Synthesis of oligomeric assemblies of a platelet-binding key disaccharide in heparin and their biological activities

Article abstract of DOI:10.1016/S0040-4039(00)01826-8

Heparin, highly sulfated glycosaminoglycan, binds to platelets. A key disaccharide unit in heparin was previously found to be responsible for the binding, and the frequency of the disaccharide unit was important for the binding potency. A newly developed method based on the reductive amination was effectively applied to prepare structurally defined oligomeric assemblies possessing multiple units of the key disaccharide. From their platelet-binding activities measured by the competitive binding assay, the enhancement of the activity was clearly observed with increasing number of the key disaccharide.

Full text of DOI:10.1016/S0040-4039(00)01826-8

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 1289–1292
Synthesis of oligomeric assemblies of a platelet-binding key
disaccharide in heparin and their biological activities
Shuhei Koshida,a Yasuo Suda,a,* Michael Sobelb and Shoichi Kusumotoa
aDepartment of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan
bDepartment of Surgery, Syracuse Veterans Administration and Upstate Medical University, State University of New York,
Syracuse, NY 13210, USA
Received 5 October 2000; accepted 19 October 2000
Abstract—Heparin, highly sulfated glycosaminoglycan, binds to platelets. A key disaccharide unit in heparin was previously found
to be responsible for the binding, and the frequency of the disaccharide unit was important for the binding potency. A newly
developed method based on the reductive amination was effectively applied to prepare structurally defined oligomeric assemblies
possessing multiple units of the key disaccharide. From their platelet-binding activities measured by the competitive binding assay,
the enhancement of the activity was clearly observed with increasing number of the key disaccharide. © 2001 Elsevier Science Ltd.
All rights reserved.
Heparin, a glycosaminoglycan (GAG) best known as
an anticoagulant drug,1 binds to human platelets and
alters their functions.2 This event is thought to be
mediated by a binding interaction between specific
structures in heparin and specific protein(s) on the
platelet surface. We previously found that a disaccha-
ride unit in heparin, O-(2-deoxy-2-sulfamido-6-O-sulfo-
deprotected. We employed a sulfated trisaccharide com-
ponent 1 (Fig. 1), which is composed of the GlcNS6S-
IdoA2S disaccharide unit and an additional
D-glucose.
Using the reducing end of the glucose moiety, the
component 1 can be assembled with aromatic amino
groups of linker molecules in one-step by the reductive
amination method as described in the accompanying
paper. The glucose unit works not only as a reducing
end donor for the reductive amination, but also as the
hydrophilic portion in the molecule to minimize any
non-specific hydrophobic interactions between the
linker and the target proteins or cells.
a-
D
-glucopyranosyl)-(1“4)-2-O-sulfo-a-L-idopyrano-
syluronic acid (abbreviated as GlcNS6S-IdoA2S), is a
key unit responsible for binding to platelets.3,4 Further-
more, using synthetic oligomer compounds containing
two or three units of GlcNS6S-IdoA2S we identified the
enhancement of the binding activity achieved by clus-
tering the units.5 In the synthesis of these compounds,
the protected oligomeric frameworks were first pre-
pared, and then converted through many steps of
deprotection and sulfation into regiospecifically sulfated
target compounds. To prepare different types of
oligomer compounds based on the above strategy, the
same tedious transformations must be repeated for each
individual compound. When more complex structures
are designed, however, it would become difficult to
complete these transformations perfectly.
The synthesis of the trisaccharide 1 was carried out as
shown in Schemes 1 and 2. Benzyl 2,3,4-tri-O-benzyl-b-
D
-glucopyranoside (5) to be used as the precursor of the
reducing-end glucose was prepared in four steps from
benzyl b-
D
-glucopyranoside 2.6 The known
L-idose
derivative 67 was converted to a trichloroacetimidate
derivative 7, which was then coupled with 5 in the
presence of tin(II) triflate at 0°C in 1,2-dichloroethane
to give the desired a-linked disaccharide 8 by the partici-
For the efficient preparation of complex and diverse
types of oligomeric compounds, a new strategy was
devised to assemble components containing the ‘com-
pleted’ GlcNS6S-IdoA2S unit already sulfated and
OSO3Na
O
O
O
O
CO2Na
OH
OH
OH
OH
HO
OSO3Na
MeO
O
NHSO3Na
OH
1
* Corresponding author.
Figure 1.
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01826-8

