Design, synthesis, FGF-1 binding, and molecular modeling studies of conformationally flexible heparin mimetic disaccharides

Article abstract of DOI:10.1016/j.bmcl.2007.10.071

Disaccharide mimetics of a heparin sequence that binds to fibroblast growth factors were prepared by coupling a d-galactose donor with a methyl β-d-gluco- or xylopyranoside acceptor. When fully sulfated, the glucose or xylose moieties exist in solution in

Full text of DOI:10.1016/j.bmcl.2007.10.071

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 344–349
Design, synthesis, FGF-1 binding, and molecular modeling
studies of conformationally flexible heparin mimetic disaccharides
Ligong Liu, Ian Bytheway, Tomislav Karoli, Jon K. Fairweather, Siska Cochran,
Caiping Li and Vito Ferro*
Drug Design Group, Progen Pharmaceuticals Limited, PO Box 2403, Toowong, Brisbane, Qld 4066, Australia
Received 17 August 2007; revised 19 October 2007; accepted 20 October 2007
Available online 25 October 2007
Abstract—Disaccharide mimetics of a heparin sequence that binds to fibroblast growth factors were prepared by coupling a D-gal-
actose donor with a methyl b-D-gluco- or xylopyranoside acceptor. When fully sulfated, the glucose or xylose moieties exist in solu-
tion in equilibrium between the 4C1 and 1C4 conformers, as confirmed by 1H NMR spectroscopy, thus mimicking the
conformationally flexible L-iduronic acid found in heparin. Docking calculations showed that the predicted locations of disaccharide
sulfo groups in the binding site of FGF-1 are consistent with the positions observed for co-crystallized heparin-derived oligosaccha-
rides. Predicted binding affinities are in accord with experimental Kd values obtained from binding assays and are similar to the
predicted values for a model heparin disaccharide.
Ó 2007 Elsevier Ltd. All rights reserved.
The fibroblast growth factors (FGFs) are a family of
structurally related proteins that play important roles
in cell proliferation, differentiation, and migration, as
well as disease processes such as tumor angiogenesis.1,2
The most extensively studied members of the FGF fam-
ily are FGF-1 and FGF-2 which function by binding to
and dimerizing a family of signal-transducing FGF
receptors (FGFRs), leading to receptor activation and
cell signaling. This process is initiated through binding
of heparan sulfate (HS) to the FGF and FGFR to form
a ternary complex. The manner in which the compo-
nents of the complex associate is not fully understood,
and several models have been proposed.3–5 Prevention
of the formation of the HS:FGF:FGFR ternary com-
plex via blocking the interaction of HS with the FGF
may, therefore, form the basis for antiangiogenic thera-
pies.6–8
rasaccharides11) bind to the FGFs, although more re-
cent studies indicate that unsulfated di- and
trisaccharides do not bind FGF,12 and longer oligosac-
charides are usually required for the promotion of
dimerization and activation. X-ray crystal structures of
heparin-derived oligosaccharides bound to FGFs
alone13,14 or in ternary complex with FGFR15,16 have
been determined. Analyses of these structures reveal that
not all oligosaccharide sulfo and carboxyl groups bind
to FGF, and that the disaccharide GlcN(2S,6S)-
IdoA(2S) (1, Fig. 1) represents a minimal heparin/HS
consensus sequence for FGF binding.17 The 2-O-sulfo
group of IdoA and the N-sulfo group of GlcN in 1 form
the primary protein contacts and in the case of FGF-1,
Heparin and HS are glycosaminoglycans (GAGs) com-
posed of repeating 1 ! 4 linked disaccharide sequences
of a-D-glucosamine (GlcN) and a uronic acid (b-D-glu-
curonic acid, GlcA, or a-L-iduronic acid, IdoA).9 Short
heparin/HS sequences (di- and trisaccharides10 and tet-
Figure 1. Structure of the GlcN(2S,6S)-IdoA(2S) disaccharide
sequence 1, which represents a minimal consensus sequence for
FGF:HS binding,17 and a model disaccharide 2 considered in the
theoretical calculations presented here.
Keywords: Fibroblast growth factors; Disaccharides; Heparin
mimetics.
*
Corresponding author. Tel.: +61 7 3273 9150; fax: +61 7 3375
1168; e-mail: vitof@progen-pharma.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.071

