Assessment through chemical synthesis of the size of the heparin sequence involved in thrombin inhibition

Article abstract of DOI:10.1016/S0008-6215(99)00068-3

Deca- to eicosasaccharides having the generic structure methyl(sodium 2,3-di-O-methyl-4-O-sodium sulfonato-α-L-idopyranosyluronate)-(1→4)-[(2,3,6-tri-O-sodium sulfonato-α-D-glucopyranosyl)-(1→4)-(sodium 2,3-di-O-methyl-α-L-idopyranosyluronate)-(1→4)](n)-2

Full text of DOI:10.1016/S0008-6215(99)00068-3

www.elsevier.nl/locate/carres
Carbohydrate Research 317 (1999) 85–99
Assessment through chemical synthesis of the size of the
heparin sequence involved in thrombin inhibitionꢀ
Philippe Duchaussoy a, Guy Jaurand b, Pierre-A. Driguez a, Isidore Lederman a,
Marie-L. Ceccato, Franc¸oise Gourvenec a, Jean-M. Strassel a, Philippe Sizun c,
Maurice Petitou a,*, Jean-M. Herbert a
a Sanofi Recherche, Haemobiology Research Department, 195 route d’Espagne, F-31036 Toulouse, France
b Sanofi Chimie, route de Gap, BP15, F-04201 Sisteron, France
c Sanofi Recherche, Exploratory Research Department, 371 rue du Professeur Joseph Blayac,
F-34184 Montpellier, France
Received 27 October 1998; accepted 5 March 1999
Abstract
Deca- to eicosasaccharides having the generic structure methyl(sodium 2,3-di-O-methyl-4-O-sodium sulfonato-a-
idopyranosyluronate)-(1“4)-[(2,3,6-tri-O-sodium sulfonato-a- -glucopyranosyl)-(1“4)-(sodium 2,3-di-O-methyl-a-
-idopyranosyluronate)-(1“4)]n-2,3,6-tri-O-sodium sulfonato-a- -glucopyranoside have been synthesized from a
L-
D
L
D
single disaccharide precursor. All of them bind to and activate antithrombin. When n56 only Factor Xa inhibition
is observed, whereas when n\6 Factor Xa and thrombin are both inhibited in the presence of antithrombin. These
results indicate that, in heparin, the sequence involved in antithrombin-catalyzed thrombin inhibition is a pentadeca-
or a hexadecasaccharide. © 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Heparin; Heparin mimetics; Antithrombin; Factor Xa; Thrombin
tion of the anion-binding exosite II of the
1. Introduction
protein by the negatively charged polysaccha-
ride [4] and, according to current understand-
ing, thrombin inhibition by antithrombin
occurs when both proteins collide at the sur-
face of a heparin molecule. It is clear from this
template mechanism that an anticoagulantly
active heparin molecule must comprise an an-
tithrombin binding domain prolonged, at least
at one end, by a thrombin-binding domain. As
shown in Fig. 1, in most molecules the DE-
FGH sequence (antithrombin-binding do-
main) is elongated at both ends, mainly by
Heparin accelerates the reaction rate of
thrombin and coagulation Factor Xa with
their physiological inhibitor antithrombin, by
several orders of magnitude [2]. Factor Xa
inhibition is catalyzed by a very precise pen-
tasaccharide sequence (Fig. 1) that corre-
sponds to the antithrombin binding domain of
heparin [3]. The structure of heparin
molecules able to inhibit thrombin is still ill-
defined. Thrombin interaction with heparin is
generally assigned to an electrostatic attrac-
repeated “4)-(2-O-sulfonato-a-
syluronate)-(1“4)-(N-sulfonato-6-O-sulfo-
-glucosaminyl)-(1“ disaccharidic se-
L-idopyrano-
nato-a-
D
ꢀ Part 2 of the series: ‘Design and synthesis of heparin
mimetics able to inhibit thrombin’. For Part 1 see Ref. [1].
* Corresponding author.
quences [5] that constitute thrombin-binding
domains.
0008-6215/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(99)00068-3

86
P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 85–99
The aim of the present work was first to
stepwise by one disaccharide unit from deca-
to eicosasaccharide.
reproduce in a simple way active thrombin
inhibitory molecules and, at the same time, to
assess precisely the minimum size of a heparin
fragment able to catalyze thrombin inhibition,
since several reports on this issue give conflict-
ing values [6].
2. Results and discussion
To facilitate understanding of the various
routes toward the different oligosaccharides, a
naive synthesis pathway with hexagons for
carbohydrate units is outlined in Scheme 1.
Our initial intention was to elongate the tetra-
and hexasaccharides 8 [1] and 9 by repeated
reaction with the di- and tetrasaccharide imi-
dates 10 [1] and 11. For reasons stated below,
we also used the hexasaccharide donor 12 (see
Schemes 1 and 2).
In the preceding paper [1], we explained
how to simplify the synthesis and decided to
avoid the problem of the relative position
(reducing or non-reducing end) of the an-
tithrombin-binding domain and the thrombin-
binding domain. We reasoned that the
antithrombin-binding domain, because it con-
tains sulfate groups, may electrostatically at-
tract thrombin, and thus serve as
a
Selective removal of the levulinic group of
the previously described hexasaccharide 13 [1],
using hydrazine hydrate in a pyridine–acetic
acid mixture [7], gave the glycosyl acceptor 9
in 85% yield (Scheme 2). The latter then re-
acted with the disaccharide imidate 10 (1.3
equiv) in the presence of tert-butyldimethylsi-
lyl trifluoromethanesulfonate (ButMe2SiOTf)
thrombin-binding domain as well. Thus, an
oligosaccharide representing a continuum of
antithrombin-binding domains should be able
to inhibit thrombin and Factor Xa, as soon as
it is long enough to accommodate both anti-
thrombin and thrombin, and provided its
affinity for antithrombin is not too high, so
that thrombin can compete for binding. As
explained in the preceding paper [1], these
considerations led us to select the hexasaccha-
ride 1 as the antithrombin-binding domain. It
is obtained from a single disaccharide syn-
thon, and its affinity for antithrombin (Kd=
0.3590.01 mM) satisfies the affinity criteria
just mentioned. In the present paper, we re-
port the synthesis of longer fragments 2–7,
homologous to 1, the size of which increases
,
[8] and 4 A molecular sieves, in toluene, at
−20 °C to give, after mere purification on
Sephadex LH-20, the pure (over 95% accord-
1
ing to H NMR) octasaccharide 14, in 95%
yield. The anomeric configuration of the new
a-
D
glycosidic bond, for this coupling reac-
tion, and also for the others cited below, was
1
confirmed by high-field H NMR (l H-1 5.10
ppm, J1,2 3.5 Hz). No b-bonded product could
be detected.
Fig. 1. Antithrombin, when bound to its binding site (DEFGH) in an anticoagulant heparin molecule, undergoes a conforma-
tional change allowing Factor Xa inhibition. To be inhibited thrombin is first attracted by the thrombin-binding domains and
collides with activated (heparin bound) antithrombin. The hexasaccharide 1 displays affinity for antithrombin in spite of lacking
glucuronic acid (E) in its sequence, and catalyzes Factor Xa inhibition. Larger homologous fragments catalyze thrombin
inhibition as well.