1290
S. Koshida et al. / Tetrahedron Letters 42 (2001) 1289–1292
TrtO
HO
HO
AcO
AcO
TrtO
BnO
O
O
OBn
O
O
OBn
OBn
OBn
a, b
c
d
OH
OBn
OAc
OBn
BnO
OH
OBn
OBn
OAc
OBn
2
3
4
5
O
O
O
O
O
g
e, f
h, i
OAc
OAc
OBn
OAc
OAc
O
O C CCl3
NH
OBn
OBn
OBn
AcO
AcO
AcO
OBn
BnO
OAc
O
OAc
OAc
OBn
8
6
7
O
O
O
O
OH
O
O
CO2Me
OBn
O
OBn
OBn
j, k
l, m, n
O
OBn
OBn
OBn
OBn
OBn
BnO
BnO
OAc
BnO
O
HO
HO
OH
OBn
OBn
OAc
OBn
9
10
11
Scheme 1. (a) 0.05 M NaOMe; (b) TrtCl/pyridine, 80°C; (c) BnBr, NaH/DMF; (d) 80% AcOH, 80°C, 66% (four steps); (e)
piperidine, AcOH/THF, 61%; (f) CCl3CN, Cs2CO3/CH2Cl2, quant.; (g) 5, Sn(OTf)2, MS4A/(CH2Cl)2, 0°C, 80%; (h) 0.05 M
NaOMe; (i) (CH3)2C(OCH3)2, CSA/acetone, 89% (two steps); (j) Ac2O, DMAP, pyridine/CH2Cl2, 95%; (k) 90% AcOH, 96%; (l)
TEMPO, NaClO, KBr; (m) NaClO2, NaH2PO4/H2O, t-BuOH; (n) TMSCHN2, 74% (three steps).
O
OAc
O
O
OAc
O
O
O
OBn
O
O
OBn
CO2Me
a
CO2Me
+
O C CCl3
NH
OBn
OBn
OBn
OBn
OBn
OBn
HO
BnO
BnO
MeO
MeO
O
MeO
OAc
OBn
OBn
OAc
OBn
O
N3
N3
11
12
b, c, d
13
OSO3Na
O
OSO3Na
O
O
O
O
O
O
e, f, g
CO2Na
CO2Na
OH
OH
OBn
OBn
OH
OH
OBn
MeO
O
BnO
O
HO
OSO3Na
OBn
OSO3Na
OH
N3
NHSO3Na
14
1
Scheme 2. (a) TBDMSOTf, MS4A/toluene, −20°C, 82%; (b) 0.05 M NaOMe, 92%; (c) SO3·pyridine complex/DMF; (d)
NaOH/MeOH; (e) 10% Pd–C, H2 1 kg/cm2/THF:H2O=2:1; (f) SO3·pyridine complex/H2O, pH 9.5; (g) 10% Pd–C, H2
kg/cm2/H2O:AcOH=5:1; 93% (five steps).
7
pation of the neighboring acetyl group at the 2-posi-
tion in 80% yield. After the transformations of protect-
ing groups in four steps, the primary 6%-hydroxy group
of the resultant 10 was selectively oxidized using
2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) fol-
lowed by NaClO2 treatment. The resulting carboxyl
group was then esterified with (trimethylsilyl)-
diazomethane to afford the disaccharide methyl ester
11.
permeation chromatography using Sephadex G-25 gave
the desired sulfated trisaccharide 1 in 93% yield (five
steps).
The assembly of the sulfated trisaccharide 1 was per-
formed using the method described in the accompany-
ing paper. Coupling of 1 with the divalent linker 15 (0.4
equiv. to 1) was performed using NaBH3CN (10 equiv.)
at pH 3 (Scheme 3). After 1 day at 37°C, NaBH3CN
(10 equiv.) was added again to complete the reaction.
After an additional 2 days of incubation, the product
was purified by gel permeation chromatography with
Sephadex G-50 to give the desired dimeric assembly 18
containing two units of GlcNS6S-IdoA2S in 60% yield
(based on the linker 15). The preparation of the dimeric
assembly 19, possessing a longer methylene bridge com-
pared to 18, was carried out in the same manner. Using
the trivalent linker 17, the trimeric assembly 20 possess-
ing three units of GlcNS6S-IdoA2S was prepared suc-
cessfully by a similar procedure. The structure of the
assemblies 18–20 were confirmed by 600 MHz 1H
NMR and ESI-MS.9
The 2-azido sugar derivative 125 was coupled with the
disaccharide 11 using t-butyldimethylsilyl triflate
(TBDMSOTf) at −20°C to give selectively an a-linked
trisaccharide 13 in 82% yield (Scheme 2).5,8 After all the
acetyl groups of 13 were removed, the resulting 2%- and
6¦-hydroxy groups were O-sulfated and the methyl
ester was hydrolyzed to give the carboxylate 14. The
transformation of the 2¦-azide into the amino group
was performed by hydrogenolysis under 1 kg/cm2 of
hydrogen with Pd/C in THF:H2O. No benzyl group
was removed under this mild and neutral conditions as
judged from the TLC analysis. The resulting 2¦-amino
group was N-sulfated and the product was hydro-
genated (7 kg/cm2) using Pd/C in H2O:AcOH=5:1 to
remove all the benzyl groups. Then, purification by gel
The platelet-binding activities of 1 and the assemblies
18–20 were evaluated using a competitive binding assay

