Sugar Chips immobilized with synthetic sulfated disaccharides of heparin/heparan sulfate partial structure

Article abstract of DOI:10.1016/j.bmcl.2008.01.069

Carbohydrate chip technology has a great potential for the high-throughput evaluation of carbohydrate-protein interactions. Herein, we report syntheses of novel sulfated oligosaccharides possessing heparin and heparan sulfate partial disaccharide structures, their immobilization on gold-coated chips to prepare array-type Sugar Chips, and evaluation of binding potencies of proteins by surface plasmon resonance (SPR) imaging technology. Sulfated oligosaccharides were efficiently synthesized from glucosamine and uronic acid moieties. Synthesized sulfated oligosaccharides were then easily immobilized on gold-coated chips using previously reported methods. The effectiveness of this analytical method was confirmed in binding experiments between the chips and heparin binding proteins, fibronectin and recombinant human von Willebrand factor A1 domain (rh-vWf-A1), where specific partial structures of heparin or heparan sulfate responsible for binding were identified.

Full text of DOI:10.1016/j.bmcl.2008.01.069

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2499–2504
Sugar Chips immobilized with synthetic sulfated disaccharides
of heparin/heparan sulfate partial structureq
Masahiro Wakao,a Akihiro Saito,a Koh Ohishi,a Yuko Kishimoto,b
Tomoaki Nishimura,a,b Michael Sobelc and Yasuo Sudaa,b,
*
aDepartment of Nanostructure and Advanced Materials, Graduate School of Science and Engineering, Kagoshima University,
1-21-40 Korimoto, Kagoshima 890-0065, Japan
bSUDx-Biotec corporation, 5-5-2 Minatojima-cho, Kobe 650-0047, Japan
cDepartment of Surgery, University of Washington and VA Puget Sound Health Care System, Seattle, WA 98108, USA
Received 21 August 2007; revised 24 December 2007; accepted 16 January 2008
Available online 19 January 2008
Abstract—Carbohydrate chip technology has a great potential for the high-throughput evaluation of carbohydrate–protein interac-
tions. Herein, we report syntheses of novel sulfated oligosaccharides possessing heparin and heparan sulfate partial disaccharide
structures, their immobilization on gold-coated chips to prepare array-type Sugar Chips, and evaluation of binding potencies of pro-
teins by surface plasmon resonance (SPR) imaging technology. Sulfated oligosaccharides were efficiently synthesized from glucosa-
mine and uronic acid moieties. Synthesized sulfated oligosaccharides were then easily immobilized on gold-coated chips using
previously reported methods. The effectiveness of this analytical method was confirmed in binding experiments between the chips
and heparin binding proteins, fibronectin and recombinant human von Willebrand factor A1 domain (rh-vWf-A1), where specific
partial structures of heparin or heparan sulfate responsible for binding were identified.
Ó 2008 Elsevier Ltd. All rights reserved.
Carbohydrate chips and related array technologies1–3
have attracted a great deal of attention as a powerful
tool for glycomics. Like DNA4 and protein chips,5 they
can rapidly and simply evaluate carbohydrate–protein
interactions in parallel, with a minimum amount of
sample. Our ongoing research involves this functional
analysis of sulfated polysaccharides such as heparin
(HP) and heparan sulfate (HS).3a HP and HS are
highly sulfated polysaccharides and belong to the gly-
cosaminoglycan (GAG) superfamily. They are among
the most complex of carbohydrates, and play a signif-
icant role in biological processes through their binding
interactions with numerous proteins,6 such as growth
factors, cytokines, viral proteins, and coagulation fac-
tors, among others. HP/HS have a basic structure com-
posed of a repeating a or b(1,4)-linked disaccharide
moiety which is derived from uronic acid (either glucu-
ronic acid or iduronic acid) and N-acetyl-glucosamine
residues. In general, HP/HS chains are very heteroge-
neous and contain innumerable substitution patterns
due in part to some randomness in the multiple enzy-
matic modifications in their biosynthesis. This hetero-
geneity makes it difficult to elucidate the structure-
function relationships of HP/HS at the molecular level.
