A synthetic heparan sulfate pentasaccharide, exclusively containing L-iduronic acid, displays higher affinity for FGF-2 than its D-glucuronic acid-containing isomers

Article abstract of DOI:10.1016/S0968-0896(99)00106-6

It has been suggested that the FGF-2 binding site on heparan sulfate chains is a trisulfated pentasaccharide containing three hexuronic acid units. The configuration at C-5 of two of them being undetermined, we have synthesized the four possible pentasaccharides, and have evaluated their FGF-2 binding affinity through in vitro biological assays. The pentasaccharide containing L-iduronic acid as the sole hexuronic acid showed higher affinity for FGF-2 than the other pentasaccharides, where one hexuronic acid unit at least is D-glucuronic acid. Copyright (C) 1999.

Full text of DOI:10.1016/S0968-0896(99)00106-6

Bioorganic & Medicinal Chemistry 7 (1999) 1567±1580
A Synthetic Heparan Sulfate Pentasaccharide, Exclusively
Containing L-Iduronic Acid, Displays Higher Anity for FGF-2
than its D-Glucuronic Acid-Containing Isomers
Jose Kovensky, a,1 Philippe Duchaussoy, b Francoise Bono, b Markku Salmivirta, c
Philippe Sizun, d Jean-Marc Herbert, b Maurice Petitou b,* and Pierre Sinay a,2
a
Â
Â
Â
Ecole Normale Superieure, Departement de Chimie, Associe au CNRS, 24 rue Lhomond, 75231 Paris Cedex 05, France
bSano® Recherche, Haemobiology Research Department, 135 route d'Espagne, 31036 Toulouse Cedex, France
cUppsala University, Department of Medical and Physiological Chemistry, Biomedical Center, PO Box 575, S-75123 Uppsala, Sweden
dSano® Recherche, Exploratory Research Department, 371 rue du Professeur Joseph Blayac, 34184 Montpellier Cedex, France
Received 16 September 1998; accepted 1 February 1999
AbstractÐIt has been suggested that the FGF-2 binding site on heparan sulfate chains is a trisulfated pentasaccharide containing
three hexuronic acid units. The con®guration at C-5 of two of them being undetermined, we have synthesized the four possible
pentasaccharides, and have evaluated their FGF-2 binding anity through in vitro biological assays. The pentasaccharide con-
taining l-iduronic acid as the sole hexuronic acid showed higher anity for FGF-2 than the other pentasaccharides, where one
hexuronic acid unit at least is d-glucuronic acid. # 1999 Published by Elsevier Science Ltd. All rights reserved.
Introduction
sulfated oligosaccharide fragments containing l-iduro-
nic acid as the only hexuronic acid. Nevertheless, in one
occasion a nonsulfated, and d-glucuronic acid contain-
ing trisaccharide, was found to bind and activate FGF-
2.8 Recently, the binding site for FGF-2 was proposed
to be a pentasaccharide (Fig. 1) containing two N-sul-
fonato groups and one O-sulfonato group.9 However,
the methods used in this work did not allow the authors
to fully elucidate the exact nature (l-iduronic or d-glu-
curonic) of two (Fig. 1, units A and C) out of the three
uronic acid units present in this pentasaccharide
sequence.
The interaction of growth factors and extracellular
matrix components is a key element for the control of
cell growth and cell di€erentiation in pluricellular
organisms. In this regard, proteoglycans have been
considered as critical modulators of ®broblast growth
factors (FGF) activity, and several models have been
proposed to explain this function of heparan sulfate.1±4
Heparan sulfate is a complex polysaccharide composed
of alternating units of partially O-sulfonated hexuronic
acid (d-glucuronic or l-iduronic) and glucosamine (N-
acetyl or N-sulfonato). Most of the structural studies5±7
dealing with the oligosaccharide sequences involved
in heparan sulfate interaction with FGF-2 pointed to
To solve this problem, we decided to synthesize the four
possible pentasaccharide isomers (Fig. 2) and to assess
their anity for FGF-2, as well as their properties
against the biological activity of FGF-2. We recently
reported the synthesis of the pentasaccharide 1 that
contains exclusively l-iduronic acid.10 In the present
article, we would like to describe the synthesis of the
three other possible isomers 14, 22, and 24. We also
would like to report the results of in vitro experiments
where the activity of the four pentasaccharides was
compared in assays involving their interaction with
FGF-2.
Key words: Heparan sulfate; growth factors; carbohydrates; glyco-
sylation.
* Corresponding author. Tel.: +33-(0)561-162390; fax: +33-(0)561-
162286; e-mail: maurice.petitou@sano®.com
Present address: Departamento de Quõmica Organica; Facultad de
Ciencias Exactas y Naturales; Ciudad Universitaria-Pabellon II-3ꢀ
piso; 1428 Buenos Aires, Argentina.
Second corresponding author. Tel.: +33-(0)144-323390; fax: +33-
(0)144-323397; e-mail: pierre.sinay@ens.fr
1
2
0968-0896/99/$ - see front matter # 1999 Published by Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00106-6

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J. Kovensky et al. / Bioorg. Med. Chem. 7 (1999) 1567±1580
Results and Discussion
Chemical synthesis of the pentasaccharides 14, 22, and 24
The synthesis of the target heparan sulfate fragments
14, 22, and 24 has been achieved through successive
additions of the appropriate hexuronic acid-glucosa-
mine disaccharide building block to the 4-position of
the hexuronic acid unit standing at the non-reducing
end of the growing chain. As an example of this strategy
the retrosynthesis of 14 is depicted in Scheme 1.
Figure 1. Pentasaccharide sequence proposed9 to represent the FGF-2
binding site on heparan sulfate. The con®guration at C-5 of units A
and C (d-gluco or l-ido) was not reported.
During the building up of the pentasaccharide back-
bone, the allyl ether (All) protecting group has been
used to temporarily protect the only hydroxyl group to
be ultimately sulfonated. This choice allowed us to use
benzyl ethers and any esters to protect other hydroxyl
groups. Four building blocks 8, 9, 12, and 19 have been
selected (Scheme 2). The synthesis of 9 and 12 has
already been described.10
On these premises, the synthesis of the glucuronic acid-
containing disaccharide donor 8 (Scheme 3) started
from 1-O-acetyl-2-azido-3,6-di-O-benzyl-2-deoxy-a,b-d-
glucopyranose11 (2) which was condensed with phenyl
2,3-di-O-benzoyl-4,6-O-benzylidene-1-thio-b-d-gluco-
pyranoside12 (3), in the presence of N-iodosuccinimide
and tri¯uoromethanesulfonic acid,13,14 to give di-
0
saccharide 4 in 92% yield. The large J1 2 coupling con-
0
1
stant (8.2 Hz) observed in the H NMR of 4 con®rmed
the expected b con®guration. Hydrolytic removal of the
benzylidene group, followed by selective silylation of the
primary alcohol and levulinoylation of the secondary
Figure 2. Structure of the four pentasaccharides 1, 14, 22, and 24.
Scheme 1. Retrosynthesis of pentasaccharide 14. In this scheme, the selected depicted conformation of the two l-iduronic acid units in 14 is the
major one (see Table 2). In protected derivatives 11, 12, and 13, l-iduronic acid units adopt the depicted 1C4 conformation.

J. Kovensky et al. / Bioorg. Med. Chem. 7 (1999) 1567±1580
1569
Scheme 2. Stucture of the four building blocks used in the present work.
Scheme 3. Synthesis of pentasaccharide 14. Reagents: (a) NIS, TfOH, CH2Cl2; (b) 70% TFA; (c) TBDMSCl, Et3N, DMAP, CH2Cl2, then Lev2O,
Et3N; (d) CrO3, aq H2SO4, acetone, then CH2N2; (e) BnNH2, ether; (f) DBU, Cl3CCN; (g) TBDMSOTf; (h) N2H4, AcOH, pyridine; (i)
TBDMSOTf, toluene; (j) IR complex, THF, H2 then HgO/HgCl2, acetone/H2O; (k) Et3N/SO3, DMF; (l) LiOH/H2O2 then NaOH; (m) H2, Pd/C; (n)
pyridine/SO3, aq NaHCO3.
alcohol, gave 5 (50% over the three steps). The latter
was oxidized under Jones conditions, and the acid was
esteri®ed to give 6. Selective anomeric deacetylation
using benzylamine15 gave 7 as a syrup, which was
immediately engaged in a reaction with trichloro-
acetonitrile and DBU16 in dichloromethane to provide,
after silica gel chromatography, the key glycosyl donor
imidate 8. A 1.5 molar excess of this imidate was con-
densed with the known10 methyl (methyl 2-O-allyl-3-O-
benzyl-a-l-idopyranosid)uronate 9 in dry toluene, in the
presence of tert-butyldimethylsilyl tri¯ate, to give, after
chromatography, the trisaccharide 10 (75%). No b-cou-
pled product could be detected. Removal of the levuli-
noyl group of 10 with hydrazine17 a€orded the
trisaccharide acceptor 11 (90%). Reaction of 11 with
(methyl 2,3,4-tri-O-acetyl-a-l-idopyranosyluronate)-
(1!4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-1-O-tri-
chloroacetimidoyl-d-glucopyranose10 12, under the con-
ditions described for 10, gave 13, the fully protected
precursor of 14, in 64% yield. Deallylation of 13 in the
presence of 1,5 cyclooctadiene-bis-[methyldiphenylphos-
phine]-iridium hexa¯uorophosphate,18 followed by
hydrolysis of the vinyl ether in the presence of mercury
oxide and mercuric chloride, yielded the expected hy-
1
droxy compound. H NMR analysis showed that dur-
ing isomerization 10% of the starting material was
converted into the undesired propyl ether. Sulfonation
of the alcohol in DMF, using sulfur trioxide-triethyl-
1
amine, was checked by mass spectrometry and by H
NMR which showed the expected 0.8 ppm down®eld
shift of the corresponding H-2 proton. Saponi®cation
was followed by hydrogenolysis of the benzyl ethers and
concomitant generation of the amino groups from the
azido groups. Selective N-sulfonation was ®nally carried
out in water under controlled pH (9.5), as previously
described.19
The synthesis of the glucuronic acid-containing glycosyl
donor disaccharide 19 (Scheme 4) started from methyl
(2,3,4-tri-O-acetyl-1-O-trichloroacetimidoyl-d-glucopyr-