P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 85–99
87
Scheme 1. Synthesis pathway toward oligomers of the disaccharide methyl 4-O-(sodium 2,3-di-O-methyl-a-
L
-idopyranosy-
luronate)-2,3,6-tri-O-sodium sulfonato-a-D-glucopyranoside. Condensation of the left column glycosyl donor imidates (white
triangle reducing end), and the centre column glycosyl acceptors (black triangle non-reducing end), gave the right column fully
protected oligo- and polysaccharides according to this scheme. The latter were further deprotected and O-sulfated to give the final
compounds of Table 1.
Another possible route to 14 was to con-
dense the two tetrasaccharides 11 and 8. To
this end, we intended to prepare the imidate
11 by acetolysis of the methyl glycoside of 15
[1] to obtain 16, followed by anomeric
deacetylation (to give 17), and classical reac-
tion with trichloroacetonitrile. However, the
poor yield of the acetolysis step (33%) and the
need for a large quantity of 11 prompted us to
devise another, more laborious, route (Scheme
3) where the acetate at the reducing end of the
tetrasaccharide was introduced at the disac-
charide level. Thus, starting from the disac-
charide 18 [1], 19 was obtained (67%) using
chloroacetic anhydride, 4-(dimethylamino)-
110 °C, cleanly removed the chloroacetyl
group without affecting the anomeric acetate,
thus giving the acceptor 21 (76%). The change
1
observed in high-field H NMR for the cou-
pling constants of the iduronic acid moiety
when 20 is converted into 21 (see Table 2)
indicates that the conformation of this unit is
1
shifted toward C4 when the substituent at
C-4, a chloroacetyl group here, is removed.
The observation of the long-range couplings
4
1
4J1,3 (1.2 Hz) and J2,4 (1.3 Hz) on the H
NMR COSY spectrum of 21 confirmed both
1
the C4 conformation of the iduronic acid ring
and the already proven a-
L
configuration of
the anomeric carbon. This change in confor-
mation is also observed when a levulinyl
group C-4 is removed. Glycosylation of 21
with 10 [1], using trimethylsilyl trifl-
uoromethanesulfonate (Me3SiOTf) [11] as
1
pyridine, and triethylamine. As expected, H
NMR analysis indicated that H-4% of 19 was
shifted downfield at 5.00 ppm (compared to
3.96 ppm in 18). Acetolysis of the methyl
glycoside using a mixture of trifluoroacetic
acid and acetic acid in acetic anhydride, at
60 °C, gave a mixture of the anomeric acetates
20 (a/b 4:1) in 60% yield. As already observed
[9], the benzyl groups at positions 3 and 6 of
the glucose residue were also acetolysed under
these conditions. Deprotection of position 4%
with thiourea in pyridine–ethanol [10], at
,
catalyst, in the presence of 4 A molecular
sieves, in dichloromethane at −20 °C, af-
forded the tetrasaccharide 16 in 73% yield.
The a-
D
configuration of the new glycosidic
1
bond was confirmed by 500 MHz H NMR
(l H-1 5.10 ppm, J1,2 3.6 Hz). The anomeric
acetate of 16 was cleaved with ethanolamine

88
P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 85–99
Surprisingly, reaction of 10 and 21 also
furnished the hexasaccharide 22 in 5% yield.
Its hexasaccharide nature was first suspected
during gel-permeation chromatography on
LH-20, since it was eluted before the tetrasac-
charide 16. The structure was confirmed un-
ambiguously by mass spectrometry {m/z
(thioglycerol+NaCl), 2072 [M+Na]+}, and
in tetrahydrofuran [12] to give 17 (79%),
which was treated with trichloroacetonitrile
and potassium carbonate in dichloromethane
1
to give the imidate 11 in 91% yield. The H
NMR spectrum of 17 was identical to that of
16 except for an upfield shift of the anomeric
protons from 6.28 and 5.60 ppm to 5.18 and
4.75 ppm for the a and b anomers, respec-
tively.
1
by H NMR spectroscopy, where two signals
Scheme 2. Reagents and conditions: (a) Me3SiOTf or ButMe2SiOTf, toluene or CH2Cl2,−20 °C; (b) 1 M NH2NH2·H2O,
pyridine/AcOH, 15 min; (c) H2, 10%, Pd/C, DMF; (d) NaOH, MeOH, 16 h; (e) pyridine–SO3, DMF, 55 °C, 20 h.

P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 85–99
89
Scheme 3. Reagents and conditions: (a) (ClCH2CO)2O, DMAP, Et3N, CH2Cl2, 67%. (b) CF3COOH, Ac2O, AcOH, 60 °C, 60%;
(c) (NH2)2CS, pyridine/EtOH, 110 °C, 79%; (d) Me3SiOTf, CH2Cl2, −20 °C, 16: 73%+22: 5%; (e) NH2CH2CH2OH, THF,+
4 °C, 17: 73%, 23: 84%; (f) CCl3CN, K2CO3, CH2Cl2, 11: 91%, 12: 93%.
for H-1 of glucose units involved in glycosidic
bonds at l 5.10 and 5.08 ppm were observed
with the same J1,2 of 3.6 Hz (Table 2). Forma-
tion of 22 might have resulted from cleavage
of the levulinyl ester of the tetrasaccharide
followed by glycosylation. One may also
imagine that the ketone of the levulinyl groups
reacts with the intermediate anomeric carbo-
cation of the glycosylating species to form an
intermediate that rearranges into the gly-
coside. No experiment was attempted to ex-
plain this reaction. As shown below, we took
advantage of this unexpected result and used
this hexasaccharide synthon for our synthesis.
Thus, transformation of 22, realized as de-
scribed for the conversion of 16 to 11, gave
first anomeric free 23 (84%), then the imidate
12 (93%).
Condensation of 11 and 8 in toluene
(Scheme 2) in the presence of Me3SiOTf, fol-
lowed by gel-permeation chromatography
(Sephadex LH-20) gave two fractions, the first
one containing unreacted 8 (32%). TLC analy-
sis of the second one showed that it was a
mixture that could be purified on silica gel to
give the expected octasaccharide 14 (49%) and
another compound that was identified as the
dodecasaccharide 26. Thus, as observed dur-
ing the synthesis of 16, overglycosylation took
place to give the homologous oligosaccharide,
once again with a yield around 5%. The struc-
1
ture of 26 was confirmed by 500 MHz H
Table 1
Biochemical data for compounds 2–7
Compound
Size
MW
Kd/antithrombin (nM)
aXa (U/mg)
aXa (U/nmol)
aIIa (IC50 ng/mL)
Heparin
15000
3606
4300
4995
5689
6384
7078
180
405
360
310
359
290
236
3.3
2
3
4
5
6
7
10-mer
12-mer
14-mer
16-mer
18-mer
20-mer
21
19
19
22
28
nd
1.5
1.6
1.6
2.0
1.9
1.7
\10000
\10000
\10000
130910
2394
6.793

90
P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 85–99
Table 2
1H NMR data: carbohydrate ring proton chemical shifts (ppm) and coupling constants (Hz)
lH-1
lH-2
lH-3
lH-4
lH-5
lH-6
lH-6%
J1,2
J2,3
J3,4
J4,5
J5,6-5,6%
J6,6%
9
IdoAVI
GlcV
4.88
1–2
5.11
3.6
4.89
6.8
5.12
3.9
5.23
6.5
3.15
3–4
3.41
9.8
2.80
7–8
3.36
9.9
2.91
8.4
3.43
9–10
3.45
3–4
5.33
9.6
3.51
7–8
5.30
9.5
3.64
8.4
3.80
9–10
3.90
2
3.60
10.0
3.81
5.7
3.51
10.0
3.78
5.8
3.78
9–10
4.60
4.01
1–4
4.47
4.35
4.33
3.72
4.21
4.13
3.68
IdoAIV
GlcIII
IdoAII
GlcI
3.97
1–4
4.42
4.56
3.5
3.69
1–4
11
IdoAIV
GlcIII
IdoAII
GlcIa
GlcIb
4.92
4.7
5.09
3.6
4.98
6.7
6.40
3.5
2.98
6.0
3.41
10.0
2.94
7.6
3.60
9.8
3.60
9.5
3.45
6.0
5.34
9.2
3.56
7.6
5.35
9.2
4.98
4.5
3.64
10.0
3.85
5.7
3.59
10.0
3.74
10.0
4.67
4.03
1.8
4.50
4.36
4.2
4.23
−12.2
4.10
1.8, 4.3
4.20
4.43
−12.4
4.50
4.5
4.20
5.90
7.5
5.19
9.2
4.14
−12.3
1.8
14
IdoAVIII
GlcVII
IdoAVI
GlcV
4.92
4.7
5.10
3.6
4.93
6.8
5.10
3.6
4.90
6.8
5.14
3.9
5.26
6.5
4.56
3.5
2.97
6.0
3.41
10.0
2.86
7–8
3.36
10.0
2.78
7–8
3.36
9.9
3.45
6.0
5.34
9.2
3.55
7–8
5.31
9.2
3.52
7–8
5.31
9.5
4.98
4.5
3.63
10.0
3.82
5.7
3.51
10.0
3.81
5–6
3.51
10.0
3.80
5.8
4.67
4.03
1.8
4.47
4.36
4.2
4.22
−12.2
4.01
1.8
4.45
4.41
4.2
4.10
−12.2
IdoAIV
GlcIII
3.98
1–4
4.43
4.43
3.75
4.13
3.69
IdoAII
GlcI
2.92
8.4
3.45
9–10
3.65
8.4
3.81
9–10
3.80
9–10
3.70
1–4
16
IdoAIV
GlcIII
IdoAII
GlcIa
GlcIb
4.92
5.0
5.10
3.6
4.96
6.7
6.27
3.6
2.98
6.0
3.42
10.0
2.95
7.6
3.48
9.8
3.44
9.0
3.45
6.0
5.34
9.2
3.57
7.6
5.33
9.2
4.98
4.5
3.64
10.0
3.85
5.7
3.60
10.0
3.64
10.0
4.67
4.03
1.8, 4.2
4.50
4.37
4.23
−12.2
3.94
1.9, 4.4
3.69
4.40
−12.4
4.43
4.18
4.18
5.60
8.1
5.16
9.2
1.8, 4.5
−12.3
17
IdoAIV
4.93
5.0
2.98
6.0
3.45
6.0
4.96
4.5
4.67

