Synthesis of heparin-like oligosaccharides on a soluble polymer support.

Article abstract of DOI:10.1039/b307259b

Based on previously developed solution phase chemistry, an effective general approach to the synthesis of heparin-like oligosaccharides on a soluble polymer support is reported.

Full text of DOI:10.1039/b307259b

Synthesis of heparin-like oligosaccharides on a soluble polymer support†
Rafael Ojeda, José-Luis de Paz and Manuel Martín-Lomas*
Grupo de Carbohidratos, Instituto de Investigaciones Químicas, CSIC, Americo Vespucio s/n, E-41092,
Sevilla, Spain. E-mail: Manuel.Martin-Lomas@iiq.cartuja.csic.es; Tel: 34 95 4489563; Fax: +34 95
4460565
Received (in Corvallis, OR, USA) 25th June 2003, Accepted 25th August 2003
First published as an Advance Article on the web 3rd September 2003
Based on previously developed solution phase chemistry, an
effective general approach to the synthesis of heparin-like
oligosaccharides on a soluble polymer support is reported.
sis conditions to construct oligosaccharides on automated
synthesisers is much needed. However, solid phase method-
ologies for the synthesis of such complex oligosaccharides have
to be further developed.9 In spite of the considerable effort
devoted to the synthesis of GAG oligosaccharides,10 only a
preliminary communication on solid phase synthesis of O-
methylated HS-like oligomers has appeared in the literature.11
As a consequence of our interest in the molecular mechanism
of FGF activation we are involved in work directed towards
developing a general solid phase synthesis approach based on
our previous solution phase syntheses.8 As a result we report in
this communication an effective preparation of heparin-like
oligosaccharides on a soluble polymer support as a key step in
simplifying the synthetic process. The effectiveness of this
approach is illustrated by the synthesis of hexasaccharide 14
which contains the structural motif of the regular region of
heparin.
The biological functions of heparin-binding proteins are
regulated by heparan sulfate glycosaminoglycans (HS-GAGs).1
The recognition that a structurally unique heparin pentasacchar-
ide binds antithrombin with high selectivity2 suggested that the
interactions of GAGs with heparin-binding proteins3 are
saccharide specific. However, the minimal structural require-
ments for GAGs to bind other heparin-binding proteins have not
been as precisely determined. Thus, for fibroblast growth
factors 1 and 2 (FGF-1 and FGF-2)4 minimal binding motifs
have been reported5 but the information available from
crystallographic analysis of FGF complexes with heparin
fragments reveals highly diverse patterns of stoichiometry,
contact sites and orientation.6 The heterogeneity of the GAG
fragments used in these studies is thought to be partially
responsible for this apparently conflicting evidence. Indeed, HS
is recognised to be a family of closely related polysaccharides
composed of unsulfated and variously sulfated sequences of
The extension of our solution phase chemistry8 to the
polymer support has involved a careful study of the compatibil-
ity of all parameters involved such as type of support, linker and
attachment as well as capping, glycosylation and cleavage
strategies. Problems associated with the solid phase reactivity of
the key disaccharide building blocks, and particularly of the
increasing oligosaccharide constructs, in the formation of the
a(1 ? 4) glycosidic linkage decided us to use a soluble polymer
support such as the readily available polyethylene glycol w-
monomethyl ether (MPEG).12 On the other hand, our solution
phase chemistry conditions were compatible with an ester
linker, the crucial choice being the attachment position.
Regarding the sulfation patterns of natural GAGs it seemed
advisable to avoid positions R1, R3, R4, and R5. Problems
alternating 1 ? 4 linked
D
-glucosamine and
L
-iduronic or -
D
glucuronic acid units.7 It is generally agreed that the availability
of specifically designed heparin-like oligosaccharides with
precisely defined molecular structures will constitute a crucial
step in studying and understanding the molecular basis of the
diverse heparin (HS) regulated biological activities.3,4
In the frame of a programme on the activation of FGFs we
have synthesised several heparin-like oligosaccharides using a
convergent n + 2 approach (Scheme 1) that permits us to control
the size, the sequence and the pattern of sulfation in the final
product.8 Effective as this approach might be, the synthesis of
the diversity of oligosaccharides required for investigating the
molecular recognition events involved in the interaction of
heparin (HS) with heparin-binding proteins demands further
simplification and automation. Here, as in most areas in
Glycobiology, the development of efficient solid phase synthe-
associated with the well established preparation of the key -
L
iduronate monosaccharide building bocks13 precluded R7 and
previous experience also advised us to avoid R8. Therefore, we
envisaged attaching the starting disaccharide building block (1)
to the support through the carboxylate group (Scheme 2). For
the sake of practicality and in order to make as much use as
possible of well established solution phase chemistry the methyl
ester group was transformed into the 2-hydroxyethyl uronate (2)
using recently reported mild transesterification conditions.14.
Compound 2 was then attached to MPEG through hemisucci-
nate 3. The MPEG-bound disaccharide 4 was then transformed
into a glycosyl acceptor using the previously established8
sequence 4 ? 5 ? 6 (Scheme 3). The outcome of this and the
ensuing reaction sequences was directly followed using NMR
spectroscopy. Glycosylation with glycosyl donor 78 gave the
MPEG bound tetrasaccharide 8.15 This glycosylation step was
Scheme 1
† Electronic supplementary information (ESI) available: NMR spectra. See
Scheme 2 a) Ethylene glycol, Bu2SnO, toluene, 135 °C, 81%; b) succinic
http://www.rsc.org/suppdata/cc/b3/b307259b/
anhydride, DMAP, Py, 98%; c) MPEG, DIC, DMAP, CH2Cl2, 100%.
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CHEM. COMMUN., 2003, 2486–2487
This journal is © The Royal Society of Chemistry 2003