Therefore, structurally defined HP/HS sequences are
necessary for the precise elucidation of the mode of
HP/HS actions on their target molecules. So far, many
synthetic efforts have been dedicated to the synthesis of
HP/HS fragments.3b–d,7,8
Previously, we have reported that a specific disaccharide
unit in HP, O-(2-deoxy-2-sulfamido-6-O-sulfo-a-D-
glucopyranosyl)-(1-4)-2-O-sulfo-a-L-idopyranosyluronic
acid (abbreviated as GlcNS6S-IdoA2S), is a key unit
for binding to human platelets9 and von Willebrand
factor (vWf),10 and that the clustering of these disac-
charides significantly enhanced the interaction.11,12 To
systematically investigate heparin’s binding properties,
we have developed a method3a for the immobilization
the sulfated oligosaccharide onto a gold-coated chip,
Keywords: Sugar; Carbohydrate; Chip; Heparin; Heparan sulfate;
Carbohydrate–protein interaction; Surface plasmon resonance; SPR;
SPR-imaging.
q
Syntheses of sulfated oligosaccharide of heparin and heparan sulfate
partial structures and their application to Sugar Chips are described.
*
Corresponding author. Tel./fax: +81 99 285 8369; e-mail:
ysuda@eng.kagoshima-u.ac.jp
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.069

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M. Wakao et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2499–2504
and have devised an analytical system using surface
plasmon resonance (SPR) technology, which permits
their real-time study without further labeling. These
systems can be applied to the investigation of the bind-
ing interactions of a variety of structurally defined
oligosaccharides.
Figure 1. Sulfated disaccharide partial structures of heparin/heparan sulfate.
Scheme 1. Synthesis of ligand conjugate 2 containing GlcNS-IdoA2S. Reagents: (a) TBDMSCl, imidazole, MS4AP in CH2Cl2, 45%; (b) 1 M NaOH,
MeOH/THF (1:1), 70%; (c) SO3Pyr in Pyr; (d) HFPyr in Pyr; (e) 10% Pd–C, H2 (1 kg/cm2) in THF/MeOH (2:1); (f) SO3Pyr in H2O; (g) 10% Pd–C,
H2 (7 kg/cm2) in H2O/AcOH (5:1), 29% (5 steps); (h) NaBH3CN in DMAc/H2O/AcOH (1:1:0.1), 82%.

M. Wakao et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2499–2504
2501
To better understand the HP/HS disaccharide structures
involved in specific protein interactions, we designed three
kinds of sulfated trisaccharide ligand conjugates 2–4 con-
taining the disaccharide units as shown in Figure 1;
GlcNS-IdoA2S (2): O-(2-deoxy-2-sulfamido-a-D-gluco-
pyranosyl)-(1-4)-2-O-sulfo-a-L-idopyranosyluronic acid,
GlcNS6S-GlcA (3): O-(2-deoxy-2-sulfamido-6-O-sulfo-
a-D-glucopyranosyl)-(1-4)-a-D-glucopyranosyluronic
acid, GlcNS-GlcA (4): O-(2-deoxy-2-sulfamido-a-D-glu-
copyranosyl)-(1-4)-a-D-glucopranosyluronic acid. The
disaccharide units contained in ligand conjugates 1–4 of
Figure 1 are frequently found in HP/HS disaccharide unit.
moieties, respectively. Selective sulfation onto glucosa-
mine and iduronic acid or glucuronic acid moieties can
be carried out by an appropriate functionalization.
The 6-OH glucose derivative 7 was used as the reducing
end for the conjugation to linker molecule 16 after
deprotection on the glucose, which works not only as
a reducing end donor for reductive amination but also
as the hydrophilic moiety in the molecule to minimize
any non-specific hydrophobic interactions between the
linker and target proteins or cells.