P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 85–99
91
Table 2 (Continued)
lH-1
lH-2
lH-3
lH-4
lH-5
lH-6
lH-6%
J1,2
J2,3
J3,4
J4,5
J5,6-5,6%
J6,6%
GlcIII
IdoAII
GlcIa
GlcIb
5.10
3.6
4.96
6.7
5.18
3.6
4.75
7.6
3.42
10.0
2.95
7.6
3.44
9.8
5.35
9.2
3.58
7.6
5.35
9.2
5.12
9.2
3.65
10.0
3.85
5.7
3.62
10.0
3.59
10.0
4.04
1.8, 4.2
4.90
4.37
−12.2
4.22
4.04
1.9, 4.4
4.12
4.37
−12.4
4.52
4.22
4.44
3.24
9.0
1.8, 4.5
−12.3
19
20
IdoAII
GlcI
5.20
5.0
4.56
7.6
3.08
6.0
3.55
9–10
3.53
6.0
5.35
9–10
5.00
4.5
4.89
9–10
1–4
IdoAII
GlcIa
GlcIb
4.98
4.8
6.30
3.6
5.63
8.0
3.06
5.2
3.55
9.8
3.50
9.2
3.49
5.8
5.39
9.7
5.23
9.7
5.04
4.4
3.77
10.1
3.78
10.1
4.76
3.99
2.4, 3.6
3.74
4.33
−12.4
4.38
4.29
4.28
1.7, 2.5
−12.3
21
22
IdoAII
GlcIa
GlcIb
4.91
1.8
6.30
3.6
5.63
7.9
3.26
3.2
3.53
9.8
3.48
8.8
3.51
3.3
5.40
9.3
5.23
9.2
3.97
2.0
3.74
10.1
3.79
10.0
4.70
3.97
3.5, 1.9
3.72
4.30
−12.4
4.30
4.24
4.24
3.5, 1.9
−12.2
IdoAVI
GlcV
4.92
5.0
5.10
3.6
4.92
6.7
5.08
3.6
4.95
6.7
6.27
3.6
5.59
8.1
2.97
6.0
3.41
10.0
2.87
7.6
3.36
10.0
2.93
7.6
3.47
9.8
3.45
6.0
5.34
9.2
3.55
7.6
5.31
9.2
3.56
7.6
5.32
9.2
5.16
9.2
4.98
4.5
3.64
10.0
3.83
5.7
3.51
10.0
3.84
5.7
3.58
10.0
3.62
10.0
4.67
4.03
1.8, 4.2
4.48
4.36
−12.2
4.22
4.16
IdoAIV
GlcIII
IdoAII
GlcIa
GlcIb
4.00
1.8, 4.2
4.49
4.41
−12.2
3.93
1.9, 4.4
3.68
4.39
−12.4
4.43
4.18
4.17
3.43
9.2
1.8, 4.5
−12.3
NMR, which showed the a configuration of
the new anomeric centers, and by mass spec-
trometry {m/z (thioglycerol+NaCl), 4032
[M+Na]+}.
The levulinyl group of the octasaccharide
14 was cleaved to give the acceptor 24 (96%)
that reacted with the disaccharide imidate 10
to provide the decasaccharide 25 (68% yield
after gel-permeation chromatography fol-
lowed by silica gel chromatography), and with
the tetrasaccharide imidate 11 to yield the
dodecasaccharide 26 (69%). Delevulinylation
of 25, as described for 13, gave the acceptor
27 (70%) that was coupled with the tetrasac-
charide imidate 11, using Me3SiOTf in
dichloromethane, to furnish the tetradecasac-
charide 28 (40%). This low yield was explained
by extensive formation of by-products, which
were difficult to isolate. Delevulinylation of 26
gave 29 (95%), which was coupled with te-

92
P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 85–99
trasaccharide 11 to furnish the hexadecasac-
charide 30 (62%) together with unreacted ac-
ceptor 29 (20%), and hemiacetal 17 (20%).
The octadecasaccharide 31 was prepared (50%
yield) by glycosylation of 29 with the hexasac-
charide imidate 12. Again, 31 was purified first
by exclusion chromatography, then over silica
gel. Delevulinylation of 30 gave the hexade-
Table 3
1H NMR data for the glycosyl acceptor hexadecasaccharide 32 (similar data were obtained for the glycosyl acceptors 29, 27, and
24), the protected eicosasaccharide 33 (similar data were obtained for the fully protected 31, 30, 28, 26, and 25) and the
eicosasaccharide 7 (similar data were obtained for 6, 5, 4, 3, and 2)
lH-1
lH-2
lH-3
lH-4
lH-5
lH-6
lH-6%
J1,2
J2,3
J3,4
J4,5
J5,6-5,6%
J6,6%
32
33
7
IdoAXVI
GlcXV
4.88
2–3
5.07
3–4
4.92
6–7
5.08
3–4
4.90
6–7
5.14
3–4
5.26
6–7
4.56
3–4
2.87
3–4
3.38
9–10
2.84
7–8
3.35
9–10
2.78
7–8
3.36
9–10
2.92
7–8
3.53
3–4
5.36
9–10
3.55
7–8
5.30
9–10
3.52
7–8
5.30
9–10
3.65
7–8
3.83
2–3
3.66
9–10
3.81
5–6
3.48
9–10
3.81
5–6
3.51
9–10
3.80
5–6
4.49
4.02
1–4
4.46
4.26
−12
4.21
4.14
4.14
3.70
IdoAXIV
GlcV,VII,IX,XI,XIII
IdoAIV,VI,VIII,X,XII
GlcIII
4.00
1–4
4.46
4.40
−12
3.98
1–4
4.42
4.33
−12
IdoAII
GlcI
3.45
9–10
3.81
9–10
3.80
9–10
3.70
1–4
3.75
−12
IdoAXX
4.92
4–5
5.10
3–4
4.92
6–7
5.10
3–4
4.90
6–7
5.14
3–4
5.26
6–7
4.56
3–4
2.97
5–6
3.41
9–10
2.86
7–8
3.35
9–10
2.78
7–8
3.36
9–10
2.92
7–8
3.45
5–6
5.34
9–10
3.55
7–8
5.30
9–10
3.52
7–8
5.30
9–10
3.66
7–8
4.98
4–5
3.64
9–10
3.83
5–6
3.48
9–10
3.81
5–6
3.51
9–10
3.79
5–6
4.67
GlcXIX
4.02
1–4
4.48
4.36
−12
4.23
4.14
4.14
3.70
IdoAXVIII
GlcV,VII,IX,XI,XIII,XV,XVII
3.99
1–4
4.46
4.40
−12
IdoAIV,VI,VIII,X,XII,XIV,XVI
GlcIII
IdoAII
GlcI
3.98
1–4
4.43
4.33
−12
3.45
9–10
3.81
9–10
3.80
9–10
3.70
1–4
3.75
−12
IdoAXX
5.09
1–2
5.40
3–4
5.06
3–4
5.41
3–4
5.07
2–3
5.15
3.5
3.51
3–4
4.30
9–10
3.48
5–6
4.32
9–10
3.49
4–5
4.36
9.5
4.04
3–4
4.59
9–10
3.72
3–4
4.54
9–10
3.75
3–4
4.65
9.5
4.77
2
4.95
GlcXIX
3.95
9–10
4.22
2–3
3.95
9–10
4.18
2–3
3.95
9.5
4.20
1–4
4.89
4.30
−12
4.15
4.15
4.26
IdoAIV,VI,VIII,X,XII,XIV,XVI,XVIII
GlcIII,V,VII,IX,XI,XIII,XV,XVII
4.16
1–4
4.76
4.30
−12
IdoAII
GlcI
4.08
1–4
4.38
−12