support. Using this approach, the diversity of disaccharide
building blocks already prepared will permit the straightforward
preparation of a diversity of GAG oligosaccharides for
biological investigation.
We thank the Ministry of Science and Technology for
financial support (Grant BQU 2002-03734) and N. C. Reichardt
for helpful discussions.
Notes and references
1 H. E. Conrad, Heparin-binding proteins, Academic Press, San Diego,
1998.
2 L. Thunberg, G. Bäckström and U. Lindahl, Carbohydr. Res., 1982,
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3 I. Capila and R. J. Linhardt, Angew. Chem., Int. Ed., 2002, 41,
390–412.
4 S. Faham and R. J. Linhardt, Curr. Opin. Struct. Biol., 1998, 8,
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5 J. Kreuger, M. Salmivirta, L. Sturiale, G. Giménez-Gallego and U.
Lindahl, J. Biol. Chem., 2001, 276, 30744–30752.
6 (a) S. Faham, R. E. Gileman, J. R. Fromm, R. J. Linhardt and D. C. Rees,
Science, 1996, 271, 1116–1120; (b) A. D. DiGabriele, I. Lax, D. I. Chen,
C. M. Svahn, M. Jaye, J. Schlessinger and W. A. Hendrickson, Nature,
1998, 393, 812–817; (c) J. Schlessinger, A. N. Plotnikov, O. A.
Ibrahimi, A. V. Eliseenkova, B. K. Yeh, A. Yayon, R. J. Linhardt and M.
Mohammadi, Mol. Cell, 2000, 6, 743–750; (d) L. Pellegrini, D. F.
Burke, F. Von Delft, B. Mulloy and T. L. Blundell, Nature, 2000, 407,
1029–1034.
7 B. Casu and U. Lindahl, Adv. Carbohydr. Chem. Biochem., 2001, 57,
159–256.
8 (a) J. L. de Paz, J. Angulo, J. M. Lassaletta, P. M. Nieto, M. Redondo
Horcajo, R. M. Lozano, G. Giménez-Gallego and M. Martín-Lomas,
ChemBioChem, 2001, 2, 673–685; (b) R. Ojeda, J. Angulo, P. M. Nieto
and M. Martín-Lomas, Can. J. Chem., 2002, 80, 917–936; (c) R. Lucas,
J. Angulo, P. M. Nieto and M. Martín-Lomas, Org. Biomol. Chem.,
2003, 1, 2253–2266.
9 (a) P. H. Seeberger and W.-C. Haase, Chem. Rev., 2000, 100,
4349–4393; (b) O. J. Plante, E. R. Palmaci and P. H. Seeberger, Science,
2001, 291, 1523–1527; (c) E. R. Palmaci, O. J. Plante and P. H.
Seeberger, Eur. J. Org. Chem., 2002, 595–606.
10 J. Tamura, Trends Glycosci. Glycotechnol., 2001, 13, 65–88 and
references therein.
11 C. M. Dreef-Tromp, H. A. M. Willems, P. Westerduin, P. van Veelen
and C. A. A. van Boeckel, Bioorg. Med. Chem. Lett., 1997, 7,
1175–1180.
12 S. P. Douglas, D. M. Whitfield and J. J. Krepinsky, J. Am. Chem. Soc.,
1991, 113, 5095–5097.
Scheme 3 a) EtSH, pTsOH, CH2Cl2; b) BzCN, Et3N (cat.), CH3CN, 220