S. Koshida et al. / Tetrahedron Letters 42 (2001) 1289–1292
1291
according to our previous method.5,10 In Fig. 2, the
competitive binding activities of 1 and 18–20 were
compared with those of a commercial heparin (average
molecular weight 17500, from porcine intestine, Nacalai
Tesque, Kyoto, Japan). The binding potency was
enhanced as the number of GlcNS6S-IdoA2S moieties
increased. The trisaccharide 1, containing only one unit
of GlcNS6S-IdoA2S, showed no significant activity at
the concentration range tested, whereas the dimeric
assemblies 18 and 19 showed distinct binding potency.
The trimeric assembly 20 showed the highest activity.
These results further establish the role of a clustering
effect, based on the subunit structure GlcNS6S-IdoA2S.
themselves. The novel methodology reported here for
the assembly of structurally defined oligosaccharides,
using reductive amination reaction, may be an effective
way to study the biology of oligosaccharides.
Acknowledgements
This study was supported in part by ‘Research for the
Future’ Program No. 97L00502 from the Japan Society
for the Promotion of Science. We are grateful to Pro-
fessor T. Tamura and Ms. K. Aoyama at Hyogo Med-
ical College for their invaluable advice and kind
assistance in the platelet competitive assay.
It is often difficult to measure the biological activity of
small oligosaccharide units that have low affinity by
OSO3Na
O
O
O
CO2Na
OH
O
H2N
H2N
H2N
O
O
O
OH
OH
OH
N
H
H2N
HO
OSO3Na
MeO
O
O
(CH2)n
O
NHSO3Na
OH
N
H
1
N
H
OH
H2N
NaBH3CN (10 +10 eq)
H2O:AcOH = 4:1
NaBH3CN (10 +10 eq)
H2O:AcOH = 9:1
N
H
N
H
pH 3, 37 °C, 4 d
pH 3, 37 °C, 3 d
15: n = 2
16: n = 5
(0.4 eq to 1)
(0.25 eq to 1)
17
OSO3Na
O
O
O
OH
NH
CO2Na
OH
OSO3Na
O
O
OH
OH
OH
OH
O
OH
HO
OSO3Na
MeO
MeO
MeO
O
O
O
O
CO2Na
NH
NH
OH
OH
OH
N
H
OH
OH
NHSO3Na
OSO3Na
HO
MeO
MeO
O
O
O
N
H
O
O
OH
NH
NHSO3Na
OSO3Na
OSO3Na
O
O
OH
OH
CO2Na
OH
OH
OH
O
O
HO
OSO3Na
O
O
(CH2)n
O
CO2Na
OH
N
H
O
H
OH
OH
NHSO3Na
OSO3Na
O
HO
O
OH
NH
N
H
OSO3Na
OH
NHSO3Na
CO2Na
OH
18: n = 2 (yield: 60%)
19: n = 5 (yield: 60%)
O
HO
OSO3Na
N
H
OH
NHSO3Na
20 (yield: 43%)
Scheme 3.
Figure 2. Binding competitive activity of synthetic assemblies and commercial heparin.