P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 85–99
93
minor undersulfated impurities, the major fully
sulfated species representing between 85 and
92% of the different compounds.
casaccharide acceptor 32, used without purifi-
cation in the glycosylation reaction with 11.
The low yield (25%) in the eicosasaccharide 33
could be explained by trapping of the imidate
by a contaminant by-product present in the
crude 32 used in this reaction.
The anti-Factor Xa and anti-thrombin in-
hibitory potencies of the compounds are re-
ported in Table 1. No significant difference was
found for the anti-Xa activities expressed in
U/nmol. On the contrary, thrombin inhibition
was only detected from hexadecasaccharide 5
and as observed in the case of heparin frag-
ments [3], increased with the size for larger
oligosaccharides. Since, as shown previously,
theseO-methylated-O-sulfonatedoligosacchar-
ides very well mimic the interaction of heparin
and antithrombin [14,15], the present results
indicate that the heparin sequence involved in
antithrombin catalyzed thrombin inhibition
has the size of a pentadeca- or a hexadecasac-
charide.
During this process of chain elongation,
1
every step was controlled by 500 MHz H
NMR, to prove the structure of the com-
pounds, and also to check their homogeneity
(all compounds were purified to over 95%
homogeneity; to save as much as possible of
these elaborated intermediates we did not per-
1
form destructive analyses). H NMR data are
shown Tables 2 and 3. Above the size of a
hexa- or octasaccharide, similar NMR spectra
were obtained for the different members in
each family of these homopolymers. We have
reported in Table 3 only the data for the larger
ones. A precise assessment of the coupling
constants in the oligomers was impossible,
except for some well-individualized signals.
However, careful analysis of COSY spectra
revealed that the coupling constants were in
the same range as those of the lower members
of the families [6]. The proof for the anomeric
configuration of all the new interglycosidic
bonds was established by NMR, and it must be
underlined that during all the above glycosyla-
tion reactions that involved reaction of gluco
3. Experimental
General methods.—All compounds were ho-
mogeneous by TLC analysis and had spectral
properties consistent with their assigned struc-
tures. Melting points were determined in capil-
lary tubes in a Mettler apparatus, and are
uncorrected. Optical rotations were measured
with a Perkin–Elmer model 241 polarimeter at
room temperature (rt) (2293 °C). Compound
purity was checked by TLC on Silica Gel 60
F254 (E. Merck) with detection by charring
with sulfuric acid. Unless otherwise stated,
column chromatography was performed on
Silica Gel 60, 40–63 or 63–200 mm (E. Merck).
1H NMR spectra were recorded with Bruker
AC 200, AM 250, AC 300 or AM 500 instru-
ments, for solution in CDCl3 or D2O. Before
analysis in D2O, samples were passed through
a Chelex (Bio-Rad) ion-exchange column, and
lyophilized three times from D2O. Chemical
shifts are relative to external Me4Si when the
spectra were recorded in CDCl3, and to exter-
nal sodium 4,4-dimethyl-4-silapentanoate
(TSP) when the spectra were recorded in D2O.
MS analyses were performed on a ZAB-2E
instrument (Fisons). Elemental analyses were
performed on a Fisons elemental analyzer.
Acti6ation of imidates with Me3SiOTf (Pro-
cedure 1).—Me3SiOTf (0.04 M in toluene; 0.06
mol/mol of imidate) was slowly added, under
imidates at C-4 of -iduronic acid derivatives,
L
we did not isolate b-bonded products.
The six oligosaccharides 25, 26, 28, 30, 31,
and 33 were then deprotected and sulfated
using the same procedure: hydrogenolysis of
benzyl esters and ethers in N,N-dimethylform-
amide, saponification of acetyl groups by treat-
ment with 5 N aqueous sodium hydroxide in
methanol, and finally O-sulfation with sulfur
trioxide–pyridine complex in DMF at 55 °C.
The desired sulfated compounds 2–7 were thus
obtained as amorphous powders after purifica-
tion on Sephadex G-25 and lyophilization. The
yields over the three final steps varied between
49 and 86%. The structures of the final com-
1
pounds were checked by H NMR and electro
1
spray ionisation mass spectrometry. H NMR
also indicated that the compounds were be-
tween 90 and 95% pure, while capillary elec-
trophoresis [13] revealed the presence of some

94
P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 85–99
O-Sulfation: pyridine/sulfur trioxide com-
plex (5 mol/mol of hydroxyl function) was
added to a solution of the compound to be
sulfated in DMF (10 mg/mL). After 20 h
heating at 55 °C the solution was layered on top
of a Sephadex G-25 column (1.6×115 cm)
eluted with 0.2 M NaCl. The fractions contain-
ing the product were concentrated, and desalted
using the same column, equilibrated in water.
Lyophilization provided the desired com-
pound.
argon, to a stirred, cooled (−20 °C) solution
of the acceptor alcohol, and the donor imidate
,
in toluene (15 mL/mmol), containing 4 A
powdered molecular sieves (1.7 g). After 15–30
min (TLC), solid NaHCO3 was added, and the
solution was filtered, washed with water, dried
(Na2SO4), and concentrated.
Clea6age of the le6ulinyl group (Procedure
2).—A solution of hydrazine hydrate (1 M in
3:2 pyridine–AcOH) was added (5 mL/mmol)
to a cooled (0 °C) solution in pyridine (5
mL/mmol) of the compound to be delevuliny-
lated. After 15–30 min (TLC), the solution was
concentrated. The residue was dissolved in
EtOAc, washed with water, 10% aq KHSO4,
water, 2% aq NaHCO3, water, dried (Na2SO4),
and concentrated.
Crude methyl(benzyl 2,3-di-O-methyl-h-
idopyranosyluronate)-(1“4)-[(3,6-di-O-acetyl-
2-O-benzyl-h- -glucopyranosyl)-(1“4)-(benz-
yl 2,3-di-O-methyl-h- -idopyranosyluronate)-
(1“4)]2 -2,3,6-tri-O-benzyl-h- -glucopyran-
L-
D
L
D
oside (9).—Compound 13 [6] (485 mg, 229
mmol) was delevulinylated according to Proce-
dure 2. Column chromatography (10:1
CH2Cl2–acetone) gave 9 (392 mg, 85%): [h]D +
20° (c 0.8, CH2Cl2). TLC, Rf 0.38, 10:1 CH2Cl2–
Acti6ation of imidates with ButM2SiOTf
(Procedure 3).—ButMe2SiOTf (0.5 mol/mol of
imidate) was slowly added under argon to a
stirred, cooled (−20 °C) solution of the accep-
tor alcohol, and the donor imidate, in toluene
1
acetone. H NMR (500 MHz): carbohydrate
,
(35 mL/mmol) containing 4 A powdered molec-
ring protons (see Table 2); 7.15–7.30 (m, 40 H,
8 Ph); 4.50–5.45 (16 H, 8 CH2Ph); 3.41, 3.38,
3.35, 3.34, 3.33, 3.24 (6 s, 21 H, 7 OMe); 2.10,
2.05, 1.89, 1.84 (4 s, 12 H, 4 CH3C(O)).
ular sieves. After 15–30 min (TLC), solid
NaHCO3 was introduced under stirring. After
5 min, toluene was added, the solution was
filtered, washed with 2% aq NaHCO3, water,
dried, and concentrated.
(Benzyl 4-O-le6ulinyl-2,3-di-O-methyl-h-
idopyranosyluronate)-(1“4)-(3,6-di-O-acetyl-
2-O-benzyl-h- -glucopyranosyl)-(1“4)-(benz-
yl 2,3-di-O-methyl-h- -idopyranosyluronate)-
(1“4) - 3,6 - di - O - acetyl - 2 - O - benzyl - 1 - tri-
chloroacetimidoyl- -glucopyranose (11).—A
L-
Deprotection and sulfation of oligo- and
polysaccharides (Procedure 4).—Hydrogenoly-
sis of benzyl ethers and benzyl esters: a solution
of the compound (5 mg/mL), in DMF or
MeOH, was stirred for 2–6 h (TLC control)
under an hydrogen atmosphere (5 bar) in the
presence of 10% Pd/C (2×mass of compound).
After filtration, the product was directly en-
gaged in the next step. Saponification of esters:
5 M aq NaOH was added (0.5 M final concen-
tration) to a solution of the above compound
in MeOH (150 mL/mmol). After 16 h (TLC)
water was introduced, followed by Dowex 50
(H+) resin, until pH 1–2. After filtration and
concentration the residue was passed through
a Sephadex G-25 column (1.6×115 cm) eluted
with water. Lyophilization finally gave the fully
deprotected compound. At this stage complete
removal of protective groups was checked by
high-field 1H NMR. If required the compound
was again submitted to hydrogenation and/or
saponification.
D
L
D
mixture of trichloroacetonitrile (151 mL, 1.5
mmol), tetrasaccharide 17 (343 mg, 249 mmol),
and K2CO3 (62 mg, 448 mmol) in CH2Cl2 (2 mL)
was stirred for 16 h at rt. Dichloromethane was
added, and after filtration, the solution was
concentrated. Column chromatography (3:1
toluene–acetone) afforded 11 (346 mg, 91%):
1
TLC, Rf 0.63, 2:1 CH2Cl2–EtOAc. H NMR
(500 MHz): carbohydrate ring protons (see
Table 2); 8.59 (1 H, NH); 7.37–7.25 (m, 20 H,
4 Ph); 4.50–5.45 (8 H, 4 CH2Ph); 3.42, 3.40,
3.39, 3.32 (4 s, 12 H, 4 OMe); 2.18–2.60 (m, 4
H, C(O)CH2CH2C(O)); 2.10, 2.05, 1.92, 1.89,
1.84 (5 s, 15 H, 5 CH3C(O)).
(Benzyl 4-O-le6ulinyl-2,3-di-O-methyl-h-
idopyranosyluronate)-(1“4)-[(3,6-di-O-acetyl-
2-O-benzyl-h- -glucopyranosyl)-(1“4)-(benz-
yl 2,3-di-O-methyl-h- -idopyranosyluronate)-
L-
D
L

