Identification of a hexasaccharide sequence able to inhibit thrombin and suitable for 'polymerisation'

Article abstract of DOI:10.1016/S0008-6215(99)00067-1

Three hexasaccharides, having from low to very high affinity for antithrombin, were synthesised from disaccharide building block precursors. One of them, 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)]₂-2,3,6-tri-O-sodium sulfonato-α-D-glucopyranoside, obtainable from a single disaccharide building block precursor, constitutes a good starting point for obtaining simple oligosaccharidic heparin mimetics able to inhibit the two coagulation factors thrombin and Factor Xa. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(99)00067-1

www.elsevier.nl/locate/carres
Carbohydrate Research 317 (1999) 63–84
Identification of a hexasaccharide sequence able to
inhibit thrombin and suitable for ‘polymerisation’ꢀ
Philippe Duchaussoy a, Guy Jaurand b, Pierre-A. Driguez a, Isidore Lederman a,
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
Three hexasaccharides, having from low to very high affinity for antithrombin, were synthesised from disaccharide
building block precursors. One of them, methyl(sodium 2,3-di-O-methyl-4-O-sodium sulfonato-a- -idopyranosy-
luronate)-(1“4)-[(2,3,6-tri-O-sodium sulfonato-a- -glucopyranosyl)-(1“4)-(sodium 2,3-di-O-methyl-a- -idopyr-
anosyluronate)-(1“4)]2-2,3,6-tri-O-sodium sulfonato-a- -glucopyranoside, obtainable from a single disaccharide
L
D
L
D
building block precursor, constitutes a good starting point for obtaining simple oligosaccharidic heparin mimetics
able to inhibit the two coagulation factors thrombin and Factor Xa. © 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Heparin; Heparin mimetics; Antithrombin; Factor Xa; Thrombin
binding domain of heparin (Fig. 1). Binding of
1. Introduction
this pentasaccharide to antithrombin induces
a conformational change, thus allowing inhi-
bition of Factor Xa [1]. Thrombin inhibition
by heparin–antithrombin occurs through a
different mechanism where the two proteins
interact at the surface of a heparin molecule
serving as a template [4]. In such a molecule,
the pentasaccharide sequence is elongated at
both ends, mainly by repeated “4)-2-O-sul-
The anticoagulant activity of heparin
reflects its ability to reinforce by several orders
of magnitude the reaction rate of thrombin
and coagulation Factor Xa with their physio-
logical inhibitor antithrombin [1]. Following
experiments on heparin fragments indicating
that thrombin inhibition was size-dependent,
whereas Factor Xa inhibition was not [2], the
search for the smallest heparin fragment able
to catalyse Factor Xa inhibition led to the
pentasaccharide sequence DEFGH [3], which
was finally identified as the antithrombin
fonato-a-
L
-idopyranosyluronate-(1“4)-N-
-glucosaminyl-
sulfonato-6-O-sulfonato-a-
D
(1“ disaccharide sequences that play a cru-
cial role, in so far as they constitute thrombin
binding domains (Fig. 1). Thrombin interac-
tion with heparin results from electrostatic
interactions of the negatively charged polysac-
charide with the anion-binding exosite II of
thrombin.
ꢀ Part 1 of the series: ‘Design and synthesis of heparin
mimetics able to inhibit thrombin’.
* Corresponding author.
0008-6215/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(99)00067-1

64
P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 63–84
Fig. 1. Structure of an anticoagulant heparin molecule. The pentasaccharide sequence DEFGH constitutes the antithrombin-bind-
ing domain. It is prolonged at both ends by thrombin binding domains (T-domains). Bound antithrombin undergoes a
conformational change allowing Factor Xa inhibition. To be inhibited, thrombin is first attracted by the thrombin binding
domains and collides with activated (heparin bound) antithrombin.
In our program devoted to the synthesis of
new antithrombotic oligosaccharides, we
wished to obtain, in a simple way, heparin-
mimetic oligosaccharides displaying this dual
anti-Factor Xa/anti-Factor IIa activity [5]. We
reasoned that the antithrombin binding do-
main, which contains sulfate groups, may elec-
trostatically attract thrombin, and thus serve
as a thrombin-binding domain as well. Thus, a
continuum of antithrombin binding domains
should be able to inhibit thrombin as well as
Factor Xa, as soon as the size of the fragment
is long enough to accommodate both an-
tithrombin and thrombin. The advantage of
this approach is that it circumvents the prob-
lem of the relative position of the two do-
mains (in most heparin molecules the
antithrombin-binding domain is prolonged at
both ends by sequences used as thrombin-
binding domains, yet only one of them is
involved in thrombin inhibition). In order to
simplify the chemistry, with a view to drug
development, our first objective was to iden-
tify a highly symmetrical antithrombin bind-
charide having affinity for antithrombin, we
started from the high affinity pentasaccharide
1 [6] (Scheme 1). The idea was to ‘symmetrise’
the structure while keeping the affinity in a
range compatible with our final goal i.e., not
too weak to observe significant Factor Xa
inhibition, and not too high to allow thrombin
to compete with antithrombin for binding (the
affinity of thrombin for heparin is in the mM
range). We decided first to synthesise the three
hexasaccharides 2–4 (Fig. 2 and Scheme 1)
obtainable from a reduced number of disac-
charide building blocks. Hexasaccharide 2 was
selected to assess the influence of introducing
a trisulfated glucose unit at the non-reducing
end of 1. The next step was to impose one
type of uronic acid only: hexasaccharide 3,
containing only 2,3-di-O-methyl-
acid b-linked to per-O-sulfated -glucose, and
hexasaccharide 4, the analogue where b- -glu-
curonic acid has been replaced by a-
D
-glucuronic
D
D
L
-
iduronic acid, were thus selected. The three
hexasaccharides were prepared using similar
strategies (Scheme 1) based on prior prepara-
tion of disaccharide building blocks that were
then coupled to each other, the resulting hex-
asaccharides being further deprotected and O-
sulfonated to give the final compounds.
ing domain obtainable from
a
single
disaccharide synthon. Polymerisation of this
synthon should deliver fragments able to in-
hibit thrombin and Factor Xa. In the present
article, we report on the selection of such a
suitable antithrombin binding domain. The
following article will be devoted to the prepa-
ration of longer fragments.
Hexasaccharide 2.—The synthesis of 2 pro-
ceeds through the fully protected hexasaccha-
ride 23 (see Scheme 3), obtainable from the
new disaccharide building block 19 and the
known compound 20 [7]. The glycosyl accep-
tor 19 was prepared first (for a more efficient
preparation, on many occasions the crude in-
termediates were directly engaged in the next
step. However, samples of the crude mixtures
were submitted to column chromatography to
allow characterisation of the compound).
2. Results and discussion
Aware of the constraints imposed by the
specific recognition of antithrombin by hep-
arin to design a highly symmetrical oligosac-

P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 63–84
65
Scheme 1. ‘Symmetrisation’ of the high-affinity binding DEFGH sequence. Replacement of the D unit by a third trisulfated
glucose residue was tried first (Fig. 2, compound 2). Then two hexasaccharides containing only glucuronic acid (Fig. 2, compound
3) or iduronic acid (Fig. 2, compound 4) were prepared. The latter can be prepared from a single disaccharide building block.
4
4
Acidic treatment of 5,6-anhydro-1,2-O-iso-
3J-interproton couplings, JH-2,H-4 0.8 Hz, JH-
1
propylidene-3-O-methyl- -idopyranose (5) [8]
L
1,H-3 a: 1.2 Hz, b: 0.6 Hz, JC-1,H-1 a: 162.2
resulted in hydrolysis of the isopropylidene
Hz, b: 154.6 Hz, H-3, H-5 coupling 50.5
Hz). The 4,6-diol system of 9 was then pro-
tected by treatment with 2,2-dimethoxypro-
pane, in the presence of camphorsulfonic acid
to give 10. At this stage column chromatogra-
phy was used, followed by crystallisation (cy-
clohexane), to obtain a mixture containing
exclusively the two anomers of 10 with an
overall yield of 24% from 5. 1H NMR analysis
of 10a and 10b (both are crystalline com-
pounds) gave similar results to those for 8 and
9 (see Table 2). The hydroxyl at position two
was then benzoylated to furnish a mixture of
the two crystalline glycosyl donors 11a and
11b in 90% yield. Once again, 1H NMR analy-
sis of 11a and 11b gave similar results as those
for 8 and 9 (see Table 2).
and opening of the epoxide, giving 3-O-
methyl- -idopyranose (6), which was directly
L
acetylated to give a mixture of the anomeric
acetates 7. Treatment of 7 with ethanethiol
and boron trifluoride gave the ethyl thiogly-
cosides 8. A pure fraction of both anomers, 8a
and 8b, was prepared by column chromatog-
1
raphy with a view to H NMR analysis. In
3
each case, the small J-interproton coupling
constants observed (see Table 2) proved the
1C4 conformation of the ring, which was confi-
4
rmed by observation of long-range JH-2,H-4
coupling (0.8 Hz). Configuration of the
anomeric carbon was assigned on the basis of
4
the chemical shifts and of the JH-1,H-3 cou-
pling constants (1.2 Hz for the a anomer, 0.8
Hz for the b anomer), and confirmed by mea-
1
Condensation of 11 and methyl 2,3,6-tri-O-
benzoyl-a- -glucopyranoside (12) [9], in
surement of the JC-1,H-1 heteronuclear cou-
D
pling constants (164.7 Hz for the a anomer,
151.4 Hz for the b anomer). A small (50.5
Hz) H-3, H-5 coupling could also be observed
for both anomers. Deacetylation of 8 gave
crude 9. The two anomers were again sepa-
toluene, using N-iodosuccinimide (NIS) and
trifluoromethanesulfonic acid (TfOH) [10],
gave the disaccharide 13, isolated in excellent
(93%) yield after column chromatography
(Scheme 2). No b-coupled product could be
1
rated and H NMR analysis gave the same
1
results as for the acetylated parents (small
detected in this reaction. H NMR analysis

66
P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 63–84
Fig. 2. The hexasaccharides 2–4.
1
conformation of the iduronic acid ring and the
confirmed the C4 conformation of the idose
configuration of the
ring (4J2,4) and a-
anomeric carbon through observation of the
usual J1,3 coupling (1 Hz). The next step
L
already proven a-
anomeric carbon.
L
configuration of the
4
Condensation of the acceptor 19 and the
imidate 20 [7] under classical conditions, at
−20 °C, in dichloromethane, using trimethyl-
silyl trifluoromethanesulfonate (Me3SiOTf) as
promoter [13], gave the tetrasaccharide 21 in
62% yield (Scheme 3). The small coupling
constant (J1,2 3.6 Hz) observed for the
anomeric proton of the unit engaged in the
new glycosidic bond proved the a configura-
tion. Delevulinylation as described for 19
yielded the acceptor tetrasaccharide 22 (87%).
Delevulinylation of 21 resulted in an upfield
shift from 5.02 to 3.68 of the H-4%%% proton,
which showed a 2.8 Hz coupling with the
hydroxylic proton. Glycosylation of 22 with
20 gave the protected hexasaccharide 23 (39%)
toward 19 was to convert the idose derivative
into an iduronic acid derivative. To this end,
the 4,6-O-isopropylidene protecting group
was hydrolysed first, with aqueous acetic acid
at 50 °C, to give the diol 14 which was not
purified. A tert-butyldimethylsilyl ether and a
levulinyl ester were successively introduced in
a one-pot procedure at position six and four,
respectively, to provide 16. Under Jones’ oxi-
dation conditions, the alcohol at position six
was oxidised without prior removal of the silyl
ether [11], to give the acid 17 which was
esterified with benzyl bromide in N,N-
dimethylformamide in the presence of potas-
sium hydrogen carbonate to give 18.
Delevulinylation with hydrazine hydrate in a
mixture of pyridine–acetic acid at 0 °C [12]
gave the glycosyl acceptor 19. At this stage, it
was necessary, for the third time in the se-
quence, to run a column chromatography to
purify 19, which was obtained in 75% overall
yield from 13. Observation of the long-range
together with some b-
D
anomer (8%), and
1
some recovered glycosyl acceptor (18%). H
NMR analysis of the anomeric proton of the
new interglycosidic bond (J1,2 3.5 Hz) estab-
lished its a configuration, and allowed us to
identify the b anomer (l H-1: 4.30 ppm, J1,2
7.7 Hz). Compared to the synthesis of 21, the
yield in 23 was low, although the reaction
conditions were similar except for the use of
4
4
1
couplings J1,3 and J2,4 on the H NMR
COSY spectrum of 19 confirmed both the C4
1

