 116.55 (2C), 100.37, 99.99, 74.15, 72.72, 72.48, 71.90, 70.41, 68.55, 68.32 (2C), 63.16, 62.75, 51.98, 40.40, 22.13. HRMS (ESI) m/z calcd for C 23 H 31 N 2 O 16 (M-Na) 591.1674, found 591.1677. 4-Nitrophenyl O-(5-acetamido-9-O-acetyl-3,5-dideoxy-D-glycero-a-D-galacto-2-nonulopyranosylonic acid)-(2? 6)-O-b-D-galactopyranoside (Neu5,9Ac 2 a2,6galbpnp, 14) Yield, 78%; light yellow foam. 1 H NMR (300 MHz, D 2 O) δ 8.26 (d, 2H, J = 9.3 Hz), 7.24 (d, 2H, J = 9.3 Hz), 5.16 (d, 1H, J = 7.2 Hz), 4.34 (dd, 1H, J = 11.7 and 2.4 Hz), 4.15-3.49 (m, 12H), 2.73 (dd, 1H, J = 12.0 and 4.8 Hz, H-3 eq ), 2.04 (s, 3H), 1.99 (s, 3H), 1.64 (t, 1H, J = 12.0 Hz, H-3 ax ); 13 C NMR (75 MHz, D 2 O) δ 175.10, 174.46, 173.65, 162.00, 142.66, 126.27 (2C), 116.54 (2C), 100.48, 100.01, 74.19, 72.47, 72.18, 70.39, 69.36, 68.53, 68.34, 65.83, 63.23, 62.58, 51.93, 40.37, 22.13, 20.33. HRMS (ESI) m/z calcd for C 25 H 33 N 2 O 17 (M-Na) 633.1779, found 633.1771. 4-Nitrophenyl O-(5-acetamido-3,5-dideoxy-9-O-lactyl-D-glycero-a-D-galacto-2-nonulopyranosylonic acid)-(2? 6)-O-b-D-galactopyranoside (Neu5Ac9Lta2,6GalbpNP, 15) Yield, 92%; light yellow foam. 1 H NMR (300 MHz, D2O) δ 8.26 (d, 2H, J = 9.3 Hz), - 3 -
7.24 (d, 2H, J = 9.3 Hz), 5.16 (d, 1H, J = 8.7 Hz), 4.39-4.31 (m, 2H), 4.08-3.50 (m, 12H), 2.74 (dd, 1H, J = 12.0 and 4.2 Hz, H-3eq ), 1.99 (s,3h), 1.65 (t, 1H, J = 12.0 Hz, H-3ax ), 1.36 (d, 3H, J = 7.2 Hz); 13 C NMR (75 MHz, D 2 O) δ 176.43, 175.12, 173.68, 162.05, 142.69, 126.31 (2C), 116.62 (2C), 100.55, 100.08, 74.17, 72.48, 72.18, 70.46, 69.32, 68.56, 68.26, 68.09, 66.70, 66.11, 63.21, 51.97, 40.39, 22.16, 19.35. HRMS (ESI) m/z calcd for C 26 H 35 N 2 O 18 (M-Na) 663.1885, found 663.1863. 4-Nitrophenyl O-(3,5-dideoxy-5-glycolylamido-D-glycero-a-D-galacto-2-nonulopyranosylonic acid)-(2? 6)-O-b-D-galactopyranoside (Neu5Gca2,6GalbpNP, 16) Yield, 85%; light yellow foam. 1 H NMR (400 MHz, D 2 O) δ 8.27 (d, 2H, J = 9.2 Hz), 7.25 (d, 2H, J = 9.2 Hz), 5.18 (d, 1H, J = 7.6 Hz), 4.08 (s, 2H), 4.01-3.50 (m, 13H), 2.76 (dd, 1H, J = 12.4 and 4.0 Hz, H-3 eq ), 1.65 (t, 1H, J = 12.4 Hz, H-3 ax ); 13 C NMR (75 MHz, D 2 O) δ 175.92, 173.70, 162.00, 142.64, 126.27 (2C), 116.53 (2C), 100.37, 99.98, 74.14, 72.46, 72.18, 71.95, 70.42, 68.54, 68.23, 68.05, 63.15, 62.60, 61.07, 51.67, 40.44. HRMS (ESI) m/z calcd for C 23 H 31 N 2 O 17 (M-Na) 607.1623, found 607.1616. 4-Nitrophenyl O-(3-deoxy-D-glycero-a-D-galacto-2-nonulopyranosylonic acid)- (2? 