Supporting Information s23

Supporting Information

Chemo-enzymatic synthesis of poly-N-Acetyllactosamine (poly-LacNAc)

structures and their characterization for CGL2-galectin-mediated binding

of ECM glycoproteins to biomaterial surfaces

Birgit Sauerzapfe[a], Karel Křenek[b], Judith Schmiedel [a], Warren W. Wakarchuk [c], Helena

Pelantová [b], Vladimir Křen[b], Lothar Elling* [a]

[a] Dipl.-Biol. Birgit Sauerzapfe, Dipl.-Biol. Judith Schmiedel, Prof. Dr. Lothar Elling

Laboratory for Biomaterials, Institute of Biotechnology and Helmholtz-Institute for

Biomedical Engineering, RWTH Aachen University, 52074 Aachen, Germany

Fax: +49 2418022387, e-mail: [email protected]

[b] Karel Křenek, Dr. Helena Pelantová, Prof. Dr. Vladimir Křen

Institute of Microbiology, Academy of Sciences of the Czech Republic, Videnska 1083,

142 20 Prague 4, Czech Republic

[c] Dr. Warren W. Wakarchuk

Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario,

K1A 0R6, Canada

* Corresponding author: Prof. Dr. Lothar Elling Capillary electrophoresis analysis CE analysis was performed on a P/ACE MDQ CE system (BeckmanCoulter, Germany) with normal polarity, using a 75 µm i.d. fused silica capillary (eCAP) with 50 cm effective length. The samples were introduced by pressure injection at 0.5 psi for 5 sec. The temperature of the capillary was maintained at 25 °C and the separation was carried out with the buffer 20 mM

Na2B4O7, containing 50 mM SDS at pH 9.4 and a constant voltage of 25 kV for 10 min. The products were monitored on-column with a UV-lamp at a wavelength of 254 nm.

HPLC/ESI-MS analysis HPLC was carried out using a Dionex System with a Licrospher® 100 RP18 column (4 x 250 mm; Chromatographie Service GmbH, Germany). The HPLC system comprised an ASI-100 autosampler, P680-pump and a UV-Detector 170 U module. The eluents were H2Obidest. (solvent A) and acetonitrile (solvent B). For analysis, the column was equilibrated with solvent A. The saccharides 7, 11; and 13 and their corresponding deprotected products carrying the free amino group in the linker structure (7a, 20, 13a) were eluted by a linear gradient increasing solvent B to 50% (v/v) in 45 min and kept constant for 15 min at a flow rate of 0.5 mL/min. The products were monitored at 254 nm due to the absorption maximum of the linker molecule. Mass spectra were recorded by a Thermo-Finnigan Surveyor MSQ quadruple mass spectrometer equipped with an electro spray ion source. The mass spectrometer was operated in the negative-ion mode for the tBoc-saccharides and in the positive ion-mode for the deprotected saccharides. The needle voltage was set to 3 kV, cone 50 V and the capillary temperature was 400 °C. The electron multiplier was set at 1200 V and the pressure of the sheath gas was 70 psi. The data

Results The CE data (Figure S1) and HPLC/ESI-MS data (Figure S2) reveal that no hydrolysis of the saccharides like the LacNAc (20) and the trisaccharide structure (13a) occur during the deprotection step. Further analysis of the integrated peaks gives for compound 7a, 20, and 13a relative deprotection yields between 81% and 92% according to CE (Table S1) and 89% to 99% in HPLC analysis (Table S2). Figure S1. CE analysis of tBoc-protected and deprotected saccharide products.

A1. GlcNAc-linker-NH2tBoc (7) A2. GlcNAc-linker-NH2 (7a)

B1. LacNAc-linker-NH2tBoc (12) B2. LacNAc- linker-NH2 (20)

C1. Trisaccharide- linker-NH2tBoc (13) C2. Trisaccharide-linker-NH2 (13a) Table S1. Relative yields for the deprotection step analyzed by CE analysis.

Migration time Relative yield for deprotection Product Peak area [min] [%] 7a 4.267 105829 92.3 7 5.05 8863

20 4.15 33605 83.7 12 4.667 6535

13a 4.392 38613 81.2 13 4.85 8917 Figure S2. HPLC/ESI-MS analysis of tBoc-protected and deprotected saccharide products.

