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: l.elling@biotec.rwth-aachen.de
[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.
7a
7
Migration time
[min]
4.267
5.05
20
12
4.15
4.667
33605
6535
83.7
13a
13
4.392
4.85
38613
8917
81.2
Product
Peak area
105829
8863
Relative yield for deprotection
[%]
92.3
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].
118 Peak #1 RT: 29.76 min NL: 8.94E3
%
250.2
T: {0,0} - c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-700.00]
454.3
50
216.0
237.2
174.1
271.0
343.0
386.9
485.6
411.0
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
50
182.1
267.1
304.7 334.9
0
118 Peak #1 RT: 30.10 min NL: 1.26E5
%
421.3
388.3
497.1
551.4 575.9
T: {0,0} - c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-700.00]
421.2
457.3
50
171.4
0
150
214.2
200
495.1 526.5 554.5
270.5
250
300
350
400
450
500
550
599.2
600
m/z
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).
118 Peak #3 RT: 46.05 min NL: 1.15E4
%
224.2
50
T: {0,0} + c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-700.00]
198.1
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
50
289.2
224.2
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
50
289.3
186.3
0
150
200
230.2 257.9
250
378.9
300
350
m/z
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].
118 Peak #1 RT: 28.90 min NL: 5.52E3
%
260.0
T: {0,0} - c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-700.00]
170.2
192.2
331.1
233.1
292.1
50
521.0
370.8
478.2
431.2
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
50
194.2
417.9
231.0
174.2
301.2
645.4
340.4
366.1
428.0
513.4 544.0
463.8
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
583.3
50
220.2
180.2
0
150
200
248.2
250
300.1
300
323.6
381.1
350
413.1
400
645.2
454.1 484.5
450
500
550
600
650
687.3
m/z
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).
118 Peak #2 RT: 48.35 min NL: 7.44E3
%
224.2
50
172.4
208.3
T: {0,0} + c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-700.00]
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
50
0
224.0
179.0
270.1
401.5
313.5
118 Peak #2 RT: 51.52 min NL: 1.21E5
%
451.2
229.0
0
150
206.9
200
507.2
485.2
50
172.3
451.5
T: {0,0} + c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-700.00]
257.0
250
305.4
300
341.2 370.4 400.0
350
400
433.6
450
574.4
500
550
m/z
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].
118 Peak #1 RT: 28.47 min NL: 4.92E3
%
269.4
T: {0,0} - c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-800.00]
227.1
50
192.1
171.2
664.0
378.4 411.2
312.4
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
%
247.2
T: {0,0} - c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-800.00]
752.5
161.1
786.6
50
283.9
201.2
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
%
164.2
T: {0,0} - c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-800.00]
176.2
786.5
249.0
50
263.8
0
150
200
250
305.1
300
351.5
350
408.9
400
466.6
450
505.8
500
549.8 582.6
550
600
623.7 658.7
650
751.0
700
750
m/z
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.
118 Peak #5 RT: 42.33 min NL: 6.89E3
%
224.1
172.2
50
T: {0,0} + c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-700.00]
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
%
212.8
171.3
T: {0,0} + c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-700.00]
688.4
326.4
224.5
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
%
223.9
T: {0,0} + c ESI corona sid=50.00 det=1200.00 Full ms [ 150.00-700.00]
180.1 208.2
50
254.2 283.8
0
150
200
250
300
358.3
350
418.9 448.8
400
450
490.4
500
534.4
550
578.9
627.7
600
682.1
m/z
650
700
Table S2: Relative yields for the deprotection step analyzed by HPLC/ESI-MS (see Figure
S2).
7a
7
Retention time
[min]
48.996
29.922
Peak area
mAU·min
50.3787
0.2925
Relative yield for deprotection
[%]
99.5
20
12
50.609
28.998
20.5120
2.3072
89.9
13a
13
44.297
28.639
14.1332
1.7605
88.9
Product