P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 85–99
95
acetyl-2-O-benzyl-
D
-glucopyranose
(22).—
(1“4)]2 -3,6-di-O-acetyl-2-O-benzyl-1-tri-
chloroacetimidoyl- -glucopyranose (12).—A
From 10 and 21: compounds 10 [6] (1.5 g, 1.7
mmol) and 21 (1.18 g, 1.7 mmol) were con-
densed according to Procedure 1 (CH2Cl2 was
used in place of toluene). Sephadex LH-20
chromatography column, equilibrated in 1:1
CH2Cl2–EtOH, yielded pure tetrasaccharide
16 (1.75 g, 73%) and hexasaccharide 22 (175
mg, 5%).
From 15: to a solution of 15 (50 mg, 0.033
mmol) in acetic anhydride (1.4 mL) at
−20 °C, was added a mixture of concentrated
sulfuric acid in acetic anhydride (285 mL, 1:10
v:v). After 1 h 30 min at−20 °C, the mixture
was slowly added to a stirred mixture of aq
NaHCO3 (2.8 g) and CH2Cl2 (10 ml). After
stirring for 16 h, the solution was decanted,
washed with water, dried (Na2SO4), filtered
and evaporated. Column chromatography (2:1
cyclohexane–acetone) yielded 16 (16 mg,
33%).
D
mixture of trichloroacetonitrile (18 mL, 174
mmol), 23 (70 mg, 34.9 mM), and Cs2CO3 (18
mg, 65 mmol) in CH2Cl2 (0.7 mL) was stirred
for 2 h at rt. The solution was diluted with
CH2Cl2, filtered and concentrated. Column
chromatography (3:2 cyclohexane–acetone
containing 0.1% Et3N) gave 12 (70 mg, 93%):
TLC, Rf 0.34, 3:2 cyclohexane–EtOAc.
LSIMS, positive mode: m/z (thioglycerol+
1
KF), 2190 [M+K]+. H NMR (200 MHz)
8.60, 8.65 (2 s, 1 H, a and b NH).
Methyl(benzyl 4-O-le6ulinyl-2,3-di-O-me-
thyl-h-
O - acetyl - 2 - O - benzyl - h -
(1“4)-(benzyl 2,3-di-O-methyl-h-
osyluronate) - (1“4)]3 - 2,3,6 - tri - O - benzyl - h-
-glucopyranoside (14).—Compounds 10 [6]
L
-idopyranosyluronate)-(1“4)-[(3,6-di-
- glucopyranosyl)-
-idopyran-
D
L
D
(332 mg, 0.373 mmol) and 9 (377 mg, 0.186
mmol) were condensed according to Proce-
dure 3 to give, after Sephadex LH-20 column
chromatography (1:1 CH2Cl2–EtOH), 14 (504
mg, 98%): [h]D+21° (c 0.20, CH2Cl2). TLC,
1
16: [h]D +19° (c 0.9, CH2Cl2). H NMR
(500 MHz): carbohydrate ring protons (see
Table 2); 7.24–7.36 (m, 20 H, 4 Ph); 4.45–
5.20 (8 H, 4 CH2Ph); 3.46, 3.35 (4 s, 12 H, 4
OMe); 2.20–2.60 (m, 4 H, C(O)CH2CH2C(O));
2.13–1.81 (18 H, 6 CH3C(O)). LSIMS, posi-
tive mode: m/z (thioglycerol+NaCl), 1441
[M+Na]+; (thioglycerol+KF), 1457 [M+
K]+. Anal. Calcd for C71H86O30: C, 60.08; H,
6.11. Found: C, 60.06; H, 6.40.
1
Rf 0.37, 3:2 cyclohexane–acetone. H NMR
(500 MHz): carbohydrate ring protons (see
Table 2); 7.25–7.35 (m, 50 H, 10 Ph); 4.50–
5.45 (20 H, 10 CH2Ph); 3.44–3.24 (9 s, 27 H,
9
OMe);
2.20–2.60
(m,
4
H,
C(O)CH2CH2C(O)); 2.12–1.86 (7 s, 21 H, 7
CH3C(O)). LSIMS, positive mode: m/z (thio-
glycerol+NaCl), 2771 [M+Na]+; (thioglyc-
erol+KF), 2787 [M+K]+.
Other method: compounds 8 [6] (326 mg,
0.23 mmol) and 11 (459 mg, 0.30 mmol) were
condensed according to Procedure 1. Column
chromatography (3:2 cyclohexane–acetone)
yielded pure octasaccharide 14 (319 mg, 49%)
and dodecasaccharide 26 (43 mg, 5%).
22: [h]D+21° (c 0.7, CH2Cl2). TLC, Rf 0.31,
3:2 cyclohexane–acetone. 1H NMR (500
MHz): carbohydrate ring protons (see Table
2); 7.22–7.36 (m, 30 H, 6 Ph); 4.45–5.20 (12
H, 6 CH2Ph); 3.44–3.34 (6 s, 18 H, 6 OMe);
2.20–2.60 (m, 4 H, C(O)CH2CH2C(O)); 2.10–
1.80 (24 H, 8 CH3C(O)). LSIMS, positive
mode: m/z (thioglycerol+NaCl), 2072 [M+
Na]+; (thioglycerol+KF), 2088 [M+K]+.
Anal. Calcd for C103H124O43: C, 60.35; H,
6.10. Found: C, 60.26; H, 6.18.
(Benzyl 4-O-le6ulinyl-2,3-di-O-methyl-h-
idopyranosyluronate)-(1“4)-(3,6-di-O-acetyl-
2-O-benzyl-h- -glucopyranosyl)-(1“4)-(benz-
yl 2,3-di-O-methyl-h- -idopyranosyluronate)-
(1“4)-1,3,6-tri-O-acetyl-2-O-benzyl- -glu-
copyranose (16) and benzyl 4-O-le6ulinyl-2,3-
di-O-methyl-h- -idopyranosyluronate)-(1“4)-
[(3,6-di-O-acetyl-2-O-benzyl-h- -glucopyran-
osyl)-(1“4)-(benzyl 2,3-di-O-methyl-h-
idopyranosyluronate) - (1“4)]2 - 1,3,6 - tri - O -
L
-
D
(Benzyl 4-O-le6ulinyl-2,3-di-O-methyl-h-
idopyranosyluronate)-(1“4)-(3,6-di-O-acetyl-
2-O-benzyl-h- -glucopyranosyl)-(1“4)-(benz-
yl 2,3-di-O-methyl-h- -idopyranosyluronate)-
(1“4)-3,6-di-O-acetyl-2-O-benzyl- -glu-
L-
L
D
D
L
L
D
D
copyranose (17).—Ethanolamine (80 mL, 1.31
mmol) was added to a solution of the tetrasac-
charide 16 (465 mg, 327 mmol) in THF (5
L
-