1292
S. Koshida et al. / Tetrahedron Letters 42 (2001) 1289–1292
3.01 (2H, dd, J=8.7 Hz, J=13.6 Hz), 2.68 (4H, s);
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ESI-MS (negative) m/z 903.23 [(M−8Na+6H)2−].
Spectral data for compound 19: 1H NMR (600 MHz,
D2O), l 7.12 (2H, t, J=8.0 Hz), 6.74–6.70 (4H, m), 6.57
(2H, d, J=6.6 Hz), 5.26 (2H, d, J=3.3 Hz), 5.03 (2H, d,
J=2.8 Hz), 4.42 (2H, d, J=2.2 Hz), 4.21 (2H, s), 4.19
(2H, dd, J=4.4 Hz), 4.11 (2H, s), 4.09 (2H, m), 3.97 (2H,
t, J=3.0 Hz), 3.89 (4H, m), 3.87 (2H, m), 3.77 (2H, m),
3.72 (2H, dd, J=5.8 Hz, J=2.2 Hz), 3.69 (2H, dd, J=2.1
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(2H, dd, J=3.6 Hz, J=10.4 Hz), 3.00 (2H, dd, J=8.7
Hz, J=13.4 Hz), 2.32 (4H, t, J=7.3 Hz), 1.62 (4H, dd,
J=7.6 Hz), 1.34 (2H, t, J=7.5 Hz); ESI-MS (negative)
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D2O), l 7.21 (3H, m), 6.95–6.89 (6H, m), 6.73 (3H, m),
5.25 (3H, d, J=3.6 Hz), 5.05 (3H, s), 4.48 (3H, s), 4.20
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Hz), 3.98 (3H, s), 3.87 (6H, m), 3.84 (3H, m), 3.76 (3H,
m), 3.71 (3H, m), 3.66 (3H, d, J=10.4 Hz), 3.62 (3H, t,
J=9.6 Hz), 3.56 (3H, m), 3.47 (9H, s), 3.36–3.21 (6H, b,
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3.17–3.02 (10H, b, overlapped with peaks at 3.15 and
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(2H, dd, J=2.6 Hz, J=5.2 Hz), 4.10 (2H, d, J=2.8 Hz),
4.09 (2H, d, J=2.8 Hz), 3.97 (2H, t, J=3.0 Hz), 3.89
(4H, m), 3.87 (2H, m), 3.77 (2H, m), 3.72 (2H, dd, J=5.5
Hz, J=2.2 Hz), 3.69 (2H, dd, J=2.2 Hz, J=8.5 Hz), 3.61
(2H, t, J=9.8 Hz), 3.58 (2H, dd, J=5.5 Hz, J=11.0 Hz),
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.