The synthesis of ligand conjugate 2 containing GlcNS-
IdoA2S unit was carried out as shown in Scheme 1. Tri-
saccharide 8, which was prepared according to the meth-
od reported previously,12 was selectively protected by t-
butyldimethylsilyl (TBDMS) group. The methyl ester of
trisaccharide 9 was hydrolyzed and the remaining 20-hy-
For efficient synthesis, four monomeric building blocks
were prepared. 2-Azido glucose derivative 5, idose deriv-
ative 6, and 4,6-benzylidene glucose derivative 17 were
used for glucosamine, iduronic acid, and glucuronic acid
Scheme 2. Synthesis of ligand conjugates 3 and 4 containing GlcNS6S-GlcA and GlcNS-GlcA, respectively. Reagents and conditions: (a) BF3OEt2,
MS4AP in CH2Cl2, ꢀ20 °C; (b) 0.1 M NaOMe, 90% (2 steps); (c) NaH, BnBr in DMF, 0 °C ! rt, 88%; (d)16% TFA, 8% MeOH in CH2Cl2,
0 °C ! rt, 93%; (e) TEMPO, KBr, NaClO in CH2Cl2; TMSCHN2, 83% (2 steps); (f) TBDMSOTf, MS4AP in toluene, ꢀ20 °C, 84%; (g) 5 M NaOH
in MeOH/THF (1:1), 89%; (h) SO3Pyr in Pyr; 10% Pd–C, H2 (1 kg/cm2) in THF/H2O (2:1); SO3Pyr in H2O (pH ꢁ 9.5); 10% Pd–C, H2 (7 kg/cm2) in
H2O/AcOH (5:1), 28% (4 steps); (i) 10% Pd–C, H2 (1 kg/cm2) in THF/H2O (2:1); SO3Pyr in MeOH/H2O (3:2); 10% Pd–C, H2 (7 kg/cm2) in H2O/
MeOH/AcOH (5:5:2), 39% (3 steps); (j) NaBH3CN in DMAc/H2O/AcOH (1:1:0.1), 62%; (k) NaBH3CN in DMAc/H2O/AcOH (1:1:0.1), 50%.

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M. Wakao et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2499–2504
droxy group was sulfated using sulfur trioxide-pyridine
complex at room temperature. After removing the
TBDMS group with HFpyridine complex, the azido
group was reduced using a catalytic amount of Pd-C un-
der hydrogen atmosphere and the resulting amino group
was N-sulfated. All benzyl protecting groups were
removed by hydrogenolysis using catalytic Pd-C to give
the desired trisaccharide 15. Finally, the reductive ami-
nation of trisaccharide 15 with linker compound 16
was performed using NaBH3CN to afford the desired li-
gand conjugate 2 in good yield. Compound 2 was puri-
fied by gel-filtration chromatography with Sephadex G-
1
25 fine and confirmed by H NMR and ESI-TOF/MS
analyses.13
The syntheses of ligand conjugates 3 and 4 were carried
out in the same fashion as the syntheses of 1 and 2
(Scheme 2). Glycosylation of 6-OH glucose 7 and imi-
date 17 with trimethylsilyl trifluoromethanesulfonate
(TMSOTf) as a promoter and treatment of the resultant
with NaOMe gave disaccharide 18 in a good yield. The
resulting hydroxy groups of 18 were then protected with
a benzyl group. After removal of the benzylidene group,
the primary 60-OH group was selectively oxidized to car-
boxylic acid using 2,2,6,6-tetramethyl-1-piperidinyloxy
(TEMPO).14 The resulting carboxyl group was esterified
with (trimethylsilyl)diazomethane to afford the disac-
charide 20. The 2-azido imidate 5 was condensed with
disaccharide 20 using TBDMSOTf at ꢀ20 °C to give
selectively an a-linked trisaccharide 21.11,15 Hydrolysis
of the acetyl group and methyl ester was then carried
out using aqueous NaOH to give the common interme-
diate 22 for trisaccharides 23 and 24. The sulfated trisac-
charide 23 was obtained by O-sulfation of the 600-
hydoxyl group and reduction and N-sulfation of 20-azi-
do group was followed by hydrogenolysis. Conversely,
the sulfated trisaccharide 24 was prepared by the same
method as the synthesis of trisaccharide 23, omitting
the O-sulfation. The ligand-conjugates 316 and 417 were
synthesized in satisfactory yields as similar to the de-
scribed procedure for compound 2.