1570
J. Kovensky et al. / Bioorg. Med. Chem. 7 (1999) 1567±1580
Scheme 4. Synthesis of pentasaccharide 22. Reagents: (a) TMSOTf, CH2Cl2; (b) BnNH2, ether; (c) K2CO3, Cl3CCN; (d) TBDMSOTf, toluene; (e)
IR complex, THF, H2 then HgO/HgCl2, acetone/H2O; (f) Et3N/SO3, DMF; (g) LiOH/H2O2 then NaOH; (h) H2, Pd/C; (i) pyridine/SO3, aq
NaHCO3.
anose)uronate20 (15) and 1,6-di-O-acetyl-2-azido-3-O-
benzyl-2-deoxy-b-d-glucopyranose11 (16), which were
condensed in dichloromethane, in the presence of tri-
methylsislyl tri¯ate, to give the disaccharide 17 (66%
fully protected precursor of the pentasaccharide 24.
This latter was then obtained (Scheme 5) after deally-
lation, O-sulfonation, catalytic hydrogenation, and N-
sulfonation, as described above for the preparation of
14.
0
0
after column chromatography). The large (8.5 Hz) J1 2
coupling constant observed in the 1H NMR spectrum of
17 con®rmed the b con®guration expected from the
participating acetate group at position 2 of the donor.
Selective anomeric deacetylation15 gave crude 18 and
treatment with trichloroacetonitrile and potassium car-
bonate21,22 as a base yielded the glycosyl donor 19 (65%
from 17) which was immediately condensed with methyl
[methyl (methyl 2-O-acetyl-3-O-benzyl-a-l-idopyrano-
syluronate)-(1!4)-(6-O-acetyl-2-azido-3-O-benzyl-2-
deoxy-a-d-glucopyranosyl)-(1!4)-2-O-allyl-3-O-benzyl-a-
l-idopyranosid]uronate10 20, in dichloromethane, in the
presence of tert-butyldimethylsilyl tri¯ate, to give 21
(64% from 20) the fully protected precursor of 22. The
latter was then obtained after deallylation, O-sulfona-
tion, catalytic hydrogenation, and N-sulfonation, as
described above for the preparation of 14.
Conformation of L-iduronic acid
l-iduronic acid and its derivatives are present in solu-
tion as an equilibrium between three iso-energetic con-
formers: 1C4, 4C1, and 2S0.23 This conformational
¯exibility is thought to play a prominent role in the
interaction of l-iduronic acid containing oligosacchar-
ides with proteins.24 The contribution of each con-
former to the conformational equilibrium can be
deduced from 1H NMR interproton coupling con-
stants.23 To do so the spectrum of all four penta-
saccharides was recorded at 500 MHz, all proton
chemical shifts were carefully assigned (Table 1), and
the interproton coupling constants were measured from
1-D spectra and, when needed, completed with selective
one dimensional COSY or TOCSY spectra25 recorded
in 128 scans. Coupling constants were further simulated
using the NMRSIM 2.6.1 software.19 The results of the
Finally, reaction of 19 and 11, under the conditions
reported for the preparation of 20,10 provided 23, the
Scheme 5. Synthesis of pentasaccharide 24. Reagents: (a) TBDMSOTf, toluene; (b) IR complex, THF, H2 then HgO/HgCl2, acetone/H2O; (c) Et3N/
SO3, DMF; (d) LiOH/H2O2 then NaOH; (e) H2, Pd/C; (f) pyridine/SO3, aq NaHCO3.

J. Kovensky et al. / Bioorg. Med. Chem. 7 (1999) 1567±1580
1571
Table 1. 1H NMR data on 1, 14, 22 and 24
A
B
C
D
E
Unit Hn
d
Jn,n+1
d
Jn,n+1
d
Jn,n+1
d
Jn,n+1
d
Jn,n+1
1
1
2
3
4
5
4.712
3.422
3.596
3.783
4.483
6.1
8.4
7.8
4.9
5.331
3.213
3.611
3.710
3.830
3.7
10.5
9.3
10.2
2.5
4.895
3.684
4.057
4.007
4.748
3.1
5.2
3.8
2.8
5.309
3.199
3.628
3.664
3.852
3.7
10.5
9.3
10.2
2.5
4.991
4.178
4.193
3.999
4.401
2.3
4.7
3.5
2.6
2.5
13.0
3.1
13.0
6
60
3.793
3.814
3.829
3.762
14
22
24
1
2
3
4
5
4.724
3.435
3.608
3.791
4.493
6.0
8.3
7.2
5.0
5.593
3.248
3.599
3.711
3.784
3.8
10.3
8.7
9.4
2.5
4.490
3.373
3.815
3.751
3.767
7.7
9.5
9.6
10.0
5.313
3.212
3.654
3.667
3.875
3.5
10.1
8.5
10.2
2.8
5.002
4.182
4.197
4.002
4.404
2.1
4.7
3.5
2.5
2.5
2.8
12.8
6
60
3.794
3.794
3.851
3.823
1
2
3
4
5
4.467
3.340
3.482
3.473
3.710
8.0
9.6
8.8
9.7
5.326
3.195
3.655
3.674
3.846
3.7
10.1
9.3
10.1
3.1
4.891
3.681
4.056
4.002
4.747
3.1
5.4
3.8
2.8
5.304
3.195
3.623
3.600
3.860
3.7
10.5
9.3
10.2
2.5
4.988
4.175
4.190
4.994
4.397
2.3
4.7
3.8
2.6
2.8
12.8
3.1
12.5
6
60
3.857
3.795
3.845
3.757
1
2
3
4
5
4.473
3.344
3.489
3.480
3.718
8.0
9.4
9.2
9.6
5.600
3.236
3.658
3.669
3.799
3.7
10.8
9.7
10.4
2.5
4.494
3.375
3.822
3.753
3.773
7.9
9.5
9.6
9.8
5.315
3.215
3.655
3.673
3.877
3.5
10.6
8.5
10.2
2.8
5.002
4.184
4.198
4.004
4.405
1.9
4.7
3.5
2.5
2.5
13.0
2.8
12.8
6
60
3.767
3.764
3.852
3.818
Table 2. Conformational state of l-iduronic acid unitsa (1C4/4C1/2S0)
Biological assays
deduced from 1H NMR coupling constants23
Binding experiments. Pentasaccharides 1, 14, 22, and 24
were studied for their ability to inhibit the binding of
soluble FGF-2 to heparin or heparan sulfates from
intestinal or aortic origins, using the Biacore1 technol-
ogy.28,29 In every case the pentasaccharides weakly
inhibited the binding of FGF-2 to both types of heparan
sulfate (Fig. 3) but pentasaccharide 1 was the most
e€ective compound, and dose dependently inhibited the
binding of heparin and both types of heparan sulfate
(Fig. 4). The three other pentasaccharides were equi-
potent and showed weaker potency compared to 1.
unit A
unit C
unit E
1a
14
22
24
18/78/04
19/68/13
nr
65/12/23
nrb
63/10/27
nr
74/07/19
75/06/19
74/12/13
76/05/19
nr
Preliminary erroneous studies10 on this compound led to slightly
di€erent conclusions.
a
b
nr: not relevant.
computed conformation equilibrium are reported in
Table 2. When unsulfated l-iduronic acid is located at
the non-reducing end, its predominant conformer is 4C1.
When it is surrounded by two N-sulfonato glucos-
Cell culture experiments
1
amines, the C4 conformer predominates and the skew-
2
Inhibition of FGF-2 binding to cultured human aortic
smooth muscle cells. During previous studies on the
binding of 125I-FGF-2 to human aortic smooth muscle
cells (HASMC), we distinguished high and low anity
binding sites.30 Heparin antagonized the binding of
FGF-2 to both the high anity binding sites and the
low anity binding sites (Fig. 5). The four penta-
saccharides tested in the present work also antagonized
the binding of 125I-FGF-2 to its low and high anity
boat S0 contributes to about 25% to the equilibrium.
Almost the same pattern is observed for the reducing
end sulfonated l-iduronic acid unit. The same pre-
dominant conformer is observed for equivalent iduronic
acid units in all four pentasaccharides, which is not
surprising since the immediate neighbors, which play a
preponderant role in determining the conformation,23,27
are identical.

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J. Kovensky et al. / Bioorg. Med. Chem. 7 (1999) 1567±1580
Figure 3. Inhibition of FGF-2 binding to aorta (A) or intestinal (B)
heparan sulfate by pentasaccharides 1, 14, 22, and 24.
Figure 5. E€ect on 125I-FGF-2 to HASMC. HASMC were incubated
for 3 h at 7ꢀC with 45 pM 125I-FGF-2 and increasing concentrations
of heparin (*), 1 (&), 14 (!), 22 (~), and 24 (^). The amount of
125I-FGF-2 bound to its high (A) and low (B) anity sites was deter-
mined (see Experimental). Each point represents the mean calculated
from three experimental determinations.
manner the growth of HASMC. The reference com-
pound heparin inhibited in a dose-dependent manner
this FGF-2 (30 nM) induced proliferation (Fig. 6;
IC50=24.0‹1.6 mg/m). Under the same experimental
conditions, the four pentassacharides only slightly
inhibited the mitogenic e€ect of FGF-2. The most sig-
ni®cant e€ect, corresponding to about 40% inhibition,
was obtained with 1 tested at 10 mg/mL (Fig. 6).
Conclusion
Figure 4. Dose response curves for pentasaccharide 1 inhibition of
FGF-2 binding to aorta heparan sulfate (*), intestinal heparan sulfate
(&), and heparin (~).
We have synthesized the four possible pentasaccharide
isomers that have been suggested9 to be the putative
FGF-2 binding site on heparan sulfate chains. These
four pentasaccharides were able to inhibit, although
weakly, FGF-2 binding to immobilized heparin or
heparan sulfate. This is in agreement with a recent
report on crystallographic studies of FGF-2 complexed
with heparin derived tetra- and hexasaccharides31 indi-
cating that the disaccharide sequence O-(2-deoxy-2-sul-
fonatamido-b-d-glucopyranosyl)-(1!4)-2-O-sulfonato-a-
sites, but to a smaller extent. From the results shown in
Figure 5, 1 appears as the most potent pentasaccharide,
particularly if one considers the binding to high anity
sites.
Inhibition of FGF-2 induced HASMC proliferation.
FGF-2 (1±100 nM) stimulated in a dose-dependent