6)-O-b-D-galactopyranose (KDNa2,6GalbpNP, 17). Yield, 79%; white foam. 1 H NMR (300 MHz, D 2 O) δ 8.27 (d, 2H, J = 9.3 Hz), 7.24 (d, 2H, J = 9.3 Hz), 5.16 (d, 1H, J = 7.2 Hz), 4.00-3.38 (m, 13H), 2.68 (dd, 1H, J = 12.0 and 4.8 Hz, H-3 ax ), 1.57 (t, 1H, J = 12.0 Hz, H-3 eq ); 13 C NMR (75 MHz, D 2 O) δ 173.88, 162.09, 142.71, 126.33 (2C), 116.55 (2C), 100.44, 100.07, 74.13, 73.67, 72.44, 72.21, 70.38, 70.23, 69.75, 68.53, 67.91, 62.58, 59.97, 40.00; HRMS (ESI) m/z calcd for C 21 H 29 NO 16 (M-Na) 550.1408, found 550.1407. 4-Nitrophenyl O-(5-acetamido-3,5-dideoxy-D-glycero-a-D-galacto-2-nonulopyranosylonic acid)-(2? 6)-O-2-acetamido-2-deoxy-b-D-galactopyranoside (Neu5Aca2,6- GalNAcbpNP, 18) Yield, 92%; light yellow foam. 1 H NMR (300 MHz, D 2 O) δ 8.20 (d, 2H, J = 9.0 Hz), 7.15 (d, 2H, J = 9.0 Hz), 5.19 (d, 1H, J = 8.4 Hz), 4.19 (dd, 1H, J = 10.8 and 8.4 Hz), 4.02-3.50 (m, 12H), 2.73 (dd, 1H, J = 12.4 and 4.2 Hz, H-3 eq ), 2.00 (s, 3H), 1.98 (s, 3H), 1.63 (t, 1H, J = 12.4 Hz, H-3ax ); 13 C NMR (75 MHz, D 2O) δ 175.24, 175.13, 173.66, 161.94, 142.62, 126.21 (2C), 116.51 (2C), 100.35, 99.09, 74.15, 72.71, 71.88, - 4 -
70.59, 69.69, 68.29, 67.68, 63.21, 62.72, 52.18, 51.95, 40.36, 22.27, 22.18. HRMS (ESI) m/z calcd for C25H34N3O16 (M-Na) 632.1939, found 632.1949. 4-Nitrophenyl O-(5-acetamido-9-O-acetyl-3,5-dideoxy-D-glycero-a-D-galacto-2- nonulopyranosylonic acid)-(2? 6)-O-2-acetamido-2-deoxy-b-D-galactopyranoside (Neu5,9Ac 2 a2,6galnacbpnp, 19) Yield, 33%; light yellow foam. 1 H NMR (300 MHz, D 2 O) δ 8.26 (d, 2H, J = 9.3 Hz), 7.21 (d, 2H, J = 9.3 Hz), 5.21 (d, 1H, J = 8.4 Hz), 4.37 (d, 1H, J = 12.0 Hz), 4.24-3.57 (m, 12H), 2.75 (dd, 1H, J = 12.0 and 4.2 Hz, H-3 eq ), 2.06 (s, 3H), 2.00 (s, 3H), 1.99 (s, 3H), 1.66 (t, 1H, J = 12.0 Hz, H-3 ax ); 13 C NMR (75 MHz, D 2 O) δ 175.25, 175.10, 174.49, 173.68, 162.01, 142.73, 126.28 (2C), 116.62 (2C), 100.49, 99.17, 74.28, 72.57, 70.63, 69.85, 69.36, 68.35, 67.74, 65.88, 63.35, 52.21, 51.96, 40.36, 22.27, 22.16, 20.35. HRMS (ESI) m/z calcd for C 27 H 36 N 3 O 17 (M-Na) 674.2045, found 674.2076. 4-Nitrophenyl O-(5-acetamido-3,5-dideoxy-9-O-lactyl-D-glycero-a-D-galacto-2- nonulopyranosylonic acid)-(2? 