Figure 2. Binding study with fibronectin. (a) SPR difference imaging
on the chip immobilized with compounds 1, 2, 3, 4, and Glca(1-6)Glc-
mono (Glc). Measurements were carried out with analyte in the range
between 0.98 and 125 nM. (b) Bar graph profiles of different
concentrations of protein. The error bars represent +/ꢀ SEM.
ferent pattern of oligosaccharide binding preference
was noted (Fig. 3). A strong interaction was observed
with compounds 1 (GlcNS6S-IdoA2S, KD = 1.0 lM)
and 2 (GlcNS-IdoA2S, KD = 0.9 lM). Weaker interac-
tion was seen with compound 3 (GlcNS6S-GlcA,
KD = 1.4 lM), while distinctly low binding was ob-
served with compound 4 (GlcNS-GlcA, KD = 4.3 lM).
Although the GlcNS6S-IdoA2S (1) disaccharide struc-
ture was considered a key binding domain of vWf,10
the exact disaccharide structure responsible for vWf
binding is still unclear. We found previously that clus-
tered compounds containing three units of GlcNS6S-
IdoA2S12 possessed higher competitive binding activity
compared to compounds containing less than two
units of GlcNS6S-IdoA2S (unpublished data). To-
gether with those data, the current results indicate
that the tri-sulfated disaccharide binds vWf best, that
loss of either the 6-sulfate of GlcN or the 2-sulfate of
IdoA reduces vWf binding significantly, and that the
N-sulfate of GlcN alone is not sufficient for binding
vWf.
Binding interactions were investigated by use of the SPR
imaging sensor.18 When fibronectin was tested (Fig. 2),
specific binding interactions were clearly observed with
compounds 1 (GlcNS6S-IdoA2S, KD = 5.5 nM) and 3
(GlcNS6S-GlcA, KD = 6.5 nM), but not with com-
pounds
2
(GlcNS-IdoA2S, KD = 30 nM) and
4
(GlcNS-GlcA, KD = 33 nM). These results indicate that
the N-sulfation and 6-O-sulfation of glucosamine in HP/
HS are important for fibronectin binding, while 2-O-sul-
fation of iduronic acid is less important. Recently,
Couchman and coworkers showed that N-sulfation of
glucosamine was essential for fibronectin binding and
2-O-sulfation of iduonic acid or 6-O-sulfation of gluco-
samine has marginal effects.19 Additionally, N-sulfation
and 6-O-sulfation of glucosamine were important for fo-
cal adhesion formation through syndecan-4, heparan
sulfate proteoglycan. Our results are in agreement with
those data.
In contrast, when recombinant human vWf A1 do-
main (rh-vWf-A1)20 was injected over the chips, a dif-

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2503
References and notes
1. For reviews, see: (a) Love, K. R.; Seeberger, P. H. Angew.
Chem. Int. Ed. 2002, 41, 3583; (b) Feizi, T.; Fazio, F.;
Chai, W.; Wong, C. H. Curr. Opin. Struct. Biol. 2003, 13,
637; (c) Wang, D. Proteomics 2003, 3, 2167; (d) Shin, I.;
Park, S.; Lee, M. Chem. Eur. J. 2005, 11, 2894; (e) Ortiz
Mellet, C.; Garcia Fernandez, J. M. ChemBioChem 2002,
3, 819.
2. For articles, see: (a) Blixt, O.; Head, S.; Mandala, T.;
Scanlan, C.; Hufflejt, M. E.; Alvarez, R.; Bryan, M. C.;
Fazio, F.; Calarese, D.; Stevens, J.; Razi, N.; Stevens, D.
J.; Skehel, J. J.; van Die, I.; Burton, D. R.; Wilson, I. A.;
Cummings, R.; Bovin, N.; Wong, C. H. Proc. Natl. Acad.
Sci. U.S.A. 2004, 101, 17033; (b) Chevolet, Y.; Martins, J.;
´
Milosevic, N.; Leonard, D.; Zeng, S.; Malissard, M.;
Berger, E. G.; Maier, P.; Mathieu, H. J.; Crout, D. H. G.;
Sigrist, H. Bioorg. Med. Chem. 2001, 9, 2943; (c) Park, S.;
Lee, M. R.; Pyo, S. J.; Shin, I. J. Am. Chem. Soc. 2004,
126, 4812; (d) Burn, M. A.; Disney, M. D.; Seeberger, H.
