Oligosaccharides corresponding to the regular sequence of heparin: Chemical synthesis and interaction with FGF-2

Article abstract of DOI:10.1016/S0968-0896(99)00113-3

It has been proposed that oligosaccharides corresponding to the so-called regular region of heparin/heparan sulfate (HS) bind to fibroblast growth factor-2 (FGF-2). In order to explore the molecular basis of FGF/HS interaction, we describe here the chemic

Full text of DOI:10.1016/S0968-0896(99)00113-3

Bioorganic & Medicinal Chemistry 7 (1999) 2003±2012
Oligosaccharides Corresponding to the Regular Sequence of
Heparin: Chemical Synthesis and Interaction with FGF-2y
Christine Tabeur, a Jean-Maurice Mallet, a Francoise Bono, b Jean-Marc Herbert, b
Maurice Petitou b,* and Pierre Sinay a,1
a
Â
Â
Â
Ecole Normale Superieure, Departement de Chimie, Associe au CNRS, 24 rue Lhomond, 75231 Paris Cedex 05, France
bHaemobiology Research Department, Sano® Recherche, 195, route d'Espagne, 31036 Toulouse Cedex, France
Received 5 February 1999; accepted 20 April 1999
AbstractÐIt has been proposed that oligosaccharides corresponding to the so-called regular region of heparin/heparan sulfate (HS)
bind to ®broblast growth factor-2 (FGF-2). In order to explore the molecular basis of FGF/HS interaction, we describe here the
chemical synthesis of a tetra and a hexasaccharide, prepared as methyl glycosides, corresponding to the regular sequence of heparin.
The strategy relies on the ecient preparation of three building blocks: a seeding block, an elongating block and a capping block.
The hexasaccharide inhibited the binding of FGF-2 on its receptor on human aorta vascular smooth muscle cells with an IC50 value
(16‹1.2 mg/mL) close to that of standard heparin (14.8‹0.5 mg/mL) whereas the tetrasaccharide was much less potent
(IC50=127‹10.5 mg/mL). The hexasaccharide and heparin, inhibited in a dose-dependent manner FGF-2 (30 nM) induced pro-
liferation (IC50=23.7‹1.6 and 30.1‹1.3 mg/mL, respectively). Under the same experimental conditions, the tetrasaccharide only
slightly inhibited the mitogenic e€ect of FGF-2 (IC50>100 mg/mL). # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
oligosaccharides having the same structure are required
to obtain an agonistic e€ect through dimerisation of the
FGFs receptors.5,6 However there is also evidence to
suggest that undersulfated sequences, and even non-
sulfated ones in heparan sulfates, might be involved in
FGFs binding.7,8
The ®broblast growth factors (FGFs) form a family of
181 structurally related polypeptides that are involved in
a large number of biological regulations.2 Their activity,
which requires dimerisation of their tyrosine kinase
membrane receptors, is critically modulated through an
interaction with heparin/heparan sulfate (HS) mole-
cules.3 How this modulation occurs is one of the most
challenging questions of current glycobiology.
We recently synthesized four pentasaccharides to study
the in¯uence of the nature of the uronic acid on FGF-2
binding, and found that higher anity was associated
with the presence of l-iduronic acid as the sole uronic
acid.9 The synthesis of the tetrasaccharide 1 and the
hexasaccharide 2 reported here constitute a step further
toward a rational exploration of the molecular basis of
FGF/HS interaction.
Several reports dealing with the interaction of FGFs
and HS have led to various conclusions as to how this
interaction takes place. Thus an hexasaccharide,
obtained by nitrous acid cleavage followed by borohy-
dride reduction, of the regular region of heparin, has
been shown to antagonize FGF-2 mediated cell
mitogenesis.4 More recent reports suggest that larger
Results and Discussion
Chemical synthesis of 1 and 2
The chemical synthesis of the tetrasaccharide 1 and the
hexasaccharide 2 reported here (Scheme 1) exempli®es a
strategy that can probably be applied to larger homo-
logous oligosaccharides of the same type. It is schema-
tically outlined in Scheme 1 and uses three disaccharidic
building blocks 4, 7, and 22 (Scheme 2).
Key words: Heparin; growth factor; carbohydrate; glycosylation.
y
Presented at the XVIth International Carbohydrate Symposium,
Paris, France, 1992.
* Corresponding author. Tel.: +33-(0)1-4432-3390; fax: +33-(0)5-
6116-2286; e-mail: maurice.petitou@sano®.com
1
Second corresponding author. Fax: +33-(0)1-4432-3397; e-mail:
pierre.sinay@ens.fr
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00113-3

2004
C. Tabeur et al. / Bioorg. Med. Chem. 7 (1999) 2003±2012
Scheme 1.
Scheme 2.
The synthesis of the oligosaccharides 1 and 2 is initiated
by the disaccharidic alcohol 4, which will thus be called
the seeding block. This disaccharide is so protected to
permit, in the ®nal step, a regioselective sulfation. It is
obtained in 90% yield after hydrazinolysis10 of the
known disaccharide 311 (Scheme 3).
(Scheme 1). No trace of b-anomer was detected in the
reaction mixture, making this critical elongation a
valuable one. The levulinyl group in 8 is then quantita-
tively removed by hydrazinolysis10 to give the tetra-
saccharidic alcohol 9. From this point on, three options
are possible as explained in Scheme 1.
In the ®rst elongating step, the alcohol 4 is glycosylated
by the so-called elongating block 7. This disaccharide
donor 7 is prepared in 73% overall yield from the
known disaccharide 511 (Scheme 4).
. If one desires to stop the process at this stage
(stopping process), that is to ultimately synthesize
the tetrasaccharide 1, a p-methoxybenzyl protect-
ing group is introduced,12 using p-methoxybenzyl
trichloroacetimidate,13,14 to give the tetrasaccharide
10. The initial use of benzyl trichloroacetimidate15±17
led to frustration.12 The p-methoxybenzyl group will
be called a stopping group. Compound 10 is then
The critical glycosylation step 4+7 is achieved at
10ꢀC in dichloromethane, in the presence of tri-
methylsilyl tri¯ate (TMSOTf) to give 8 in 82% yield

C. Tabeur et al. / Bioorg. Med. Chem. 7 (1999) 2003±2012
2005
Scheme 3. Reagents and conditions: (a) N2H4, AcOH, Pyr, rt, 90%.
converted in high yield into the expected tetra-
saccharide 1 using a series of reactions which are
now very classical in the ®eld18 (see Experimental).
. If one desires to oligomerize ahead, a second
upstream elongation has thus to be achieved. This
can in turn be done in two ways. In the ®rst route,
9 is glycosylated again by the elongating block 7
(elongating process) to give stereoselectively in
high yield the protected hexasaccharide 24. This
process can probably be iterated after delevuliny-
lation. For this reason, the levulinyl group is called
the elongating group.
. Another obvious option is to use for this second
elongation the so-called capping block 22 wherein
the stopping group has been introduced on this
disaccharidic block. The chemical synthesis of this
block from known 1111 is depicted in the self
explanatory Schemes 5 and 6 (see Experimental).
During the glycosylation of 14 with the thiophe-
nylglycoside 13, in the presence of NIS, formation
of the side product 16 was, not unexpectedly,
observed.
Scheme 5. Reagents and conditions: (a) MeONa, MeOH, then
Me2C(OMe)2, CSA, rt, 84% (two steps); (b) Ac2O, Pyr. rt, 96%; (c)
NIS, TfOH, 4 A MS, CH2Cl2, 0ꢀC, 15 (80%), 16 (10%).
Inhibition of FGF-2-induced HASMC proliferation.
FGF-2 (1±100 nM) stimulated in a dose-dependent
manner the growth of HASMC (not shown). The hexa-
saccharide 2 and the reference compound heparin,
inhibited in a dose-dependent manner FGF-2 (30 nM)
induced proliferation (Fig. 2; IC50=23.7‹1.6 and
30.1‹1.3 mg/mL, respectively). Under the same experi-
mental conditions, the tetrasacharide 1 only slightly
inhibited the mitogenic e€ect of FGF-2 (IC50>100 mg/
mL).
Experimental
General
All solvents and reagents were of the best commercially
available grade or were puri®ed and dried according to
standard procedures. Reactions were monitored by
TLC on silica gel 60F254 (Merck) with detection by
charring with H2SO4. Unless otherwise stated, column
chromatography was performed on silica gel 60 (E.
