Electronic Supplementary Information

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Electronic Supplementary Information

Chemoenzymatic Synthesis of Biotin-Appended Analogues of

Gangliosides GM2, GM1, GD1a and GalNAc-GD1a for Solid-Phase Applications and Improved ELISA Tests

Aliaksei V. Pukin,

^a

Dion E. A. Florack,

^b

Denis Brochu,

^c

Barend van Lagen,

^a

Gerben M. Visser,

^a

a Laboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB, Wageningen, The Netherlands; Tel.: +31317482361; Fax: +31317484914; E-mail: [email protected];

b Plant Research International, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands;

c Institute for Biological Sciences, National Research Council Canada, 100 Sussex Drive, Ottawa, Ontario, Canada.

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Includes H- and C- NMR spectra of the synthesized compounds.

NMR spectra were recorded on a Bruker DPX 400, Bruker Avance 400 or Bruker Avance 900 (for the compound 11) spectrometer.

Tom Wennekes, Michel Gilbert, Han Zuilhof

a c a,*

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10 20

30 40

50 60

70 80

90 100

110 120

130 140

150 160

170 180

29.114 28.892 28.833 28.573 26.400 25.470 20.806 20.633 20.581 20.459 20.400

170.156 170.117 169.934 169.819 169.597 169.334 168.871 100.826 100.355 76.116 72.631 72.369 71.511 70.777 70.439 69.865 68.913 66.448 61.877 60.643 51.195

168.80 169.00 169.20 169.40 169.60 169.80 170.00 170.20

Nucleus 13C Number of Transients 1499 Origin dpx_400 Original Points Count 32768 Owner veldhuis

Points Count 32768 Pulse Sequence zgpg30 Receiver Gain 32768.00 SW(cyclical) (Hz) 31847.13

Solvent CHLOROFORM-d Spectrum Offset (Hz) 12710.3887 Sweep Width (Hz) 31846.16 Temperature (degree C) 27.000

Acquisition Time (sec) 1.0289 Comment 5 mm Dual 13C/1H Date 06 Jun 14:49:36

Date Stamp 06 Jun 14:49:36 File Name D:\OBC \ 1000fid Frequency (MHz) 100.63

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Acquisition Time (sec) 1.9923 Comment 5 mm Dual 13C/1H Date 10 Jun 14:11:12

Date Stamp 10 Jun 14:11:12 File Name D:\OBC\data _181000fid Frequency (MHz) 400.13

Nucleus 1H Number of Transients 64 Origin dpx_400 Original Points Count 16384 Owner veldhuis

Points Count 16384 Pulse Sequence zg30 Receiver Gain 90.50 SW(cyclical) (Hz) 8223.68 Solvent CHLOROFORM-d

Spectrum Offset (Hz) 2471.0088 Sweep Width (Hz) 8223.18 Temperature (degree C) 27.000

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0

Chemical Shift (ppm) 0

0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00

Normalized Intensity

2.17 1.00 0.972.00 2.95 3.98 3.08 1.051.21 2.00 22.48 2.05 10.07

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)4* +, #-./0.123!450..67898..:: );2381< =>? @

+31*35 !@0 +3A; 7B ;,854865 C;4D48 E6FG9 C;4D48,* H74865 7386 CI8; /'0 H74865 7386

H3*5 J2381 KELGF9M6EN J7*O86 #/ JIP Q4R6S >? @T

P;,63; RD; 46* &U$VNW9NX

123!450Y&UYVNW9NXFE6

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01 21

31 41

51 61

71 81

91 011

001 021

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0 10

20 30

40 50

60 70

80 90

100 110

120 130

140 150

160 170

180

41.110 40.414 38.586 36.880 30.772 30.475 30.437 30.388 29.828 29.568 27.965 27.005 23.816 22.602

83.083 81.632 79.092 76.521 76.408 75.960 75.673 75.153 74.820 74.627 73.432 72.554 71.124 70.929