L. Liu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 344–349
345
the GlcN 6-O-sulfo group also interacts favorably with
the protein.17 In these structures the GlcN residue of 1
adopts the normal C1 chair conformation whilst the
ligand binding. Glycosyl donor 4 allows for sulfonation
at O-6 enabling examination of the effects of the 6-O-sul-
fo group that is present in 1.
4
conformationally flexible18 IdoA is found in the 1C4
conformation when bound only to the protein13,14 or
in a skew-boat (2SO) conformation when part of a ter-
nary complex.15,16 Recent NMR studies indicate that
FGF-1 can bind both conformations of IdoA in a bioac-
tive hexasaccharide.19
The selection criteria for IdoA mimics as glycosyl accep-
tors included ease of synthesis and ability to adopt the
1C4 conformation in solution, thus presenting a suitably
orientated 20-O-sulfo group for binding to FGF. Highly
sulfated b-D-gluco- and -xylopyranosides are known to
exist in a conformational equilibrium between the two
1
A number of structurally simple heparin mimetics also
bind to the FGFs in the HS binding site and inhibit
FGF mitogenic activity, for example, sulfonated naph-
thalenes20 and sulfated oligosaccharides6,21 and their
glycosides.22,23 Taken together, these observations lead
us to conclude that structural mimics of disaccharide 1
might be profitably explored as potential inhibitors of
FGF-mediated angiogenesis.
chair conformers in favor of the C4.29,30 The tribenzyl
b-D-glucoside 5 and dibenzyl b-D-xyloside 6 were thus
selected as glycosyl acceptors because, once deprotected
and sulfonated, the glucose or xylose ring should exhibit
the desired conformational flexibility. It was hypothe-
sized that the additional sulfates in the ring beyond
that required at O-20 would not unduly interfere with
binding.
The synthesis of heparin/HS oligosaccharides is chal-
lenging,24 and syntheses of ‘non-regular’ disaccharides,
that is, with GlcN at the non-reducing end, have only
been reported in the past decade. Despite recent pro-
gress,25–27 a major hurdle in this area continues to be
synthetic access to IdoA. Simple disaccharides, there-
fore, were sought that could be readily synthesized while
mimicking the essential features of 1, namely the a-
(1 ! 4) linkage between the two monosaccharide units,
the spatial orientation of the two key sulfo groups
[GlcN(2S) and IdoA(2S)], and the conformational
flexibility of the IdoA residue. It was considered that
suitable disaccharides might be synthesized by glycosyl-
ation of an IdoA mimic with a differentially 2-O-pro-
tected glycosyl donor.
The Gal-Glc disaccharides were prepared as shown in
Scheme 1. The acceptor alcohol 531 was glycosylated
at ꢀ20 °C with thioglycoside donor 3,32 using N-iodo-
succinimide/triflic acid (NIS/TfOH) as promoter, to give
a 4:1 a/b mixture of anomers from which pure disaccha-
ride 7 was obtained by flash chromatography in satisfac-
tory yield (61%).33 Hydrogenolysis of the benzyl ethers
(H2/Pd(OH)2 on C) proceeded in good yield (91%) and
subsequent sulfonation (sulfur trioxide trimethylamine
complex) and deacetylation (1 M NaOH) gave the de-
sired tetrasulfate 13, which was purified by size exclusion
chromatography (Bio-Gel P-2). The purity (P96%) was
determined by capillary electrophoresis (CE)34 and
NMR spectroscopy. The trimethylated derivative 11
was prepared by quantitative deacetylation and methyl-
ation (NaOMe/MeOH and NaH/MeI) of 7. Hydrogen-
olysis of the benzyl ether protecting groups (H2/Pd on
C) gave tetrol 16 in good yield (79%), and subsequent
sulfonation afforded trimethylated tetrasulfate 17. The
corresponding pentasulfates 14 and 18 were prepared
analogously starting from glycosyl donor 4.35
The D-thiogalactosides 3 and 4 were chosen as glycosyl
donors (Fig. 2) because of the ease with which the 2-
OH can be differentially protected. It was assumed that
N-sulfo groups could be substituted for by O-sulfo, as
previously demonstrated with analogues of the AT III-
binding heparin pentasaccharide.28 It was also hypothe-
sized that D-galactose (Gal) in the place of GlcN at the
non-reducing end would not significantly reduce FGF
binding. The b-configuration and the non-participating
2-O-benzyl group were expected to favor the stereoselec-
tive formation of the desired a-(1 ! 4) linkage. Like the
naturally occurring GlcN, the Gal residue should adopt
For the synthesis of the xylose analogue of 13 (Scheme
2), glycosylation of xylose acceptor 636 with donor 3 gave
an inseparable mixture of anomers (a/b = 5:1) that was
converted into the corresponding mixture of tribenzoates
from which the pure a-anomer 19 was obtained by flash
chromatography. Hydrogenolysis to give triol 20, fol-
lowed by sulfonation/debenzoylation, afforded the trisul-
fate 21 in moderate yield (24%, three steps from 19).
4
a C1 conformation in solution. Importantly, the pro-
tecting group strategy also permits the other hydroxyl
groups of this residue to remain either unprotected or
derivatized as non-polar methyl ethers28 which allows
for the testing of small hydrophobic groups in FGF
Purification of the sulfated disaccharides was challeng-
ing and the final products were obtained in low yields
(3–24%), in part, due to contamination by inorganic
salts and the presence of undersulfated species which
were difficult to separate chromatographically. The
magnitude of the vicinal coupling constants observed
1
in the H NMR spectra indicates that the Glc rings of
13, 14, 17, and 18, and the D-xylose ring of 21, exist in
1
a chair–chair equilibrium in favor of the C4 chair con-
formation (Table 1), as anticipated.29,30
The binding affinities of the sulfated disaccharides 13,
14, 17, 18, and 21 for FGF-1, measured using a surface
Figure 2. Structures of glycosyl donors (3 and 4) and acceptors (5 and
6) used in this study.