Merck 63±200 mm). 1H NMR spectra were recorded
with Bruker AM100, AC 250, AM400 or AM 500
instruments for solution in CDCl3 (internal Me4Si)
unless otherwise stated. In tetrasaccharides, sugar units
are named GHIJ starting from non reducing end, simi-
larly in hexasaccharides, sugar units are named
G0H0GHIJ. MS analyses were performed on Nermag
R10-10 instrument using chemical ionisation (NH3) and
detection of positive ions. Melting points were deter-
mined in capillary tubes with a Buchi 510 apparatus,
and are uncorrected. Optical rotations were measured
The resulting hexasaccharide 23 is now classically
deprotected to give the expected hexasaccharide 2. The
replacement of the elongating group by a stopping
group is clearly a prerequisite for a successful deprotection
functionalisation sequence, inasmuch as the levulinyl group
would not be stable on basic treatment, resulting into an
undesired sulfation of the corresponding hydroxyl group.
Biological activities
Inhibition of FGF-2 binding to cultured human aortic
smooth muscle cells (HASMC). As shown in Figure 1,
the hexasaccharide 2 representing the so-called regular
region of heparin, antagonised the binding of 125I-FGF-
2 to HASMC with an IC50 value of 16‹1.2 mg/mL.
Similar results were obtained with the reference com-
pound heparin (IC50=14.8‹0.5 mg/mL). In contrast,
the tetrasaccharide 1 only weakly antagonised the bind-
ing (IC50=127‹10.5 mg/mL).
with
a Perkin±Elmer Model 241 polarimeter at
23‹3ꢀC. Elemental analyses were carried out at the
Service Central d'Analyses (CNRS, Vernaison, France).
Scheme 4. Reagents and conditions: (a) BnNH2, Et2O, rt, 83%, (b) CCl3CN, K2CO3, 4 A MS, CH2Cl2, rt, 81%.

2006
C. Tabeur et al. / Bioorg. Med. Chem. 7 (1999) 2003±2012
Scheme 6. Reagents and conditions: (a) 80% aq AcOH, 100ꢀC, 1 h, 72%; (b) TBSCl, DMAP, Et3N, CH2Cl2, rt, 95%; (c) PMBO-C(NH)CCl3, TfOH,
CH2Cl2±Et2O, rt, 2 h, 90%; (d) CrO3, H2SO4, acetone, then MeI, KHCO3, NBu4NI, DMF, 40%; (e) BnNH2, Et2O, rt, 74%; (f) CCl3CN, K2CO3,
4 A MS, CH2Cl2, rt, 85%.
Figure 1. E€ect of heparin (*), 1 (&) or 2 (*) on the binding of 125I-FGF-2 to HASMC (left panel) and on the mitogenic e€ect of FGF-2 for
HASMC (right panel).
Methyl 6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-
(methyl 2-O-acetyl-3-O-benzyl-ꢀ-L-idopyranosyluronate)-
3H, H-3,4,40), 3.78±3.71 (m, 1H, H-30), 3.49 (s, 3H,
CO2Me), 3.45 (s, 3H, OMe), 3.50±3.42 (m, 1H, H-2),
2.62 (d, 1H, J4,OH=11.4 Hz, OH), 2.11 and 2.09 (2s, 6H,
2 Ac). Mass spectra: m/z 691 (M+NH4)+. Anal. calcd
for C32H39N3O13 (673.24): C, 57.04; H, 5.84; N, 6.24.
Found: C, 56.77; H, 5.87; N, 6.06.
ꢀ-D-glucopyranoside (4).
A solution of hydrazine
hydrate (1.77 mL, 36 mmol) in pyridine:acetic acid (3:2,
40 mL) was added to a solution of 3 (601 mg,
7.79 mmol) in pyridine (20 mL) at room temperature.
The reaction mixture was stirred during 5 min, then
diluted with CH2Cl2. The organic layer was washed
with 1 M aq HCl, water, dried (MgSO4) and concd.
Flash chromatography (toluene:ethyl acetate, 1:1) of the
residue gave 4 (472 mg, 90%). [a]d+6ꢀ (c 2.1, chloro-
form); 1H NMR (200 MHz) d 7.43±7.18 (m, 10H,
6-O-Acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-(methyl 2-O-
acetyl-3-O-benzyl-4-O-levulinyl-ꢀ-L-idopyranosyluronate)-
D-glucopyranose (6). A solution of 5 (696mg, 0.87mmol),
and benzylamine (4.6 mL, 33 mmol) in dry ether (5 mL)
was stirred overnight at room temperature, washed with
1 M aq HCl, water, dried (MgSO4) and concd. Chro-
matography (toluene:ethyl acetate, 5:6) of the residue
gave 6 (546 mg, 83%). 1H NMR (400 MHz) (A: a
anomer; B: b anomer) d 7.45±7.24 (m, 20H, arom.), 5.35
arom.), 5.09 (s, 1H, H-10), 4.95 (br.s, 1H, H-20), 4.92 (d,
0
0
0
1H, J4 ,5 =1.9 Hz, H-5 ), 4.82 (d, 1H, J1,2=3.6 Hz, H-1),
4.78 and 4.60 (2d, 2H, Jgem=11.2 Hz, CH2Ph), 4.78 and
4.70 (2d, 2H, Jgem=11.2 Hz, CH2Ph), 4.46 (dd, 1H,
0
0
=
J5,6a=1.2, J6a,6b=12.5 Hz, H-6a), 4.25 (dd, 1H, J5,6b=
3.2 Hz, H-6b), 4.01±3.92 (m, 1H, H-5), 3.92±3.78 (m,
(d, 1H, J1A,2A=3.5 Hz, H-1A), 5.19 (br.d, 1H, J1 A,2 A
1.6 Hz, H-10A), 5.17 (br.d, 1H, J1 B,2 B=1.6 Hz, H-10B),
0
0

C. Tabeur et al. / Bioorg. Med. Chem. 7 (1999) 2003±2012
2007
5.13 (dd, 1H, J3 B,4 B=J4 B,5 B=3.0 Hz, H-40B), 5.10 (dd,
arom), 5.26 (d, 1H, J1I,2I=3.0 Hz, H-1I), 5.17 (d, 1H,
J1G,2G=2.7 Hz, H-1G), 5.11 (dd, 1H, J3G,4G=J4G,5G
0
0
0
0
1H, J3 A,4 A=J4 A,5 A=3.0 Hz, H-40A), 4.99 (d, 1H, H-
=
0
0
0
0
50B), 4.96 (d, 1H, H-50A), 4.93 (dd, 1H, J2 A,3 A=2.5 Hz,
3.5 Hz, H-4G), 4.96 (dd, 1H, J2I,3I=3.0 Hz, H-2I), 4.96
(d, 1H, J1H,2H=3.0 Hz, H-1H), 4.91±4.62 (m, 11H, 4
CH2Ph and H-2G,5G,5I), 4.82 (d, 1H, J1J,2J=3.5 Hz, H-
1J), 4.45 (2dd, 2H, J5H,6aH=1.5, J6aH,6bH=12.0 Hz, H-
6aH; J5J,6aJ=1.5, J6aJ,6bJ=12.0 Hz, H-6aJ), 4.29 (dd,
0
0
H-20A), 4.89 (dd, 1H, J2 B,3 B=2.5 Hz, H-20B), 4.80±4.67
(m, 8H, 4 CH2Ph A and B), 4.65 (d, 1H, J1B,2B=8.0 Hz,
H-1B), 4.55 (dd, 1H, J5B,6aB=2.5, J6aB,6bB=12.5 Hz, H-
6aB), 4.54 (dd, 1H, J5A,6aA=2.5, J6aA,6bA=12.5 Hz, H-
6aA), 4.25 (dd, 1H, J5A,6bA=3.5 Hz, H-6bA), 4.21 (dd,
1H, J5B,6bB=4.0 Hz, H-6bB), 4.18±4.13 (m, 1H, H-5A),
3.97 (dd, 1H, J3A,4A=J4A,5A=9.0 Hz, H-4A), 3.95 (dd,
1H, J3B,4B=J4B,5B=9.0 Hz, H-4B), 3.88 (dd, 1H,
J2A,3A=9.0 Hz, H-3A), 3.87±3.82 (m, 2H, H-30A,30B),
3.57±3.52 (m, 1H, H-5B), 3.52±3.47 (m, 1H, H-2A), 3.50
and 3.49 (2s, 6H, 2 CO2Me A and B), 3.43 (dd, 1H, H-
2B), 3.34 (dd, 1H, H-3B), 3.21±3.14 (m, 1H, OH), 2.85±
2.26 (m, 8H, 2 C: OCH2CH2C: O A and B), 2.21 and
2.15 (2s, 12H, 4 Ac A and B), 2.12 and 2.11 (2s, 6H, 2
CH3C: O A and B), 1.70±1.60 (m, 1H, OH). Mass
spectra: m/z 775 (M+NH4)+. Anal. calcd for C36H43
N3O15.1/3H2O (763.72): C, 57.05; H, 5.72; N, 5.55.