- A1. GlcNAc-linker-NH2tBoc (7): m/z at 29.91 min and 30.10 min: 421.2 for [M - H] and - 457.2 for [M - H + (H2O)2] ; calculated m/z: 422.2 for [M].

220 TBOC-SERIE #48 [modified by User] 0.5mM_GlcNAc-tBoc UV_VIS_2 mAU WVL:254 nm

1 - 29.916

-20 min 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0

118 Peak #1 RT: 29.76 min NL: 8.94E3 T: {0,0} - c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-700.00] % 250.2 454.3

216.0 50 174.1 237.2 271.0 411.0 485.6 386.9 343.0 537.3 470.7 591.9 642.8 0 118 Peak #1 RT: 29.91 min NL: 3.74E4 T: {0,0} - c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-700.00] % 457.2

182.1 267.1 334.9 421.3 304.7 388.3 497.1 551.4 575.9 0 118 Peak #1 RT: 30.10 min NL: 1.26E5 T: {0,0} - c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-700.00] % 421.2

171.4 214.2 270.5 495.1 526.5 554.5 599.2 0 m/z 150 200 250 300 350 400 450 500 550 600 650 700 + A2. GlcNAc-linker-NH2 (7a): m/z at 49.0 min and 50.11 min: 323.2 for [M + H] ; calculated m/z: 322.1 for [M]. The peak 2 at 29.922 min is residual GlcNAc-linker-NH2-tBoc (7).

25.0 TBOC-SERIE #43 [modified by User] 0.5mM_enGlcNAc UV_VIS_2 mAU WVL:254 nm

3 - 48.996 20.0

1 - 17.157 5.0 2 - 29.922

-5.0 min 0.0 10.0 20.0 30.0 40.0 50.0 60.0

118 Peak #3 RT: 46.05 min NL: 1.15E4 T: {0,0} + c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-700.00] % 224.2

198.1 50

265.8 289.7 342.5 416.3 452.1 492.5 526.0 568.6 607.0 675.3 0 118 Peak #3 RT: 49.00 min NL: 2.97E6 T: {0,0} + c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-700.00] % 323.2

224.2 289.2 0 118 Peak #3 RT: 50.11 min NL: 1.12E5 T: {0,0} + c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-700.00] % 323.2

289.3 186.3 230.2 257.9 378.9 0 m/z 150 200 250 300 350 400 450 500 550 600 650 700 - B1. LacNAc-linker-NH2tBoc (12): m/z at 29.07 min and 29.28 min: 583.3 for [M - H] and

- 619.3 for [M - H + (H2O)2] ; calculated m/z: 584.2 for [M].

200 TBOC-SERIE #47 [modified by User] 0.5mM_LacNAc-tBoc UV_VIS_2 mAU WVL:254 nm

1 - 29.076 175

-20 min 0.0 10.0 20.0 30.0 40.0 50.0 60.0

118 Peak #1 RT: 28.90 min NL: 5.52E3 T: {0,0} - c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-700.00] % 260.0 170.2 331.1 192.2 233.1 292.1 50 521.0 370.8 431.2 478.2 583.7 617.8 664.4 0 118 Peak #1 RT: 29.07 min NL: 1.17E4 T: {0,0} - c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-700.00] % 583.3

619.4 248.3 194.2 50 231.0 417.9 645.4 174.2 301.2 340.4 366.1 428.0 463.8 513.4 544.0 670.5 0 118 Peak #1 RT: 29.28 min NL: 5.71E4 T: {0,0} - c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-700.00] % 619.3

248.2 323.6 645.2 180.2 220.2 300.1 381.1 413.1 454.1 484.5 687.3 0 m/z 150 200 250 300 350 400 450 500 550 600 650 700 + B2. LacNAc-linker-NH2 (20): m/z at 50.61 min and 51.52 min: for 485.2 [M + H] ; calculated m/z: 484.2 for [M]. Peak 1 at 28.998 min is residual LacNAc-linker-tBoc (12).