P. ChemBioChem 2006, 7, 421; (e) Houseman, B. T.;
Mrksich, M. Chem. Biol. 2002, 9, 443; (f) Fazio, F.; Bryan,
M. C.; Blixt, O.; Paulson, J. C.; Wong, C. H. J. Am. Chem.
Soc. 2002, 124, 14397; (g) Lee, M.; Shin, I. Angew. Chem.
Int. Ed. 2005, 44, 2881; (h) Schwarz, M.; Spector, L.;
Gargir, A.; Shtevi, A.; Gortler, M.; Altstock, R. T.;
Dukler, A. A.; Dotan, N. Glycobiology 2003, 13, 749; (i)
Zhou, X. C.; Zhou, J. H. Biosens. Bioelectron. 2006, 21,
1451; (j) Manimala, J. C.; Roach, T. A.; Li, Z. T.;
Gildersleeve, J. C. Angew. Chem. Int. Ed. 2006, 45, 3607;
(k) Wang, D.; Liu, S.; Trummer, B. J.; Deng, C. Nat.
Biotechnol. 2002, 20, 275; (l) Chevolet, Y.; Bouillon, C.;
Vidal, S.; Morvan, F.; Meyer, A.; Cloarec, J. J.; Jochum,
A.; Parly, J. P.; Vasseur, J. J.; Souteyrand, E. Angew.
Chem. Int. Ed. 2007, 46, 2398.
Figure 3. Binding study with rh-vWf-A1. (a) SPR difference imaging
on the chip immobilized with compounds 1, 2, 3, 4, and Glca(1-6)Glc-
mono (Glc). Measurements were carried out with analyte in the range
between 0.23 and 1.80 lM. (b) Bar graph profiles of different
concentrations. The error bars represent +/ꢀ SEM.
In conclusion, we have designed new, precisely sulfated
oligosaccharides of HP/HS partial structures. These oli-
gosaccharides were efficiently synthesized using appro-
priate monosaccharide intermediates. Their application
in an array type Sugar Chip, using SPR imaging analysis
has been shown to be an efficient and specific technology
to elucidate the interactions between a protein and mul-
tiple sulfated disaccharides, on a real time scale. These
techniques can be used for high-throughput screening
of protein samples, as well as for solving the struc-
ture–function relations of an individual protein–glycos-
aminoglycan interaction at the molecular and nano-
scale.
3. Recent carbohydrate chips immobilized sulfated oligosac-
charide, see: (a) Suda, Y.; Arano, A.; Fukui, Y.; Koshida,
S.; Wakao, M.; Nishimura, T.; Kusumoto, S.; Sobel, M.
Bioconjugate Chem. 2006, 17, 1125; (b) de Paz, J. L.; Noti,
C.; Seeberger, P. H. J. Am. Chem. Soc. 2006, 128, 2766; (c)
de Paz, J. L.; Spillmann, D.; Seeberger, P. H. Chem.
Commun. 2006, 3116; (d) Noti, C.; de Paz, J. L.; Polito, L.;
Seeberger, P. H. Chem. Eur. J. 2006, 12, 8664; (e) Tully, S.
E.; Rawat, M.; Hsieh-Wilson, L. C. J. Am. Chem. Soc.
2006, 128, 7740.
4. (a) Perou, C. M. Nature 2000, 406, 747; (b) Ramsey, G.
Nat. Biotechnol. 1998, 16, 40; (c) Marshall, A.; Hodgson,
J. Nat. Biotechnol. 1998, 16, 27; (d) DeRisi, J. L.; Lyer, V.
R.; Brown, P. O. Science 1997, 278, 680.
5. (a) Templin, M. F.; Stoll, D.; Schrenk, M.; Traub, P. C.;
Vchringer, C. F.; Joos, T. O. Trends Biotechnol. 2002, 20,
160; (b) Weinberger, S. R.; Dalmasso, E. A.; Fung, E. T.
Curr. Opin. Chem. Biol. 2002, 6, 86; (c) Fung, E. T.;
Thulasiraman, V.; Weinberger, S. R.; Dalmasso, E. A.
Curr. Opin. Biotechnol. 2001, 12, 65; (d) Zhu, H. Science
2001, 293, 2101; (e) MacBeath, G.; Schreiber, S. L. Science
2000, 289, 1760.