96
P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 85–99
mL), and the solution was stirred for 16 h at
4 °C. The solution was neutralized with HCl
(1 M, 2 mL), CH2Cl2 (20 mL) was added, and
the solution was washed with water, dried
(Na2SO4), and concentrated. Column chro-
matography (3:1 toluene–acetone) yielded 17
(326 mg, 79%): TLC, Rf 0.33, 3:1 toluene–ace-
(Benzyl 2,3-di-O-methyl-h-
uronate)-(1“4)-1,3,6-tri-O-acetyl-2-O-benz-
yl- -glucopyranose (21).—Thiourea (678 mg,
L
-idopyranosyl-
D
8.9 mmol) was added to a solution of 20 (1.71
g, 2.23 mmol) in pyridine (108 mL) and EtOH
(22 mL), and the mixture was heated at
110 °C for 30 min. After cooling and evapora-
tion, the residue was dissolved in CH2Cl2. The
solution was washed with satd aq NaHCO3,
5% aq KHSO4, dried (Na2SO4), and concen-
trated. Column chromatography (1:1, then 1:2
cyclohexane–EtOAc) afforded pure 21 (7:3
1
tone. H NMR (200 MHz): carbohydrate ring
protons (see Table 2); 7.24–7.36 (m, 20 H, 4
Ph); 4.45–5.20 (8 H, 4 CH2Ph); 3.46, 3.35 (4 s,
12 H,
4
OMe); 2.20–2.60 (m,
4
H,
C(O)CH2CH2C(O)); 2.13–1.81 (15 H, 5
CH3C(O)). LSIMS, positive mode: m/z (thio-
glycerol+NaCl), 1399 [M+Na]+; (thioglyc-
erol+KF), 1415 [M+K]+.
1
a/b, 1.17 g, 76%): H NMR (500 MHz): car-
bohydrate ring protons (see Table 2); 7.22–
7.36 (m, 10 H, 2 Ph); 4.45–5.20 (4 H, 2
CH2Ph); 3.31 (d, 1 H, JH4-OH 11.1 Hz, OH);
3.26, 3.36, 3.50 (3 s, 9 H, 3 OMe); 1.90, 1.97,
2.06, 2.08 (4 s, 12 H, 3 Ac). ESIMS positive
mode: m/z+NaCl 713 [M+Na]+;+KF 729
[M+K]+. Anal. Calcd for C34H42O15: C,
59.12; H, 6.13. Found: C, 59.20; H, 6.20.
Methyl(benzyl
methyl-h- -idopyranosyluronate)-(1“4)-2,3,6-
tri-O-benzyl-h- -glucopyranoside (19).—A so-
4-O-chloroacetyl-2,3-di-O-
L
D
lution of 18 [6] (326 mg, 0.43 mmol),
chloroacetic anhydride (103 mg, 0.6 mmol),
4-(dimethylamino)pyridine (5.3 mg, 42 mmol),
and Et3N (90 mL, 64 mmol) in CH2Cl2 (96 mL)
was stirred at rt for 30 min. Methanol (0.5
mL) was then added, the solution was diluted
with CH2Cl2, washed with water, dried
(Na2SO4), and concentrated. Column chro-
matography (2:3 then 1:2 cyclohexane–Et2O)
gave pure 19 (242 mg, 67%): [h]D+25° (c 1,
CH2Cl2); TLC, Rf 0.35, 1:2 cyclohexane–
(Benzyl 4-O-le6ulinyl-2,3-di-O-methyl-h-
idopyranosyluronate)-(1“4)-[(3,6-di-O-acetyl-
2-O-benzyl-h- -glucopyranosyl)-(1“4)-(benz-
yl 2,3-di-O-methyl-h- -idopyranosyluronate)-
(1“4)]2-3,6-di-O-acetyl-2-O-benzyl- -gluco-
L-
D
L
D
pyranose (23).—A solution of ethanolamine
(11 mL, 176 mmol) and compound 22 (90 mg,
44 mmol) in THF (0.66 mL) was stirred for 16
h at 4 °C. Ethanolamine (5.5 mL, 88 mmol)
was then added, and the mixture was left at rt
for 4 h. Dichloromethane (50 mL) was added.
The solution was washed with 0.1 M aq HCl,
water, dried (Na2SO4), and concentrated.
Column chromatography (3:2 cyclohexane–
acetone) afforded pure 23 (74 mg, 84%): TLC,
Rf 0.27, 3:2 cyclohexane–acetone. LSIMS,
positive mode: m/z (thioglycerol+NaCl),
2030 [M+Na]+; (thioglycerol+KF), 2046
[M+K]+.
1
Et2O. H NMR (200 MHz): carbohydrate ring
protons (see Table 2); 7.22–7.36 (m, 20 H, 4
Ph); 4.45–5.20 (8 H, 4 CH2Ph); 3.85 (2 H,
ClCH2C(O)); 3.26, 3.36, 3.50 (3 s, 9 H, 3
OMe).
(Benzyl 4-O-chloroacetyl-2,3-di-O-methyl-
h-
L
-idopyranosyluronate)-(1“4)-1,3,6-tri-O-
acetyl-2-O-benzyl-D-glucopyranose
(20).—
Trifluoroacetic acid (183 mL, 2.4 mmol) was
added to a solution of 19 (50 mg, 0.06 mmol)
in Ac2O (1.28 mL, 13.5 mmol) and AcOH (52
mL, 0.9 mmol). After heating at 60 °C for 4 h,
the solution was cooled to 0 °C and neutral-
ized with Et3N. After evaporation, column
chromatography of the residue (1:2 then 2:5
cyclohexane–Et2O) afforded a mixture (8:2
a/b) of anomers 20 (28 mg, 60%): TLC, Rf
Methyl(benzyl 2,3-di-O-methyl-h-
anosyluronate)-(1“4)-[(3,6-di-O-acetyl-2-O-
benzyl-h- -glucopyranosyl)-(1“4)-(benzyl 2,-
3 - di - O - methyl - h - - idopyranosyluronate)-
(1“4)]3 -2,3,6-tri-O-benzyl-h- -glucopyran-
L
-idopyr-
D
L
D
1
oside (24).—Compound 14 (275 mg, 0.1
mmol) was delevulinylated according to Pro-
cedure 2 to give 24 (265 mg, 96%): [h]D +27°
(c 0.56, CH2Cl2). TLC, Rf 0.43, 2:1 cyclohex-
0.31, 2:5 cyclohexane–Et2O. H NMR (500
MHz): carbohydrate ring protons (see Table
2); 7.22–7.36 (m, 10 H, 2 Ph); 4.45–5.20 (4 H,
2 CH2Ph); 3.85 (2 H, ClCH2C(O)); 3.26, 3.36,
3.50 (3 s, 9 H, 3 OMe); 1.90, 1.97, 2.06, 2.08 (4
s, 12 H, 3 Ac).
1
ane–Et2O. H NMR (500 MHz): carbohy-
drate ring protons (see Table 3); 7.20–7.40 (m,