Found: C, 56.62; H, 5.76; N, 5.50.
0
0
1H, J5J,6bJ=4.0 Hz, H-6bJ), 4.22 (dd, 1H, J5H,6bH
=
3.0 Hz, H-6bH), 4.03 (dd, 1H, J3I,4I=J4I,5I=4,5 Hz, H-
4I), 3.98±3.81 (m, 7H, H-3G,3I,3J,4H,4J,5H,5J), 3.68
(dd, 1H, J2H,3H=9.0, J3H,4H=10.0 Hz, H-3H), 3.51, 3.48
and 3.46 (3s, 9H, 2 CO2Me and OMe), 3.46 (dd, 1H,
J2J,3J=10.0 Hz, H-2J), 3.32 (dd, 1H, J2H,3H=10.0 Hz, H-
2H), 2.82±2.45 (m, 4H, C: OCH2CH2C: O), 2.21, 2.20,
2.12 and 2.10 (4s, 12H, 4 Ac), 2.08 (s, 3H, CH3C: O); 13
C
NMR (400 MHz) d 205.0 (CH3C: O (lev)); 171.0, 170.4,
170.3, 169.7, 169.5, 169.3 and 168.9 (4 CH3COO (Ac), 2
CO2Me and CH2COO (lev)); 98.3 and 97.8 (2 C-1 idur-
onate); 97.3 and 96.9 (2 C-1 glucosamine); 61.9 and 61.4
(2 CN3); 55.1 (OCH3); 51.8 and 51.6 (2 CO2CH3). Anal.
calcd for C68H80N6O27 (1413.42): C, 57.78; H, 5.70; N,
5.94. Found: C, 57.72; H, 5.62; N, 5.83.
[6-O-Acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-(methyl 2-
O-acetyl-3-O-benzyl-4-O-levulinyl-ꢀ-L-idopyranosyluron-
Methyl [(methyl 2-O-acetyl-3-O-benzyl-ꢀ-L-idopyrano-
syluronate)-(1!4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-
ꢀ-D-glucopyranosyl)-(1!4)-(methyl 2-O-acetyl-3-O-
benzyl-ꢀ-L-idopyranosyluronate)]-(1!4)-6-O-acetyl-2-
azido-3-O-benzyl-2-deoxy-ꢀ-D-glucopyranoside (9).
Compound 8 (320 mg, 0.22 mmol) was treated as
described for 4 to give 9 (300 mg, 100%) after column
chromatography (cyclohexane:ethyl acetate, 1:1). [a]d
ate)-ꢁ-D-glucopyranosyl] trichloroacetimidate (7).
A
solution of 6 (445 mg, 0.59 mmol) in dry dichloro-
methane (3 mL) was stirred at room temperature for
30 min in the presence of 4 A powdered molecular
sieves, then cooled at 0ꢀC. Dry K2CO3 (153 mg,
1.0 mmol) and trichloroacetonitrile (0.37 mL, 3.6 mmol)
were added. The reaction mixture was stirred at room
temperature for 5 h then concentrated. Flash chroma-
tography of the residue (toluene:ethyl acetate:Et3N,
10:7:0.1) gave 7 (430 mg, 81%). [a]d 25ꢀ (c 1.0, chloro-
+23ꢀ (c 1.0, chloroform). H NMR (400 MHz) d 7.40±
1
7.25 (m, 20H, arom.), 5.25 (d, 1H, J1I,2I=3.0 Hz, H-1I),
5.10 (d, 1H, J1G,2G<1.0 Hz, H-1G), 4.98±4.95 (m, 1H,
H-2I), 4.96 (d, 1H, J1H,2H=3.0 Hz, H-1H), 4.93±4.90
(m, 1H, H-2G), 4.87 (d, 1H, J4G,5G=2.0 Hz, H-5G),
4.85±4.64 (m, 10H, 4 CH2Ph and H-1J, 5I), 4.48±4.42
1
form). H NMR (250 MHz) d 8.75 (s, 1H, NH), 7.42±
7.16 (m, 10H, arom.), 5.64 (d, 1H, J1,2=8.2 Hz, H-1),
5.13 (br. s, 1H, H-10), 5.11 (dd, J3 ,4 =J4 ,5 =2.6 Hz, H-
0
0
0
0
40), 4.99 (d, 1H, H-50), 4.88 (br.s, 1H, H-20), 4.79-4.64
(m, 4H, 2 CH2Ph), 4.51 (dd, 1H, J5,6a=2.5, J6a,6b=
(m, 2H, H-6aH,6aJ), 4.30 (dd, 1H, J5J,6bJ=4.0, J6aJ,6bJ
12.0 Hz, H-6bJ), 4.21 (dd, 1H, J5H,6bH=3.0, J6aH,6bH
=
=
12.3 Hz, H-6a), 4.25 (dd, 1H, J5,6b=3.8 Hz, H-6b), 4.02
0
12.5 Hz, H-6bH), 4.03 (dd, 1H, J3I,4I=J4I,5I=4.0 Hz, H-
4I), 4.02±3.97 (m, 1H, H-4G), 3.95 (dd, 1H, J2I,3I
4.0 Hz, H-3I), 3.93±3.83 (m, 4H, H-3J,4H,4J,5J), 3.85±
0
=
(dd, 1H, J3,4=J4,5=9.3 Hz, H-4), 3.82 (dd, 1H, J2 ,3
=
1.5 Hz, H-30), 3.72 (dd, 1H, J2,3=9.6 Hz, H-2), 3.68±
3.60 (m, 1H, H-5), 3.49 (s, 3H, CO2Me), 3.40 (dd, 1H,
H-3), 2.85±2.43 (m, 4H, C: OCH2CH2C: O), 2.20 and
2.11 (2s, 6H, 2 Ac), 2.10 (s, 3H, CH3C: O). Anal. calcd
for C38H43Cl3N4O15 (902.14): C, 50.59; H, 4.80; N, 6.21.
Found: C, 50.83; H, 4.84; N, 5.96.
3.80 (m, 1H, H-5H), 3.75 (dd, 1H, J2G,3G=J3G,4G
3.5 Hz, H-3G), 3.70 (dd, 1H, J2H,3H=9.0, J3H,4H
=
=
10.0 Hz, H-3H), 3.52, 3.47 and 3.46 (3s, 9H, 2 CO2Me
and OMe), 3.46 (dd, 1H, J1J,2J=4.0, J2J,3J=10.0 Hz, H-
2J), 3.32 (dd, 1H, H-2H), 2.86±2.56 (m, 1H, OH), 2.15,
2.11, 2.10 and 2.07 (4s, 12H, 4 Ac); 13C NMR
(400 MHz) d 170.5, 169.8, 169.4, 169.0 (4 CH3COO
(Ac), 2 CO2Me); 98.3 and 97.9 (2 C-1 iduronate); 97.8
and 97.0 (2 C-1 glucosamine); 63.3 and 63.2 (2 CN3);
55.2 (OCH3); 51.9 and 51.7 (2 CO2CH3). Anal. calcd for
C63H74N6O25 (1315.30): C, 57.51; H, 5.67; N, 6.39.
Found: C, 57.74; H, 5.73; N, 6.24.
Methyl [(methyl 2-O-acetyl-3-O-benzyl-4-O-levulinyl-ꢀ-
L-idopyranosyluronate)-(1!4)-(6-O-acetyl-2-azido-3-O-
benzyl-2-deoxy-ꢀ-D-glucopyranosyl)-(1!4)-(methyl 2-O-
acetyl-3-O-benzyl-ꢀ-L-idopyranosyluronate)]-(1!4)-6-O-
acetyl-2-azido-3-O-benzyl-2-deoxy-ꢀ-D-glucopyranoside
(8). A solution of 7 (323 mg, 0.36 mmol) and 4 (201 mg,
0.30) in dichloromethane (5 mL) was stirred for 30 min
at room temperature in the presence of 4 A molecular
sieves. A 0.1 M solution of TBDMSOTf in dichloro-
methane (1.1 mL, 0.11 mmol) was added at 10ꢀC.
Stirring was continued at 10ꢀC for 20 min. The reac-
tion mixture was neutralised (iPr2NH) and concentrated.