P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 63–84
67
Scheme 2. Reagents and conditions: (a–e) aq H2SO4, 60 °C; then Ac2O, pyridine; then EtSH, BF3·Et2O, toluene; then MeONa,
MeOH/CH2Cl2; then (CH3)2C(OCH3)2, CSA, 24% from 5; (f) BzCl, DMAP, pyridine, 90%; (g) NIS, TfOH, toluene, −20 °C,
93%; (h–m) 70% aq AcOH, 50 °C; then tert-BDMSCl, DMAP, Et3N, CH2Cl2; then Lev2O, DMAP, Et3N; then aq H2SO4, CrO3,
acetone; then BnBr, KHCO3, DMF; then NH2NH2:H2O, AcOH, pyridine, 0 °C, 75% from 13.
furnish 25 (Scheme 4), which was not isolated.
The benzylidene group was then hydrolysed,
using p-toluenesulfonic acid in a water–
methanol mixture to give, after column chro-
matography, the diol 26 in 81% yield from 24.
1H NMR analysis confirmed the structure of
26, particularly the presence of the diol pro-
tons observed at 2.00 ppm (OH-6) and 2.64
ppm (OH-4). Conversion into the correspond-
ing uronic acid derivative 30 was effected as
above: protection of positions four and six
(27, 28), followed by Jones oxidation, dir-
ectly on the crude residue, and esterification
of the acid 29 followed by partial purifica-
tion. Anomeric deallylation was realised in
tetrahydrofuran, using 1,5-cyclooctadiene-
bis[methyldiphenylphosphine]-iridium hexaflu-
orophosphate under H2 atmosphere [15] to
toluene instead of dichloromethane. Prior pi-
lot experiments showed, based on thin layer
chromatography analysis, that this change
was advantageous. The hexasaccharide 23 was
submitted to catalytic hydrogenation in
dichloromethane–methanol, acyl groups were
then saponified using aqueous sodium hydrox-
ide in methanol, and finally the resulting
polyol was sulfated at 55 °C with pyridine–
sulfur trioxide complex in N,N-dimethylform-
amide to generate 2 in 89% yield over the last
three steps. The structure of 2 was confirmed
1
by 500 MHz H NMR (Table 2), which also
allowed its purity (:90%) to be assessed.
1
Two-dimensional H NMR experiments indi-
cated that the impurities were several minor
undersulfated compounds, the structure of
which was not investigated.
Hexasaccharide 3.—The synthesis of 3 pro-
ceeds through the key hexasaccharide 38
(Scheme 5), the synthesis of which can be
achieved from the already used glycosyl donor
20 and the glycosyl acceptor 35. The synthesis
of the latter started from allyl 4,6-O-benzyli-
Table 1
Affinities of compounds 1–4 for antithrombin
Compound
Kd for antithrombin (mM)
1
2
3
4
0.001990.0001 [21]
0.001490.0002
3.490.3
dene-a- -glucopyranoside (24) [14] that was
first methylated in N,N-dimethylformamide
D
0.3590.01
with methyl iodide and sodium hydride to

68
P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 63–84
Table 2
1H NMR data: carbohydrate ring proton chemical shifts (ppm) and coupling constants (Hz)
lH-1
J1,2
lH-2
J2,3
lH-3
J3,4
lH-4
J4,5
lH-5
J5,6-5,6%
lH-6
J6,6%
lH-6%
4.24
4.18
4.18
3.99
3.94
3.88
3.97
3.93
4.03
7a
6.00
1.7
5.00
3.2
3.64
3.2
4.82
1.5
4.29
4.24
8a
5.26
1.6
4.94
2.6
3.49
2.6
4.82
1.6
4.75
7.5, 5.4
4.20
−11.4
8b
4.95
1.7
4.94
2.6
3.57
2.6
4.81
1.5
4.12
7.5, 5.7
4.22
−11.4
9a
5.30
1.5
3.90
3.2
3.48
3.2
4.02
1.8
4.31
3.5, 3.0
4.03
−12.0
9b
4.92
1.3
3.80
3.0
3.60
3.0
3.91
1.2
3.74
3.5, 4.5
3.98
−12.0
10a
10b
11a
11b
13
5.31
1.5
3.86
2.9
3.39
2.9
4.00
1.0
4.10
2.2, 2.0
4.08
−13.0
4.84
1.0
3.74
2.9
3.46
2.9
3.95
1.0
3.52
2.2, 1.5
4.01
−12.8
5.47
1.5
5.22
2.7
3.48
2.5
3.97
1.5
4.16
2.2, 2.3
4.15
−13.5
5.00
1.5
5.12
2.6
3.59
2.4
3.88
2.0
3.55
2.3, 1.5
4.08
−12.9
IdoII
GlcI
5.08
1.5
5.17
3.5
5.95
3–4
5.15
9.8
3.41
3–4
6.00
9.0
3.79
2–3
4.12
10.0
3.76
–, 2.3
4.16
3.40
3.15
4.53
4.78
−11.0
1.5, 3.3
19
21
IdoAII
GlcI
5.24
2.0
5.13
3.6
5.12
3.5
5.11
9.6
3.59
3.5
6.04
9.3
3.86
2.6
4.20
9.9
4.64
4.21
1.5, 3.5
4.83
−12.0
4.60
GlcAIV
GlcIII
IdoAII
GlcI
4.13
7.8
4.97
3.6
5.50
6.1
ꢀ5.1
3.6
2.93
8.9
3.37
9.6
5.08
7–8
5.09
9.5
3.19
9.1
5.33
9.3
3.75
7–8
5.96
9.5
5.02
10
3.56
9.7
3.77
ꢀ5
4.00
9.8
3.78
3.81
1–4
4.00
4.19
4.58
4.19
4.46
4.05
1–4
22
GlcAIV
GlcIII
IdoAII
GlcI
4.13
7.9
4.96
3.6
5.46
6.1
ꢀ5.05
3.6
2.86
9.0
3.36
10.0
5.07
7.7
3.05
9.0
5.32
9.3
3.71
7.7
5.95
9.3
3.68
9.7
3.58
10.1
3.76
5.8
3.72
3.82
2.0, 3.3
3.93
4.18
−12.3
4.14
4.44
5.08
10.2
3.98
9.9
4.05
1.7, 4.6
4.56
−12.1

P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 63–84
69
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%
23
GlcAVI
GlcV
4.19
7.9
5.41
3.6
4.07
7.9
4.92
3.6
5.46
6.1
5.05
3.6
2.94
9.0
3.44
10.2
2.88
9.0
3.33
10.2
5.06
7.7
5.08
10.2
3.21
9.0
5.39
9.3
3.25
9.0
5.28
9.3
3.71
7.7
5.94
9.3
4.97
10.0
3.64
10.1
3.92
9.7
3.53
10.1
3.74
5.8
3.80
3.74
1.9, 3.0
3.82
4.61
−12.3
4.25
4.07
4.43
GlcAIV
GlcIII
IdoAII
GlcI
3.78
2.1, 3.0
3.89
4.18
−12.3
3.98
9.9
4.04
1.7, 4.6
4.58
−12.1
2
GlcAVI
GlcV
4.71
7.7
5.59
3.6
4.69
7.7
5.54
3.6
5.17
4.7
5.17
3.6
3.30
9.2
4.32
10.0
3.23
9.4
4.38
10.0
4.45
9.1
4.38
10.0
3.49
9.0
4.64
9.3
3.61
9.0
4.57
9.3
3.82
4.5
4.63
9.3
4.43
9.7
4.02
9.9
3.95
9.7
4.02
9.9
4.30
3.6
4.06
9.9
3.81
4.16
1.7, 1.9
3.81
4.41
−11.0
4.30
4.35
GlcAIV
GlcIII
IdoAII
GlcI
4.32
3.5, 1.5
4.88
4.44
−11.0
4.03
3.5, 2.0
4.51
−11.6
4.39
3.79
26
5.00
3.6
3.21
10.1
3.49
9.3
3.50
9.7
3.68
3.9, 4.6
3.83
−11.7
31a
31b
32a
32b
34
5.39
3.5
3.30
ꢀ9.5
3.64
ꢀ9.5
5.03
10.0
4.49
3.97
4.40
4.10
3.60
4.64
7.0
3.11
ꢀ9.5
3.28
ꢀ9.5
5.05
10.0
6.62
3.4
3.50
9.5
3.70
9.5
5.12
10.3
5.78
7.1
3.43
ꢀ9.5
3.92
ꢀ9.5
5.20
ꢀ10.0
GlcAII
GlcI
4.36
7.9
4.55
3.6
2.99
9.2
3.50
9.9
3.04
9.0
3.90
9.2
5.00
10.0
3.90
9.9
3.70
3.5, 1.7
3.90
−10.9
3.70
3.69
4.27
35
36
GlcAII
GlcI
4.39
7.9
4.59
3.6
2.93
9.2
3.50
9.7
2.94
9.0
3.87
9.2
3.69
10.0
3.92
10.0
3.52
3.72
3.5, 1.7
3.89
−10.9
GlcAIV
GlcIII
4.21
7.8
5.36
3.6
4.24
7.9
2.98
9.0
3.43
9.5
2.90
9.0
3.22
9.3
5.45
9.5
3.01
9.0
4.98
10.0
3.65
9.5
3.92
9.0
3.81
3.80
–, 3.0
3.52
9.6
4.56
GlcAII