16.0 TBOC-SERIE #40 [modified by User] 0.5mM_ENLacNAc UV_VIS_2 mAU WVL:254 nm

2 - 50.609

12.5 1 - 28.998

-2.0 min 0.0 10.0 20.0 30.0 40.0 50.0 60.0

118 Peak #2 RT: 48.35 min NL: 7.44E3 T: {0,0} + c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-700.00] % 224.2

50 172.4 208.3 247.3 311.2 349.8 384.7 442.6 483.6 538.8 585.1 658.4 0 118 Peak #2 RT: 50.61 min NL: 4.96E5 T: {0,0} + c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-700.00] % 485.2

179.0 224.0 270.1 313.5 401.5 451.5 507.2 0 118 Peak #2 RT: 51.52 min NL: 1.21E5 T: {0,0} + c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-700.00] % 485.2

50 451.2 229.0 172.3 206.9 257.0 305.4 341.2 370.4 400.0 433.6 574.4 0 m/z 150 200 250 300 350 400 450 500 550 600 650 700 C1. GlcNAc(1-3)Gal(1-4)GlcNAc1-linker-NH2tBoc (13): m/z at 28.64 min and 28.83 min: 786.6 for [M - H]-; calculated m/z: 787.3 for [M].

70.0 TBOC-SERIE #50 [modified by User] 0.5mM_Tri-tBoc UV_VIS_2 mAU WVL:254 nm 1 - 28.636

-10.0 min 0.0 10.0 20.0 30.0 40.0 50.0 60.0

118 Peak #1 RT: 28.47 min NL: 4.92E3 T: {0,0} - c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-800.00] % 269.4

192.1 50 171.2 664.0 312.4 378.4 411.2 474.0 502.3 568.6 604.9 699.0 739.9 768.0 0 118 Peak #1 RT: 28.64 min NL: 5.62E3 T: {0,0} - c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-800.00] % 247.2 752.5 161.1

786.6 50 201.2 283.9 347.2 391.5 438.3 481.8 518.8 582.6 630.4 676.1 716.8 0 118 Peak #1 RT: 28.83 min NL: 6.68E3 T: {0,0} - c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-800.00] %164.2 176.2 249.0 786.5 50

263.8 305.1 351.5 408.9 466.6 505.8 549.8 582.6 623.7 658.7 751.0 0 m/z 150 200 250 300 350 400 450 500 550 600 650 700 750 800 C2. GlcNAc(1-3)Gal(1-4)GlcNAc1-linker-NH2 (13a): m/z at 44.3 min: 688.4 for [M + H] +; calculated m/z: 687.3 for [M]. Peak 2 at 28.639 min is residual 13.

14.0 TBOC-SERIE #45 [modified by User] 0.5mM_enTri UV_VIS_2 mAU WVL:254 nm

12.0 5 - 44.297

10.0 2 - 28.639

4.0 8 - 50.694 6 - 747.712 - 49.247 1 - 11.238 3 - 430.876 - 32.297 2.0

-2.0 min 0.0 10.0 20.0 30.0 40.0 50.0 60.0

118 Peak #5 RT: 42.33 min NL: 6.89E3 T: {0,0} + c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-700.00] % 224.1 172.2

50 597.7 207.4 249.4 280.1 349.7 382.4 451.9 485.8 554.8 636.7 666.1 0 118 Peak #5 RT: 44.30 min NL: 7.97E3 T: {0,0} + c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-700.00] % 688.4 171.3 212.8

224.5 326.4 530.0 50 186.4 261.5 311.1 350.8 385.7 484.8 562.8 601.6 0 118 Peak #5 RT: 44.97 min NL: 1.44E4 T: {0,0} + c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-700.00] % 223.9

180.1 208.2 50

254.2 283.8 358.3 418.9 448.8 490.4 534.4 578.9 627.7 682.1 0 m/z 150 200 250 300 350 400 450 500 550 600 650 700 Table S2: Relative yields for the deprotection step analyzed by HPLC/ESI-MS (see Figure S2).

Retention time Peak area Relative yield for deprotection Product [min] mAU·min [%] 7a 48.996 50.3787 99.5 7 29.922 0.2925

20 50.609 20.5120 89.9 12 28.998 2.3072

13a 44.297 14.1332 88.9 13 28.639 1.7605