J. Kovensky et al. / Bioorg. Med. Chem. 7 (1999) 1567±1580
1573
heteronuclear correlation; A, B, C, D, and E refer to
monosaccharide units in the ®nal pentasaccharide
(A=non reducing end unit). Reactions were monitored
by thin-layer chromatography (TLC) on precoated
plates of silica gel 60 F254 (layer thickness, 0.2 mm; E.
Merck, Darmstadt, Germany) and detection by char-
ring with sulfuric acid. Unless otherwise stated, ¯ash
column chromatography was performed on silica gel 60
(230±400 mesh, Merck).
2,3-Di-O-benzoyl-4,6-O-benzylidene-ꢀ-D-glucopyranosyl-
(1!4)-1-O-acetyl-2-azido-3,6-di-O-benzyl-2-deoxy-ꢁ,ꢀ-
D-glucopyranose (4). A solution of 1-O-acetyl-2-azido-
3,6-di-O-benzyl-2-deoxy-a,b-d-glucopyranose (2, 1.67 g,
3.90 mmol) and phenyl 2,3-di-O-benzoyl-4,6-O-benzyl-
idene-1-thio-b-d-glucopyranoside (3, 3.0 g, 5.28 mmol)
in dry dichloromethane (17 mL) containing 4 A mole-
cular sieves (3.0 g), was stirred 30 min at room tem-
perature and then cooled to 0ꢀC. N-iodosuccinimide
(2.6 g, 11.6 mmol) and tri¯uoromethanesulfonic acid
(0.1 mL of a 0.12 M solution in dichloromethane) were
added, and the mixture was allowed to reach room
temperature. After 1 h, sat aq NaHCO3 (1 mL) was
added, and the mixture was ®ltered through Celite. The
®ltrate was diluted with dichloromethane and the solu-
tion was washed with 10% aq Na2S2O3, water, and
concentrated. Column chromatography (toluene:ethyl
acetate, 10:1) yielded 4 (3.2 g, 92%) as a white foam. 1H
NMR (400 MHz) b anomer: d 7.95±7.82 and 7.56±7.34
(m, 25 H, Ar.), 5.62 (dd, 1 H, J2C,3C=J3C,4C=9.5 Hz,
H-3C), 5.49 (s, 1H, CHPh), 5.43 (dd, 1H, J1C,2C=8.2
Hz, H-2C), 5.32 (d, 1H, J1D,2D=8.4 Hz, H-1D), 5.12
and 4.78 (two d, 2H, Jgem=10.5 Hz, CH2Ph), 4.80 and
4.40 (two d, 2H, Jgem=12.0 Hz, CH2Ph), 4.78 (d, 1H,
H-1C), 4.27 (dd, 1H, J5C,6aC=4.5 Hz, J6aC,6bC=10.0
Hz, H-6aC), 4.20 (dd, 1H, J3D,4D=9.0 Hz, J4D,5D=10.0
Hz, H-4D), 3.88 (dd, 1H, J2D,3D=10.0 Hz, H-3D), 3.82
(dd, 1H, J4C,5C=9.5 Hz, H-4C), 3.71 (dd, 1H,
J5D,6aD=2.0 Hz, J6aD,6bD=11.2 Hz, H-6aD), 3.65±3.38
(m, 5H, H-2D, H-5C, H-5D, H-6bC, H-6bD), 2.18 (s, 3
H, Ac); a anomer: 6.20 (d, 1H, J1D,2D=3.5 Hz, H-1D),
5.50 (s, 1H, CHPh), 5.10 and 4.77 (two d, 2H,
Jgem=10.5 Hz, CH2Ph), 4.78 and 4.38 (two d, 2H,
Jgem=12.0 Hz, CH2Ph), 4.27 (dd, 1H, J5C,6aC=4.5 Hz,
J6aC,6bC=10.0 Hz, H-6aC), 4.18 (dd, 1H, J3D,4D=9.0
Hz, J4D,5D=10.0 Hz, H-4D), 3.68 (dd, 1H, J5D,6aD=2.0
Hz, J6aD,6bD=11.2 Hz, H-6aD), 2.15 (s, 3H, Ac);
Figure 6. E€ect on the mitogenic e€ect of FGF-2 for HASMC.
Growth arrested HASMC were cultured for 1 day in the presence
FGF-2 (30 nM) and increasing concentrations of heparin (*), 1 (&),
14 (!), 22 (~), and 24 (^). Cells were treated with trypsin and
counted. Data are reported as a percent inhibition of proliferation
compared with the cells grown in the controls (n=9).
l-idopyranosiduronate, that constitutes the reducing
end part, DE, of all four compounds described here,
constitutes the key element for binding to FGF-2. It is
therefore not surprising that all the present penta-
saccharides, sharing this common DE sequence, display
some anity for FGF-2. Interestingly, when bound to
FGF-2, the l-iduronate ring involved (E here) adopted
1
the C4 conformation,31 which is the preferred one we
have observed here in solution.
While 14, 22, and 24 were almost equipotent, 1 clearly
showed a higher anity. The present results are thus in
agreement with previous studies5,7 on the structure of
hypothetical FGF-2 binding sequences, which identi®ed
l-iduronic acid as the only uronic acid involved in the
interaction between heparan sulfate and FGF-2.
Experimental
General
Melting points (mp) were determined with a Buchi,
model 510, apparatus and are uncorrected. Optical
rotations were measured at 20‹2ꢀC with a Perkin±
Elmer, model 241, digital polarimeter, using a 10 cm/
1 mL cell. Chemical Ionisation (C.I.; ammonia) mass
spectra were obtained with a Nermag R10-10 spectro-
meter. Elemental analyses were performed by the Centre
Regional de Microanalyse (Paris VI University,
13C NMR (100.57 MHz)
d 168.85, 165.42, 165.02
(COO ), 138.25-126.10 (Ph), 101.40, 100.66 (C-1), 92.56
(D-1b), 90.31 (D-1a), 66.72, 66.47 (D-6), 64.33, 62.03
(D-2), 20.93 (Ac); MS (CI) m/z 903 (M + NH+4 ), 886
(M + H+). Anal. calcd for C49H47N3O13: C, 66.43; H,
5.35; N, 4.74. Found: C, 66.41; H, 5.39; N, 4.85.
1
France). H NMR spectra were recorded with a Bruker
2,3-Di-O-benzoyl-6-O-tert-butyldimethylsilyl-4-O-levulin-
oyl-ꢀ-D-glucopyranosyl-(1!4)-1-O-acetyl-2-azido-3,6-
di-O-benzyl-2-deoxy-ꢁ,ꢀ-D-glucopyranose (5). Aqueous
70% tri¯uoroacetic acid (6.4 mL) was added to a
solution of 4 (2.2 g, 2.48 mmol) in dichloromethane
(120 mL), and the mixture was stirred 2 h at room tem-
perature. After neutralization with triethylamine, the
solution was washed with water, dried (MgSO4), and
concentrated to an oil (1.54 g). The crude diol was dried
AC 250, a Bruker AM 400 and/or a Bruker AM 500
spectrometer for solutions in CDCl3 (reference: internal
TMS) or in D2O (reference: external TSP) at ambient
temperature. 13C NMR spectra were recorded at
62.89 MHz with a Bruker AC 250 and at 100.57 MHz
with a Bruker AM 400 for solutions in CDCl3 adopting
77.00 ppm for the central line of CDCl3. Assignments
were made using J-mode technique, and homo- and