70
P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 63–84
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%
GlcI
4.57
7.8
3.46
10.0
3.80
9.5
3.86
10.0
3.66
1–4
3.88
3.66
37
38
GlcAIV
GlcIII
GlcAII
GlcI
4.24
7.8
5.39
3.6
4.28
7.8
4.61
3.5
2.94
9.0
3.47
9.7
2.94
9.0
3.48
9.8
3.11
9.0
5.47
9.7
3.05
9.0
3.83
9.3
3.79
3.69
3.96
3.89
3.77
3.83
3.57
3.90
4.60
3.92
4.29
3.68
GlcAVI
GlcV
4.17
7.8
5.39
3.8
4.20
7.8
5.33
3.6
4.14
7.8
4.56
ꢀ3.5
2.94
9.2
3.42
9–10
2.89
9.2
3.40
9–10
2.92
9.2
3.45
9–10
3.19
9.2
5.37
9–10
2.98
9.2
5.40
9–10
3.27
9.2
3.78
9–10
4.96
9.9
3.62
9–10
3.92
9.7
3.60
9–10
3.90
9.7
3.84
9–10
3.79
3.72
1–4
3.49
4.59
4.57
3.89
4.24
4.19
3.86
GlcAIV
GlcIII
GlcAII
GlcI
3.75
1–4
3.84
3.63
3.81
3
GlcAVI
GlcV
4.71
7.8
5.60
3.6
4.69
7.7
5.58
3.6
4.67
7.7
5.19
3.6
3.30
9.4
4.32
9.8
3.28
9.4
4.33
10.0
3.29
9.3
3.49
9.0
4.63
9.3
3.60
9.0
4.61
9.3
3.61
9.0
4.66
9.3
4.43
9.7
4.02
10.1
3.95
9.7
4.02
10.1
3.94
9.7
4.02
9.9
4.16
2.1, 1.9
3.81
4.41
−11.2
4.30
4.30
4.38
GlcAIV
GlcIII
GlcAII
GlcI
4.15
2.1, 1.9
3.80
4.41
−11.2
4.41
10.0
4.13
2.0, 4.6
4.44
−11.3
39
40
46
47
IdoII
GlcI
5.06
2.2
4.59
3.6
5.09
4.2
3.58
9.8
3.40
3.1
3.83
9.4
3.73
2.1
3.88
9.9
3.88
2.3, 2.8
3.73
3.43
−13.0
3.65
3.07
3.58
3.7, 2.6
−11.0
IdoII
GlcI
4.94
2.0
4.60
3.7
3.59
3.5
3.58
9.6
3.32
3.5
3.80
9.3
3.71
1.5
3.85
9.9
3.85
2.2, 2.8
3.75
3.39
−12.8
3.64
3.06
3.63
3.3, 3.0
−12.3
IdoAII
GlcI
5.21
3.6
4.57
3.6
3.06
5.5
3.53
9.8
3.52
4.6
3.85
9.1
5.00
3.7
3.92
9.8
4.85
3.78
1–4
3.70
IdoAII
GlcI
5.18
1.5
4.55
3.7
3.19
3.3
3.53
9.6
3.50
3.3
3.85
9.3
3.94
2.0
3.88
9.9
4.88
3.76
4.0, 2.6
3.68
−11.1
3.64

P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 63–84
71
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%
48
49
IdoAII
GlcIa
GlcIb
4.94
4.7
6.30
3.6
5.62
7.6
3.04
5.1
3.57
9.8
3.50
ꢀ10.0
3.46
5.2
5.39
9.6
5.22
ꢀ9.0
5.04
4.4
3.75
9.8
3.77
ꢀ10.0
4.71
3.97
1–4
4.70
1–4
4.34
4.20
4.27
4.40
IdoAII(a)
IdoAII(b)
GlcIa
4.98
4–5
4.97
4–5
5.21
3.5
3.06
5–6
3.05
5–6
3.49
9.5
3.29
9–10
ꢀ3.48
5–6
ꢀ3.48
5–6
5.40
9.5
5.18
9–10
ꢀ5.02
4–5
ꢀ5.02
4–5
3.72
10.0
4.72
4.69
4.17
1–4
3.62
1–4
4.40
4.28
4.47
4.22
GlcIb
4.79
8.0
3.74
9–10
50
51
52
IdoAII(a)
IdoAII(b)
GlcIa
4.98
4–5
4.98
4–5
6.46
3.6
3.07
5–6
3.06
5–6
3.65
9.5
3.66
9–10
ꢀ3.48
5–6
ꢀ3.48
5–6
5.49
9.5
5.23
9–10
ꢀ5.03
4–5
ꢀ5.02
4–5
3.81
10.0
4.72
4.69
4.09
1–4
3.81
1–4
4.41
4.44
4.27
4.28
GlcIb
5.85
7.3
3.91
9–10
IdoAIV
GlcIII
IdoAII
GlcI
4.90
5–6
5.14
3.6
5.27
3.6
2.91
ꢀ7
3.42
9–10
2.94
ꢀ6
3.42
ꢀ7
5.34
9–10
3.68
ꢀ6
4.98
4–5
3.66
9–10
3.81
4–5
4.66
4.02
1–4
4.46
4.24
4.24
4.56
3–4
3.48
9–10
3.80
9–10
IdoAIV
GlcIII
IdoAII
GlcI
4.86
2–3
5.10
3.7
5.19
6.2
3.21
3–4
3.38
9–10
2.94
7.5
3.47
3–4
5.36
9–10
3.62
7.5
3.92
2–3
3.66
9–10
3.79
4.5
4.68
4.04
1–4
4.48
4.22
3.72
4.16
3.67
4.55
3.5
3.45
9–10
3.82
9–10
3.82
9–10
3.70
1–4
53
IdoAVI
GlcV
4.90
4.3
5.10
3.9
4.89
6.8
5.12
3.9
5.23
6.5
4.56
3.5
2.95
5–6
3.40
9.8
2.80
7–8
3.36
9.9
2.91
8.4
3.43
9–10
3.43
5–6
5.32
9.6
3.51
7–8
5.30
9.5
3.64
8.4
3.80
9–10
4.97
4.65
3.62
10.0
3.81
5.7
3.51
10.0
3.78
5.8
4.02
1–4
4.47
4.36
4.33
3.72
4.21
4.13
3.68
IdoAIV
GlcIII
IdoAII
GlcI
3.97
1–4
4.42
3.78
9–10
3.69
1–4

72
P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 63–84
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%
4
IdoAVI
GlcV
5.07
2.2
5.43
3.6
5.08
3.7
5.41
3.6
5.08
2.8
5.17
3.6
3.51
4.0
4.31
10.0
3.51
5.5
4.32
10.0
3.51
4.9
4.37
9.8
4.03
3.0
4.58
9.3
3.75
3.3
4.57
9.4
3.78
3.3
4.69
9.4
4.79
2.2
3.95
9.9
4.22
2.8
3.96
10.1
4.19
2.2
4.97
4.21
2.1, 2.3
4.89
4.29
−11.5
4.25
4.29
4.26
IdoAIV
GlcIII
IdoAII
GlcI
4.16
2.1, 1.9
4.75
4.30
−11.5
3.95
9.9
4.12
2.3, 5.1
4.40
−11.3
isomerise the allyl group, followed by treat-
ment with a mixture of mercuric oxide–mer-
curic chloride in acetone–water. The
hemiacetal 31 (7:3 a/b ratio) was thus isolated
after column chromatography (30% from 26).
A small portion of the pure a and b anomers
was isolated for NMR analysis (Table 2).
Treatment of 31 with trichloroacetonitrile in
the presence of potassium carbonate [16] pro-
vided the imidate 32 (93%). Again, a small
portion of the pure a and b anomers was
isolated for NMR analyses, which were in
agreement with the expected structures (Table
2). Coupling of 31 and methyl 2,3,6-tri-O-ben-
NMR analysis (Table 2). Some b-
D
anomer (l
H-1: 4.32 ppm, J1,2 7.6 Hz) was also isolated
(7%). Deprotection and O-sulfonation, similar
to that used for 2, furnished 3 as an amor-
phous powder after freeze-drying (94% from
38). The structure of 3 was confirmed by 500
1
MHz H NMR (Table 2), which also allowed
its purity (:95%) to be assessed. As for 2,
1
two-dimensional H NMR experiments indi-
cated that the impurities were several minor
undersulfated compounds, the structure of
which was not investigated.
Hexasaccharide 4.—The fully protected
hexasaccharide 53 is a key intermediate in the
synthesis of 4 (Scheme 6). Its preparation was
based on condensation of the glycosyl donor
disaccharide 50 and the glycosyl acceptor 47,
both derived (Scheme 7) from the pivotal syn-
thon 46. The synthesis of 46 started by con-
densing the thioglycoside 11 and the acceptor
33 [17], as described for the synthesis of 13, to
zyl-a- -glucopyranoside (33) [17] furnished
D
the disaccharide 34 in 52% yield after column
1
chromatography. High-field H NMR analysis
proved that the major compound was the
expected b- anomer (J1,2 7.5 Hz). Purification
D
on silica gel followed by delevulinylation
1
(confirmed, using H NMR, by an upfield
1
yield the disaccharide 39. H NMR analysis of
shift from 5.00 to 3.69 ppm of the H-4% pro-
ton) then gave the acceptor 35 (95%).
Condensation of 35 and 20 (−20 °C,
dichloromethane, Me3SiOTf) provided the te-
trasaccharide 36 in 69% yield (Scheme 5). The
a purified sample confirmed the 1C4 conforma-
tion of the idose ring (4J2,4 0.8 Hz), and the
a-
L
configuration of the anomeric carbon
(4J1,3 1 Hz). Crude 39 was used as such in the
next step, where the benzoate at position two
was removed using sodium methoxide in a
mixture of methanol–dichloromethane to give
40 in 86% yield over the two steps. The hy-
droxyl group of 40 was then methylated to
provide 41, and removal of the 4,6-O-iso-
propylidene group with aqueous trifl-
uoroacetic acid in dichloromethane gave 42.
Treatment of the diol, as described above for
small J1,2 coupling of 3.6 Hz proved the a-
D
configuration of the anomeric carbon. Re-
moval of the levulinyl group of 36 (confirmed,
1
using H NMR, by an upfield shift from 4.98
to 3.79 ppm of the H-4%%% proton) provided the
new glycosyl acceptor 37 (95%), and a new
reaction with 20 in toluene yielded the hexa-
saccharide 38 (63%). The structure and the
1
homogeneity of 38 were confirmed by H

P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 63–84
73
Coupling of the imidate 50 and the glycosyl
acceptor 47 was realised under the conditions
used for the synthesis of 21, except for tert-
butyldimethylsilyl trifluoromethanesulfonate
(ButMe2SiOTf; [19]) replacing Me3SiOTf as
activator (Scheme 6). This change followed
thin-layer chromatography analyses of pilot
reactions indicating that the formation of by-
products was reduced. The tetrasaccharide 51
14 and 26, gave the desired iduronic acid
containing disaccharide 46. For the first time
since compound 40, a column chromatogra-
phy was performed, and gave the key 46 in
52% yield over the six steps. The acceptor 47
was then prepared by delevulinylation, as de-
scribed above for 19 (91%). The structure of
1
47 was confirmed by H NMR analysis (Table
1
2) that also clearly showed the C4 conforma-
1
was thus obtained in 54% yield. H NMR
tion of the iduronic acid unit (JH-2,H-4 1.2 Hz).
analysis (J1,2 3.6 Hz) confirmed the a- nature
D
3
The value of the long-range JH-1,H-3 coupling
of the new interglycosidic bond. Delevulinyla-
(1.0 Hz) is in agreement with the a- configu-
L
tion of 51 provided 52 in quantitative yield
1
ration of the anomeric carbon. Interestingly,
the coupling constants observed for the pro-
(confirmed, using H NMR by an upfield shift
from 4.98 to 3.92 ppm of the H-4III proton),
tons of the -ido ring in 47 are weaker than in
L
which in turn, was coupled with 50 to give the
1
46. This probably reflects a shift of the confor-
hexasaccharide 53 (64%). H NMR analysis
4
mation of this unit toward the C1 conforma-
again confirmed the configuration of the new
glycosidic bond (J1,2 3.9 Hz). Note, in contrast
to the synthesis of 38 where the position four
tion in 46 [18]. This observation can be
extended to the non-reducing end units of 48,
49, 50, 51, and 53, where the measured cou-
pling constants were larger than in 52. Most
probably, the introduction of a substituent at
position four induces a shift of the conforma-
of -glucuronic acid was glycosylated, no for-
D
mation of the b anomer was observed during
the synthesis of 53 where the position four of
L-iduronic acid was involved. This is in agree-
ment with previous observations, in our labo-
ratory, about the absence of b anomer
formation in similar situations. Deprotection
and O-sulfonation were realised as with the
synthesis of 2, and furnished 4 in 60% yield
tional equilibrium of the corresponding
iduronic acid toward the C1 conformer.
L
-
4
For the preparation of the glycosyl donor
50, 46 was acetolysed into 48 (67%, Scheme 7)
using a mixture of trifluoroacetic acid, acetic
acid and acetic anhydride. Selective anomeric
deacetylation was then performed with
ethanolamine in tetrahydrofuran to give 49
(74%), and finally the imidate 50 was obtained
(94%) in the same way as 32.
1
from 53. High-field H NMR analysis confi-
rmed the structure of 4, and indicated that this
compound was around 95% pure. It also indi-
cated that the impurities were constituted by
several minor undersulfated compounds, the
structure of these were not investigated.
Scheme 3. Reagents and conditions: (a) Me3SiOTf, CH2Cl2, −20 °C, 62%; (b) NH2NH2:H2O, AcOH, pyridine, 0 °C, 87%; (c)
Me3SiOTf, toluene, −20 °C, 39%; (d–f) Pd/C 10%, H2, MeOH/CH2Cl2; then aq NaOH, MeOH; then pyridine–SO3 complex,
DMF, 55 °C, 89% from 23.