Puri®cation on Sephadex LH20 (dichloromethane:
ethanol, 1:1) gave 8 (345 mg, 82%). [a]d +9ꢀ (c 1.1,
Methyl [(methyl 2-O-acetyl-3-O-benzyl-4-O-p-methoxy-
benzyl-ꢀ-L-idopyranosyluronate)-(1!4)-(6-O-acetyl-2-
azido-3-O-benzyl-2-deoxy-ꢀ-D-glucopyranosyl)-(1!4)-
(methyl 2-O-acetyl-3-O-benzyl-ꢀ-L-idopyranosyluron-
ate)]-(1!4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-ꢀ-D-
glucopyranoside (10). To a solution of 9 (124 mg,
0.09mmol) in 3:1 diethyl ether:dichloromethane (0.2mL),
was added p-methoxybenzyl trichloroacetimidate (52 mg)
1
chloroform). H NMR (400 MHz) d 7.40±7.25 (m, 20H,

2008
C. Tabeur et al. / Bioorg. Med. Chem. 7 (1999) 2003±2012
followed by a solution of TfOH in ether (0.01 M,
10 mL). The reaction mixture was stirred at room tem-
perature for 2 h, neutralised (Et3N) and concentrated.
The residue was chromatographed on Sephadex LH20
(dichloromethane:ethyl acetate, 1:1) to give 10 (114 mg,
86%). [a]d +11ꢀ (c 0.8, chloroform). 1H NMR
(500 MHz) d 7.34±7.27 (m, 23H, arom.), 7.12±6.81 (m,
2H, arom.), 5.27 (d, 1H, J1I,2I=5.6 Hz, H-1I), 5.24 (d,
N-sulfation. The residue (41 mg) obtained above was
dissolved in water (1.5 mL). Pyridine:SO3 (10 mg,
0.06 mmol) was added in ®ve portions at t=0; 0.5; 1.0;
1.5; 2.0, and 3.0 h, the pH being maintained at 9.5 by
addition of 4 M aq NaOH. After 3 h, the reaction mix-
ture was layered onto a Sephadex G25 column eluted
with 0.2 M aq NaCl. After concentration and desalting
(Sephadex G25) 2 was lyophilized from water (55 mg,
1H, J1G,2G=3.6 Hz, H-1G), 4.94±4.88 (m, 2H, J1H,2H
=
98%). 1H NMR (500 MHz, D2O) d 5.38 (d, 1H, J1H,2H
3.8 Hz, H-1H), 5.17 (d, 1H, J1I,2I=3.0 Hz, H-1I), 5.14
(d, 1H, J1G,2G<1.0 Hz, H-1G), 4.98 (d, 1H, J1J,2J
=
3.0 Hz, H-1H, 2G), 4.88±4.80 (m, 1H, H-2I), 4.88±4.60
(m, 8H, 4 CH2Ph), 4.77 (d, 1H, J1J,2J=3.6 Hz, H-1J),
4.69 (d, 1H, J4G,5G=4.2 Hz, H-5G), 4.58 (d, 1H,
J4I,5I=5.0 Hz, H-5I), 4.46 (m, 2H, CH2Ph (p-methoxy-
benzyl), 4.40 (dd, 1H, J5J,6aJ=1.5, J6aJ,6bJ=12.6 Hz,
H-6aJ), 4.34 (dd, 1H, J5H,6aH=1.5, J6aH,6bH=12.4 Hz,
H-6aH), 4.24 (dd, 1H, J5J,6bJ=3.9 Hz, H-6bJ), 4.14 (dd,
=
3.5 Hz, H-1J), 4.80 (d, 1H, J4G,5G=2.2 Hz, H-5G), 4.69
(d, 1H, J4I,5I=2.4 Hz, H-5I), 4.35±4.20 (m, 6H, H-
2G,2I,6aH,6bH,6aJ,6bJ), 4.18±4.13 (m, 1H, H-3I),
4.06±4.04 (m, 2H, H-3G,4I), 4.01 (d, 1H, J4H,5H
=
9.8 Hz, H-5H), 3.89±3.84 (m, 2H, H-4G,5J), 3.75±3.60
(m, 4H, H-3H,3J,4H,4J), 3.40 (s, 3H, OMe), 3.26±3.20
(m, 2H, H-2H,2J); 13C NMR (200 MHz, D2O) d 102.08,
101.83, 100.93 and 99.19 (4 C-1); 60.69 and 60.40 (2
CNHSO3 ); 58.16 (OCH3).
1H, J5H,6bH=3.1 Hz, H-6bH), 4.00 (dd, 1H, J3G,4G
=
4.6 Hz, H-4G), 3.93 (dd, 1H, J3H,4H=J4H,5H=9.6 Hz,
H-4H), 3.91 (dd, 1H, J2G,3G=4.9 Hz, H-3G), 3.88±3.72
(m, 9H, CO2Me and H-3I,3J,4I,4J,5H,5J), 3.64 (dd, 1H,
J2H,3H=9.7 Hz, H-3H), 3.58 (s, 3H, CO2Me), 3.49 and
3.42 (2s, 6H, OMe), 3.38 (dd, 1H, J2J,3J=9.5 Hz, H-2J),
3.26 (dd, 1H, H-2H), 2.11, 2.05, 2.02 and 2.01 (4s, 12H,
4 Ac).
Phenyl 3-O-benzyl-4,6-O-isopropylidene-1-thio-ꢀ-L-ido-
pyranoside (12). Phenyl 2,4,6-tri-O-acetyl-3-O-benzyl-1-
thio-a-l-idopyranoside 11 (1.35 g) was dissolved in a 0.1
M solution of MeONa in MeOH. The solution was
stirred overnight, neutralised (IR120, H+), and con-
centrated. Camphorsulfonic acid (catalytic amount) was
added to a solution of the residue (1 g, 3 mmol) in 2,2-
dimethoxypropane (15 mL). The solution was stirred for
2 h, neutralized (Et3N), and concentrated. Puri®cation
on silica gel (cyclohexane:ethyl acetate, 6:1) gave 12
(926 mg, 84%). [a]d 173ꢀ (c 2.65, chloroform). 1H
NMR (250 MHz) d 7.55±7.20 (m, 10H, 2Ph), 5.64 (s,
1H, H-1), 4.90 and 4.60 (2d, 2H, J=12 Hz, CH2Ph),
4.30 (s, 1H, H-4), 4.14±3.91 (m, 5H, H-6a, H-2, H-5, H-
6b and OH), 3.67 (s, 1H, H-3), 1.46 (s, 6H, C(CH3)2).
Anal calcd for C22H26O5S (402.45): C, 65.66; H, 6.51.
Found: C, 65.65; H, 6.56.
Methyl [(sodium 2-O-sodium sulfonato-ꢀ-L-idopyrano
syluronate)-(1!4)-(2-deoxy-2-N-sodium sulfonato-6-O-
sodium sulfonato-ꢀ-D-glucopyranosyl)-(1!4)-(sodium 2-
O-sodium sulfonato-ꢀ-L-idopyranosyluronate)]-(1!4)-2-
deoxy-2-N-sodium sulfonato-6-O-sodium sulfonato-ꢀ-D-
glucopyranoside (1).
Saponi®cation. To a cooled ( 5ꢀC) solution of 10
(59 mg, 0.041 mmol) in THF (4.2 mL) was added drop-
wise 30% aq H2O2 (2.06 mL, 67.1 mmol) and 0.7 M aq
lithium hydroxyde (0.939 mL, 0.65 mmol). The reaction
mixture was stirred for 1 h at 5ꢀC, 2 h at 0ꢀC, then
overnight at room temperature. Methanol (3.75 mL),
then 4 N aq NaOH (1.02 mL, 4.08 mmol) were added
dropwise to the cooled (0ꢀC) solution. The reaction
mixture was stirred at room temperature for 12 h; acid-
i®ed with 6 N aq HCl (pH 1.5), and extracted with
CH2Cl2. The organic layer was washed with acidi®ed
(pH 3.5) aq sodium sul®te, water, dried (MgSO4), and
concentrated. The residue was puri®ed on Sephadex
LH20 (dichloromethane:ethanol, 1:1).
Phenyl 2-O-acetyl-3-O-benzyl-4,6-O-isopropylidene-1-thio-
ꢀ-L-idopyranoside (13). 12 (17.1 g, 0.04 mol) was acety-
lated (Ac2O, pyridine) to give 13 (17.1 g, 96%) after
column chromatography on silica gel (cyclohexane:ethyl
acetate, 6:1). [a]d +30ꢀ (c 0.4, chloroform); H NMR
1
(250 MHz) d 7.54±7.19 (m, 10H, 2Ph), 5.66 (s, 1H, H-1),
5.21 (s, 1H, H-2), 4.91 and 4.64 (2d, 2H J=11 Hz,
CH2Ph), 4.30 (s, 1H, H-4), 4.16 (dd, 1H, J5,6a=2.3,
J6a,6b=7 Hz, H-6a), 3.97±3.92 (m, 2H, H-6b and H-5),
3.69 (s, 1H, H-3), 2.09 (s, 3H, OAc), 1.46 (s, 6H,
C(CH3)2). Anal calcd for C24H28O6S (444.55): C, 64.85;
H, 6.35. Found: C, 64.90; H, 6.36.