1574
J. Kovensky et al. / Bioorg. Med. Chem. 7 (1999) 1567±1580
overnight over P2O5 and then dissolved in dry dichloro-
methane (16 mL). Triethylamine (0.534 mL, 3.8 mmol),
dimethylaminopyridine (18 mg, 0.14 mmol) and tert-
butyldimethylsilyl chloride (384 mg, 2.5 mmol) were
added under argon. After 2 h at room temperature, a
solution of levulinic anhydride (621 mg, 2.9 mmol) in
dichloromethane (2 mL) was added, together with tri-
ethylamine (0.8 mL). After stirring overnight at room
temperature, the mixture was diluted with dichloro-
methane, washed with water, dried, and concentrated.
Column chromatography (cyclohexane:ethyl acetate,
6.19 (d, 1H, J1D,2D=3.5 Hz, H-1D), 4.18 (dd, 1H,
J3D,4D=8.7 Hz, J4D,5D=10.0 Hz, H-4D), 3.95 (d, 1H,
J4C,5C=10.0 Hz, H-5C), 3.71 (dd, 1H, J5D,6aD=2.5 Hz,
J6aD,6bD=11.5 Hz, H-6aD), 3.62 (s, 3H, OMe), 2.12
and 2.09 (two s, 6H, Ac and CH3 Lev); 13C NMR
(100.57 MHz) d 205.63 (CO Lev), 171.14, 169.05, 166.74,
164.47, 164.57 (COO ), 138.03±127.52 (Ph), 100.22,
100.01 (C-1), 92.60 (D-1b), 90.40 (D-1a), 66.70 (D-6),
64.32, 61.96 (D-2), 52.84 (COOMe), 37.65, 29.55, 27.65
(Lev), 20.97, 20.93 (Ac); MS (CI) m/z 941 (M + NH+4 ).
Anal. calcd for C48H49N3O16: C, 62.39; H, 5.35; N, 4.55.
Found: C, 62.45; H, 5.46; N, 4.56.
1
4:1) yielded 5 (1.26 g, 50% from 4) as a white foam: H
NMR (400 MHz) b anomer: d 7.95±7.80 and 7.56±7.30
(m, 20H, Ar.), 5.46 (dd, 1H, J2C,3C=J3C,4C=10.0 Hz,
H-3C), 5.36 (dd, 1H, J1C,2C=8.0 Hz, H-2C), 5.28 (d,
1H, J1D,2D=8.0 Hz, H-1D), 5.21 (dd, 1H, J4C,5C=10.0
Hz, H-4C), 5.09, 4.83, 4.81 and 4.39 (four d, 4H,
Jgem=10.5 and 12.0 Hz, CH2Ph), 4.74 (d, 1H, H-1C),
4.15 (dd, 1H, J3D,4D=8.5 Hz, J4D,5D=10.0 Hz, H-4D),
3.82 (dd, 1H, J2D,3D=10.0 Hz, H-3D), 3.77 (dd, 1H,
J5C,6aC=3.0 Hz, J6aC,6bC=11.5 Hz, H-6aC), 3.70±3.60
(m, 2H, H-6bC, H-6aD), 3.56±3.40 (m, 3H, H-5C, H-
2D, H-6bD), 3.22 (m, 1H, H-5D), 2.63±2.45 (m, 4 H,
CH2 Lev), 2.15 and 2.08 (two s, 6H, Ac and CH3 Lev),
0.90 (s, 9H, Me3C), 0.05 (s, 6 H, Me2Si); (anomer: d 6.18
Methyl [methyl (methyl 2,3-di-O-benzoyl-4-O-levulinoyl-
ꢀ-D-glucopyranosyluronate)-(1!4)-(2-azido-3,6-di-O-
benzyl-2-deoxy-ꢁ-D-glucopyranosyl)-(1!4)-2-O-allyl-3-
O-benzyl-ꢁ-L-idopyranosid]uronate (10). Benzylamine
(2.0 mL) was added to a solution of 6 (500 mg,
0.53 mmol) in dry ether (20 mL). After 6 h at 0ꢀC the
mixture was concentrated, diluted with dichloro-
methane (300 mL), washed with 1 M HCl, water,
dried, and concentrated to a syrup to give crude 7 that
was dissolved in dry dichloromethane (13 mL) contain-
ing 4 A molecular sieves (0.4 g). After 30 min stirring at
room temperature the solution was cooled to 0ꢀC,
and 1,8-diazabicyclo-(5.4.0)-undec-7-ene (0.025 mL,
0.16mmol) and trichloroacetonitrile (1.1mL, 10.6mmol)
were added. The temperature was allowed to rise, and
after 30 min stirring the mixture was ®ltered through
Celite and concentrated. Column chromatography
(toluene:ethyl acetate, 5:1) yielded 8 (383.2 mg, 69%
from 6) as a white solid. This latter (308 mg, 0.30 mmol)
and methyl (methyl 2-O-allyl-3-O-benzyl-a-l-idopyr-
anosid)uronate 9 (70.0 mg, 0.20 mmol), in dry toluene
containing 4 A molecular sieves (220 mg), were stirred
30 min at room temperature, then cooled to 20ꢀC, and
tert-butyldimethylsilyl tri¯uoromethanesulfonate (1 M
in dichloromethane; 0.06 mL) was added. After 2 h, the
mixture was neutralized with N,N-di-isopropyl-N-ethyl-
amine, diluted with dichloromethane, ®ltered through
Celite, and concentrated. Column chromatography
(toluene:ethyl acetate, 10:3) gave 10 (180 mg, 75%) as a
white foam: [a]d +10ꢀ (c 1.47, CHCl3); 1H NMR
(400 MHz) d 7.95±7.80 and 7.60±7.30 (m, 25H, Ar.),
5.90 (m, 1H, -CH=), 5.49±5.39 (m, 4H, H-2C, H-3C,
H-4C, CHPh), 5.29±5.16 (m, 3H,ˆCH2, CHPh), 5.14 (d,
1H, J1D,2D=3.5 Hz, H-1D), 4.99 (d, 1H, J1E,2E=5.2 Hz,
H-1E), 4.85 and 4.73 (three d, 3H, CHPh), 4.66 (m, 2H,
H-1C, CHPh), 4.38 (m, 2H, H-5E, CHPh), 4.22±4.04
(m, 3H, OCH2, H-4D), 3.96 (dd, 1H, J3E,4E=6.5,
J4E,5E=5.0 Hz, H-4E), 3.92 (d, 1H, J4C,5C=9.5 Hz, H-
5C), 3.89 (dd, 1H, J2E,3E=6.5 Hz, H-3E), 3.74±3.45 (m,
3H, H-3D, H-5D, H-6aD), 3.63, 3.53, and 3.47 (three s,
9H, COOMe, OMe), 3.38 (dd, 1H, J5D,6bD=1.5,
J6aD,6bD=11.0 Hz, H-6bD), 3.31±3.25 (m, 2H, H-2D,
H-2E), 2.70±2.41 (m, 4H, CH2 Lev), 2.10 (s, 3H, CH3
Lev); 13C NMR (100.57 MHz) d 205.62 (CO Lev),
171.13, 169.85, 166.73, 164.45, 164.52 (C-6, E-6,
COO ), 138.21±137.52 (Ph), 134.55 (-CHˆ), 133.50±
127.47 (Ph), 116.99 (ˆCH2), 101.70 (E-1), 100.00 (C-1),
97.55 (D-1), 66.71 (D-6), 62.35 (D-2), 56.47 (OMe),
52.81, 51.88 (COOMe), 37.66, 29.54, 27.66 (Lev); MS
(CI) m/z 1233 (M + NH+4 ), 1216 (M + H+). Anal.
(d, 1H, J1D,2D=4.0 Hz, H-1D), 4.19 (dd, 1H, J3D,4D
=
8.5 Hz, J4D,5D=10.0 Hz, H-4D), 2.23 and 2.09 (two s,
6H, Ac and CH3 Lev), 0.89 (s, 9H, Me3C), 0.03 (s, 6H,
Me2Si); 13C NMR (100.57 MHz) d 205.59 (CO Lev),
171.25, 168.88, 165.59, 164.59 (COO ), 137.99±127.40
(Ph), 99.67, 99.51 (C-1), 92.50 (D-1b), 90.34 (D-1a),
66.74 (D-6), 64.33, 62.03 (D-2), 62.34, 62.29 (C-6),
37.63, 29.40, 27.73 (Lev), 25.72 (Me3C), 20.84, 20.80 (Ac),
18.09 (Me3C), 5.58, 5.61 (Me2Si); MS (CI) m/z 1027
(M + NH+4 ). Anal. calcd for C53H63N3O15: C, 63.01; H,
6.29; N, 4.16. Found: C, 63.14; H, 6.41; N, 4.07.
Methyl 2,3-di-O-benzoyl-4-O-levulinoyl-ꢀ-D-glucopyr-
anosyluronate-(1!4)-1-O-acetyl-2-azido-3,6-di-O-benzyl-
2-deoxy-ꢁ,ꢀ-D-glucopyranose (6). A solution of CrO3
(0.26 g, 2.6 mmol) in 3.5 M H2SO4 (1.14 mL) was slowly
added at 0ꢀC to a solution of 5 (1.0 g, 0.99 mmol) in
acetone (20 mL). After 4 h the mixture was poured into
ice water and extracted with dichloromethane. The
organic layer was dried (MgSO4) and the solvent was
evaporated. The residue was dissolved in ethyl acetate
(15 mL) and treated with an ethereal solution of diazo-
methane. After 10 min the mixture was concentrated in
vacuo to ꢁ1 mL, and diluted with dichloromethane.
The solution was washed with water, dried, and con-
centrated. Column chromatography (cyclohexane:ethyl
1
acetate, 3:1) yielded pure 6 (559.6 mg, 61%). H NMR
(400MHz) b anomer: d 7.95±7.80 and 7.60±7.30 (m, 20H,
Ar.), 5.53±5.39 (m, 3H, H-2C, H-3C, H-4C), 5.30 (d,
1H, J1D,2D=8.0 Hz, H-1D), 5.20, 4.85, 4.73, and 4.35
(four d, 4H, Jgem=10.5 Hz and 12.0 Hz, CH2Ph), 4.75
(d, 1H, J1C,2C=8.0 Hz, H-1C), 4.15 (dd, 1H, J3D,4D
8.5 Hz, J4D,5D=10.0 Hz, H-4D), 3.90 (d, 1H, J4C,5C
=
=
10.0 Hz, H-5C), 3.87 (dd, 1H, J2D,3D=10.0 Hz, H-3D),
3.67 (dd, 1H, J5D,6aD=2.5 Hz, J6aD,6bD=11.5 Hz, H-
6aD), 3.65 (s, 3H, OMe), 3.54±3.45 (m, 2H, H-2D, H-
6bD), 3.24 (m, 1H, H-5D), 2.70±2.43 (m, 4 H, CH2 Lev),
2.16 and 2.10 (two s, 6H, Ac and CH3 Lev); a anomer: d