74
P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 63–84
Scheme 4. Reagents and conditions: (a–b) MeI, NaH, DMF, 0 °C then RT; then p-TsOH, MeOH/H2O, 81% from 24; (c–g)
tert-BDMSCl, DMAP, Et3N, CH2Cl2; then Lev2O, DMAP, Et3N; then aq H2SO4, CrO3, acetone; then BnBr, KHCO3, DMF;
then H2, 1,5-cyclooctadiene-bis[methyldiphenylphosphine]-iridium hexafluorophosphate, THF, then HgO, HgCl2, acetone, H2O,
30% from 26; (h) CCl3CN, K2CO3, CH2Cl2, 93%; (i) Me3SiOTf, CH2Cl2, −20 °C, 52%; (j) NH2NH2:H2O, AcOH, pyridine, 0 °C,
95%.
The affinity for antithrombin of the three
hexasaccharides 2–4 was assessed by fluores-
cence spectroscopy [20]. The dissociation
constants are presented in Table 1. As as-
sumed, modification of the non-reducing ex-
tremity of the reference pentasaccharide 1 by
introducing a third sulfated glucose unit did
not affect the recognition process with an-
affinity was partly recovered. Interestingly,
the affinity of 4 toward antithrombin was in
the range expected for the affinity of
thrombin and an oligosaccharide bearing
four negative charges per disaccharide unit
[4]. This situation should allow thrombin to
compete efficiently with antithrombin to bind
to such antithrombin binding domains. For
this reason, and because of synthetic conve-
nience (as already mentioned, no formation
of the other anomer during the coupling re-
action was observed), the hexasaccharide 4
was selected for further elongation [5].
tithrombin. On the contrary, introducing
D-
glucuronic acid instead of
L
-iduronic acid,
like in 3, resulted in a dramatic loss of activ-
ity. If the glucuronic acid units were replaced
by
L
-iduronic acid (com-pound 4), the
Scheme 5. Reagents and conditions: (a) Me3SiOTf, CH2Cl2, −20 °C, 69%; (b) NH2NH2:H2O, AcOH, pyridine, 0 °C, 95%; (c)
Me3SiOTf, toluene, −20 °C, 63%; (d–f) Pd/C 10%, H2, MeOH/CH2Cl2; then aq NaOH, MeOH; then pyridine–SO3 complex,
DMF, 55 °C, 94% from 38.

P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 63–84
75
The crude acid, dissolved in DMF (7.5 mL/
mmol), was treated for 2.5–20 h (TLC) at rt
with BnBr (10 equiv), and KHCO3 (5 equiv).
Methanol was then added, and the product was
extracted with Et2O, washed with water, dried
(Na2SO4), and concentrated.
3. Experimental
General methods.—Melting points were de-
termined in capillary tubes in a Mettler appara-
tus, and are uncorrected. Optical rotations
were measured with a Perkin–Elmer model 241
polarimeter at room temperature (rt) (229
3 °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 per-
formed on Silica Gel 60, 40–63 or 63–200 mm
Acti6ation of imidates with Me3SiOTf (Pro-
cedure
3).—Under
argon
atmosphere,
Me3SiOTf (0.04 M in CH2Cl2, 0.06 mol/mol of
imidate) was slowly added to a stirred, cold
(−20 °C) solution of imidate and glycosyl
acceptor in CH2Cl2 (15 mL/mmol), in the
,
presence of finely grounded 4 A molecular
1
(E. Merck). H NMR spectra were recorded
sieves. After 10–40 min (TLC), solid NaHCO3
was added until neutral, the reaction mixture
was diluted in CH2Cl2, filtered, washed with 2%
aq NaHCO3, water, dried (Na2SO4), and con-
centrated.
with Bruker AC 200, AM 250, AC 300 or AM
500 instruments, for solution in CDCl3 or D2O.
Before analysis in D2O, samples were passed
through a Chelex (Bio-Rad) ion exchange
column, and lyophilised three times from D2O.
Chemical shifts were relative to external Me4Si
when the spectra were recorded in CDCl3, and
to external sodium 4,4-dimethyl-4-silapen-
tanoate (TSP) when the spectra are recorded in
D2O. Coupling constants for compounds hav-
Deprotection and O-sulfonation (Procedure
4).—Hydrogenolysis of benzyl esters and ben-
zyl ethers: a solution of the protected com-
pound in a mixture of 1:4 CH2Cl2 –MeOH (5
g/L) was stirred under H2, in the presence of
10% Pd/C (2×the weight of the compound)
for 2–6 h (TLC). After filtration, the crude
compound was engaged as such in the next
step. Saponification of esters: 5 M aq NaOH
(final concentration: 0.5 M) was added to a
solution of ester in MeOH (150 mL/mmol).
After 2–5 h (TLC), the solution was acidified
with Dowex 50WX4 (H+) resin (pH 2). After
filtration, the solution was loaded on top of a
Sephadex G25F column (1.6×115 cm) equili-
brated with water. The fractions containing the
compound were collected, and freeze-dried. At
this stage, complete removal of protecting
ing the -ido configuration were simulated us-
L
ing the NMRSIM software. MS analyses were
performed on a ZAB-2E instrument (Fisons).
Elemental analyses were performed on a Fisons
elemental analyser.
Silylation and le6ulinylation of 4,6-diols (Pro-
cedure 1).—To a solution of 4,6-diol in CH2Cl2
(2.6 mL/mmol) were added, at 0 °C, tert-
butyldimethylsilyl chloride (1.45 equiv), tri-
ethylamine (1.5 equiv), and 4-(dimethylamino)-
pyridine (DMAP, 0.12 equiv). After 2–4 h of
stirring at 50 °C (TLC), levulinic anhydride
(1.5 equiv) was added, as well as DMAP (0.2
equiv), and triethylamine (1.5 equiv). After
1–20 h (TLC), the reaction mixture was diluted
with CH2Cl2, washed with aq 10% KHSO4,
water, aq 2% NaHCO3, water, dried (Na2SO4),
and concentrated to give the desired com-
pound.
Oxidation of primary alcohol into carboxylic
acid, and benzyl ester formation (Procedure
2).—Crude silyl ether was dissolved in acetone
(7.3 mL/mmol), and a solution of CrO3 (2.6
equiv) in H2SO4 (3.5 M, 1.11 mL/mmol) was
added at 0 °C. After 4–6 h (TLC) of stirring at
rt, the reaction mixture was diluted with
CH2Cl2 and washed with water. The organic
layer was dried (Na2SO4), and concentrated.
1
groups was controlled by 500 MHz H NMR.
O-Sulfonation: pyridine/sulfur trioxide com-
plex (5 mol/mol of hydroxyl) was added to a
solution of fully deprotected compound in
DMF (10 mg/mL). The solution was then
heated at 55 °C with protection from light for
20 h. After cooling to rt, the solution was
diluted with aq 0.2 M NaCl, and layered on top
of a Sephadex G25F gel column (1.6×115 cm)
equilibrated in aq 0.2 M NaCl. The fractions
containing the hexasaccharide were pooled to-
gether and the compound was desalted using
the same column equilibrated in water. After
freeze-drying, the desired compound was ob-
tained.

76
P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 63–84
Scheme 6. Reagents and conditions: (a) ButMe2SiOTf, toluene, −20 °C, 54%; (b) NH2NH2:H2O, AcOH, pyridine, 0 °C, 100%;
(c) like a, 64%; (d–f) Pd/C 10%, H2, MeOH/CH2Cl2; then aq NaOH, MeOH; then pyridine–SO3 complex, DMF, 55 °C, 60%
from 53.
Crude 3-O-methyl-
L
-idopyranose (6).—A
matography (3:7 EtOAc–cyclohexane) partly
suspension of 5 (92 g, 0.43 mol) in aq H2SO4
(0.1 M, 2.4 L) was heated at 60 °C for 5 h. The
solution was then neutralised with barium hy-
droxide and filtered through Celite. Concentra-
tion gave crude 6 that was used as such in the
next step.
purified the anomers. 8a: [h]D −121° (c 0.5,
1
CH2Cl2). H NMR (500 MHz): carbohydrate
ring protons (see Table 2); 3.53 (s, 3 H, OMe);
2.71 (q, SCH2CH3); 2.08, 2.13, 2.14 (3 s, 9 H,
3 CH3C(O)); 1.33 (t, SCH2CH3). 8b: [h]D +13°
(c 0.4, CH2Cl2). 1H NMR (500 MHz): carbohy-
drate ring protons (see Table 2); 3.55 (s, 3 H,
OMe); 2.74 (q, SCH2CH3); 2.07, 2.11, 2.13 (3
s, 9 H, 3 CH3C(O)); 1.31 (t, SCH2CH3). ESIMS,
positive mode: m/z (+NaCl), 387.1 [M+
Na]+; (+KF), 403.0 [M+K]+.
Crude 1,2,4,6-tetra-O-acetyl-3-O-methyl- -
L
idopyranose (7).—To a solution of the above
residual syrup in pyridine (400 mL) acetic
anhydride (323 mL, 8 equiv) was slowly added,
at 0 °C, followed by DMAP (1.04 g, 0.02 equiv).
After 16 h at rt, MeOH (138 mL, 8 equiv) was
added at 0 °C, and after 1 h the solution was
concentrated. The residue, dissolved in CH2Cl2,
was successively washed with 10% aq KHSO4,
water, aq satd NaHCO3, water, dried (Na2SO4),
and concentrated. Column chromatography
(1:2 cyclohexane–Et2O) allowed the a anomer
Ethyl 3-O-methyl-1-thio-h-
(9a),andethyl3-O-methyl-1-thio-i-
L
-idopyranoside
-idopyran-
L
oside (9b).—To a solution of the above crude
8 in a 1:3 mixture of CH2Cl2–MeOH (2.6 L),
was added a 2 M solution of MeONa in MeOH
(189 mL, 0.38 mol). After 1 h, the reaction
mixture was neutralised with Dowex 50WX4
(H+), filtered, and concentrated to give crude
9. A sample of both anomers could be isolated
for characterisation, after column chromatog-
raphy (3:7 toluene–EtOAc): 9a: [h]D−219° (c
1
7a to be partly purified. H NMR (250 MHz):
carbohydrate ring protons (see Table 2); 3.55 (s,
3 H, OMe); 2.02, 2.07, 2.09, 2.10 (4 s, 4
CH3C(O)). ESIMS, positive mode: m/z (+
NaCl), 385 [M+Na]+; (+KF), 401 [M+
K]+.
1
1.00, CH2Cl2). H NMR (500 MHz): carbohy-
drate ring protons (see Table 2); 4.32 (d, 1 H,
JOH,H-2 9.3 Hz, OH-2); 3.46 (s, 3 H, OMe);
2.65–2.60 (q, SCH2CH3); 2.47 (1 H, OH-6);
1.29 (t, SCH2CH3). ESIMS, positive mode: m/z
(+NaCl), 261.2 [M+Na]+. Anal. Calcd for
C9H18O5S (238.30): C, 45.36; H, 7.61. Found:
C, 45.52; H, 7.71. 9b: [h]D +55° (c 1.00,
Crude ethyl 2,4,6-tri-O-acetyl-3-O-methyl-1-
thio- -idopyranoside (8).—Thioethanol (29
L
mL, 0.39 mol) was added to a solution of the
above residue in toluene (1.7 L), and a 1 M
solution of BF3·Et2O complex in toluene (194
mL, 0.19 mol) was added slowly. After 4.5 h,
aq satd NaHCO3 was introduced, and the
solution was diluted with CH2Cl2, washed with
water, brine, dried (Na2SO4), and concentrated
to give a mixture of crude 8. Silica gel chro-
1
CH2Cl2). H NMR (500 MHz): carbohydrate
ring protons (see Table 2); 3.46 (s, 3 H, OMe);
2.75 (q, SCH2CH3); 1.32 (t, SCH2CH3). ESIMS,