Sulfonation. A solution of the above residue in DMF
(1.5 mL) was heated at 50ꢀC overnight in the presence of
Et3N:SO3 (purity 40%; 178 mg, 0.393 mmol). TLC
(ethyl acetate:pyridine:acetic acid:water, 26:12:2.6:7)
showed complete conversion of the starting compound.
Aqueous 10% NaHCO3 (3.69 mL, 2.9 mmol) was
added. The reaction mixture was stirred for 5 h at room
temperature and concentrated. A solution of the residue
in dichloromethane:ethanol (1:1); was ®ltered and con-
centrated. The residue was puri®ed on Sephadex LH20
(dichloromethane:ethanol, 1:1).
1,6-Di-O-acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-(2-O-
acetyl-3-O-benzyl-4,6-O-isopropylidene-ꢀ-L-idopyrano-
syl)-D-glucopyranose (15). A solution of 13 (95.3 mg,
0.21 mmol) and 14 (68 mg, 0.18 mmol) in dry dichloro-
methane (1.5 mL) was stirred for 30 min at room tem-
perature, in the presence of 4 A molecular sieves, and
cooled (0ꢀC). NIS (125 mg, 0.55 mmol), then a 0.15 M
solution of tri¯uoromethanesulfonic acid in dichlor-
omethane (0.37 mL, 0.055 mmol) were added. Sat aq
NaHCO3 was added after 10 min, the reaction mixture
was ®ltered through Celite, diluted with CH2Cl2,
Hydrogenolysis. A solution of the above obtained com-
pound in 2:3 tert-butanol:water (3 mL) was treated by
hydrogen (60 bar) in the presence of Pd/C catalyst
(10%, 69 mg) for three days, ®ltered, and concentrated.

C. Tabeur et al. / Bioorg. Med. Chem. 7 (1999) 2003±2012
2009
washed with sat aq Na2S2O3 then water, dried (MgSO4)
and concentrated. The residue was puri®ed on silica gel
(cyclohexane:ethyl acetate; 3:1) to give ®rst 15 (102 mg,
80%) then N-succinimidyl 2-O-acetyl-3-O-benzyl-4,6-O-
isopropylidene-a-l-idopyranoside 16 (9.1 mg, 10%).
0.09 and 0.02 (2s, 12H, 2 (CH3)2Si A and B). Anal.
calcd for C38H53N3O13Si.0.5H2O (796.91): C, 57.27; H,
6.83. Found: C, 57.48; H, 7.19.
1,6-Di-O-acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-(2-O-
acetyl-3-O-benzyl-4-O-p-methoxybenzyl-6-O-tert-butyl-
dimethylsilyl-ꢀ-L-idopyranosyl)-D-glucopyranose (19).
Compound 18 (720 mg, 0.91 mmol) was treated with p-
methoxybenzyl trichloroacetimidate as described for 10
1
15: H NMR (200 MHz) d (A: a anomer; B: b anomer)
7.42±7.20 (m, 20H, arom), 6.22 (d, 1H, J1A,2A=3.6 Hz,
H-1A), 5.46 (d, 1H, J1B,2B=8.4 Hz, H-1B), 5.03±4.91
(m, 2H, H-20A,20B), 4.92±4.82 (m, 2H, H-10A,10B),
4.90±4.60 (m, 8H, 4 CH2Ph), 4.42 (dd, 1H, J5B,6aB=2.0,
J6aB,6bB=9.8 Hz, H-6aB), 4.40 (dd, 1H, J5A,6aA<1.0,
J6aA,6bA=12.3 Hz, H-6aA), 4.07 (dd, 1H, J5B,6bB< 1.0 Hz,
H-6bB), 4.05 (dd, 1H, J5A,6bA<1.0 Hz, H-6bA), 3.90±
3.70 (m, 7H, H-4A,4B,40A,40B,5A,50A,50B), 3.74±3.50
1
to give 19 (745 mg, 90%). H NMR (200 MHz) (A: a
anomer, B: b anomer) d 7.33±7.17 (m, 24H, arom.),
7.13±6.78 (m, 4H, arom.), 6.17 (d, 1H, J1A,2A=3.6 Hz,
H-1A), 5.41 (d, 1H, J1B,2B=8.4 Hz, H-1B), 5.05±4.40
(m, 16H, 6 CH2Ph A and B and H-10A,10B,20A,20B),
4.40 (dd, 1H, J5B,6aB=2.2, J6aB,6bB=12.3 Hz, H-6aB),
4.31 (dd, 1H, J5A,6aA<1.0, J6aA,6bA=12.1 Hz, H-6aA),
4.22 (dd, 1H, J5A,6bA<1.0 Hz, H-6bA), 4.20 (dd, 1H,
J5B,6bB=4.0 Hz, H-6bB), 4.15±4.09 (m, 2H, H-50A,50B),
3.90±3.65 (m, 17H, 2 OCH3 A and B and H-
3A,3B,30A,30B,4A,4B,5A,60aA,60bA,60aB,60bB), 3.60±
3.42 (m, 5H, H-2A,2B,40A,40B,5B), 2.15, 2.14, 2.06 and
2.03 (4s, 18H, 6 Ac A and B), 0.89 (s, 18H, 2 (CH3)3C A
and B), 0.09 and 0.02 (2s, 12H, 2 (CH3)2Si A and B).
(m, 6H, H-2A,2B,3A,30A,30B,5B), 3.40 (dd, 1H, J5 B,6 aB
=
=
=
0
0
2.5, J6 aB,6 bB=6.4 Hz, H-60aB), 3.40 (dd, 1H, J2B,3B
0
0
0
9.5 Hz, H-3B), 3.35 (dd, 1H, J5 A,6 aA=2.8, J6 aA,6 bA
0
0
0
6.8 Hz, H-60aA), 3.17 (dd, 1H, J5 A,6 bA=3.1 Hz, H-60bA),
0
0
3.10 (dd, 1H, J5 B,6 bB=3.1Hz, H-60bB), 2.17, 2.15, 2.06,
2.04 and 2.03 (5s, 18H, 6 Ac A and B), 1.42, 1.32, 1.25 and
1.24 (4s, 12H, 2 (CH3)2C A and B). Anal. calcd for C45H43
N3O13 (713.74): C, 58.90; H, 6.08; N, 5.89. Found: C,
59.20; H, 6.36; N, 5.65. 16: 1H NMR (250 MHz) d 7.40±
0
0
7.20 (m, 5H, arom.), 5.88 (dd, 1H, J1,2=8.8, J2,3
=
1,6-Di-O-acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-(methyl
2-O-acetyl-3-O-benzyl-4-O-p-methoxybenzyl-ꢀ-L-idopyr-
anosyluronate)-D-glucopyranose (20). Chromium tri-
oxide (245 mg) in 3.5 M aq H2SO4 (1 mL) was added
slowly to a solution of 19 (825 mg, 0.91 mmol) in acetone
(7 mL) at 0ꢀC. The reaction mixture was poured in icy
water and extracted with CH2Cl2. The organic layer was
washed with water, dried, and concentrated. MeI
(0.260 mL) was added slowly to the solution of the resi-
due in DMF (1 mL) in the presence of KHCO3 (410 mg)
at 0ꢀC. The reaction mixture was stirred for 2 h at room
temperature, concentrated. A solution of the residue in
CH2Cl2 was washed with water, dried, and chromato-
graphed (cyclohexane:ethyl acetate, 3:1) to give 20
(269 mg, 40% two steps). Anal. calcd for C41H47N3O15
(821.83): C, 59.90; H, 5.76; N, 5.11. Found: C, 59.97; H,
5.82; N, 4.81.
10.2 Hz, H-2), 5.57 (d, 1H, H-1), 4.77 and 4.64 (2d, 2H,
Jgem=12.1 Hz, CH2Ph), 4.68±4.61 (m, 1H, H-5), 4.26
(dd, 1H, J3,4=J4,5=3.2 Hz, H-4), 4.01 (dd, 1H, J5,6a=
4.2, J6a,6b=12.8 Hz, H-6a), 3.81±3.72 (m, 2H, H-3,6b),
2.70±2.65 (m, 4H, C: OCH2 CH2C: O), 2.00 (s, 3H, Ac),
1.43 (s, 6H, (CH3)2C). Mass spectra: m/z 451 (M+NH4)+.