P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 85–99
97
oside (27).—Compound 25 (99.7 mg, 2.95
mmol) was delevulinylated according to Proce-
dure 2. Column chromatography (5:2
toluene–acetone) gave 27 (68 mg, 70%): TLC,
50 H, 10 Ph); 4.45–5.20 (10 CH2Ph); 3.25–
3.50 (s, 27 H, 9 OMe); 1.90–2.10 (s, 18 H, 6
Ac). LSIMS, positive mode: m/z (thioglyc-
erol+NaCl), 2673.3 [M+Na]+; (thioglyc-
erol+KF), 2688.8 [M+K]+.
1
Rf 0.32, 3:1 toluene–acetone. H NMR (500
MHz): carbohydrate ring protons (see Table
3); 7.20–7.40 (m, 60 H, 12 Ph); 4.45–5.20 (12
CH2Ph); 3.25–3.50 (s, 33 H, 11 OMe); 1.90–
2.10 (s, 24 H, 8 Ac).
Methyl(benzyl 4-O-le6ulinyl-2,3-di-O-me-
thyl-h-
O - acetyl - 2 - O - benzyl - h -
(1“4)-(benzyl 2,3-di-O-methyl-h-
osyluronate)-(1“4)]4-2,3,6-tri-O-benzyl-h-
L
-idopyranosyluronate)-(1“4)-[(3,6-di-
- glucopyranosyl)-
-idopyran-
D
L
Methyl(benzyl 4-O-le6ulinyl-2,3-di-O-me-
D-
thyl-h-
O - acetyl - 2 - O - benzyl - h -
(1“4)-(benzyl 2,3-di-O-methyl-h-
osyluronate) - (1“4)]6 - 2,3,6 - tri - O - benzyl - h-
-glucopyranoside (28).—Compounds 11 (22
L
-idopyranosyluronate)-(1“4)-[(3,6-di-
- glucopyranosyl)-
-idopyran-
glucopyranoside (25).—Compounds 10 (134
mg, 151 mmol) and 24 (200 mg, 75 mmol) were
condensed as described in Procedure 3. Sep-
hadex LH-20 column chromatography (195×
3.7 cm; 1:1 CH2Cl2–EtOH) followed by silica
gel column chromatography gave pure de-
casaccharide 25 (173 mg, 68%): [h]D +21° (c
0.56, CH2Cl2). TLC, Rf 0.37, 3:1 toluene–ace-
D
L
D
mg, 14.6 mmol) and 27 (32 mg, 9.8 mmol) were
condensed as described in Procedure 1.
Column chromatography (3:2 cyclohexane–
acetone) yielded pure 28 (18 mg, 40%): [h]D +
1
tone. H NMR (500 MHz): carbohydrate ring
1
21° (c 0.3, CH2Cl2). H NMR (500 MHz):
protons (see Table 3); 7.20–7.40 (m, 60 H, 12
Ph); 4.45–5.20 (12 CH2Ph); 3.25–3.50 (s, 33
carbohydrate ring protons (see Table 3); 7.20–
7.40 (m, 80 H, 16 Ph); 4.45–5.20 (16 CH2Ph);
3.25–3.50 (s, 45 H, 15 OMe); 2.20–2.60 (m, 4
H, C(O)CH2CH2C(O)); 1.90–2.10 (s, 39 H, 13
CH3C(O)). LSIMS, positive mode: m/z (thio-
glycerol+NaCl), 4664 [M+Na]+; (thioglyc-
erol+KF), 4680 [M+K]+.
H, 11 OMe); 2.20–2.60 (m,
4
H,
C(O)CH2CH2C(O)); 1.90–2.10 (s, 27 H,
9CH3C(O)). LSIMS, positive mode: m/z (thio-
glycerol+NaCl), 3402 [M+Na]+; (thioglyc-
erol+KF), 3418 [M+K]+.
Methyl(benzyl 4-O-le6ulinyl-2,3-di-O-me-
Methyl(benzyl 2,3-di-O-methyl-h-
anosyluronate)-(1“4)-[(3,6-di-O-acetyl-2-O-
benzyl-h- -glucopyranosyl)-(1“4)-(benzyl 2,-
3 - di - O - methyl - h - - idopyranosyluronate)-
(1“4)]5 -2,3,6-tri-O-benzyl-h- -glucopyran-
L
-idopyr-
thyl-h-
O - acetyl - 2 - O - benzyl - h -
(1“4)-(benzyl 2,3-di-O-methyl-h-
osyluronate) - (1“4)]5 - 2,3,6 - tri - O - benzyl - h-
-glucopyranoside (26).—Compounds 24
L
-idopyranosyluronate)-(1“4)-[(3,6-di-
- glucopyranosyl)-
-idopyran-
D
D
L
L
D
D
oside (29).—Compound 26 (216 mg, 54 mmol)
was delevulinylated according to Procedure 2
to give 29 (199 mg, 95%): [h]D +20° (c 0.9,
CH2Cl2). TLC, Rf 0.56, 1:1 cyclohexane–ace-
(97.3 mg, 36 mmol) and 11 (83.8 mg, 55 mmol)
were condensed according to Procedure 1.
Column chromatography (2:1, 7:4 then 3:2
cyclohexane–acetone) yielded 26 (102 mg,
69%): [h]D +22° (c 0.51, CH2Cl2). TLC, Rf
1
tone; Rf 0.55, 2:1 toluene–acetone. H NMR
(500 MHz): carbohydrate ring protons (see
Table 3); 7.20–7.40 (m, 70 H, 14 Ph); 4.45–
5.20 (14 CH2Ph); 3.25–3.50 (s, 39 H, 13
OMe); 1.90–2.10 (s, 30 H, 10 Ac). LSIMS,
positive mode: m/z (thioglycerol+NaCl),
3934 [M+Na]+; (thioglycerol+KF), 3950
[M+K]+.
1
0.18, 3:2 cyclohexane–acetone. H NMR (500
MHz): carbohydrate ring protons (see Table
3); 7.20–7.40 (m, 70 H, 14 Ph); 4.45–5.20 (14
CH2Ph); 3.25–3.50 (s, 39 H, 13 OMe); 2.20–
2.60 (m, 4 H, C(O)CH2CH2C(O)); 1.90–2.10
(s, 33 H, 11 CH3C(O)). LSIMS, positive
mode: m/z (thioglycerol+NaCl), 4032 [M+
Na]+; (thioglycerol+KF), 4047 [M+K]+.
Methyl(benzyl 4-O-le6ulinyl-2,3-di-O-me-
thyl-h-
O - acetyl - 2 - O - benzyl - h -
(1“4)-(benzyl 2,3-di-O-methyl-h-
osyluronate) - (1“4)]7 - 2,3,6 - tri - O - benzyl - h-
-glucopyranoside (30).—Compounds 11 (33
mg, 21.4 mmol) and 29 (52.4 mg, 13.3 mmol)
L
-idopyranosyluronate)-(1“4)-[(3,6-di-
- glucopyranosyl)-
-idopyran-
Crude methyl(benzyl 2,3-di-O-methyl-h-
idopyranosyluronate)-(1“4)-[(3,6-di-O-acetyl-
2-O-benzyl-h- -glucopyranosyl)-(1“4)-(benz-
yl 2,3-di-O-methyl-h- -idopyranosyluronate)-
(1“4)]4 -2,3,6-tri-O-benzyl-h- -glucopyran-
L
-
D
L
D
L
D
D