P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 63–84
77
Scheme 7. Reagents and conditions: (a–b) NIS, TfOH, toluene, −20 °C; then MeONa, MeOH/CH2Cl2, 86% from 11 and 33;
(c–h) MeI, NaH, DMF, 0 °C then RT; then aq CF3CO2H, CH2Cl2; then tert-BDMSCl, DMAP, Et3N; then Lev2O, DMAP,
Et3N; then aq H2SO4, CrO3, acetone; then BnBr, KHCO3, DMF, 52% from 40; (i) NH2NH2:H2O, AcOH, pyridine, 0 °C, 91%;
(j) CF3CO2H, Ac2O, AcOH, 60 °C, 67%; (k) HO(CH2)2NH2, THF, +4 °C then RT, 74%; (l) CCl3CN, K2CO3, CH2Cl2, 94%.
Ethyl 2-O-benzoyl-4,6-O-isopropylidene-3-
O-methyl-1-thio-h- -idopyranoside (11a) and
ethyl 2-O-benzoyl-4,6-O-isopropylidene-3-O-
methyl-1-thio-i- -idopyranoside (11b).—Ben-
positive mode: m/z (+NaCl), 261.2 [M+
Na]+; (+KF), 277.2 [M+K]+. Anal. Calcd
for C9H18O5S (238.30): C, 45.36; H, 7.61.
Found: C, 45.66; H, 7.77.
L
L
zoyl chloride (4.60 mL, 39.2 mmol) and
DMAP (0.44 g, 3.92 mmol) were added to a
solution of 10 (3.02 g, 10.8 mmol) in pyridine
(50 mL). After 1.5 h the mixture was concen-
trated, diluted with CH2Cl2 and successively
washed with 5% aq KHSO4 and water, dried
(Na2SO4), filtered and concentrated to give,
after column chromatography (5:1 n-hexane–
EtOAc), a mixture (7:3 a/b) of 11 (4.61 g,
90 %). 11a: mp 111 °C. [h]D −110° (c 1.05,
Ethyl 4,6-O-isopropylidene-3-O-methyl-1-
thio-h-
L
-idopyranoside (10a) and ethyl 4,6-O-
isopropylidene-3-O-methyl-1-thio-i-
L
-idopyr-
anoside (10b).—Crude 9 was dissolved in 2,2-
dimethoxypropane (231 mL), acidified with
camphorsulfonic acid (2.92 g, 12.6 mmol), and
stirred for 1 h. The mixture was neutralised
with triethylamine (3.5 mL), and concen-
trated. Pure 10 (28 g, 24% over the five steps)
could be isolated by column chromatography
(4:1 cyclohexane–EtOAc) and crystallisation.
10a: mp 87 °C (cyclohexane). [h]D −199° (c
1
CH2Cl2). H NMR (500 MHz): carbohydrate
ring protons (see Table 2); 7.40–8.20 (m, 5 H,
Ph); 3.58 (s, 3 H, OMe); 2.64 (q, SCH2CH3);
1.48, 1.47 (2 s, 6 H, Me2C); 1.29 (t,
SCH2CH3). Anal. Calcd for C19H26O6S
(382.46): C, 59.66; H, 6.85; S, 8.4. Found: C,
59.66; H, 6.82; S, 8.28. 11b: mp 98 °C. [h]D +
1
1.00, CH2Cl2). H NMR (500 MHz): carbohy-
drate ring protons (see Table 2); 3.90 (d, 1 H,
JOH,H-2 11.3 Hz, OH); 3.48 (s, 3 H, OMe); 2.62
(q, SCH2CH3); 1.47, 1.46 (2 s, 6 H, Me2C);
1.28 (t, SCH2CH3). ESIMS, positive mode:
m/z (+NaCl), 301.4 [M+Na]+; (+KF),
317.4 [M+K]+. Anal. Calcd for C12H22O5S
(278.37): C, 51.78; H, 7.97. Found: C, 51.76;
H, 8.05. 10b: mp 95 °C (cyclohexane). [h]D
+106° (c 1.00, CH2Cl2). 1H NMR (500
MHz): carbohydrate ring protons (see Table
2); 3.48 (d, 1 H, JOH,H-2 12.2 Hz, OH); 3.48 (s,
3 H, OMe); 2.75 (q, SCH2CH3); 1.47, 1.46 (2
s, 6 H, Me2C); 1.30 (t, SCH2CH3). ESIMS,
positive mode: m/z (+NaCl), 301.4 [M+
Na]+; (+KF), 317.4 [M+K]+. Anal. Calcd
for C12H22O5S (278.37): C, 51.78; H, 7.97.
Found: C, 51.75; H, 8.15.
1
90° (c 0.91, CH2Cl2). H NMR (500 MHz):
carbohydrate ring protons (see Table 2); 7.40–
8.20 (m, 5 H, Ph); 3.60 (s, 3 H, OMe); 2.76 (q,
SCH2CH3); 1.41, 1.35 (s, 6 H, Me2C); 1.35 (t,
SCH2CH3). Anal. Calcd for C19H26O6S
(382.46): C, 59.66; H, 6.85; S, 8.4. Found: C,
59.71; H, 6.80; S, 8.28.
Methyl(2-O-benzoyl-4,6-O-isopropylidene-3-
O-methyl-h-
L
-idopyranosyl)-(1“4)-2,3,6-tri-
-glucopyranoside (13).—Triflic
O-benzoyl-h-
D
acid in toluene (0.15 M, 6.5 mL) was added,
under argon, to a stirred, cold (−20 °C) solu-
tion of 11 (13 g, 34 mmol), 12 [9] (17.2 g, 34
mmol), and NIS (19.1 g, 85 mmol), in toluene

78
P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 63–84
,
(480 mL) containing finely ground 4 A
molecular sieves. After 1 h, solid NaHCO3
(125 mg) was introduced, and after 15 min,
the solution was filtered, diluted with CH2Cl2,
washed with 10% aq Na2S2O3, dried (Na2SO4),
and evaporated. Column chromatography
(5:1, 5:2, then 2:1 n-hexane–EtOAc) gave 13
Methyl(benzyl 4-O-le6ulinyl-2,3-di-O-me-
thyl-i- -glucopyranosyluronate)-(1“4)-(3,6-
di-O-acetyl-2-O-benzyl-h- -glucopyranosyl)-
(1“4)-(benzyl 2-O-benzoyl-3-O-methyl-h-
idopyranosyluronate)-(1“4)-2,3,6-tri-O-benz-
oyl-h- -glucopyranoside (21).—A mixture of
D
D
L-
D
20 (1.20 g, 1.35 mmol) and 19 (1.18 g, 1.32
mmol) was treated according to Procedure 3
to give, after column chromatography (5:2
toluene–acetone), pure 21 (1.33 g, 62%):
1
(26.4 g, 93%): [h]D +92° (c 1.09, CHCl3). H
NMR (200 MHz): carbohydrate ring protons
(see Table 2); 7.25–8.0 (m, 20 H, 4 Ph); 3.40,
3.41 (2 s, 6 H, 2 OMe); 1.25, 1.28 (2 s, 6 H,
Me2C). Anal. Calcd for C45H46O15 (826.86): C,
65.37; H, 5.61. Found: C, 65.11; H, 5.64.
1
[h]D +66° (c 1.00, CH2Cl2). H NMR (200
MHz): carbohydrate ring protons (see Table
2); 7.16–8.01 (m, 35 H, 7 Ph); 4.50, 4.95, 5.05
(6 H, 3 CH2Ph); 3.49, 3.35, 3.26, 3.22 (4 s, 12
Crude methyl(2-O-benzoyl-3-O-methyl-h-
L
-
idopyranosyl)-(1“4)-2,3,6-tri-O-benzoyl-h-
D-
H,
4
OMe); 2.20–2.50 (m,
4
H,
glucopyranoside (14).—Compound 13 (26.2 g,
31.7 mmol) was treated with aq AcOH (70%,
565 mL) at 50 °C for 4 h, and coevaporated
with toluene to give crude 14: TLC Rf 0.48,
20:1 CH2Cl2 –MeOH.
C(O)CH2CH2C(O)); 1.89, 2.02, 2.12 (3 s, 9 H,
3 CH3C(O)). Anal. Calcd for C86H90O31
(1619.66): C, 63.78; H, 5.60. Found: C, 63.60;
H, 5.66.
Methyl(benzyl 2,3-di-O-methyl-i-
pyranosyluronate)-(1“4)-(3,6-di-O-acetyl-2-
O-benzyl-h- -glucopyranosyl)-(1“4)-(benzyl
2-O-benzoyl-3-O-methyl-h- -idopyranosyluro-
nate)-(1“4)-2,3,6-tri-O-benzoyl-h- -gluco-
D-gluco-
Crude methyl(2-O-benzoyl-4-O-le6ulinyl-3-
O-methyl-6-O-tert-butyldimethylsilyl-h-
L
-ido-
D
pyranosyl)-(1“4)-2,3,6-tri-O-benzoyl-h-
D-
L
glucopyranoside (16).—The above crude 14
(27 g) was treated according to Procedure 1 to
yield 16 (38.5 g): TLC Rf 0.78, 1:1 n-hexane–
EtOAc.
D
pyranoside (22).—Compound 21 (1.00 g, 0.62
mmol) was delevulinylated as described for 19
to give, after column chromatography (2:1
toluene–acetone), pure 22 (858 mg, 87%):
Crude methyl(benzyl 2-O-benzoyl-4-O-le6-
1
ulinyl-3-O-methyl-h-
L
-idopyranosyluronate)-
-glucopyranos-
[h]D +64° (c 1.00, CH2Cl2). H NMR (200
(1“4)-2,3,6-tri-O-benzoyl-h-
D
MHz): carbohydrate ring protons (see Table
2); 7.15–8.00 (m, 35 H, 7 Ph); 4.50, 5.05 (4 H,
2 CH2Ph); 3.60, 3.34, 3.27, 3.22 (4 s, 12 H, 4
ide (18).—The above crude 16 was treated as
described in Procedure 2 to give first the acid
17 (TLC Rf 0.41, 18:1 CH2Cl2–MeOH), then
the benzyl ester 18: TLC Rf 0.47, 1:1 n-
hexane–EtOAc.
OMe); 2.58 (d, JOH,H4 2.8 Hz, OH); 1.87,
III
2.00 (2 s, 6 H, 2 CH3C(O)). Anal. Calcd for
C81H84O29 (1521.56): C, 63.94; H, 5.56.
Found: C, 63.62; H, 5.75.
Methyl(benzyl 2-O-benzoyl-3-O-methyl-h-
idopyranosyluronate)-(1“4)-2,3,6-tri-O-benz-
oyl-h- -glucopyranoside (19).—A solution of
L
-
Methyl(benzyl 4-O-le6ulinyl-2,3-di-O-me-
D
thyl-i-
di-O-acetyl-2-O-benzyl-h-
(1“4)-(benzyl 2,3-di-O-methyl-i-
anosyluronate)-(1“4)-(3,6-di-O-acetyl-2-O-
benzyl-h- -glucopyranosyl)-(1“4)-(benzyl 2-
O-benzoyl-3-O-methyl-h- -idopyranosyluro-
nate)-(1“4)-2,3,6-tri-O-benzoyl-h- -gluco-
D
-glucopyranosyluronate)-(1“4)-(3,6-
-glucopyranosyl)-
-glucopyr-
hydrazine hydrate (1 M in 3:2 pyridine–
AcOH, 156 mL) was added to a cooled (0 °C)
solution of crude 18 in pyridine (159 mL).
After 1 h at 0 °C, concentration followed by
column chromatography (16:1 CH2Cl2–
EtOAc) yielded 19 (21 g, 75% from 13):
D
D
D
L
D
1
[h]D+103° (c 1.32, CHCl3). H NMR (500
pyranoside (23).—A mixture of imidate 20
(351 mg, 394 mmol) and acceptor 22 (600 mg,
394 mmol) was treated according to Procedure
3 except for CH2Cl2 being replaced by toluene,
to give, after column chromatography (4:3
cyclohexane–acetone), pure 23 (431 mg, 39%)
together with some b anomer (91 mg, 8%),
MHz): carbohydrate ring protons (see Table
2); 7.23–8.05 (m, 25 H, 5 Ph); 4.56 and 4.96 (2
H, CH2Ph); 3.41, 3.45 (2 s, 6 H, 2 OMe); 2.55
(d, 1 H, JH-4 ,OH 11 Hz, OH). Anal. Calcd for
C49H46O16 (8I90.90): C, 66.06; H, 5.20. Found:
C, 65.95; H, 5.26.