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L. Liu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 344–349
Scheme 1. Reagents and conditions: (a) NIS, TfOH, CH2Cl2, ꢀ20 °C; (b) H2, Pd(OH)2 or Pd/C; (c) i—NaOMe, MeOH, ii—NaH, MeI; (d)
SO3ÆMe3N, DMF, 60 °C; (e) 1 M NaOH.
Scheme 2. Reagents and conditions: (a) NIS, TfOH, CH2Cl2, ꢀ20 °C; (b) i—NaOMe, MeOH; ii—BzCl, Py; iii—chromatography; (c) H2, Pd(OH)2,
CHCl3; (d) i—SO3ÆMe3N, DMF, 60 °C; ii—1 M NaOH.
Table 1. Experimental data for synthesized disaccharides
Disaccharide
J1,2
J2,3
Kd (lM)b
DG(obs)
Gscore (1C4)
Gscore (4C1)
13
14
17
18
21
2.8
<4.0
3.2
77.5 2.5
21.8 0.6
233 23
ꢀ5.60
ꢀ6.36
ꢀ4.95
ꢀ5.89
ꢀ3.89
ꢀ4.18
ꢀ4.43
ꢀ4.22
ꢀ4.70
ꢀ4.13
ꢀ2.72
ꢀ4.94
ꢀ3.58
ꢀ4.06
ꢀ3.97
3.6
2.2
1.1
2.8
n.o.a
3.6
<2.8
47.9 1.3
1400 170
Selected vicinal coupling constants J (Hz) for the reducing end b-D-glucopyranosyl or b-D-xylopyranosyl moiety of sulfated disaccharides (data
extracted from 1H NMR spectra determined in D2O at 400 MHz.)
Dissociation constants (Kd) and corresponding DG values measured for sulfated disaccharides binding to FGF-1 and calculated XPGlide Gscores for
disaccharides docked with FGF-1.
a Not observed.
b All values are means and standard deviation of at least two independent measurements.
plasmon resonance solution affinity assay,21 are shown
in Table 1. The affinities are in the lM range and com-
pare well with reported binding affinities for FGF-1 of
the synthetic heparin tetrasaccharides GlcN(2S,6S)-
IdoA(2S)-GlcN(2S,6S)-IdoA(2S)-OPr (IC50 = 0.24 lM)
and
GlcN(2S)-IdoA(2S)-GlcN(2S,6S)-IdoA(2S)-OPr