106.711 105.011 104.265 104.180 103.540 176.020 175.668 175.295 174.870 166.166

101.5 102.0 102.5 103.0 103.5 104.0 104.5 105.0 105.5 106.0 106.5 107.0 107.5 108.0 108.5

F r e q u e n c y ( M H z ) 1 0 0 .6 3 N u c le u s 1 3 C N u m b e r o f T r a n s ie n t s 7 6 8 0 0 O r ig in d p x _ 4 0 0

O r ig in a l P o in t s C o u n t 3 2 7 6 8 O w n e r v e ld h u is P o in t s C o u n t 3 2 7 6 8 P u l s e S e q u e n c e z g p g 3 0

S p e c t r u m O f f s e t ( H z ) 1 2 8 9 7 .4 6 1 9 S w e e p W id t h ( H z ) 3 1 8 4 6 . 1 6 T e m p e r a t u r e ( d e g r e e C ) 2 7 .0 0 0

A c q u is it io n T im e ( s e c ) 1 .0 2 8 9 C o m m e n t 5 m m D u a l 1 3 C / 1 H D a t e 0 9 S e p 0 9 :0 1 :5 2

R e c e i v e r G a in 3 2 7 6 8 .0 0 S W ( c y c l ic a l) ( H z ) 3 1 8 4 7 . 1 3 S o lv e n t CD3OD

D a t e S t a m p 0 9 S e p 0 9 : 0 1 :5 2 F il e N a m e D : \N F \ 0 1 4 0 0 0 f id

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Acquisition Time (sec) 3.4210 Comment 5 mm Multinuclear inverse Z-grad Date 21 Apr 09:42:24

Date Stamp 21 Apr 09:42:24 File Name D:\NF\060000fid

Frequency (MHz) 400.13 Nucleus 1H Number of Transients 256 Origin dpx_400

Original Points Count 16384 Owner veldhuis Points Count 16384 Pulse Sequence zg30

Receiver Gain 71.80 SW(cyclical) (Hz) 4789.27 Solvent DEUTERIUM OXIDE

Spectrum Offset (Hz) 1996.2163 Sweep Width (Hz) 4788.98 Temperature (degree C) 27.000

4.5 4.0 3.5 3.0 2.5 2.0 1.5

Chemical Shift (ppm) 0

0.005 0.010 0.015 0.020 0.025 0.030 0.035 0.040 0.045 0.050 0.055

Normalized Intensity

1.98 1.10 1.03 1.21 1.98 2.95 2.00 8.91 1.22 1.17 2.45 9.92

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20 30

40 50

60 70

80 90

100 110

120 130

140 150

160 170

180

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01234546478 4 51 786 !"#$%$&'#%("

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+31*35 !@0 +3A; 7B ;,854865 C;4D48 E6FG9 C;4D48,* H74865 7386 CI8; /'0 H74865 7386

H3*5 J2381 KELGF9M6EN J7*O86 #/ JIP Q4R6S >? @T

P;,63; RD; @ 46* &8UV0$&#8$WNX9NY

123!450Z&8UVG&#8ZWNX9NYFE6

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20 30

40 50

60 70

80 90

100

SF=226.33 SW=45454.55 AQ=0.36 TD=131072 Units=ppm

Acquisition Time (sec) 0.3604 Comment 1D 13C, 298K Date 10 Jun 11:22:40

Date Stamp 10 Jun 11:22:40 File Name D:\NF\data900\LSF_Han_150408\LSF_Han_150408_008000fid

Frequency (M Hz) 226.35 Nucleus 13C Number of Transients 102400 Origin spect Original Points Count 16384

Owner psm Points Count 16384 Pulse Sequence zgpg30 Receiver Gain 8192.00 SW(cyclical) (Hz) 45454.54

Solvent Spectrum Offset (Hz) 22637.5723 Sweep Width (Hz) 45451.77 Temperature (degree C) 31.000