P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 63–84
79
give 26 (12.9 g, 81%) as a solid: mp 55 °C.
and some recovered glycosyl acceptor (138
mg, 18%). 23: [h]D +76° (c 0.27, CH2Cl2). H
1
1
[h]D +159° (c 0.65, CH2Cl2). H NMR (500
MHz): carbohydrate ring protons (see Table
NMR (500 MHz): carbohydrate ring protons
(see Table 2); 7.14–8.02 (m, 45 H, 9 Ph); 4.40,
4.55, 4.90, 5.03, 5.05 (10 H, 5 CH2Ph); 3.46,
3.44, 3.38, 3.43, 3.22, 3.21 (6 s, 18 H, 6 OMe);
2.20–2.60 (m, 4 H, C(O)CH2CH2C(O)); 2.11,
2.09, 2.02, 1.92, 1.78 (5 s, 15 H, 5 CH3C(O)).
ESIMS, positive mode: monoisotopic mass=
2248.78; chemical mass=2250.29; experimen-
tal mass=2250.08.
2); 5.94 (m, 1 H, CH2ꢀCHCH2); 5.32 (m, 1 H,
CH2ꢀCHCH2 trans); 5.22 (m,
1
H,
CH2ꢀCHCH2 cis); 4.20 and 4.06 (m, 2 H,
CH2ꢀCHCH2); 3.68, 3.51 (2 s, 6 H, 2 OMe);
2.64 (d, 1 H, JOH,H-4 1.7 Hz, 4-OH); 2.00 (m, 1
H, JOH,H-6 5.2 Hz, JOH,H-6% 7.1 Hz, 6-OH).
Anal. Calcd for C18H24O6: C, 53.22; H, 8.12.
Found: C, 53.15; H, 8.07.
Methyl(sodium 2,3-di-O-methyl-4-O-sodium
Crude allyl-4-O-le6ulinyl-2,3-di-O-methyl-
sulfonato-i-
(2,3,6-tri-O-sodium sulfonato-h-
osyl)-(1“4)-(sodium 2,3-di-O-methyl-i-
glucopyranosyluronate) - (1“4) - (2,3,6 - tri - O-
sodium sulfonato-h- -glucopyranosyl)-(1“4)-
(sodium 3-O-methyl-2-O-sodium sulfonato-h-
- idopyranosyluronate) - (1“4) - 2,3,6 - tri - O-
sodium sulfonato-h- -glucopyranoside (2).—
D
-glucopyranosyluronate)-(1“4)-
6-O-tert-butyldimethylsilyl-h- -glucopyranose
D
D
-glucopyran-
(28).—A solution of 26 (13.3 g, 53.6 mmol)
was treated according to Procedure 1 to give
28, which was used in the next step without
purification.
D
-
D
Crude benzyl (allyl 4-O-le6ulinyl-2,3-di-O-
L
methyl-h-D-glucopyranosid)uronate
(30).—
D
Crude 28 was treated according to Procedure
2 to give 30, after partial purification by chro-
matography (5:2 cyclohexane–acetone).
Compound 23 (204 mg, 91 mmol) was hydro-
genated and saponified according to
Procedure 4 to give fully deprotected hexasac-
charide (92 mg, 91%). The latter (39 mg, 35
mmol) was then sulfated as described in Proce-
dure 4 to give 2 (39 mg, 98%) as a white
Benzyl 4-O-le6ulinyl-2,3-di-O-methyl- -glu-
D
copyranuronate (31).—(1,5–Cyclooctadiene)-
bis(methyldiphenylphosphine)-iridium(I)hexa-
fluorophosphate (130 mg, 15.7 mmol) was
added under H2 atmosphere to a solution of
the previous compound (24.8 g, 55.2 mmol) in
THF (550 mL). After 16 h stirring, the mix-
ture was concentrated, diluted in CH2Cl2 (1
L), and successively washed with 2% aq
NaHCO3, water, dried (Na2SO4), and concen-
trated. The residue was then dissolved in a 1:5
water–acetone mixture (600 mL), and HgO
(23.8 g, 110 mmol) then HgCl2 (29.8 g, 110
mmol) were added. After 50 min stirring, the
solvent was partly removed under reduced
pressure, and the residue was diluted in
CH2Cl2 (2 L), and successively washed with
water, satd aq KI, water, brine, dried
(Na2SO4), and concentrated. Column chro-
matography (2:1 cyclohexane–acetone) gave
1
powder: [h]D +30° (c 1.00, H2O). H NMR
(500 MHz): carbohydrate ring protons (see
Table 2); 3.67, 3.66, 3.65, 3.62, 3.61, 3.48 (6 s,
18 H, 6 OMe). LSIMS, negative mode: m/z
2280.4 [M−Na]−.
Crude
methyl-h-
allyl-4,6-O-benzylidene-2,3-di-O-
-glucopyranoside (25).—To a solu-
D
tion of 24 [14] (19.7 g, 61.4 mmol) in DMF
(450 mL) were added, at 0 °C, NaH (5.8 g,
192.0 mmol) and MeI (10.4 mL, 166 mmol).
After 16 h, MeOH was introduced, and the
reaction mixture was concentrated. The
residue was dissolved in EtOAc, washed with
water, dried (Na2SO4), filtered and concen-
trated to give crude 25, which was directly
used in the next step.
1
Allyl-2,3-di-O-methyl-h-D-glucopyranoside
31 (a/b 7:3; 5.89 g, 30% from 26). H NMR
(26).—p-Toluenesulfonic acid (9.51 g, 50.0
mmol) was added to a solution of crude 25 in
1:2.6 water–MeOH (1.15 L). After 1.25 h the
solution was cooled to 0 °C, and Et3N (7.7
mL) was added. The mixture was then con-
centrated, and codistillations with toluene
were realised. The residue was purified by
chromatography (3:2 cyclohexane–acetone) to
(200 MHz): carbohydrate ring protons (see
Table 2); 7.32 (m, 5 H, Ph); 5.10 (2 H,
CH2Ph); 3.49, 3.48 (2 s, 6 H, 2 OMe); 2.20–
2.50 (m, 4 H, C(O)CH2CH2C(O)); 2.10 (s, 3
H, CH3C(O)). DCIMS, positive mode: m/z
(+NH3), 428 [M+NH4]+. Anal. Calcd for
C20H26O9: C, 58.53; H, 6.39. Found: C, 58.28;
H, 6.60.

80
P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 63–84
Benzyl(4 - O - le6ulinyl - 2,3 - di - O - methyl - 1-
O-trichloroacetimidoyl- -glucopyranos)uronate
Methyl(benzyl 4-O-le6ulinyl-2,3-di-O-me-
thyl-i- -glucopyranosyluronate)-(1“4)-(3,6-
di-O-acetyl-2-O-benzyl-h- -glucopyranosyl)-
(1“4)-(benzyl 2,3-di-O-methyl-i- -gluco-
pyranosyluronate)-(1“4)-2,3,6-tri-O-benzyl-h-
-glucopyranoside (36).—A mixture of im-
D
D
(32).—To a solution of 31 (1.02 g, 2.27 mmol)
in CH2Cl2 (5 mL) were added CCl3CN (1.62
mL, 16.2 mmol) and K2CO3 (652 mg, 4.7
mmol). After 16 h, the mixture was filtered
over silica gel (2:1 cyclohexane–EtOAc) to
give 32 (1.17 g, 93%). An analytical fraction
where the a anomer largely predominated
D
D
D
idate 20 (900 mg, 1.01 mmol) and acceptor 35
(663 mg, 0.87 mmol) was treated according to
Procedure 3 to give, after column chromatog-
raphy (1:1 cyclohexane–EtOAc), pure 36 (900
1
could be obtained for analysis: H NMR (250
1
mg, 69%): [h]D +71° (c 1.00, CH2Cl2). H
MHz): carbohydrate ring protons (see Table
2); 8.68 (s, 1 H, NH); 7.34 (m, 5 H, Ph); 5.12
(2 H, CH2Ph); 3.55, 3.49 (2 s, 6 H, 2 OMe);
2.20–2.50 (m, 4 H, C(O)CH2CH2C(O)); 2.12
(s, 3 H, CH3C(O)). LSIMS, positive mode:
m/z (+thioglycerol+NaCl), 576 [M+
Na]+,(+KCl), 592 [M+K]+. Anal. Calcd
for C22H26Cl3NO9: C, 47.63; H, 4.72; N, 2.52.
Found: C, 47.83; H, 4.82; N, 2.48.
NMR (500 MHz): carbohydrate ring protons
(see Table 2); 7.18–7.39 (m, 30 H, 6 Ph);
4.40–5.15 (12 H, 6 CH2Ph); 3.47, 3.43, 3.41,
3.36 (4 s, 15 H, 5 OMe); 2.26–2.58 (m, 4 H,
C(O)CH2CH2C(O)); 1.93, 2.06, 2.11 (3 s, 9 H,
3 CH3C(O)). LSIMS, positive mode: m/z (+
thioglycerol+KF), 1525.5 [M+K]+. Anal.
Calcd for C80H94O27: C, 64.59; H, 6.37.
Found: C, 64.15; H, 6.34.
Methyl(benzyl 4-O-le6ulinyl-2,3-di-O-me-
thyl-i-
D
-glucopyranosyluronate)-(1“4)-2,3,6-
Methyl(benzyl 2,3-di-O-methyl-i-
pyranosyluronate)-(1“4)-(3,6-di-O-acetyl-2-
O-benzyl-h- -glucopyranosyl)-(1“4)-(benzyl
2,3-di-O-methyl-i- -glucopyranosyluronate)-
(1“4)-2,3,6-tri-O-benzyl-h- -glucopyranoside
D-gluco-
tri-O-benzyl-h-
D
-glucopyranoside (34).—A
mixture of imidate 32 (1.17 g, 2.11 mmol) and
acceptor 33 [17] (1.22 g, 2.64 mmol) was
treated according to Procedure 3 to give, after
column chromatography (5:2 toluene–ace-
tone), pure 34 (0.94 g, 52%): [h]D +32° (c
D
D
D
(37).—Compound 36 (861 mg, 0.58 mmol)
was delevulinylated like for the synthesis of 19
to give, after column chromatography (10:1
CH2Cl2–acetone), pure 37 (769 mg, 95%):
1
1.00, CH2Cl2). H NMR (300 MHz): carbohy-
drate ring protons (see Table 2); 7.22–7.39 (m,
20 H, 4 Ph); 4.40–5.10 (8 H, 4 CH2Ph); 3.48,
3.46, 3.37 (3 s, 9 H, 3 OMe); 2.20–2.70 (m, 4
H, C(O)CH2CH2C(O)); 2.11 (s, 3 H, CH3C-
(O)). LSIMS, positive mode: m/z (+thioglyc-
erol), 857 [M+H]+. Anal. Calcd for C48-
H56O14: C, 67.28; H, 6.59. Found: C, 67.30; H,
6.59.
1
[h]D −76° (c 1.00, CH2Cl2). H NMR (500
MHz): carbohydrate ring protons (see Table
2); 7.25–7.36 (m, 30 H, 6 Ph); 4.50–5.15 (12
H, 6 CH2Ph); 3.59, 3.47, 3.46, 3.44, 3.36 (5 s,
15 H, 5 OMe); 2.06, 1.93 (2 s, 6 H, 2
CH3C(O)). LSIMS, positive mode: m/z (+
thioglycerol+KF), 1427.6 [M+K]+.
Methyl(benzyl 2,3-di-O-methyl-i-
pyranosyluronate)-(1“4)-2,3,6-tri-O-benzyl-
h- -glucopyranoside (35).—Compound 34
D
-gluco-
Methyl (benzyl 4-O-le6ulinyl-2,3-di-O-me-
thyl-i-
di-O-acetyl-2-O-benzyl-h-
(1“4)-(benzyl 2,3-di-O-methyl-i-
anosyluronate)]2-(1“4)-2,3,6-tri-O-benzyl-h-
-glucopyranoside (38).—A mixture of imi-
D
-glucopyranosyluronate)-[(1“4)-(3,6-
-glucopyranosyl)-
-glucopyr-
D
D
(876 mg, 1.02 mmol) was delevulinylated as
for the synthesis of 19 to give, after column
chromatography (1.7:1 cyclohexane–EtOAc),
pure 35 (750 mg, 95%): [h]D +35° (c 0.6,
D
D
date 20 (600 mg, 675 mmol) and acceptor 37
(625 mg, 450 mmol) was treated according to
Procedure 3, except for CH2Cl2 replaced by
toluene, to give, after column chromatography
(12:1 CH2Cl2–acetone), pure 38 (600 mg,
1
CH2Cl2). H NMR (300 MHz): carbohydrate
ring protons (see Table 2); 7.20–7.40 (m, 20
H, Ph); 4.50–5.20 (8 H, 4 CH2Ph); 3.65, 3.52,
3.42 (3 s, 9 H, 3 OMe); 2.70 (d, JOH,H4% 2.8 Hz,
OH). LSIMS, positive mode: m/z (+thioglyc-
erol+KF), 797 [M+K]+. Anal. Calcd for
C43H50O12: C, 68.59; H, 6.74. Found: C, 68.06;
H, 6.64.
1
63%): [h]D +86° (c 0.6, CH2Cl2). H NMR
(500 MHz): carbohydrate ring protons (see
Table 2); 7.22–7.36 (m, 40 H, 8 Ph); 4.35–
5.25 (16 H, 8 CH2Ph); 3.46, 3.42, 3.38, 3.35 (4