L. Liu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 344–349
347
Figure 3. Sulfate groups of the relaxed, FGF-1-docked poses of the synthesized disaccharide ligands. (a) The disaccharides with Glc in the 1C4
conformation. (b) The disaccharides with Glc in the 4C1 conformation. The molecular surface of the sulfate and carboxylate groups of the
cocrystallized hexasaccharide ligand that are involved in hydrogen bonding interactions with the protein is shown, and the backbone a-carbon atoms
of FGF-1 are represented by the orange tube. Carbon, nitrogen, sulfur, oxygen, and hydrogen atoms are shown in gray, blue, yellow, red and green,
respectively.
(IC50 = 290 lM).11 The extra sulfo group of 14 resulted
in a small (3.5-fold) improvement in affinity over 13,
while capping the free hydroxyl groups as methyl ethers
(i.e., 17 and 18) resulted in decreased affinity compared
with their non-methylated counterparts. Disaccharide
21 bound poorly to FGF-1 suggesting that the 60-O-sul-
fo group may be important in binding, perhaps by mim-
icking the carboxylate in IdoA. These results suggest
that it may be possible to tailor the specificity of binding
to different HS-binding growth factors by altering the
substituents around the two key 2-O-sulfo groups.
Table 1) and importantly, the binding modes of the
disaccharides mimic the co-crystallized hexasaccharide
ligand by positioning their sulfate groups in similar sites.
This is illustrated in Figure 3 where the positions of the
disaccharide sulfates are shown relative to the negatively
charged functional groups of the hexasaccharide (sulfate
and carboxylate) involved in hydrogen bonding interac-
tions with the protein. In all cases the bound disaccha-
ride conformation involves placement of at least one
sulfate group in a region also occupied by a charged
group from the hexasaccharide. While it is not possible
for a disaccharide to present all sulfate groups to the
protein in the preferred locations, a consequence of
the reduced size and flexibility of the disaccharide com-
pared to the hexasaccharide, the preference for nega-
tively charged functional groups in specific locations
within the FGF-1 binding site is clear, as noted
previously.41,42
Molecular docking calculations were performed using
the extra precision mode37 (XP) of the Glide pro-
gram38–40 to examine the FGF-1 binding modes of model
(2) and synthesized (13, 14, 17, 18, and 21) disaccharides.
The protein structure used for this study was of FGF-1
co-crystallized with a heparin-derived hexasaccharide li-
gand (PDB accession code 2AXM). The disaccharides
4
were constructed with the Glc moiety in both the C1
FGF-1 docking of the model disaccharide 2 yielded a
best Gscore of ꢀ4.01 kcal/mol, which is similar to those
obtained for the synthesized disaccharides (Table 1).
The bound conformation of 2 corresponding to this
Gscore is shown in Figure 4a. Three of the four charged
groups interact in formal hydrogen bonding interactions
with the protein with only the 6-O-sulfo group not par-
ticipating in hydrogen bonding. If, however, the geome-
try of 2 and the surrounding protein residues are relaxed
and 1C4 conformers, with ring flipping disallowed during
the conformer generation stage of docking. After dock-
ing, the best docked pose (i.e., lowest Gscore) for each li-
gand was relaxed in the presence of the frozen protein to
remove any unfavorable intra-ligand contacts.
The calculated Gscores are indicative of modest binding
affinity and similar in magnitude to those observed (see
Figure 4. Conformations of the model disaccharide 2 bound to FGF-1. (a) The XPGlide docked pose after relaxation of the ligand. (b) The bound
conformation after relaxation of both the ligand and nearby protein residues. The molecular surface and color scheme are the same as in Figure 3.
Hydrogen bonding interactions between FGF-1 and 2 are depicted by dotted lines.

348
L. Liu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 344–349
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together, then all of the disaccharide’s charged func-
tional groups are able to participate in hydrogen bond-
ing interactions with the protein. This is shown in Figure
4b and illustrates how an induced fit might occur in
the heparin binding site of FGF-1 upon ligand binding.
Together these results indicate that the most active
disaccharides synthesized here have predicted binding
affinities and binding modes similar to the heparin-de-
rived disaccharide they were designed to mimic.
In conclusion, simple disaccharide mimetics of a heparin
sequence that bind to FGF-1 were prepared to mimic
the conformational flexibility of IdoA. Docking calcula-
tions showed that the predicted locations of disaccharide
sulfo groups in the binding site of FGF-1 are consistent
with the positions observed for co-crystallized heparin-
derived oligosaccharides. Predicted binding affinities
are in accord with experimental Kd values obtained from
binding assays and are similar to the predicted values for
a model heparin disaccharide. These results may aid in
the design of potential inhibitors of FGF-mediated
angiogenesis.
´
20. Fernandez-Tornero, C.; Lozano, R. M.; Redondo-Hor-
cajo, M.; Gomez, A. M.; Lopez, J. C.; Quesada, E.; Uriel,
´
´
´
C.; Valverde, S.; Cuevas, P.; Romero, A.; Gimenez-
Acknowledgments
Gallego, G. J. Biol. Chem. 2003, 278, 21774.
21. Cochran, S.; Li, C.; Fairweather, J. K.; Kett, W. C.;
Coombe, D. R.; Ferro, V. J. Med. Chem. 2003, 46,
4601.
We thank Drs. Robert Don and Edward Hammond for
useful discussions.
22. Karoli, T.; Liu, L.; Fairweather, J. K.; Hammond, E.; Li,
C. P.; Cochran, S.; Bergefall, K.; Trybala, E.; Addison, R.
S.; Ferro, V. J. Med. Chem. 2005, 48, 8229.
23. Ferro, V.; Liu, L.; Hammond, E.; Dredge, K.; Bytheway,
I.; Li, C.; Johnstone, K.; Karoli, T.; Copeman, E.; Davis,
K.; Gautam, A. Semin. Thromb. Hemost. 2007, 33, 557.
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Supplementary data
General experimental procedures and details of the syn-
thesis, purification, and characterization of disaccha-
rides 13, 14, 17, 18, and 21, and of the computational
methods used in this study. Supplementary data associ-
ated with this article can be found, in the online version,
at doi:10.1016/j.bmcl.2007.10.071.
25. Orgueira, H. A.; Bartolozzi, A.; Schell, P.; Litjens, R. E. J.
N.; Palmacci, E. R.; Seeberger, P. H. Chem. Eur. J. 2003,
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