J. Kovensky et al. / Bioorg. Med. Chem. 7 (1999) 1567±1580
1575
calcd for C64H69N3O21: C, 63.20; H, 5.72; N, 3.45.
Found: C, 63.19; H, 5.75; N, 3.34.
Hz, CHPh), 4.56 (dd, 1H, J5B,6aB=2.0, J6aB,6bB=12.5
Hz, H-6aB), 4.45 (d, 1H, J=12.0 Hz, CHPh), 4.39 (d,
1H, J4E,5E=5.0 Hz, H-5E), 4.30 (dd, 1H, J4C,5C=9.5
Hz, H-4C), 4.22±4.13 (m, 2H, OCH, H-6bB), 4.11±4.05
(m, 2H, OCH, H-4D), 3.97 (dd, 1H, J3E,4E=6.5 Hz, H-
4E), 3.92±3.87 (m, 2H, H-4B, H-3E), 3.82 (d, 1H, H-
5C), 3.78±3.67 (m, 4H, H-3B, H-3D, H-5B, H-6aD),
3.53 (m, 1H, H-5D), 3.65, 3.52, 3.50, 3.48 (four s, 12H,
Methyl [methyl (methyl 2,3-di-O-benzoyl-ꢀ-D-glucopyr-
anosyluronate)-(1!4)-(2-azido-3,6-di-O-benzyl-2-deoxy-
ꢁ-D-glucopyranosyl)-(1!4)-2-O-allyl-3-O-benzyl-ꢁ-L-ido-
pyranosid]uronate (11). Hydrazine hydrate (0.03 mL,
0.6 mmol) was added at 0ꢀC to a solution of 10
(150.0 mg, 0.12 mmol) in 3:2 pyridine:acetic acid
(1.2 mL). After 15 min, acetone (5 mL) was added, and
the mixture was stirred for 15 min at room temperature.
After evaporation, column chromatography (toluene:
ethyl acetate, 7:1) gave 11 (124.2 mg, 90%) as a
colorless oil. [a]d +2 ꢀ (c 1.0, CHCl3); 1H NMR
(400 MHz) d 7.95±7.80 and 7.60±7.30 (m, 25H, Ar.),
5.90 (m, 1H, -CHˆ), 5.49±5.09 (m, 8H, H-2C, H-3C, H-
4C, H-1D,=CH2, CH2Ph), 4.98 (d, 1H, Jgem=12.5 Hz,
CHPh), 4.97 (d, 1H, J1E,2E=5.2 Hz, H-1E), 4.80 (m,
3H, CHPh), 4.68±4.59 (m, 2H, H-1C, CHPh), 4.39±4.31
(m, 2H, H-5E, CHPh), 4.22±4.02 (m, 3H, OCH2, H-
4D), 3.96 (dd, 1H, J3E,4E=6.5, J4E,5E=5.0 Hz, H-4E),
3.87 (dd, 1H, J2E,3E=6.5 Hz, H-3E), 3.79 (d, 1H,
J4C,5C=9.8 Hz, H-5C), 3.74±3.45 (m, 3H, H-3D, H-5D,
H-6aD), 3.62, 3.51, and 3.47 (three s, 9H, COOMe,
OMe), 3.38 (dd, 1H, J5D,6bD=1.5, J6aD,6bD=11.0 Hz,
H-6bD), 3.31±3.25 (m, 2H, H-2D, H-2E); 13C NMR
(100.57 MHz) d 169.84, 168.87, 166.50, 164.75 (C-6, E-6,
COO ), 138.33±137.43 (Ph), 134.54 (-CHˆ), 133.51±
127.45 (Ph), 117.02 (ˆCH2), 101.71 (E-1), 100.08 (C-1),
97.52 (D-1), 66.72 (D-6), 62.29 (D-2), 56.45 (OMe),
52.76, 51.84 (COOMe); MS (CI) m/z 1135 (M + NH+4 ).
Anal. calcd for C59H63N3O19: C, 63.37; H, 5.68; N, 3.76.
Found: C, 63.29; H, 5.67; N, 3.70.
COOMe, OMe), 3.42 (dd, 1H, J5D,6bD=1.5, J6aD,6bD
=
12.0 Hz, H-6bD), 3.31±3.25 (m, 3 H, H-2B, H-2D, H-
2E), 2.21, 2.13, 2.07, (three s, 12H, Ac); 13C NMR
(100.57 MHz) d 170.39±164.67 (A-6, C-6, E-6, COO ),
138.10±137.23 (Ph), 134.52 (-CHˆ), 133.46±127.51 (Ph),
116.99 (ˆCH2), 101.69 (E-1), 100.25 (C-1), 99.04 (D-1),
97.46, 97.16 (A-1, B-1), 66.85 (D-6), 63.63, 62.34 (B-2,
D-2), 61.09 (B-6), 56.43 (OMe), 52.90, 52.13, 51.82
(COOMe), 20.87, 20.81, 20.68, 20.50 (Ac); MS (CI) m/z
1770 (M + NH+4 ). Anal. calcd for C87H96N6O33: C,
59.58; H, 5.52; N, 4.79. Found: C, 59.42; H, 5.61; N, 4.73.
Sodium [ methyl (ꢁ-L-sodium idopyranosyluronate)-
(1!4)-(2-deoxy-2-sodium sulfonatamido-ꢁ-D-glucopyr-
anosyl)-(1!4)-(ꢀ-D-sodium glucopyranosyluronate)-
(1!4)-(2-deoxy-2-sodium sulfonatamido-ꢁ-D-glucopyr-
anosyl)-(1!4)-2-O-sodium sulfonato-ꢁ-L-idopyranosid]-
uronate (14).
Deallylation. Compound 13 (66.2 mg, 0.037 mmol) and
1,5 cyclooctadiene-bis-[methyldiphenylphosphine]-iri-
dium hexa¯uorophosphate (0.51 mg, previously actived
by hydrogen) were dissolved in peroxide-free THF
(2.6 mL), and the mixture was stirred at room tempera-
ture under argon. After 5 min the solvent was evapo-
rated, the residue was dissolved in dichloromethane,
washed with aq NaHCO3, dried, and concentrated to
give a colorless glass (66 mg). The crude propenyl ether
thus obtained was dissolved in 5:1 acetone:water
(3 mL). HgO (20.4 mg, 0.094 mmol) and HgCl2 (25.6 mg,
0.094 mmol) were added, and the mixture was stirred at
room temperature for 10 min, then diluted with
dichloromethane, washed with aq 5% KI, water, dried,
and concentrated. Column chromathography (cyclo-
hexane:ethyl acetate, 1:1) yielded a syrup (47.6 mg,
73.8%). [a]d 19 ꢀ (c 1.0, CH2Cl2); ESI MS, positive
mode: + NaCl, monoisotopic mass: 1712.57, average
mass: 1713.69, experimental mass: 1713.39‹0.17. 1H
NMR (500 MHz) d 7.91±7.82 and 7.60±7.20 (m, 30H,
Ar.), 5.50 (dd, 1H, J2C,3C=J3C,4C=9.5 Hz, H-3C), 5.33
Methyl [methyl (methyl 2,3,4-tri-O-acetyl-ꢁ-L-idopyr-
anosyluronate)-(1!4)-(6-O-acetyl-2-azido-3-O-benzyl-2-
deoxy-ꢁ-D-glucopyranosyl)-(1!4)-(methyl 2,3-di-O-
benzoyl-ꢀ-D-glucopyranosyluronate)-(1!4)-(2-azido-3,6-
di-O-benzyl-2-deoxy-ꢁ-D-glucopyranosyl)-(1!4)-2-O-
allyl-3-O-benzyl-ꢁ-L-idopyranosid]uronate (13). A solu-
tion of 11 (40.0 mg, 0.036 mmol) and methyl 2,3,4-tri-O
-acetyl-a-l-idopyranosyluronate-(1!4)-6-O-acetyl-2-
azido-3-O-benzyl-2-deoxy-1-O-trichloroacetimidoyl-d-
glucopyranose 12 (37.3 mg, 0.046 mmol), in dry toluene
(2.5 mL) containing 4 A molecular sieves (110 mg), was
stirred at room temperature for 30 min, cooled to
20ꢀC, and tert-butyldimethylsilyl tri¯uoromethane-
sulfonate (1 M in dichloromethane, 0.011 mL) was
added. After 3.5 h, the mixture was neutralized with
N,N-di-isopropyl-N-ethylamine, diluted with dichloro-
methane, ®ltered through Celite, and concentrated.
Column chromatography (toluene-ethyl:acetate, 5:1)
yielded 13 (40.3 mg, 64%) as a white foam. [a]d 10 ꢀ (c
1.0, CHCl3); 1H NMR (400 MHz) d 8.00±7.85 and 7.60±
7.20 (m, 30H, Ar.), 5.90 (m, 1H, -CHˆ), 5.58 (dd, 1H,
J2C,3C=10.0, J3C,4C=9.5 Hz, H-3C), 5.41 (dd, 1H,
J1C,2C=8.0 Hz, H-2C), 5.35 (d, 1H, J1A,2A=4.0 Hz, H-
(dd, 1H, J1C,2C=8.0 Hz, H-2C), 5.30 (d, 1H, J1A,2A
=
4.4 Hz, H-1A), 5.18 (dd, 1H, J2A,3A=J3A,4A=5.5 Hz,
H-3A), 5.04 (dd, 1H, J3A,4A=5.6 Hz, J4A,5A=4.2 Hz,
H-4A), 4.92 (d, 1H, J1B,2B=3.7 Hz, H-1B), 4.87 (d, 1H,
J1E,2E=ꢁ1 Hz, H-1E), 4.85 (d, 1H, J1D,2D=ꢁ1 Hz, H-
1D), 4.82 (m, 1H, H-2A), 4.78 (d, 1H, J4A,5A=4.2 Hz,
H-5A), 4.71 (H-5E), 4.70 (d, 1H, J1C,2C=8.1 Hz, H-1C),
4.50 (d, J6aB,6bB=11.3 Hz, 1H, H-6aB), 4.23 (t, 1H,
J3C,4C=J4C,5C=9.5 Hz, H-4C), 4.11 (dd, J5B,6bB=2.2 Hz,
1H, H-6bB), 4.03 (m, H-4D), 4.02 (m, H-4E), 3.84 (dd,
1H, J3B,4B=J4B,5B=9.6 Hz, H-4B), 3.74 (H-3C), 3.70
(H-2E), 3.69 (H-6aD and H-5B), 3.67 (H-3B), 3.65 (H-
3D), 3.58, 3.46, 3.40. 3.09 (4 s, 12 H, COOMe and
OMe), 3.54 (H-2D), 3.44 (H-6bD), 3.24 (m, H-5D), 3.23
(m, H-2B), 2.14, 2.08, 2.07, 2.02 (4 s, 12 H, 3 Ac).
1A), 5.31±5.24 (m, 1H,=CH), 5.22 (dd, 1H, J2A,3A
=
J3A,4A=5.5 Hz, H-3A), 5.21±5.11 (m, 3H, H-1D,=CH,
CHPh), 5.10 (dd, 1H, J4A,5A=4.0 Hz, H-4A), 4.99 (d,
1H, J1B,2B=3.5 Hz, H-1B), 4.98 (d, 1H, J1E,2E=5.0 Hz,
H-1E), 4.90±4.82 (m, 5H, H-2A, H-5A, 3 CHPh), 4.77±
4.70 (m, 3H, H-1C, 2CHPh), 4.62 (d, 1 H, Jgem=10.5