P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 63–84
81
s, 21 H, 7 OMe); 2.20–2.60 (m, 4 H,
C(O)CH2CH2C(O)); 2.16, 2.10, 2.08, 2.07,
1.92 (5 s, 15 H, 5 CH3C(O)). LSIMS, positive
mode: m/z (+thioglycerol), 2177.6 [M+
H]+, (+KF), 2156.6 [M+K]+.
4.55–5.05 (6 H, 3 CH2Ph); 3.42, 3.35 (2 s, 6
H, 2 OMe); 3.26 (d, 1 H, JOH,H%-2 10.3 Hz,
OH); 1.27, 1.36 (2 s, 6 H, Me2C). ESIMS,
positive mode: m/z (+NaCl), 703 [M+
Na]+; (+KF), 719 [M+K]+. Anal. Calcd
for C38H48O11: C, 67.04; H, 7.11. Found: C,
67.05; H, 7.16.
Methyl (sodium 2,3-di-O-methyl-4-O-so-
dium
[(1“4)-(2,3,6-tri-O-sodium sulfonato-h-
copyranosyl)-(1“4)-(sodium 2,3-di-O-methyl-
i- -glucopyranosyluronate)]2-(1“4)-2,3,6-tri-
O-sodium sulfonato-h- -glucopyranoside (3).
sulfonato-i-
D
-glucopyranosyluronate)-
Crude methyl (4,6-O-isopropylidene-2,3-di-
D-glu-
O-methyl-h-
O-benzyl-h-
L
-idopyranosyl)-(1“4)-2,3,6-tri-
D-glucopyranoside (41).—Iodome-
D
thane (3.2 mL, 50.8 mmol) was added, at
0 °C, to a solution of 40 (26.6 g, 39.1 mmol),
and NaH (1.48 g, 58.7 mmol), in DMF (60
mL). Another addition of MeI (1.6 mL, 25.4
mmol) and NaH (0.74 g, 29.3 mmol) was
made after 5 h at rt. After 16 h, MeOH (10
mL) was slowly introduced, and after 1.5 h
the reaction mixture was concentrated. The
product was extracted with EtOAc (1.5 L).
The solution was washed with water, dried
(Na2SO4), and concentrated. Crude 41 thus
obtained (32.1 g) was used as such in the next
step: TLC, Rf 0.55, 3:2 cyclohexane–EtOAc.
D
—Compound 38 (185 mg, 87.3 mmol) was
hydrogenated and transesterified according to
Procedure 4 to give fully deprotected hexasac-
charide (98 mg, 99%). The latter (58 mg, 35
mmol) was then sulfated as described in Proce-
dure 4 to give 3 (99 mg, 95%) as a white
1
powder: [h]D +43° (c 0.72, water). H NMR
(500 MHz): carbohydrate ring protons (see
Table 2); 3.63, 3.62, 3.61, 3.58, 3.56, 3.44 (6 s,
21 H, 7 OMe). LSIMS, negative mode: m/z
2192.8 [M−Na]−.
Methyl(2-O-benzoyl-4,6-O-isopropylidene-3-
O-methyl-h-
O-benzyl-h-
L
-idopyranosyl)-(1“4)-2,3,6-tri-
Crudemethyl(2,3-di-O-methyl-h-
L
-idopyran-
-gluco-
D
-glucopyranoside (39).—Thiogly-
osyl)-(1“4)-2,3,6-tri-O-benzyl-h-
D
coside 11 (1.1 g, 2.87 mmol), and glycosyl
acceptor 33 (1.34 g, 2.87 mmol) were treated
like for the synthesis of 13. The same amount
of acid was added after 25 and 50 min. After
1.5 h, work-up as for 13 provided crude 39
(2.49 g), that was directly used for the next
step. Column chromatography (3:1 cyclohex-
ane–EtOAc) of a portion gave pure 39:
[h]D+31° (c 1, CH2Cl2). TLC, Rf 0.36, 3:1
pyranoside (42).—Aqueous CF3COOH (70%,
43 mL) was slowly added over 10 min to a
solution of the above crude 41 (32.1 g) in CH2Cl2
(215 mL). After 25 min at rt the solution was
diluted with CH2Cl2 (1 L), washed with cold satd
aq NaHCO3, water, and dried (Na2SO4). Crude
42 obtained after concentration (27.5 g), was
used as such in the next step: TLC, Rf 0.29, 2:3
cyclohexane–EtOAc.
1
cyclohexane–EtOAc. H NMR (500 MHz):
Crude methyl (6-O-tert-butyldimethylsilyl-4-
carbohydrate ring protons (see Table 2); 7.25–
7.40 (m, 20 H, 4 Ph); 4.55–5.05 (6 H, 3
CH2Ph); 3.41, 3.34 (2 s, 6 H, 2 OMe); 1.40,
1.29 (2 s, 6 H, Me2C). ESIMS, positive mode:
m/z (+NaCl), 345 [M+Na]+; (+KF), 361
[M+K]+. Anal. Calcd for C45H52O12: C,
68.86; H, 6.68. Found: C, 68.61; H, 6.75.
Methyl (4,6-O-isopropylidene-3-O-methyl-h-
O-le6ulinyl-2,3-di-O-methyl-h-
L
-idopyranosyl)-
(1“4)-2,3,6-tri-O-benzyl-h-
D
-glucopyranoside
(44).—A solution of crude 42 was treated
according to Procedure 1 to yield 44 (2.45 g),
which was used as such in the next step: TLC,
Rf 0.5, 12:1 cyclohexane–EtOAc.
Methyl
methyl-h-
tri - O - benzyl - h -
(benzyl
-idopyranosyluronate)-(1“4)-2,3,6-
- glucopyranoside (46).—
4-O-le6ulinyl-2,3-di-O-
L
L
-idopyranosyl)-(1“4)-2,3,6-tri-O-benzyl-h-
-glucopyranoside (40).—Crude 39 (2.34 g)
D
D
Crude 44 (2.45 g) was treated as described in
Procedure 2 to give, first 45 (TLC, Rf 0.56,
12:1 CH2Cl2–MeOH), then its benzyl ester 46
isolated (1.07 g, 52% from 40) after column
chromatography (2:1 then 3:2 cyclohexane–
EtOAc): [h]D +18° (c 1, CH2Cl2). TLC, Rf
0.53, 5:1 CH2Cl2–EtOAc. 1H NMR (250
was treated as for the synthesis of 9 to give,
after column chromatography (3:1 then 2:1
cyclohexane–EtOAc), pure 40 (1.74 g, 86%
over the two steps): [h]D +23° (c 1, CH2Cl2).
1H NMR (500 MHz): carbohydrate ring pro-
tons, (see Table 2); 7.25–7.33 (m, 15 H, 3 Ph);