6)-O-2-acetamido-2-deoxy-b-D-galactopyranoside (Neu5Ac9Lta2,6GalNAcbpNP, 20) Yield, 42%; light yellow foam. 1 H NMR (300 MHz, D 2 O) δ 8.25 (d, 2H, J = 9.3 Hz), 7.20 (d, 2H, J = 9.3 Hz), 5.21 (d, 1H, J = 8.7 Hz), 4.40-3.57 (m, 14H), 2.75 (dd, 1H, J = 12.0 and 4.2 Hz, H-3eq ), 2.00 (s, 3H), 1.99 (s, 3H), 1.66 (t, 1H, J = 12.0 Hz, H-3 ax ), 1.37 (d, 3H, J = 6.6 Hz); 13 C NMR (75 MHz, D 2 O) δ 176.40, 175.26, 175.10, 173.68, 162.01, 142.72, 126.28 (2C), 116.64 (2C), 100.54, 99.19, 74.27, 72.53, 70.64, 69.32, 68.30, 68.17, 67.76, 66.77, 66.13, 63.34, 52.24, 52.00, 40.36, 22.30, 22.15, 19.38. HRMS (ESI) m/z calcd for C 28 H 38 N 3 O 18 (M-Na) 704.2150, found 704.2067. 4-Nitrophenyl O-(3,5-dideoxy-5-glycolylamido-D-glycero-a-D-galacto-2-nonulopyranosylonic acid)-(2? 6)-O-2-acetamido-2-deoxy-b-D-galactopyranoside (Neu- 5Gca2,6GalNAcbpNP, 21) Yield, 85%; light yellow foam. 1 H NMR (300 MHz, D 2 O) δ 8.23 (d, 2H, J = 9.6 Hz), 7.17 (d, 2H, J = 9.6 Hz), 5.21 (d, 1H, J = 8.4 Hz), 4.19 (dd, 1H, J = 10.8 and 8.4 Hz), 4.07 (s, 2H), 4.02-3.50 (m, 12H), 2.75 (dd, 1H, J = 12.0, 3.9 Hz, H- 3 eq ), 1.98 (s, 3H), 1.65 (t, 1H, J = 12.0 Hz, H-3 ax ); 13 C NMR (75 MHz, D 2 O) δ 175.94, 175.27, 173.71, 162.01, 142.74, 126.28 (2C), 116.61 (2C), 100.44, 99.15, 74.22, 72.48, - 5 -
71.97, 70.65, 68.28, 68.08, 67.74, 63.28, 62.74, 61.10, 52.24, 51.70, 40.43, 22.92. HRMS (ESI) m/z calcd for C25H34N3O17 (M-Na) 648.1888, found 648.1904. 4-Nitrophenyl O-(3-deoxy-D-glycero-a-D-galacto-2-nonulopyranosylonic acid)- (2? 6)-O-2-acetamido-2-deoxy-b-D-galactopyranoside (KDNa2,6GalNAcbpNP, 22). Yield, 80%; white foam. 1 H NMR (300 MHz, D2O) δ 8.28 (d, 2H, J = 9.3 Hz), 7.22 (d, 2H, J = 9.3 Hz), 5.24 (d, 1H, J = 8.4 Hz), 4.22 (dd, 1H, J = 10.8 and 8.4 Hz), 4.05 (d, 1H, J = 3.6 Hz), 4.00-3.36 (m, 11H), 2.72 (dd, 1H, J = 12.0 and 4.8 Hz, H-3 ax ), 2.02 (s, 3H), 1.62 (t, 1H, J = 12.0 Hz, H-3 eq ); 13 C NMR (75 MHz, D 2 O) δ 175.25, 173.84, 162.00, 142.71, 126.25 (2C), 116.56 (2C), 100.34, 99.14, 74.23, 73.72, 72.24, 70.63, 70.26, 70.02, 67.98, 67.76, 63.49, 62.83, 52.22, 40.01, 22.27; HRMS (ESI) m/z calcd for C 23 H 31 N 2 O 16 (M-Na) 591.1674, found 591.1668. - 6 -
Neu5Acα2,3GalβpNP (8) - 7 -
Neu5,9Ac 2 α2,3galβpnp (9) - 8 -
Neu5Ac9Ltα2,3GalβpNP (10) - 9 -
Neu5Gcα2,3GalβpNP (11) - 10 -
KDNα2,3GalβpNP (12) - 11 -
Neu5Acα2,6GalβpNP (13) - 12 -
Neu5,9Ac 2 α2,6galβpnp (14) - 13 -
Neu5Ac9Ltα2,6GalβpNP (15) - 14 -
Neu5Gcα2,6GalβpNP (16) - 15 -
KDNα2,6GalβpNP (17) - 16 -
Neu5Acα2,6GalNAcβpNP (18) - 17 -
Neu5,9Ac 2 α2,6galnacβpnp (19) - 18 -
Neu5Ac9Ltα2,6GalNAcβpNP (20) - 19 -
Neu5Gcα2,6GalNAcβpNP (21) - 20 -
KDNα2,6GalNAcβpNP (22) - 21 -