1,6-Di-O-acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-(2-O-
acetyl-3-O-benzyl-ꢀ-L-idopyranosyl)-D-glucopyranose
(17). A solution of 15 (1.16 g; 1.6 mmol) in 80% aq
AcOH (20 mL) was heated for 1 h at 100ꢀC and con-
centrated. Chromatography of the residue on silica gel
(toluene:ethyl acetate, 1:1) gave 17 (743 mg, 72%). Anal.
calcd for C32H39N3O13.1/3H2O (679.67): C, 56.54; H,
5.88. Found: C, 56.94; H, 5.82.
1,6-Di-O-acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-(2-O-
acetyl-3-O-benzyl-6-O-tert-butyldimethylsilyl-ꢀ-L-ido-
pyranosyl)-D-glucopyranose (18). tBDMSCl (230 mg),
Et3N (0.18 mL), and DMAP (5 mg) were added to a
solution of 17 (723 mg, 1.07 mmol) in CH2Cl2 (5 mL).
The reaction mixture was stirred overnight at room
temperature, diluted with water and with CH2Cl2. The
organic layer was dried (MgSO4) and concentrated.
Column chromatography of the residue on silica gel
(cyclohexane:ethyl acetate, 1:1) gave 18 (801 mg, 95%).
1H NMR (200 MHz) d7.40±7.20 (m, 20H, arom.), 6.21
6-O-Acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-(methyl 2-
O-acetyl-3-O-benzyl-4-O-p-methoxybenzyl-ꢀ-L-idopyr-
anosyluronate)-D-glucopyranose (21). Compound 20
(183 mg, 0.22 mmol) was treated with BnNH2 as
described for 6 to give 21 (128 mg, 74%) after chroma-
tography (toluene:ethyl acetate, 5:3). 1H NMR
(200 MHz): (A: a anomer, B b anomer) d 7.40±7.30 (m,
28H, arom.), 5.29 (d, 2H, J1 A,2 A=J1 B,2 B=5.0 Hz, H-
10A,10B), 5.26 (d, 1H, J1A,2A=3.0 Hz, H-1A), 4.95±4.82
(m, 2H, H-20A,20B), 4.80±4.40 (m, 14H, 6 CH2Ph and
H-50A,50B), 4.65 (d, 1H, J1B,2B=8.0 Hz, H-1B), 4.45±
4.35 (m, 2H, H-6aA,6aB), 4.25±4.10 (m, 2H, H-6bA,
0
0
0
0
(d, 1H, J1A,2A=4.1 Hz, H-1A), 5.46 (d, 1H, J1B,2B
=
8.2 Hz, H-1B), 4.95±4.55 (m, 12H, 4 CH2Ph and H-
10A,10B,20A,20B), 4.40 (dd, 1H, J5B,6aB=2.2, J6aB,6bB
12.3 Hz, H-6aB), 4.31 (dd, 1H, J5A,6aA<1.0, J6aA,6bA
=
=
6bB), 4.17±4.15 (m, 1H, H-5A), 3.97 (dd, 1H, J3A,4A
J4A,5A=8.5 Hz, H-4A), 3.95 (dd, 1H, J3B,4B=J4B,5B
=
=
12.1 Hz, H-6aA), 4.22 (dd, 1H, J5A,6bA<1.0 Hz, H-
6bA), 4.22 (dd, 1H, J5B,6bB=4.0 Hz, H-6bB), 4.20±4.12
(m, 2H, H-50A,50B), 3.90±3.70 (m, 6H, H-3A,4A,
40A,4B,40B,5A), 3.70±3.40 (m, 9H, H-2A,2B,30A,30B,
9.0 Hz, H-4B), 3.85±3.70 (m, 5H, H-3A,30A,30B,
40A,40B), 3.80 (s, 6H, 2 OCH3 (p-methoxybenzyl, A and
B), 3.55 (s, 6H, 2 CO2Me A and B), 3.52±3.30 (m, 4H,
H-2A,2B,3B, 5B), 3.00 (br.s, 1H, OH), 2.10, 2.05, 2.02
and 2.01 (4s, 12H, 4 Ac A and B), 1.70±1.60 (m, 1H,
OH). Anal. calcd for C39H45N3O14 (779.79): C, 60.07;
H, 5.89; N, 5.39. Found: C, 60.42; H, 5.88; N, 5.32.
5B,60aA,60bA,60aB,60bB), 3.40 (dd, 1H, J2B,3B=J3B,4B
=
9.8 Hz, H-3B), 2.17, 2.15, 2.06 and 2.05 (4s, 18H, 6 Ac A
and B), 0.91 and 0.86 (2s, 18H, 2 (CH3)3C A and B),

2010
C. Tabeur et al. / Bioorg. Med. Chem. 7 (1999) 2003±2012
[6-O-Acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-(methyl 2-
O-acetyl-3-O-benzyl-4-O-p-methoxybenzyl-ꢀ-L-idopyr-
anosyluronate)-ꢁ-D-glucopyranosyl] trichloroacetimidate
(22). Compound 21 (96 mg, 0.12 mmol) was treated with
CCl3CN as described for 7 to give 22 (89 mg, 85%) after
column chromatography (toluene:ethyl acetate:Et3N,
deoxy-2-N-sodium sulfonato-6-O-sodium sulfonato-ꢀ-D-
glucopyranosyl)-(1!4)-(sodium 2-O-sodium sulfonato-ꢀ-
L-idopyranosyluronate)]-(1!4)-2-deoxy-2-N-sodium sul-
fonato-6-O-sodium sulfonato-ꢀ-D-glucopyranoside (2).
Saponi®cation. To a cooled ( 5ꢀC) solution of 23
(116 mg, 0.056 mmol) in THF (5.8 mL) were added
dropwise 30% aq H2O2 (2.87 mL, 93.6 mmol) and 0.7 M
aq LiOH (1.31 mL, 0.92 mmol) The reaction mixture
was stirred for 1 h at 5ꢀC then 2 h at 0ꢀC, and over-
night at room temperature. Methanol (5.2 mL) followed
by 4 N aq NaOH (1.4 mL, 5.72 mmol) were added
dropwise to the cooled (0ꢀC) solution. The reaction
mixture was stirred at room temperature overnight,
acidi®ed (pH 1.5) with aq 6 M HCl, diluted with
CH2Cl2. The organic layer was washed with aq (acid-
i®ed to pH 3.5) Na2SO3, with water, dried (MgSO4),
and concentated. The residue was puri®ed on Sephadex
LH20 (dichloromethane:ethanol, 1:1).
5:3:0.1). [a]d
20ꢀ (c 1.2, chloroform). 1H NMR
(200 MHz) d 8.69 (s, 1H, NH), 7.40±6.80 (m, 14H,
arom.), 5.58 (d, 1H, J1,2=8.2 Hz, H-1), 5.23 (d, 1H,
0
0
0
J1 ,2 =4.8 Hz, H-1 ), 4.95±4.30 (m, 9H, 3 CH2Ph and H-
20,50,6a), 4.18 (dd, 1H, J5,6b=4.0, J6a,6b=12.3 Hz, H-
6b), 4.04 (dd, 1H, J3,4=J4,5=9.1 Hz, H-4), 3.85±3.70
(m, 2H, H-30,40), 3.80 (s, 3H, CO2Me), 3.70±3.55 (m,
2H, H-2,5), 3.55 (s, 3H, OCH3), 3.45 (dd, 1H, J2,3
=
J3,4=9.2 Hz, H-3), 2.03 and 2.01 (2s, 6H, 2 Ac). Anal.
calcd for C41H45Cl3N4O14 (924.18): C, 53.28; H, 4.90;
N, 6.06. Found: C, 53.51; H, 5.05; N, 6.06.