1576
J. Kovensky et al. / Bioorg. Med. Chem. 7 (1999) 1567±1580
O-Sulfonation. A solution of the deallylated product
(44.2 mg, 0.0258 mmol) and sulfur trioxide triethyl-
amine complex (38.6 mg, 0.129 mmol) in dry DMF
(2.5 mL) was stirred overnight at 50ꢀC. After cooling,
chromatography on Sephadex LH-20 (ethanol:dichloro-
methane, 1:1), followed by lyophilization, gave a white
powder (46.4 mg): ESI MS, negative mode: m/z 1792
[M-(C2H5)3NH] . 1H NMR (500 MHz) d 7.91±7.82 and
1254.02, average mass: 1254.82, experimental mass:
1
1254‹0.24. H NMR: see Table 1.
Methyl 2,3,4-tri-O-acetyl-ꢀ-D-glucopyranosyluronate-
(1!4)-1,6-di-O-acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-
ꢀ-D-glucopyranose (17). A solution of methyl (2,3,4-tri-
O-acetyl-1-O-trichloroacetimidoyl-b-d-glucopyranosyl)-
uronate (15, 500 mg, 1.04 mmol) and 1,6-di-O-acetyl-2-
azido-3-O-benzyl-2-deoxy-d-d-glucopyranose (16, 303mg,
0.8 mmol) in dry dichloromethane (10 mL) containing
4 A molecular sieves (0.5 g) was stirred at room tem-
perature for 30 min, then cooled to 20ꢀC. Trimethyl-
silyl tri¯uoromethanesulfonate (0.24 mL of a 1 M solu-
tion in dichloromethane) was added. After 4 h, the
solution was neutralized by addition of N,N-diisopropyl-
N-ethylamine, ®ltered through Celite, and concentrated.
Column chromatography (5:1.5 to 5:2 toluene:ethyl
acetate) gave 17 as a white solid (370 mg, 66%). mp
137.5ꢀC; [a]d -5 ꢀ (c 1.0, CHCl3); 1H NMR (400 MHz) d
7.40±7.20 (m, 5H, Ar.), 5.45 (d, 1H, J1B,2B=8.5 Hz, H-
1B), 5.23 (m, 2H, H-3A, H-4A), 5.08 and 4.82 (two d,
2H, Jgem=11.5 Hz, CH2Ph), 5.05 (dd, 1H, J1A,2A=7.8
Hz, J2A,3A=9.5 Hz, H-2A), 4.73 (d, 1H, H-1A), 4.47
(dd, 1H, J5B,6aB=2.0, J6aB,6bB=12.5 Hz, H-6aB), 4.14
7.60±7.20 (m, 30H, Ar.), 5.54 (dd, 1H, J2C,3C=J3C,4C
=
9.5 Hz, H-3C), 5.33 (dd, 1H, J1C,2C=8.1 Hz, H-2C),
5.30 (d, 1H, J1A,2A=4.4 Hz, H-1A), 5.17 (dd, 1H,
J2A,3A=J3A,4A=5.6 Hz, H-3A), 5.06 (2 d, 2H, J1D,2D=
4.0 Hz, H-1D and J1E,2E=ꢁ 1 Hz, H-1E), 5.04 (d, 1H,
J4A,5A=4.2 Hz, H-4A), 4.93 (d, 1H, J1B,2B=3.7 Hz, H-
1B), 4.85 and 4.59 (2 d, 2H, J=12 Hz, PhCH2), 4.80
and 4.68 (2 d, 2H, J=10.5 Hz, PhCH), 4.80 (m, H-2A),
4.79 (m, H-5A), 4.76 (d, 1H, J=8.1 Hz, H-1C), 4.64 (d,
1H, J4E,5E=2.7 Hz, H-5E), 4.53 (br. s, 1H, J1E,2E=1 Hz,
H-2E), 4.49 (dd, 1H, J6aB,6bB=11.4 Hz, H-6aB), 4.24
(m, H-4C), 4.22 (m, H-3E), 4.11 (m, H-6bB), 4.02 (dd,
1H, J3D,4D=J4D,5D=9.3 Hz, H-4D), 3.94 (br. s, 1H,
J3E,4E=1±2 Hz, H-4E), 3.84 (dd, 1H, H-4B), 3.78 (d,
1H, J4C,5C=9.7 Hz, H-5C), 3.70 (m, H-3D), 3.69 (m, H-
5B), 3.67 (m, H-3B and H-6aD), 3.54, 3.45, 3.38, 3.36 (4
s, 12H, COOMe and OMe), 3.40 (m, 2H, H-5D and H-
6bD), 3.23 (dd, 1H, J1B,2B=3.7 Hz, J2B,3B=10.2 Hz, H-
2B), 3.15 (m, H-2D), 3.13 (q, 6H, (CH2-CH3)3NH+),
2.14, 2.08, 2.07, 2.02 (4 s, 12 H, 3 Ac), 1.32 (t, 9 H,
(CH2-CH3)3NH+).
(dd, 1H, J5D,6bD=5.0 Hz, H-6bB), 3.91 (d, 1H, J4A,5A
=
9.8 Hz, H-5A), 3.85 (dd, 1H, J3B,4B=J4B,5B=10.0 Hz,
H-4B), 3.63 (ddd, 1H, H-5B), 3.56 (s, 3H, COOMe),
3.57±3.53 (m, 2H, H-2B, H-3B), 2.19, 2.14, 2.09, 2.04,
and 2.03 (®ve s, 15H, Ac); 13C NMR (100.57 MHz) d
170.31, 170.00, 169.30, 169.16, 168.74, 166.58 (A-6, 5
COO ), 138.01, 129.00±127.48 (Ph), 100.69 (A-1), 92.49
(B-1), 61.78 (B-6), 52.74 (COOMe), 20.90, 20.81, 20.51,
20.41 (Ac); MS (CI) m/z 713 (M + NH+4 ), 668 (M+
CH2CO). Anal. calcd for C30H37N3O16: C, 51.79; H,
5.36; N, 6.04. Found: C, 51.97; H, 5.38; N, 5.92.
Saponi®cation. The O-sulfonated product (44 mg,
0.0232 mmol) was dissolved in THF (4 mL), cooled to
5ꢀC, and 30% H2O2 (1.5 mL) followed by 0.7 M aq
LiOH (0.7 mL) were added. After 20 h stirring at room
temperature, methanol (3.3 mL), and 4 N NaOH
(0.9 mL) were added at 0ꢀC, and stirring was prolonged
overnight at room temperature. After neutralization
with 1 M HCl and concentration to ꢁ4 mL, chromato-
graphy on Sephadex LH-20 (ethanol:water, 1:1) yielded
a colorless glass (34 mg, 100%).
Methyl [methyl (methyl 2,3,4-tri-O-acetyl-ꢀ-D-glucopyr-
anosyluronate)-(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-glucopyranosyl)-(1!4)-
Hydrogenation. A solution of the above crude com-
pound in 1:1 tert-butyl alcohol:water (2 mL) was stirred
overnight under H2 in the presence of 10% Pd/C
(60 mg), ®ltered, and concentrated. The treatment was
repeated until no signals of residual benzyl groups could
2-O-allyl-3-O-benzyl-ꢁ-L-idopyranosid]uronate
(21).
Benzylamine (1.26 mL, 11.4 mmol) was added to a
solution of 17 (200.0 mg, 0.29 mmol) in dry ether
(10.0 mL) at 0ꢀC. After 1 h, the mixture was con-
centrated, and dichloromethane (150 mL) was added.
The solution was washed with 1 M HCl, water, dried
(MgSO4), and the solvent was evaporated. After over-
night drying over P2O5, crude 18 (195mg) was dissolved in
dry dichloromethane (2.5mL) containing 4 A (molecular
sieves (0.2 g). Potassium carbonate (60 mg, 0.43mmol)
and trichloroacetonitrile (0.17 mL, 1.65 mmol) were
added, and the mixture was stirred until no more start-
ing material was detected. The suspension was ®ltered
through Celite, and after ¯ash chromatography (tolue-
ne:ethyl acetate, 5:2, containing 1% of triethylamine) 19
was obtained (148 mg, 65% from 17). A solution of 19
(117 mg, 0.15 mmol) and methyl [methyl O-(methyl 2-O-
acetyl-3-O-benzyl-a-l-idopyranosyl-uronate)-(1!4)-O-(6-
O-acetyl-2-azido-3-O-benzyl-2-deoxy-a-d-glucopyr-
anosyl)-(1!4)-O-methyl 2-O-allyl-3-O-benzyl-a-l-ido-
pyranosid]uronate 20 (112 mg, 0.11 mmol) in dry
dichloromethane (4.2 mL) containing 4 A molecular
1
be detected by H NMR. Chromatography of the resi-
due on Sephadex G-25 in water, yielded the deprotected
compound (23 mg). [a]d +42 ꢀ (c 1.0, H2O).
N-Sulfonation.
The
above
product
(22.5 mg,
0.0214 mmol) was dissolved in water (5 mL), and the pH
of the solution was adjusted to 9.5 with aq 0.5 M
NaOH. Sulfur trioxide±pyridine complex (136 mg,
0.86 mmol) was added, and the pH was maintained at
9.5 by addition of 0.5 N NaOH. After 1 h at room
temperature, the mixture was chromatographed on
Sephadex G-25, equilibrated with 0.2 N NaCl. Frac-
tions containing the desired product were pooled and
desalted on the same column, using water as eluent.
Pooled fractions were lyophilized to give 14 (18 mg,
55% from the deallylated compound). [a]d+33 ꢀ (c 0.48,
H2O); ESI MS, negative mode, monoisotopic mass:

J. Kovensky et al. / Bioorg. Med. Chem. 7 (1999) 1567±1580
1577
sieves (0.16 g) was stirred 30 min at room temperature
under argon and then cooled to 20ꢀC. tert-Butyldi-
methylsilyl tri¯uoromethanesulfonate (0.034 mL of a 1
M solution in dichloromethane) was added, and after
1.5 h the mixture was neutralized with triethylamine,
diluted with dichloromethane, ®ltered through Celite,
and concentrated. Column chromatography (tolue-
ne:ethyl acetate, 5:1) yielded 21 (117 mg, 64%) as a
white foam: [a]d +16 ꢀ (c 1.47, CHCl3); 1H NMR
(400 MHz) d 7.42±7.28 (m, 20H, Ar.), 5.90 (m, 1H,
-CHˆ), 5.33 (d, 1H, J1C,2C=4.5 Hz, H-1C), 5.31±5.12
(m, 5H,ˆCH2, H-3A, H-4A, H-1D), 5.08 (dd, 1H,
PhCH2), 4.89 and 4.62 (d, 2H, PhCH2), 4.79 (d, 1H,
J4E,5E=2.5 Hz, H-5E), 4.65 (d, 1H, J1A,2A=8.0 Hz, H-
1A), 4.57 (d, 1H, J4C,5C=5.6 Hz, H-5C),4.56 (br. signal,
1H, J2E,3E=2.5 Hz, H-2E), 4.45 (dd, 1H, J6aD,6bD=10.8
Hz, H-6aD), 4.35 (m, 1H, H-6aB), 4.34 (m, 1H, H-3E),
4.18 (dd, 1H, J5B,6bB=3.4 Hz, J6aB,6bB=12.3 Hz, H-
6bB), 4.10 (dd, 1H, J5D,6bB=3.4 Hz, J6aD,6bD=10.8 Hz,
H-6bD), 4.02 (br. signal, 1H, J4E,5E=2.5 Hz, H-4E),
3.99 (dd, 1H, J3C,4C=J4C,5C=5.6, Hz, H-4C), 3.90 (H-
5A and H-3C), 3.86 (H-5D), 3.80 (H-4B and H-4D),
3.72 (H-3B), 3.70 (H-5B), 3.67 (H-3D), 3.67, 3.65, 3.57,
3.44 (4 s, 12H, COOMe and OMe), 3.22 (H-2B), 3.21
(H-2D), 3.06 (q, 6H, CH2±CH3)3NH+), 2.10, 2.06, 2.05,
2.02, 2.00, 1.99 (6 s, 18 H, 6 Ac), 1.28 (t, 9 H, (CH2±
CH3)3NH+).
J1A,2A=8.0, J2A,3A=9.0 Hz, H-2A), 5.03 (d, 1H, J1E,2E
=
4.5 Hz, H-1E), 5.01 (d, 1H, J1B,2B=3.5 Hz, H-1B), 4.96
(dd, 1H, J2C,3C=5.0 Hz, H-2C), 4.90±4.67 (m, 10H, 4
CH2Ph, H-1A, H-5E), 4.65 (d, 1H, J4C,5C=4.5 Hz, H-
5C), 4.50 (dd, 1H, J5B,6aB=2.0 Hz, J6aB,6bB=12.0 Hz,
H-6aB), 4.44 (dd, 1H, J5D,6aD=2.0 Hz, J6aD,6bD=12.0
Hz, H-6aD), 4.27 (dd, 1H, J5D,6bD=3.5 Hz, H-6bD),
4.22±4.07 (m, 4H, CH2CHˆ, H-6bB, H-4E), 4.05 (dd,
1H, J3C,4C=4.5 Hz, H-4C), 3.99±3.81 (m, 7H, H-3C, H-
3E, H-4B, H-4D, H-5A, H-5B, H-5D), 3.77 (s, 3H,
COOMe), 3.74 (dd, 1H, J2D,3D=10.0, J3D,4D=9.0 Hz,
H-3D), 3.71 (dd, 1H, J2B,3B=10.0, J3B,4B=8.0 Hz, H-
3B), 3.62, 3.55, and 3.52 (three s, 9H, COOMe, OMe),
3.38 (dd, 1H, J2E,3E=6.5 Hz, H-2E), 3.30 (dd, 1H,
J1D,2D=3.5 Hz, H-2D), 3.26 (dd, 1H, H-2B), 2.16, 2.10,
2.08, 2.05, and 2.04 (®ve s, 18H, Ac); 13C NMR
(100.57 MHz) d 170.67±166.59 (A-6, C-6, E-6, COO ),
137.95±137.37 (Ph), 134.51 (-CH=), 128.51±127.57 (Ph),
117.11 (ˆCH2), 101.72 (E-1), 100.55 (A-1), 97.95 (C-1),
97.58, 97.35 (B-1, D-1), 78.31 (E-2), 77.97 (B-3), 77.82
(D-3), 62.98 (D-2), 62.64 (B-2), 61.91 (B-6), 61.39 (D-6),
56.44 (OMe), 52.73, 52.17, 52.12 (COOMe), 20.82, 20.80,
20.68, 20.53, 20.46 and 20.43 (Ac); MS (CI) m/z 1646 (M
+ NH+4 ). Anal. calcd for C77H92N6O33: C, 56.75; H,
5.69; N, 5.16. Found: C, 56.70; H, 5.71; N, 5.07.
Saponi®cation. The O-sulfonated product (38 mg,
0.0214mmol) was treated as described for the preparation
of 14 to give the saponi®ed derivative (31.3 mg, 100%).
Hydrogenation. The above compound was treated as
described for the preparation of 14 to give quantita-
tively the hydrogenated derivative.
N-sulfonation. The hydrogenated product (14 mg,
0.013 mmol) was treated as described for the prepara-
tion of 14 to give 22 (13.8 mg, 54% from the deallylated
compound): [a]d +32 ꢀ (c 0.99, H2O); ESI MS, negative
mode: monoisotopic mass: 1254.02, average mass:
1254.82, experimental mass: 1254.4‹0.17. 1H NMR:
see Table 1.
Methyl [methyl (methyl 2,3,4-tri-O-acetyl-ꢀ-D-glucopyr-
anosyluronate)-(1!4)-(6-O-acetyl-2-azido-3-O-benzyl-2-
deoxy-ꢁ-D-glucopyranosyl)-(1!4)-(methyl 2,3-di-O-benz-
oyl-ꢀ-D-glucopyranosyluronate)-(1!4)-(2-azido-3,6-di-O-
benzyl-2-deoxy-ꢁ-D-glucopyranosyl)-(1!4)-2-O-allyl-3-
O-benzyl-ꢁ-L-idopyranosid]uronate (23). A solution of
11 (42.0 mg, 0.038 mmol) and 19 (44.0 mg, 0.055 mmol)
in dry toluene (2.5 mL) containing 4 A molecular sieves
(150 mg) were stirred 30 min at room temperature and
then cooled to 20ꢀC. tert-butyldimethylsilyl tri¯uoro-
methanesulfonate (0.011 mL of a 1 M solution in
dichloromethane) was added. After 2 h, the mixture was
neutralized with N,N-diisopropyl-N-ethylamine, diluted
with dichloromethane, ®ltered through Celite, and con-
centrated. Column chromatography (toluene:ethyl ace-
tate, 5:1) gave 23 (35.6 mg, 54%) as a white foam: [a]d
Sodium [methyl (ꢀ-D-sodium glucopyranosyluronate)-
(1!4)-(2-deoxy-2-sodium sulfonatamido-ꢁ-D-glucopyr-
anosyl)-(1!4)-(ꢁ-L-sodium idopyranosyluronate)-(1!4)-
(2-deoxy-2-sodium sulfonatamido-ꢁ-D-glucopyranosyl)-
(1!4)-2-O-sodium sulfonato-ꢁ-L-idopyranosid]uronate
(22)
Deallylation. Compound 21 (62 mg, 0.038 mmol) was
treated as described for the preparation of 14. Column
chromathography (cyclohexane:ethyl acetate, 1:1) yiel-
ded a syrup (43 mg, 71%): [a]d +7 ꢀ (c 0.96, CH2Cl2);
LSIMS, positive mode: m/z thioglycerol + NaCl, 1611
(M + Na)+; thioglycerol + KF, 1627 (M + K)+.
+7 ꢀ (c 0.7, CHCl3); H NMR (400 MHz) d 7.97±7.85
1
and 7.60±7.20 (m, 30H, Ar.), 5.90 (m, 1H, -CHˆ), 5.53
(dd, 1H, J2C,3C=10.0, J3C,4C=9.5 Hz, H-3C), 5.37 (dd,
1H, J1C,2C=8.0 Hz, H-2C), 5.30±5.22 (m, 3H, H-3A, H-
4A,=CH), 5.20±5.02 (m, 6H, H-1D, H-2A,=CH,
3CHPh), 4.99 (d, 1H, J1E,2E=5.0 Hz, H-1E), 4.97 (d,
1H, J1B,2B=3.5 Hz, H-1B), 4.83 (d, 2H, Jgem=11.2 Hz,
2CHPh), 4.75±4.70 (m, 2H, H-1A, CHPh), 4.68±4.56
(m, 4H, H-1C, H-6aB, 2CHPh), 4.43 (d, 1H, Jgem=12.0
Hz, CHPh), 4.38 (d, 1H, J4E,5E=5.0 Hz, H-5E), 4.29
(dd, 1H, J4C,5C=9.0 Hz, H-4C), 4.22±4.03 (m, 4H,
OCH2, H-4D, H-6bB), 3.99±3.91 (m, 2H, H-4E, H-5A),
3.89 (dd, 1H, J2E,3E=J2E,3E=7.0 Hz, H-3E), 3.83±3.75
(m, 4H, H-3B, H-4B, H-5B, H-5C), 3.73±3.66 (m, 2H,
H-3D, H-6aD), 3.55±3.50 (m, 1H, H-5D), 3.63, 3.52,
O-sulfonation. The deallylated product (43 mg,
0.02 mmol) was treated as described for the preparation
of 14 to give a white powder (42 mg): ESI MS, negative
mode: m/z 1668.7 [M-(C2H5)3NH+] . 1H NMR
(500 MHz) d 7.38±7.24 (m, 20H, Ar.), 5.29 (d, 1H,
J1C,2C=5.0 Hz, H-1C), 5.22 (m, 1H, H-3A), 5.20 (m,
1H, H-4A), 5.13 (br. signal, 2H, J1B,2B=3.7 Hz, H-1B
and J1E,2E=ꢁ1 Hz, H-1E), 5.09 and 4.63 (2 d, 2H,
J=11 Hz, PhCH2), 5.03 (dd, 1H, J1A,2A=8.0 Hz,
J2A,3A=8.9 Hz, H-2A), 4.97 (d, 1H, J1D,2D=3.7 Hz, H-
1D), 4.90 (m, H-2C), 4.90 and 4.62 (2 d, 2H, J=11 Hz,