6. (a) Conrad, H. E. Heparin-Binding Proteins; Academic
Press: San Diego, 1998; (b) Capila, I.; Lindhardt, R. J.
Angew. Chem. Int. Ed. 2002, 41, 390; (c) Turnbull, J.;
Powell, A.; Guimond, S. Trends Cell Biol. 2001, 11, 75; (d)
Bernfield, M.; Go¨tte, M.; Park, P. W.; Reizes, O.;
Fitzgerald, M. L.; Lincecum, J.; Zako, M. Annu. Rev.
Biochem. 1999, 68, 729; (e) Rabenstein, D. A. Nat. Prod.
Rep. 2002, 19, 312; (f) Casu, B.; Lindahl, U. Adv.
Carbohydr. Chem. Biochem. 2001, 57, 159.
Acknowledgments
The present work was financially supported by the Pre-
venture program and the Core Research for Evolutional
Science and Technology (CREST) of the Japan Science
and Technology Agency (JST) to Y.S., and the National
Institutes of Health (Grant HL079182) and the Depart-
ment of Veterans Affairs Research Service to M.S.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2008.01.069.
7. For comprehensive review on the synthesis on GAGs, see:
Yeung, B. K. S.; Chong, P. Y. C.; Petillo, P. A.
J. Carbohydr. Chem. 2002, 21, 799.

2504
M. Wakao et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2499–2504
8. Recent articles, see: (a) Lu, L.-D.; Shie, C.-R.; Kulkarni,
S. S.; Pan, G.-R.; Lu, X.-A.; Hung, S.-C. Org. Lett. 2006,
8, 5995; (b) Zhou, Y.; Lin, F.; Chen, J.; Yu, B. Carbohydr.
(1H, m), 2.30–2.27 (2H, m), 1.88–1.80 (1H, m), 1.65–1.46
(4H, m), 1.35–1.33 (2H, m), ESI-MS (negative mode);
Found: m/z 484.65 [(M-3Na+H)2ꢀ], Calcd. for
C33H50N3O22S4Na3: 1037.15.
´
Res. 2006, 341, 1619; (c) Codee, J. D. C.; Stubba, B.;
Schiattarella, M.; Overkleeft, H. S.; van Boeckel, C. A. A.;
van Boom, J. H.; van der Marel, G. A. J. Am. Chem. Soc.
2005, 127, 3767, And references are therein.
17. Spectral data for compound 4: 1H NMR (600 MHz, D2O),
d 7.09 (1H, t, J = 7.9 Hz), 7.04 (1H, s), 6.67 (1H, d,
J = 7.9 Hz), 6.52 (1H, d, J = 7.9 Hz), 5.43 (1H, d,
J = 4.1 Hz), 4.30 (1H, d, J = 8.2 Hz), 3.91 (1H, d, J = 8.9
Hz), 3.83–3.79 (1H, m), 3.79–3.74 (1H, m), 3.70–3.50
(11H, m), 3.38 (3H, s), 3.25–3.18 (2H, m), 3.14 (1H, dd,
J = 9.6 and 9.6Hz), 3.11–2.96 (4H, m), 2.35–2.28 (1H, m),
2.26 (2H, t, J = 6.9 Hz), 1.86–1.80 (1H, m), 1.65–1.45 (4H,
m), 1.45–1.27 (2H, m), ESI-MS (negative mode); Found:
m/z 444.71 [(M-2Na)2ꢀ], Calcd. for C33H51N3O19S2Na2:
935.21.
9. (a) Suda, Y.; Marques, D.; Kermode, J. C.; Kusumoto, S.;
Sobel, M. Thromb. Res. 1993, 69, 501; (b) Suda, Y.; Bird,
K.; Shiyama, T.; Koshida, S.; Marques, D.; Fukase, K.;
Sobel, M.; Kusumoto, S. Tetrahedron Lett. 1996, 37, 1053.
10. Poletti, L. F.; Bird, K. E.; Marques, D.; Harris, R. B.;
Suda, Y.; Sobel, M. Arterioscler. Thromb. Vasc. Biol.
1997, 17, 925.
11. Koshida, S.; Suda, Y.; Fukui, Y.; Ormsby, J.; Sobel, M.;
Kusumoto, S. Tetrahedron Lett. 1999, 40, 5725.