82
P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 63–84
added, and the mixture was stirred at rt for 3
h. After cooling to 0 °C, 1 M aq HCl was
added until acidic pH, followed by CH2Cl2
(150 mL). The solution was washed with wa-
ter, dried (Na2SO4), and concentrated.
Column chromatography (2:1 then 1:1
CH2Cl2–EtOAc) afforded pure 49 (3 g, 74%):
[h]D +3° (c 1, CH2Cl2). TLC, Rf 0.21, 1:1
MHz): carbohydrate ring protons (see Table
2); 7.20–7.52 (m, 20 H, 4 Ph); 4.60–5.15 (8 H,
4 CH2Ph); 3.49, 3.32, 3,32 (3 s, 9 H, 3 OMe);
2.10–2.52 (m, 4 H, C(O)CH2CH2C(O)); 2.11
(1 s, 3 H, 1 CH3C(O)). ESIMS, positive mode:
m/z (+NaCl), 879 [M+Na]+; (+KF), 895
[M+K]+. Anal. Calcd for C48H56O14: C,
67.27; H, 6.55. Found: C, 67.07; H, 6.73.
1
Methyl (benzyl 2,3-di-O-methyl-h-
anosyluronate)-(1“4)-2,3,6-tri-O-benzyl-h-
-glucopyranoside (47).—Disaccharide 46
L
-idopyr-
toluene–EtOAc. H NMR (300 MHz): carbo-
hydrate ring protons (see Table 2); 7.25–7.36
(m, 10 H, 2 Ph); 4.60–5.25 (4 H, 2 CH2Ph);
3.45, 3.42 (2 s, 6 H, 2 OMe); 2.23–2.56 (m, 4
H, C(O)CH2CH2C(O)); 2.12, 2.10, 1.94 (3 s, 3
CH3C(O)). ESIMS, positive mode: m/z (thio-
glycerol+NaCl), 769 [M+Na]+; (thioglyc-
erol+KF), 785 [M+K]+. Anal. Calcd for
C37H46O16, 0.5 H2O: C, 58.80; H, 6.27. Found:
C, 58.87; H, 6.13.
D
(0.65 g, 0.76 mmol) was treated as for the
synthesis of 19 to give 47 (0.52 g, 91%) after
column chromatography (2:1 then 3:1 cyclo-
hexane–EtOAc): [h]D +34° (c 0.97, CH2Cl2).
1H NMR (500 MHz): carbohydrate ring pro-
tons (see Table 2); 7.20–7.34 (m, 20 H, 4 Ph);
4.55–5.15 (8 H, 4 CH2Ph); 3.40, 3.35, 3.23 (3
s, 9 H, 3 OMe). LSIMS, positive mode: m/z
(thioglycerol+NaCl), 781 [M+Na]+; (thio-
glycerol+KF), 797 [M+K]+. Anal. Calcd
for C43H50O12: C, 68.06; H, 6.64. Found: C,
68.09; H, 6.92.
(Benzyl 4-O-le6ulinyl-2,3-di-O-methyl-h-
idopyranosyluronate)-(1“4)-3,6-di-O-acetyl-
2-O-benzyl-1-O-trichloroacetimidoyl- -gluco-
L-
D
pyranose (50).—Compound 49 (1.03 g, 1.38
mmol) was treated as for the synthesis of 32 to
give, after column chromatography (4:1
toluene–acetone), pure 50 (1.16 g, 94%): TLC,
(Benzyl 4-O-le6ulinyl-2,3-di-O-methyl-h-
idopyranosyluronate) - (1“4) - 1,3,6 - tri - O -
acetyl-2-O-benzyl- -glucopyranose (48).—Tri-
L
-
D
1
Rf 0.31 and 0.48, 2:3 cyclohexane–EtOAc. H
fluoroacetic acid (28 mL, 0.364 mol) was
added to a solution of 46 (7.8 g, 9.1 mmol) in
Ac2O (194 mL, 2.06 mol) and AcOH (7.8 mL,
0.136 mol). After heating at 60 °C for 4 h the
solution was cooled to 0 °C and water (30
mL) was slowly introduced followed by Et3N
(69 mL). After evaporation, the residue was
dissolved in CH2Cl2, and washed with satd aq
NaHCO3, water, dried (Na2SO4), and concen-
NMR (300 MHz): carbohydrate ring protons
(see Table 2); 7.25–7.37 (m, 10 H, 2 Ph);
4.50–5.30 (4 H, 2 CH2Ph); 3.46, 3.45 (2 s, 6
H, 2 OMe); 2.23–2.57 (m, 4 H, C(O)CH2-
CH2C(O)); 2.13, 2.09, 2.08, 1.94, 1.92 (5 s, 9
H, 3 CH3C(O)). LSIMS, positive mode: m/z
(thioglycerol+LiCl), 896 [M+Li]+; (thio-
glycerol+NaCl), 912 [M+Na]+; (thioglyc-
erol+KF), 928 [M+K]+. Anal. Calcd for
C39H46O16NCl3: C, 52.56; H, 5.20; N, 1.57.
Found: C, 52.42; H, 5.20; N, 1.54.
trated.
Column
chromatography
(5:1
CH2Cl2 –EtOAc) afforded a mixture (4:1 a/b)
of the anomers of 48 (4.7 g, 67%): TLC, Rf
1
Methyl (benzyl 4-O-le6ulinyl-2,3-di-O-me-
0.35, 3:2 cyclohexane–acetone. H NMR (200
thyl-h-
-O-acetyl-2-O-benzyl-h-
(1“4)-(benzyl 2,3-di-O-methyl-h-
osyluronate)-(1“4)-2,3,6-tri-O-benzyl-h-
L
-idopyranosyluronate)-(1“4)-(3,6-di-
-glucopyranosyl)-
-idopyran-
MHz): carbohydrate ring protons (see Table
2); 7.20–7.37 (m, 10 H, 2 Ph); 4.50–5.20 (4 H,
2 CH2Ph); 3.44, 3.41 (2 s, 6 H, 2 OMe);
2.23–2.56 (m, 4 H, C(O)CH2CH2C(O)); 2.12,
2.08, 2.06, 1.93 (4 s, 4 CH3C(O)).
D
L
D
-
glucopyranoside (51).—A mixture of 47 (2.90
g, 3.83 mmol) and 50 (4.16 g, 4.67 mmol) was
treated according to Procedure 3 except that
CH2Cl2 was replaced by toluene, and
Me3SiOTf by ButMe2SiOTf (0.5 mol/mol of
imidate). Column chromatography (1:1 cyclo-
hexane–EtOAc) afforded pure 51 (3.2 g,
54%): [a]D +24° (c 1, CH2Cl2). TLC, Rf 0.52,
(Benzyl 4-O-le6ulinyl-2,3-di-O-methyl-h-
idopyranosyluronate)-(1“4)-3,6-di-O-acetyl-
2-O-benzyl- -glucopyranose (49).—A solution
L
-
D
of ethanolamine (1.3 mL, 21.6 mmol) and 48
(4.25 g, 5.28 mmol) in THF (80 mL) was
stirred for 16 h at 4 °C. Another portion of
ethanolamine (0.65 mL, 10.8 mmol) was then

P. Duchaussoy et al. / Carbohydrate Research 317 (1999) 63–84
83
2:3 cyclohexane–EtOAc. 1H NMR (200
MHz): carbohydrate ring protons (see Table
2); 7.21–7.36 (m, 30 H, 6 Ph); 4.45–5.20 (12
H, 6 CH2Ph); 3.43, 3.39, 3.35, 3.25 (4 s, 15 H,
5 OMe); 2.25–2.55 (m, 4 H, C(O)CH2CH2-
C(O)); 2.12, 2.00, 1.92 (3 s, 9 H, 3 CH3C(O)).
ESIMS, positive mode: m/z (+NaCl), 1510.3
[M+Na]+; (+KF), 1526.4 [M+K]+. Anal.
Calcd for C80H94O27: C, 64.59; H, 6.37.
Found: C, 64.61; H, 6.49.
pound 53 (39.0 mg, 18.4 mmol) was hydro-
genated and transesterified according to
Procedure 4 to give fully deprotected hexasac-
charide (98 mg, 99%). The latter (16.4 mg,
14.5 mmol) was then sulfated as described in
Procedure 4 to give 4 (24.9 mg, 60%): [h]D +
28° (c 1.0, H2O).1H NMR (500 MHz): carbo-
hydrate ring protons (see Table 2); 3.52, 3.49,
3.46, 3.43 (4 s, 21 H, 7 OMe). LSIMS negative
mode, 2192.6 [M−Na]−.
Methyl (benzyl 2,3-di-O-methyl-h-
anosyluronate)-(1“4)-(3,6-di-O-acetyl-2-O-
benzyl-h- -glucopyranosyl)-(1“4)-(benzyl2,3-
di-O-methyl-h- -idopyranosyluronate)-(1“4)-
2,3,6-tri-O-benzyl-h- -glucopyranoside (52).—
L
-idopyr-
D
Acknowledgements
L
D
This work is part of a collaboration be-
tween N.V. Organon (Oss, The Netherlands),
and Sanofi Recherche on antithrombotic
oligosaccharides. The authors thank T. Barzu
and J.-P. He´rault for the determination of the
binding constants, and the Sanofi Recherche
Service d’Analyse de Recherche staff (C. Pi-
card, Head) for elemental and HPLC analyses
(M. Maftouh, S. Albugues); NMR analyses
(C. Ponthus, D. Albene, M. Rival) and MS
analyses (F. Uzabiaga, V. Videau).
Compound 51 (1 g, 0.672 mmol) was
delevulinylated as for the synthesis of 19 to
give 52 in quantitative yield after column
chromatography (1:1 cyclohexane–EtOAc):
[h]D+28° (c 1, CH2Cl2). TLC, Rf 0.56, 2:3
1
cyclohexane–EtOAc. H NMR (500 MHz):
carbohydrate ring protons (see Table 2); 7.19–
7.32 (m, 30 H, 6 Ph); 4.50–5.15 (12 H, 6
CH2Ph); 3.40, 3.38, 3.37, 3.34, 3.25 (5 s, 15 H,
5 OMe); 2.01, 1.88 (2 s, 6 H, 2 CH3C(O)).
ESIMS, positive mode: m/z (+NaCl), 1412.1
[M+Na]+; (+KF), 1428.0 [M+K]+.
Methyl(benzyl 4-O-le6ulinyl-2,3-di-O-me-
References
thyl-h-
O - acetyl - 2 - O - benzyl - h -
(1“4)-(benzyl 2,3-di-O-methyl-h-
osyluronate) - (1“4)]2 - 2,3,6 - tri - O - benzyl - h-
-glucopyranoside (53).—A mixture of 50
L
-idopyranosyluronate)-(1“4)-[(3,6-di-
- glucopyranosyl)-
-idopyran-
[1] D.A. Lane, U. Lindahl (Eds.), Heparin, E. Arnold, Lon-
don, 1989.
[2] L.O. Andersson, T.W. Barrowcliffe, E. Holmer, E.A.
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G. Gatti, Biochem. Biophys. Res. Commun., 116 (1983)
492–499.
D
L
D
(386 mg, 434 mmol) and 52 (500 mg, 360
mmol) was treated as for the synthesis of 51.
Sephadex LH-20 column chromatography
(195×3.7 cm; 1:1 CH2Cl2–EtOH) gave pure
hexasaccharide 53 (495 mg, 64%): [h]D+22°
(c 1.2, CH2Cl2). TLC, Rf 0.36, 10:1 CH2Cl2–
[4] S.T. Olson, I. Bjo¨rk, Semin. Thromb. Hemostasis, 20
(1994) 373–409.
[5] P. Duchaussoy, G. Jaurand, P.-A. Driguez, I. Lederman,
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J.-M. Herbert, Carbohydr. Res., 317 (1999) 85–99.
[6] P. Westerduin, C.A.A. van Boeckel, J.E.M. Basten, M.A.
Broekhoven, H. Lucas, A. Rood, H. van der Heijden,
R.G.M. van Amsterdam, T.G. van Dinther, D.G.
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He´rault, J.-C. Lormeau, A. Bernat, J.-M. Herbert, J.
Med. Chem., 40 (1997) 1600–1607.
1
acetone. H NMR (500 MHz): carbohydrate
ring protons (see Table 2); 7.19–7.33 (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);
2.18–2.60 (m, 4 H, C(O)CH2CH2C(O)); 2.10,
2.05, 1.92, 1.89, 1.84 (5 s, 15 H, 5 CH3C(O)).
Methyl(sodium 2,3-di-O-methyl-4-O-sodium
[8] L.A.G.M. van den Broek, D.J. Vermaas, B.M.
Heskamp, C.A.A. van Boeckel, Recl. Tra6. Chim. Pays-
Bas, 112 (1993) 82–94.
sulfonato-h-
3,6-tri-O-sodium
osyl)-(1“4)-(sodium 2,3-di-O-methyl-h-
pyranosyluronate)-(1“4)]2-2,3,6-tri-O-sodium
sulfonato-h- -glucopyranoside (4).—Com-
L
-idopyranosyluronate)-(1“4)-[(2,
sulfonato-h- -glucopyran-
-ido-
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