Methyl [(methyl 2-O-acetyl-3-O-benzyl-4-O-p-methoxy-
benzyl-ꢀ-L-idopyranosyluronate)-(1!4)-(6-O-acetyl-2-
azido-3-O-benzyl-2-deoxy-ꢀ-D-glucopyranosyl)-(1!4)-
(methyl 2-O-acetyl-3-O-benzyl-ꢀ-L-idopyranosyluronate)-
(1!4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-ꢀ-D-gluco-
pyranosyl)-(1!4)-(methyl 2-O-acetyl-3-O-benzyl-ꢀ-L-
idopyranosyluronate)]-(1!4)-6-O-acetyl-2-azido-3-O-
benzyl-2-deoxy-ꢀ-D-glucopyranoside (23). A solution of
22 (179 mg; 0.2 mmol) and 9 (200 mg; 0.15 mmol) in dry
toluene (5 mL) was stirred for 30 min in the presence of
4 A molecular sieves. A 1 M solution of TBDMSOTf
in CH2Cl2 (60 mL) was added to the cooled ( 20ꢀC)
reaction mixture. The solution was stirred at 20ꢀC for
3 h, neutralised (iPr2NH), ®ltered through Celite, and
concentrated. Chromatography on LH20 (dichloro-
methane:methanol, 1:1) gave 23 (237 mg, 80%). [a]d
+11ꢀ (c 0.45, chloroform). 1H NMR (500 MHz) d 7.40±
7.20 (m, 32H, arom.), 7.15±6.80 (m, 2H, arom.), 5.30±
Sulfonation. A solution of the above residue in DMF
(2.0 mL) was heated overnight at 50ꢀC in the presence of
Et3N:SO3 (purity 40%, 321.7 mg, 0.704 mmol). 10% aq
NaHCO3 (6.65 mL, 5.29 mmol) was added at the cooled
solution. The reaction mixture was stirred at room
temperature for 5 h, concentrated. A solution of the
residue in dichloromethane:ethanol (1:1) was ®ltered
and concentrated. The residue was chromatographied
on Sephadex LH20 (dichloromethane:ethanol, 1:1).
Hydrogenolysis. A solution of the above compound
(106 mg) in 2/3 tert-butanol/water (3 mL) was stirred
under hydrogen (60 bar) in the presence of Pd/C cata-
lyst (10%, 106 mg) for three days, ®ltered, and con-
centrated.
5.26 (m, 2H, H-1G,1G0), 5.24 (d, 1H, J1I,2I=3.8 Hz, H-
N-Sulfonation. The above residue (80 mg) was dissolved
in water (1.5 mL). Pyridine:SO3 (22 mg, 0.14 mmol) was
added in ®ve portions at t=0; 0.5; 1.0; 1.5; 2.0 h. During
this time the pH was maintained to 9.5 by addition of
4 M aq NaOH. After 3 h, the reaction mixture was
poured onto Sephadex G25 eluted with 0.2 M aq NaCl.
The combined fractions were lyophilized and desalted
on Sephadex G25 to give 2 (54 mg, 46% over the four
steps).
0
0
0
1I), 4.96 (d, 1H, J1H ,2H =3,4 Hz, H-1H ), 4.94±4.88 (m,
2H, H-2G,2I), 4.92 (d, 1H, J1H,2H=3.0 Hz,0H-1H), 4.87
0
0
0
0
(dd, 1H, J1G ,2G =J2G ,3G =5.2 Hz, H-2G ), 4.87±4.60
(m, 12H, 6 CH2Ph), 4.82 (d, 1H, J4I,5I=3.0 Hz, H-5I),
0
0
=
4.76 (d, 1H, J1J,2J=3.0 Hz, H-1J), 4.58 (d, 1H, J4G ,5G
5.4 Hz, H-5G0), 4.56 (d, 1H, J4G,5G=6.0 Hz, H-5G),
4.47±4.41 (m, 2H, CH2Ph (p-methoxybenzyl)), 4.40 (dd,
1H, J5J,6aJ<1.0, J6aJ,6bJ 12.1 Hz, H-6aJ), 4.36 (dd, 1H,
J5H,6aH<1.0, J6aH,6bH=12.0 Hz, H-6aH), 4.33 (dd, 1H,
0
1H, J5J,6bJ=3.0 Hz, H-6bJ), 4.18 (dd, 1H, J5H,6bH
1H NMR (500 MHz-D2O) d 5.39 (d, 1H, J1H ,2H
3.5 Hz, H-1H0), 5.36 (d, 1H, J1H,2H=3.5 Hz, H-1H),
5.17 (d, 1H, J1I,2I=3.8 Hz, H-1I), 5.16 (d, 1H, J1G,2G=
0
0
0
0
J5H ,6aH <1.0, J6aH ,6bH =11.5 Hz, H-6aH ), 4.23 (dd,
0
0
=
=
3.1 Hz, H-6bH), 4.15 (dd, 1H, J5H ,6bH=4.1 Hz, H-
0
6bH0), 4.02±3.97 (m, 2H, H-4G,4I), 3.94 (dd, 1H,
0
0
3.8 Hz, H-1G), 5.14 (d, 1H, J1G ,2G <1.0 Hz, H-1G ),
0
0
0
0
0
0
0
0
=
J3H ,4H =J4H ,5H =10.2 Hz, H-4H ), 3.93±3.89 (m, 2H,
H-3G,3I), 3.89±3.72 (m, 11H, OMe, H-3G0,3J,4G0,
4H,4J,5H,5H0,5J), 3.65 (dd, 1H, J2H,3H=J3H,4H0 9.8 Hz,
4.98 (d, 1H, J1J,2J=3.5 Hz, H-1J), 4.81 (d, 1H, J4G ,5G
2.2 Hz, H-5G0), 4.77 (d, 1H, J4I,5I=2.7 Hz, H-5I), 4.69
0
0
=
(d, 1H, J4G,5G=3.0 Hz, H-5G), 04.36 (dd, 1H, J5H ,6aH
0
0
0
0
H-3H), 3.62 (dd, 1H, J2H ,3H =9.6 Hz, H-3H ), 3.56,
3.55 and 3.51 (3s, 9H, 3 CO2Me), 3.43 (s, 3H, OMe),
3.38 (dd, 1H, J2J,3J=9.7 Hz, H-2J), 3.27 (dd, 1H, H-
2H0), 3.25 (dd, 1H, H-2H), 2.11, 2.05, 2.02, 2.01 and
2.00 (5s, 18H, 6 Ac).
2.8, J6aH ,6bH =11.6 Hz, H-6aH ), 4.34±4.20 (m, 8H, H-
2G,2G0,2I,6aH,6bH,6aJ,6bJ,6bH0), 4.18±4.12 (m, 2H,
H-3G,3I), 4.12±4.04 (m, 3H, H-3G0,4G,4I), 4.03±3.91
(m, 4H, H-4G0,4J,5H,5H0), 3.75±3.60 (m, 6H, H-
3H,3H0,3J,4H,4H0,5J), 3.40 (s, 3H, OMe), 3.24 (2dd,
0
0
0
2H, J2H ,3H =10.1 Hz, H-2H and J2J,3J=10.1 Hz, H-
2J), 3.23 (dd, 1H, J2H,3H=9.0 Hz, H-2H); 13C NMR
(200 MHz-D2O) d 98.08, 97.60, and 96.86 (3 C-1 idur-
onate); 95.47 and 95.24 (3 C-1 glucosamine); 63.77 and
63.51 (3 CNHSO3 ); 54.76 (OCH3).
Methyl [(sodium 2-O-sodium sulfonato-ꢀ-L-idopyrano-
syluronate)-(1!4)-(2-deoxy-2-N-sodium sulfonato-6-O-
sodium sulfonato-ꢀ-D-glucopyranosyl)-(1!4)-(sodium 2-
O-sodium sulfonato-ꢀ-L-idopyranosyluronate)-(1!4)-(2-

C. Tabeur et al. / Bioorg. Med. Chem. 7 (1999) 2003±2012
2011
Methyl [(methyl 2-O-acetyl-3-O-benzyl-4-O-levulinyl-ꢀ-
L-idopyranosyluronate)-(1!4)-(6-O-acetyl-2-azido-3-O-
benzyl-2-deoxy-ꢀ-D-glucopyranosyl)-(1!4)-(methyl 2-
O-acetyl-3-O-benzyl-ꢀ-L-idopyranosyluronate)-(1!4)-
(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-ꢀ-D-glucopyr-
anosyl)-(1!4)-(methyl 2-O-acetyl-3-O-benzyl-ꢀ-L-ido-
pyranosyluronate)]-(1!4)-6-O-acetyl-2-azido-3-O-benzyl-
2-deoxy-ꢀ-D-glucopyranoside (24). A solution of 7
(40 mg, 0.044 mmol), and 9 (57.1 mg, 0.042 mmol) in dry
dichloromethane (1 mL) was stirred for 30 min at room
temperature in the presence of 4 A molecular sieves. A 1
sulfate and 4 mM of glutamine. For cell growth mea-
surements, HASMC were seeded in DMEM +0.2%
FCS at 3.104 cells per well (24-well plates) for 3 days in
a humidi®ed CO2 incubator maintained at 37ꢀC. Cul-
ture medium was then removed and cells were seeded in
DMEM+0.2% FCS with 30 nM of FGF-2. The inhi-
bition of proliferation induced by FGF-2 by various
concentrations of heparin and compounds 1 and 2 was
studied. After 24 h in culture, triplicate plates were
trypsinized and the cells counted with a Coulter counter
(Coultronics, France).