18. Binding interactions were measured by use of the SPR
imaging sensor, Multi SPRinter (TOYOBO Co. Ltd.,
Osaka, Japan), under the recommended manufacture’s
guidelines with slight modification. Array-type Sugar
Chips were prepared with the purified ligand-conjugates
1, 2, 3, and 4. An aGlc-containing ligand-conjugate
(Glca4Glc-mono) was also included in the chips as a
non-sulfated control. Typical procedures were as follows.
After cleaning the chip surface by UV/O3 treatment, 1 ll
of each sample solution (0.5 mM) in H2O containing 10%
glycerol was spotted on the chip by a spotter (TOYOBO),
and left to stand overnight at room temperature. The
resulting chip was then washed with water, treated with
TEG conjugate21 to mask the unmodified Au surface, and
washed with 0.05% Tween 20 aqueous solution and water
in an ultrasonic cleaner. A protein solution in PBS
containing 0.05% Tween 20 was injected over the surface
at a flow rate of 150 ll/min at various concentrations. The
binding interaction was monitored at 25 °C as the change
in luminance intensity.
12. Koshida, S.; Suda, Y.; Sobel, M.; Kusumoto, S. Tetrahe-
dron Lett. 2001, 42, 1289.
13. Spectral data for compound 2: 1H NMR (600 MHz, D2O),
d 7.11 (1 H, t, J = 7.9 Hz), 6.77–6.75 (2 H, m), 6.58 (1H, d,
J = 7.9 Hz), 5.22 (1H, d, J = 3.4 Hz), 4.97 (1H, brs), 4.14
(1H, brs), 4.05 (1H, brs), 3.92 (1H, brs), 3.82–3.71 (2H, m),
3.70–3.61 (5H, m), 3.59–3.55 (5H, m), 3.38 (3H, s), 3.28
(1H, dd, J = 9.6 and 3.4 Hz), 3.16 (1H, dd, J = 9.6 and
10.3 Hz), 3.09–3.00 (4H, m), 2.34–2.31 (1H, m), 2.27 (2H,
t, J = 6.9), 1.87-1.83 (1H, m), 1.63–1.50 (4H, m), 1.37–1.33
(2H, m), ESI-MS (negative mode); Found: m/z 484.62
[(M-3Na+H)2ꢀ], Calcd. for C33H50N3O22S4Na3: 1037.15.
14. (a) Davis, N. J.; Flitsch, S. L. Tetrahedron Lett. 1993, 34,
1181; (b) Anelli, P. L.; Biffi, C.; Montanari, F.; Quici, S.
J. Org. Chem. 1987, 52, 2559.
15. Kovensky, J.; Duchaussoy, P.; Petitou, M.; Sinay, P.
¨
Tetrahedron: Asymmetry 1996, 7, 3119.
16. Spectral data for compound 3: 1H NMR (600 MHz, D2O),
d 7.13 (1H, t, J = 8.2 Hz), 7.11 (1H, s), 6.77 (1H, d,
J = 8.2 Hz), 6.60 (1H, d, J = 8.2 Hz), 5.44 (1H, d,
J = 3.4 Hz), 4.30 (1H, d, J = 7.6 Hz), 4.15 (1H, d,
J = 10.3 Hz), 4.01 (1H, d, J = 10.3 Hz), 3.90 (1H, d,
J = 11.0 Hz), 3.83–3.82 (1H, m), 3.76–3.71 (3H, m),
3.66–3.60 (6H, m), 3.52 (1H, dd, J = 10.3 Hz,
J = 9.6 Hz), 3.42 (3H, s), 3.27–3.18 (3H, m), 3.14 (1H,
dd, J = 3.4 Hz, J = 10.3 Hz), 3.07–3.02 (3H, m), 2.34–2.30
19. Mahalingam, Y.; Gallagher, J. T.; Couchman, J. R.
J. Biol. Chem. 2007, 282, 3221.
20. Cruz, M. A.; Handin, H. I.; Wise, R. J. J. Biol. Chem.
1993, 268, 21238.
21. TEG conjugate is easily prepared by coupling of thioctic
acid and 2-{2-[2-(2-hydroxy-ethoxy)-ethoxy]-ethoxy}-
ethylamine.