°C; c) i. TMSOTf, CH2Cl2 (four cycles); ii. PS-Suc-COOH, DMAP, DIC,
CH2Cl2; d) EtSH, pTsOH, CH2Cl2; e) BzCN, Et3N (cat.), CH3CN, 215 °C;
f) i. TMSOTf, CH2Cl2 (four cycles); ii. PS-Suc-COOH, DMAP, DIC,
CH2Cl2; g) i. LiOH, H2O2, THF; ii. MeOH, KOH 3M, 37% from 3, 8 steps;
h) see reference [8a].
13 (a) J. C. Jacquinet, M. Petitou, P. Duchaussoy, I. Lederman, J. Choay,
G. Torri and P. Sinaÿ, Carbohydr. Res., 1984, 130, 221–241; (b) R.
Ojeda, J. L. de Paz, M. Martín-Lomas and J. M. Lassaletta, Synlett,
1999, 8, 1316–1318; (c) A. Lubineau, O. Gavard, J. Alais and D.
Bonnaffé, Tetrahedron Lett., 2000, 41, 307–311.
followed by capping using a Merrifield type resin functionalised
with an acid-ended tether to esterify unreacted hydroxyl
groups.15 The MPEG-bound tetrasaccharide glycosyl acceptor
was then generated using the sequence 8 ? 9 ? 10.
Glycosylation of 10 with donor 118 afforded bound hex-
asaccharide 12.15 Although cleavage from the support could be
performed by dibutyltin oxide mediated transesterification with
methanol,14 cleaner results were obtained by directly submitting
12 to basic conditions (Scheme 3). The obtained hexasaccharide
13 showed an NMR spectrum that was identical in all respects
to that of a sample previously prepared using solution phase
chemistry.8 From 13 the target hexasaccharide 14 can be
directly prepared.8a
14 P. Baumhof, R. Mazitschek and A. Giannis, Angew. Chem., Int. Ed.,
2001, 40, 3672–3674.
15 The glycosylation reactions were carried out using the following general
protocol: To a mixture of the MPEG-bound glycosyl acceptor (0.1
mmol) and glycosyl donor (0.2 mmol) in dry CH2Cl2 (3 mL), TMSOTf
(0.03 mmol) was added. After stirring for 1–2 h, Et3N was added and the
volume reduced to 2 mL. Et2O (30 mL) was added and the precipitate
removed. Capping was then performed by swelling a mixture of the
formed MPEG-bound oligosaccharide, PS-Suc-COOH (0.49 mmol) and
DMAP in dry CH2Cl2 (7 mL), then adding DIC (0.8 mmol) and shaking
overnight. The mixture was filtered and the filtrate concentrated to 3–4
mL. Et2O (40–50 mL) was added until precipitation of the pure MPEG-
bound oligosaccharide occurred.
In conclusion, the solution phase synthetic approach pre-
viously developed by us has allowed for the construction of the
basic heparin-like hexasaccharide backbone on a polymer
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