98
P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 85–99
D
-glucopyranoside (33).—Compounds 11 (31
were condensed as described in Procedure 3.
Column chromatography (7:4 cyclohexane–
acetone) yielded pure 30 (43.8 mg, 62%):
[h]D+19° (c 0.5, CH2Cl2). TLC, Rf 0.36, 3:2
mg, 21.7 mmol) and 32 (94.5 mg, 18.3 mmol)
were condensed according to Procedure 1. Re-
peated column chromatography finally pro-
vided pure 33 (29.2 mg, 25%): [h]D +22° (c
0.33, CH2Cl2). TLC, Rf 0.26, 4:3 cyclohexane–
1
cyclohexane–acetone. H NMR (500 MHz):
carbohydrate ring protons (see Table 3); 7.20–
7.40 (m, 90 H, 18 Ph); 4.45–5.20 (18 CH2Ph);
3.25–3.50 (s, 51 H, 17 OMe); 2.20–2.60 (m, 4
H, C(O)CH2CH2C(O)); 1.90–2.10 (s, 45 H, 15
CH3C(O)). LSIMS, positive mode: m/z (thio-
glycerol+KF), 5310 [M+K]+.
1
acetone. H NMR (500 MHz): carbohydrate
ring protons (see Table 3); 7.20–7.40 (m, 110
H, 22 Ph); 4.45–5.20 (22 CH2Ph); 3.25–3.50
(s, 63 H, 21 OMe); 2.20–2.60 (m, 4 H,
C(O)CH2CH2C(O)); 1.90–2.10 (s, 57 H, 19
CH3C(O)).
Methyl(benzyl 4-O-le6ulinyl-2,3-di-O-me-
thyl-h-
O - acetyl - 2 - O - benzyl - h -
(1“4)-(benzyl 2,3-di-O-methyl-h-
osyluronate) - (1“4)]8 - 2,3,6 - tri - O - benzyl - h-
-glucopyranoside (31).—Compounds 29
L
-idopyranosyluronate)-(1“4)-[(3,6-di-
- glucopyranosyl)-
-idopyran-
Methyl(sodium 2,3-di-O-methyl-h-
anosyluronate)-(1“4)-[(2,3,6-tri-O-sodium sul-
fonato-h- -glucopyranosyl)-(1“4)-(sodium
2,3 - di - O - methyl - h - - idopyranosyluronate)-
(1“4)]4-2,3,6-tri-O-sodium sulfonato-h- -glu-
L
-idopyr-
D
L
D
L
D
D
(55.5 mg, 14.2 mmol) and 12 (36.7 mg, 17.1
mmol) were condensed according to Procedure
3. Column chromatography (3:1 toluene–ace-
tone) yielded pure 31 (42 mg, 50%): [h]D +20°
(c 0.5, CH2Cl2). TLC, Rf 0.52, 2:1 toluene–
copyranoside (2).—Compound 25 (64.6 mg,
1.91 mmol) was treated according to Procedure
4 to give 2 (57 mg, 83% over the three steps):
[h]D +30° (c 0.43, water). 1H NMR (500
MHz): carbohydrate ring protons (see Table
3); 3.46–3.56 (s, 33 H, 11 OMe). ESIMS,
negative mode: monoisotopic mass=3603.5,
average mass=3606.3, experimental mass=
3605.1390.9 a.m.u.
1
acetone. H NMR (500 MHz): carbohydrate
ring protons (see Table 3); 7.20–7.40 (m, 100
H, 20 Ph); 4.45–5.20 (20 CH2Ph); 3.25–3.50
(s, 57 H, 19 OMe); 2.20–2.60 (m, 4 H,
C(O)CH2CH2C(O)); 1.90–2.10 (s, 51 H, 17
CH3C(O)). LSIMS, positive mode: m/z (thio-
glycerol+KF), 5940 [M+K]+.
Methyl(sodium 2,3-di-O-methyl-h-
anosyluronate)-(1“4)-[(2,3,6-tri-O-sodium sul-
fonato-h- -glucopyranosyl)-(1“4)-(sodium
2,3 - di - O - methyl - h - - idopyranosyluronate)-
(1“4)]5-2,3,6-tri-O-sodium sulfonato-h- -glu-
L
-idopyr-
D
Crude methyl(benzyl 2,3-di-O-methyl-h-
idopyranosyluronate)-(1“4)-[(3,6-di-O-acetyl-
2-O-benzyl-h- -glucopyranosyl)-(1“4)-(benz-
yl 2,3-di-O-methyl-h- -idopyranosyluronate)-
(1“4)]7 -2,3,6-tri-O-benzyl-h- -glucopyran-
L
-
L
D
D
copyranoside (3).—Compound 26 (30 mg,
0.74 mmol) was treated according to Procedure
4 to give 3 (11.7 mg, 75% over the three steps):
[h]D +29° (c 0.42, water). 1H NMR (500
MHz): carbohydrate ring protons (see Table
3); 3.45–3.55 (s, 39 H, 13 OMe). ESIMS,
negative mode: monoisotopic mass=4297.5,
average mass=4300.7, experimental mass=
4296.890.9 a.m.u.
L
D
oside (32).—The hexadecasaccharide 30 (100
mg, 19 mmol) was delevulinylated according to
Procedure 2 to give crude 32 that was used as
such in the next step: [h]D +24° (c 0.36,
CH2Cl2). TLC, Rf 0.31, 4:3 cyclohexane–ace-
1
tone. H NMR (500 MHz): carbohydrate ring
protons (see Table 3); 7.20–7.40 (m, 90 H, 18
Ph); 4.45–5.20 (18 CH2Ph); 3.25–3.50 (s, 51
H, 17 OMe); 1.90–2.10 (s, 42 H, 14 Ac).
LSIMS, positive mode: m/z (thioglycerol+
NaCl), 5196 [M+Na]+; (thioglycerol+KF),
5212 [M+K]+.
Methyl(sodium 2,3-di-O-methyl-h-
anosyluronate)-(1“4)-[(2,3,6-tri-O-sodium sul-
fonato-h- -glucopyranosyl)-(1“4)-(sodium
2,3 - di - O - methyl - h - - idopyranosyluronate)-
(1“4)]6-2,3,6-tri-O-sodium sulfonato-h- -glu-
L
-idopyr-
D
L
D
copyranoside (4).—Compound 28 was treated
according to Procedure 4 to give 4 (49% over
Methyl(benzyl 4-O-le6ulinyl-2,3-di-O-me-
1
thyl-h-
O - acetyl - 2 - O - benzyl - h -
(1“4)-(benzyl 2,3-di-O-methyl-h-
osyluronate) - (1“4)]9 - 2,3,6 - tri - O - benzyl - h-
L
-idopyranosyluronate)-(1“4)-[(3,6-di-
- glucopyranosyl)-
-idopyran-
the three steps): [h]D +27° (c 0.6, water). H
D
NMR (500 MHz): carbohydrate ring protons
(see Table 3); 3.43–3.55 (s, 45 H, 15 OMe).
ESIMS, negative mode: monoisotopic mass=
L

P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 85–99
99
4991.4, average mass=4995.2, experimental
mass=4993.092.2 a.m.u.
and Sanofi Recherche on antithrombotic
oligosaccharides. The authors thank J.-P. He´r-
ault for the determination of the biological
activities. They also thank the Sanofi
Recherche Service d’Analyse de Recherche
staff (C. Picard, Head) for elemental, HPLC,
and capillary electrophoresis analyses (M.
Maftouh, S. Albugues); NMR analyses (C.
Ponthus, D. Albene, M. Rival); and MS
analyses (F. Uzabiaga, V. Videau).
Methyl(sodium 2,3-di-O-methyl-h-
anosyluronate)-(1“4)-[(2,3,6-tri-O-sodium sul-
fonato-h- -glucopyranosyl)-(1“4)-(sodium
2,3 - di - O - methyl - h - - idopyranosyluronate)-
(1“4)]7-2,3,6-tri-O-sodium sulfonato-h- -glu-
L
-idopyr-
D
L
D
copyranoside (5).—Compound 30 (37.9 mg,
0.71 mmol) was treated according to Procedure
4 to give 5 (30.2 mg, 74% over the three steps):
[h]D+34° (c 0.55, water). 1H NMR (500
MHz): carbohydrate ring protons (see Table
3); 3.45–3.55 (s, 51 H, 17 OMe). ESIMS,
negative mode: monoisotopic mass=5685.3,
average mass=5689.6, experimental mass=
5687.692.3 a.m.u.
References
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[12] G. Grynkiewicz, I. Fokt, W. Szeja, H. Fitak, J. Chem.
Res. (S), (1989) 152–153.
Methyl(sodium 2,3-di-O-methyl-h-
anosyluronate)-(1“4)-[(2,3,6-tri-O-sodium sul-
fonato-h- -glucopyranosyl)-(1“4)-(sodium
2,3 - di - O - methyl - h - - idopyranosyluronate)-
(1“4)]8-2,3,6-tri-O-sodium sulfonato-h- -glu-
L
-idopyr-
D
L
D
copyranoside (6).—Compound 31 (35.2 mg,
0.59 mmol) was treated according to Procedure
4 to give 6 (27.9 mg, 73% over the three steps):
[h]D+27° (c 0.4, water). 1H NMR (500 MHz):
carbohydrate ring protons (see Table 3); 3.44–
3.54 (s, 57 H, 19 OMe). ESIMS, negative
mode: monoisotopic mass=6379.2, average
mass=6384.1, experimental mass=6381.49
3.2 a.m.u.
Methyl(sodium 2,3-di-O-methyl-h-
anosyluronate)-(1“4)-[(2,3,6-tri-O-sodium sul-
fonato-h- -glucopyranosyl)-(1“4)-(sodium
2,3 - di - O - methyl - h - - idopyranosyluronate)-
(1“4)]9-2,3,6-tri-O-sodium sulfonato-h- -glu-
L
-idopyr-
D
L
D
copyranoside (7).—Compound 33 (25.4 mg,
0.38 mmol) was treated according to Procedure
4 to give 7 (16 mg, 60% over the three steps):
[h]D+27° (c 0.4, water). 1H NMR (500 MHz):
carbohydrate ring protons (see Table 3); 3.45–
3.55 (s, 63 H, 21 OMe). ESIMS, negative
mode: monoisotopic mass=7073.1, average
mass=7078.5, experimental mass=7077.39
3.2 a.m.u.
[13] M. Maftouh, C. Azalbert, Abstr., 12th Int. Symp. High
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(1997) 14683–14688.
This work is part of a collaboration be-
tween N.V. Organon (Oss, The Netherlands),
.