M
solution of TBDMSOTf in dichloromethane
(46.2 mL, 45.3 mmol) was added to the cooled ( 20ꢀC)
solution. The reaction mixture was stirred for 20 min,
neutralised (di-isopropylamine) ®ltered through Celite,
and concentrated. The residue was puri®ed using
Sephadex LH20 chromatography (dichloromethane:
methanol, 1:1) to give 24 (55 mg, 81%). [a]d +8ꢀ (c 2.0,
125I-FGF-2 binding to HASMC. HASMC were cultured
as described above and seeded in 24-well cluster plates
(3.104 cells/well) in DMEM+10% FCS. Cells were
routinely used between the 3rd and 10th passage. Sub-
con¯uent cultures (about 3.105 cells/well) were washed
twice with ice-cold DMEM supplemented with 25 mM
HEPES (pH 7.4), 0.3 mg/mL of soybean trypsin inhi-
bitor (STI), 0.5 mg/mL of bacitracin and 0.2% of gela-
tin. Incubations were carried out in a total 200 mL
volume of HEPES 25 mM (pH 7.4) supplemented with
0.3 mg/mL of STI, 0.5 mg/mL of bacitracin and 0.2% of
gelatin (bu€er A) that contained 45 pM 125I-FGF-2
(110 mCi/mg) and increasing concentrations of the tested
compounds. Triplicate incubations were carried out at
7ꢀC for 3 h. Nonspeci®c binding was considered as the
value obtained in the presence of a 100-fold excess on
FGF-2. In all experiments, representative wells were
trysinizsed and cells were counted with a Coulter coun-
ter (Coultronics, France).
1
chloroform). H NMR (500 MHz) (7.40±7.30 (m, 30H,
arom), 5.30 (d, 1H, J1I,2I=4.8 Hz, H-1I), 5.28 (2d, 2H,
0
5.12 (dd, 1H, J3G ,4G =J4G ,5G =4.0 Hz, H-4G ), 5.11 (d,
0
J1G,2G=4.0 Hz, H-1G and J1G ,2G =3.0 Hz, H-1G ),
0
0
1H, J1H ,2H =3.5 Hz, H-1H ), 4.97 (dd, 1H, J2G,3G
0
0
0
0
0
3.7 Hz, H-2G and d, 1H, J1H,2H=3.5 Hz, H-1H), 4.94
0
0
=
(dd, 1H, J2I,3I=5.0 Hz, H-2I), 4.91±4.87 (m, 1H, H-
0
2G0), 4.87 (d, 1H, J4G ,5G =2.5 Hz, H-5G ), 4.82 (d, 1H,
J1J,2J=3.7 Hz, H-1J), 4.64 (d, 1H, J4G,5G=4,5 Hz, H-
5G), 4.91±4.64 (m, 13H, 6 CH2Ph and H-5I), 4.45 (dd,
0
0
0
0
0
0
0
1H, J5H ,6aH <1.0, J6aH ,6bH =12.0 Hz, H-6aH ), 4.44
(dd, 1H, J5J,6aJ<1.0, J6aJ,6bJ=12.0 Hz, H-6aJ), 4.41 (dd,
1H, J5H,6aH<1.0, J6aH,6bH=10.5 Hz, H-6aH), 4.28 (dd,
1H, J5H ,6bH 3.5 Hz, H-6bH0), 4.26±4.18 (m, 2H, H-
6bH,6bJ), 4.05±4.01 (m, 2H, H-4G,4I), 3.98±3.81 (m,
10H, H-3G,3G0,3I,3J,4H,4H0,4J,5H,5H0,5J), 3.70 (dd,
1H, J2H,3H=9.0, J3H,4H=10.0 Hz, H-3H), 03.66 (dd, 1H,
0
0
References
0
0
0
0
J2H ,3H =9.0 Hz, J3H ,4H =10.0 Hz, H-3H ), 3.52, 3.51
and 3.46 (3s, 12H, 3 CO2Me and OMe), 3.46 (dd, 1H,
J2J,3J=10.0 Hz, H-2J), 3.34 (dd, 1H, H-2H0), 3.32 (dd,
1H, H-2H), 2.82±2.45 (m, 4H, C: OCH2CH2C: O), 2.21,
2.16, 2.13, 2.11 and 2.10 (5s, 18H, 6 Ac), 2.06 (s, 3H,
CH3C: O).
1. Hu, M. C. T.; Qiu, W. R.; Wang, Y. P.; Hill, D.; Ring, B.
D.; Scully, S.; Bolon, B.; DeRose, M.; Luethy, R.; Simonet,
W. S.; Arakawa, T.; Danilenko, D. M. Mol. Cell. Biol. 1998,
18, 6063.
2. Basilico, C.; Moscatelli, D. Adv. Cancer Res. 1992, 59, 115.
3. Brickman, Y. G.; Ford, M. D.; Gallagher, J. T.; Nurcombe,
V.; Bartlett, P. F.; Turnbull, J. E. J. Biol. Chem. 1998, 273,
4350.
4. Tyrrell, D. J.; Isihara, M.; Rao, N.; Horne, A.; Kiefer, M.
C.; Stauber, G. B.; Lam, L. H.; Stack, R. J. J. Biol. Chem.
1993, 268, 4684.
5. DiGabriele, A. D.; Lax, I.; Chen, D. I.; Svahn, C.-M.; Jaye,
M.; Schlessinger, J.; Hendrickson, W. A. Nature 1998, 393,
812.
6. Moy, F. J.; Safran, M.; Seddon, A. P.; Kitchen, D.; Bohlen,
P.; Aviezer, D.; Yayon, A.; Powers, R. Biochemistry 1997, 36,
4782.
7. Maccarana, M.; Casu, B.; Lindahl, U. J. Biol. Chem. 1993,
268, 23898.
8. Ornitz, D. M.; Herr, A. B.; Nilsson, M.; Westman, J.;
Svahn, C.-M.; Walksman, G. Science 1995, 268, 432.
9. Kovensky, J.; Duchaussoy, P.; Bono, F.; Salmivirta, M.;
Sizun, P.; Herbert, J.-M.; Petitou, M.; Sinay, P. Bioorg. Med.
Chem. 1998, 7, 1567.
Cell culture experiments
Materials. Human aortic smooth muscle cells
(HASMC) were purchased from Clonetics (Tebu, Le
Perray, France). Dulbecco's modi®ed Eagle medium
(DMEM) and phosphate bu€ered saline (PBS) were
from Biochrom KG (Poly-Labo, Strasbourg, France).
Foetal calf serum (FCS), penicillin, streptomycin, and
glutamine were from Boerhinger Mannheim (Meylan,
France). 24-well cluster plates were purchased from
Falcon (Becton Dickinson, Le Pont de Claix, France).
FGF-2 and 125I-FGF-2 were from Amersham (Les Ulis,
France). Heparin was from Sano® Recherche (Tou-
louse, France), HEPES, soybean trypsin inhibitor (STI),
bacitracin were from Sigma (L'Isle d'Abeau, France),
and gelatin, NaCl and NaOH were from Prolabo (Gra-
dignan, France).
10. Koeners, H. J.; Verhoeven, J.; van Boom, J. H. Recl. Trav.
Chim. Pays Bas 1981, 100, 65.
11. Tabeur, C.; Machetto, F.; Mallet, J.-M.; Petitou, M.;
Sinay, P. Carbohydr. Res. 1996, 281, 253.
12. Jaurand, G.; Tabeur, C.; Petitou, M. Carbohydr. Res.
1994, 255, 295.
Cell culture and growth measurements. HASMC were
routinely cultured in DMEM supplemented with 10%
FCS, 50 U/mL of penicillin, 50 mg/mL of streptomycin

2012
C. Tabeur et al. / Bioorg. Med. Chem. 7 (1999) 2003±2012
13. Takaku, H.; Ueda, S.; Ito, T. Tetrahedron Lett. 1983, 24,
5363.
14. Nakajima, N.; Horita, K.; Abe, R.; Yonemitsu, O. Tetra-
hedron Lett. 1988, 29, 4139.
15. Iversen, T.; Bundle, K. R. J. Chem. Soc., Chem. Commun.
1981, 1240.
16. White, J. D.; Reddy, G. N.; Spessard, G. O. J. Am. Chem.
Soc. 1988, 110, 1624.
17. Widmen, U. Synthesis 1987, 568.
18. Lei, P. S.; Duchaussoy, P.; Sisun, P.; Mallet, J.-M.; Petitou,
M.; Sinay, P. Bioorg. Med. Chem. 1998, 6, 1337.