1578
J. Kovensky et al. / Bioorg. Med. Chem. 7 (1999) 1567±1580
and 3.47 (three s, 12H, COOMe, OMe), 3.40 (dd, 1H,
J5D,6bD=1.5, J6aD,6bD=11.0 Hz, H-6bD), 3.31±3.24 (m,
2H, H-2D, H-2E), 3.20 (dd, 1H, J2B,3B=10.1 Hz, H-
2B), 2.15, 2.13, 2.06, 2.04 (four s, 12H, Ac); 13C NMR
(100.57 MHz) d 170.22±164.72 (A-6, C-6, E-6, COO ),
138.17±137.44 (Ph), 134.54 (-CHˆ), 133.45±127.41 (Ph),
116.98 (ˆCH2), 101.69 (E-1), 100.62 (A-1), 100.16 (C-1),
98.82 (D-1), 97.46 (B-1), 66.74 (D-6), 63.09, 62.34 (B-2,
D-2), 61.05 (B-6), 56.43 (OMe), 52.79, 52.70, 51.83
(COOMe), 20.87, 20.53, 20.42 (Ac); MS (CI) m/z 1770
(M + NH+4 ). Anal. calcd for C87H96N6O33: C, 59.58; H,
5.52; N, 4.79. Found: C, 59.27; H, 5.87; N, 4.86.
(m, J3E,4E=1±2 Hz, H-3E), 4.09 (dd, 1H, J5B,6aB=2.7
Hz, J6aB,6bB=12.5 Hz, H-6bB), 4.00 (dd, 1H, J3D,4D
=
J4D,5D=9.5 Hz, H-4D), 3.91 (m, H-E4), 3.89 (m, H-5A),
3.78 (d, 1H, J4C,5C=9.7 Hz, H-5C), 3.73 (H-4B), 3.72
(H-3B), 3.66 (H-5B), 3.65 (H-3D and H-6aD), 3.55,
3.47, 3.38, 3.36 (4 s, 12H, COOMe and OMe), 3.38 (H-
6bD), 3.35 (H-5D), 3.18 (dd, 1H, J2D,3D=10 Hz, H-
2D), 3.14 (dd, 1H, J2B,3B=9.5 Hz, H-2B), 3.07 (q, 6H,
(CH2±CH3)3NH+), 2.09, 2.08, 2.01, 1.98 (4 s, 12H, 3
Ac), 1.29 (t, 9 H, (CH2±H3)3NH+).
Saponi®cation. The O-sulfonated product (26 mg,
0.0138 mmol) was treated as described for the prepara-
tion of 14 to give a colorless glass (20 mg, 100%).
Sodium [methyl (ꢀ-D-sodium glucopyranosyluronate)-
(1!4)-(2-deoxy-2-sodium sulfonatamido-ꢁ-D-glucopyr-
anosyl)-(1!4)-(ꢀ-D-sodium glucopyranosyluronate)-
(1!4)-(2-deoxy-2-sodium sulfonatamido-ꢁ-D-glucopyr-
anosyl)-(1!4)-2-O-sodium sulfonato-ꢁ-L-idopyranosid]-
uronate (24).
Hydrogenation. The above crude compound was treated
as described for the preparation of 14 to give quantita-
tively the hydrogenated derivative.
N-sulfonation. The hydrogenated product (12 mg,
0.0114 mmol) was treated as described for the prepara-
tion of 14 to give 24 (8 mg, 38% from the deallylated
compound): [a]d +17 (c 0.47, H2O); ESI MS, negative
mode, monoisotopic mass: 1254.02, average mass:
Deallylation. Compound 23 (43 mg, 0.0248 mmol) was
treated as described for the preparation of compound
14. Column chromathography (cyclohexane:ethyl ace-
tate, 1:1) gave a syrup (32.9 mg, 77%): [a]d 5 ꢀ (c 1,
CH2Cl2); ESI MS, positive mode: monoisotopic mass:
1712.57, average mass: 1713.69, experimental mass:
1
1254.82, experimental mass: 1255‹0.52. H NMR: see
Table 1.
1
1739‹0.37. H NMR (500 MHz) d 7.91±7.82 and 7.60±
7.20 (m, 30H, Ar.), 5.46 (t, 1H, J2C,3C=J3C,4C=9.5 Hz,
H-3C), 5.29 (dd, 1H, J1C,2C=8.1 Hz, J2C,3C=9.5 Hz, H-
2C), 5.22 (m, H-4A), 5.21 (m, H-3A), 5.12 and 4.55 (2 d,
2H, J=10.5 Hz, PhCH2), 5.08 and 4.60 (2 d, 2H, J=11
Hz, PhCH2), 5.04 (m, 1H, H-2A), 4.91 (d, 1H,
J1B,2B=3.7 Hz, H-1B), 4.87 (d, 1H, J1E,2E=ˆ 1 Hz, H-
1E), 4.86 (d, 1H, J1D,2D=4.0 Hz, H-1D), 4.85 and 4.42
(2 d, 2H, J=11 Hz, PhCH2), 4.71 (d, 1H, J4E,5E=1.6
Hz, H-5E), 4.67 (1 d, 1H, J1A,2A=8.1 Hz, H-1A), 4.65
Binding experiments: biotinylation of polysaccharides.
The polysaccharide preparations used in the binding
studies were as follows: heparin from pig intestinal
mucosa (stage 14) was from Inolex Pharmaceutical
Division (Park Forest South, IL). Heparan sulfate from
bovine aorta and intestine were gifts from Dr Keiichi
Yoshida, Seikagaku Corporation, Tokyo, Japan. Cap-
sular polysaccharide from the Escherichia coli strain K5
(designated below as K5 PS) was provided by Dr Ker-
stin Lidholt (Uppsala University). K5 PS was used as a
negative control of the binding analysis because it has
the same backbone structure as the primary polymers
produced upon heparin/HS biosynthesis, that is,
(GlcAb1,4-GlcNAca1,4)n. For biotinylation, 500 mg of
polysaccharide was dissolved in 0.1 M 4-morpholi-
neethanesulfonic acid, pH 5.6, containing 2.5 mm EZ-
Linka biotin hydrazide (Pierce, Rockford, IL), and
0.63mm N-ethyl-N0-(dimethylaminopropyl)carbodiimide
(EDC; Biacore1, Uppsala, Sweden). Following incubation
for 7 h at room temperature, the reaction mixtures were
passed through PD-10 desalting columns (Pharmacia
Biotech, Uppsala, Sweden) in water and the fractions
containing polysaccharides dried in a centrifugal evap-
orator. Biotinylated polysaccharides were then dissolved
in 0.3 M NaCl at a concentration of 1 mg/mL.
(1 d, 1H, J1C,2C=8.1 Hz, H-1C), 4.78 (d, 1H, J4A,5A
=
4.2 Hz, H-5A), 4.53 (dd, 1H, J6aB,6bB=11.5 Hz, H-6aB),
4.23 (t, 1H, J3C,4C=J4C,5C=9.5 Hz, H-3C), 4.09 (m, 1H,
H-6bB), 4.02 (m, 2H, H-4D and H-4E), 3.89 (m, 1H, H-
5A), 3.74 (H-3E and H-5C), 3.73 (H-3B), 3.72 (H-4B),
3.71 (H-2E), 3.68 (H-6aD), 3.65 (H-5B), 3.64 (H-3D),
3.53 (H-2D), 3.58, 3.46, 3.40. 3.09 (4 s, 12H, COOMe
and OMe), 3.43 (H-6bD), 3.23 (br. d, 1H, J4,5=10 Hz,
H-5D), 3.14 (dd, 1H, J2B,3B=9.8 Hz, H-2B), 2.14, 2.08,
2.07, 2.02 (4 s, 12 H, 3 Ac).
O-sulfonation. The deallylated product (29 mg,
0.0169 mmol) was treated as described for the prepara-
tion of 14 to give the sulfonated derivative as a white
powder (30.8 mg): ESI MS, negative mode: m/z 1792
[M-(C2H5)3NH+] . 1H NMR (500 MHz) d 7.89±7.82
and 7.60±7.25 (m, 30H, Ar.), 5.51 (t, 1H, J2C,3C
=
J3C,4C=9.4 Hz, H-3C), 5.30 (dd, 1H, J1C,2C=8.1 Hz, H-
2C), 5.22 (m, 1H, H-4A), 5.21 (m, 1H, H-3A), 5.08 (d,
1H, 1H, J1D,2D=3±4 Hz, H-1D), 5.07 and 4.60 (2 d, 2H,
PhCH2), 5.07 (d, 1H, 1H, J1E,2E=ꢁ1 Hz, H-1E), 5.04
and 4.58 (2 d, 2H, PhCH2), 5.03 (m, H-2A), 4.91 (d, 1H,
J1B,2B=3.7 Hz, H-1B), 4.83 and 4.56 (2 d, 2H, J=11.7
Hz, PhCH2), 4.77 and 4.40 (2 d, 2H, J=12 Hz, PhCH2),
4.72 (d, 1H, J1C,2C=8.1 Hz, H-1C), 4.67 (d, 1H,
J1A,2A=8.1 Hz, H-1A), 4.64 (d, 1H, J4E,5E=2.6 Hz, H-
5E), 4.53 (m, 2H, H-6aB, H-2E), 4.23 (m, H-4C), 4.22
Binding experiments: surface plasmon resonance (SPR)
binding analysis. Binding analyses were performed
using Biacore1 2000 biosensor (Biacore1) and CM-5
sensor chips at 30ꢀC. Sensor chip surfaces were acti-
vated by injecting 190 mL of a mixture of 200 mm EDC
and 50 mm N-hydroxysuccinimide (Biacore1) at a ¯ow
rate of 5 mL/min followed by equilibration of the sur-
faces with the running bu€er (150 mm NaCl, 10 mm
HEPES, 0.01% Tween-20, pH 7.4). The activated sur-
faces were then perfused with a 35 mL injection of

J. Kovensky et al. / Bioorg. Med. Chem. 7 (1999) 1567±1580
1579
streptavidin (200 mg/mL in 10 mm sodium acetate; ICN
Biochemicals). The remaining active groups on the chip
surfaces were inactivated by injection of 190 mL of 1 M
ethanolamine hydrochloride (Biacore1). The biotin-
ylated polysaccharide preparations were then immobi-
lized onto the sensor chips by injecting 30 mL of
polysaccharide over the streptavidin-coated surface.
Finally, the sensor chip surfaces were washed by three
20-mL injections of 3 M KSCN. For binding experi-
ments, 200 nM recombinant human FGF-2 (FGF-2;
PeproTech) in running bu€er containing 1 mg/mL car-
boxymethyl dextrane (Fluka, Buchs, Switzerland; used
in order to diminish the nonspeci®c binding of FGF-2
to the chip surface) was injected over the various poly-
saccharide-coated surfaces at a ¯ow rate of 5 mL/min.
To assess the e€ects of the synthetic pentasaccharides,
they were mixed with FGF-2 prior to the injection.
After each injection, the sensor chip surfaces were
regenerated by injection of 3 M KSCN (20 mL) in order
to remove the polysaccharide-bound FGF-2. The spe-
ci®c binding of FGF-2 was calculated by subtracting the
binding to K5 polysaccharide from the binding to
heparin and HS. Quanti®cation of the binding data was
performed using the BiaEvaluation software (Biacore1).
mL of bacitracin and 0.2% of gelatin. 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) which
contained 45 pM 125I-FGF-2 (110 mCi/mg) and increas-
ing concentrations of the tested compounds. Triplicate
incubations were carried out at 7ꢀC for 3 h. Low anity
binding of FGF-2 was determined speci®cally, by salt
extraction (5 min with cold 1 mL NaCl 2M) before
treatment by NaOH 1 M (high anity binding of FGF-
2). 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 trypsinized
and cells were counted with a Coulter counter (Coul-
tronics, France).
Acknowledgements
J. Kovensky thanks the Consejo Nacional de Investiga-
ciones Cientõ®cas y Tecnicas (CONICET), Argentina,
for a postdoctoral fellowship.
References
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