Orthogonal sulfation strategy for synthetic heparan sulfate ligands

Article abstract of DOI:10.1021/ol052130o

(Chemical Equation Presented) An orthogonal sulfation strategy involving six different protecting groups has been developed for generating sulfated carbohydrate libraries based on heparan. Chemoselective cleavage conditions (optimized for a heparan disacc

Full text of DOI:10.1021/ol052130o

ORGANIC
LETTERS
2005
Vol. 7, No. 22
5095-5098
Orthogonal Sulfation Strategy for
Synthetic Heparan Sulfate Ligands
Ren-Hua Fan, Jihane Achkar, Jesu´s M. Herna´ndez-Torres, and Alexander Wei*
Department of Chemistry, Purdue UniVersity, 560 OVal DriVe,
West Lafayette, Indiana 47907-2084
alexwei@purdue.edu
Received September 3, 2005
ABSTRACT
An orthogonal sulfation strategy involving six different protecting groups has been developed for generating sulfated carbohydrate libraries
based on heparan. Chemoselective cleavage conditions (optimized for a heparan disaccharide) can be performed in the presence of sulfate
esters as well as the remaining protecting groups.
The carbohydrate ligands referred to as heparan sulfate (HS)
are a subclass of anionic linear polysaccharides known as
the glycosaminoglycans.1 HS sequences are based on repeat-
ing-unit disaccharides comprised of glucosamines linked
R1f4 to pyranosyluronic acids and thus structurally related
to heparin, but differ in that they are typically expressed on
cell surfaces and tethered to core membrane proteins.2 HS
ligands are well-known to recruit signaling proteins such as
growth factors and chemokines and are directly implicated
in the activation of cell-surface receptors with key roles in
vascular development and immunological response.3 HS
recognition can also be exploited pathogenically: many viral
coat proteins have high affinity for heparin, and tumor cells
can hijack HS-mediated signaling pathways to facilitate
growth and metastatic invasion.4,5
The diversity of biological signaling events which rely on
HS recognition is matched by the structural complexity of
the sequences themselves. While the parent heparan poly-
saccharide is comprised of regularly repeating units of
N-acetyl-R-D-glucosamine and â-D-glucuronic acid (R-D-
GlcNAc and â-D-GlcA), several biosynthetic modifications
occur in stages within localized domains, often with variable
order and conversion (50-80% for any given step) to
produce a prodigious number of possible stereoisomers and
sulfation patterns.6,7 These structurally complex segments are
thought to be responsible for most of the biological activity
in HS. Although well over 100 HS-binding proteins have
been identified so far,7 the great majority of these have yet
to be matched with high-affinity ligands due to chal-
(4) (a) Herold, B. C.; Gerber, S. I.; Polonsky, T.; Belval, B. J.; Shaklee,
P. N.; Holme, K. Virology 1995, 206, 1108-1116. (b) Feyzi, E.; Trybala,
E.; Bergstrom, T.; Lindahl, U.; Spillmann, D. J. Biol. Chem. 1997, 272,
24850-24857.
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(b) Spillmann, D.; Lindahl, U. Curr. Opin. Struct. Biol. 1994, 4, 677-682.
(2) Ho¨o¨k, M.; Kjelle´n, L.; Johansson, S.; Robinson, J. Annu. ReV.
Biochem. 1984, 53, 847-869.
(3) (a) Arenberg, D. A.; Polverini, P. J.; Kunkel, S. L.; Shanafelt, A.;
Strieter, R. M. In Methods in Enzymology; Horuk, R., Ed.; Academic
Press: San Diego, 1997; Vol. 288, pp 190-220. (b) Lever, R.; Page, C. P.
Nature ReV. 2002, 1, 140-148.
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Nature ReV. 2002, 2, 521-528.
(6) Gallagher, J. T. Biochem. Soc. Trans. 1997, 25, 1206-1209.
(7) Conrad, H. E. Heparin-Binding Proteins; Academic Press: San Diego,
1998.
10.1021/ol052130o CCC: $30.25
© 2005 American Chemical Society
Published on Web 10/06/2005

lenges in the isolation and characterization of HS se-
quences.8,9
phthalimide group with an azide by Cu-mediated diazo
transfer onto the free amine,16 and protection of the C3
hydroxyl as a levulinate (Lev) ester (see Scheme 1).
The discovery of biologically active ligands may be
accelerated by the synthesis and screening of HS-like
oligosaccharides with variable sulfation profiles. Most
synthetic efforts related to HS have been focused on the
oligomerization of protected carbohydrate units with pre-
designated sulfation sites,10,11 whereas less attention has been
paid toward orthogonal protecting group systems which can
be used to produce diverse sulfation patterns.12 Both ap-
proaches have merit and are in fact quite complementary,
but the challenge of the latter increases rapidly with the
number of differentiable sites. To date, a focused library of
eight chondroitin sulfate disaccharides has been produced,13
and a heparan disaccharide with up to four orthogonal
protecting groups has been recently reported.12 These en-
couraging achievements set the stage for developing sulfation
patterns of greater complexity.
Scheme 1. Synthesis of Orthogonally Protected Heparan
Disaccharide 5a
Here we demonstrate an orthogonal sulfation strategy using
a heparan disaccharide unit with six different protecting
groups and a set of cleavage conditions that are also
compatible with neighboring O-sulfate esters. The chemose-
lectivity of these conditions is demonstrated by preparing a
subset of six disaccharide monosulfates, followed by depro-
tection of a neighboring hydroxyl group or conversion of
azide to NHAc. The synthetic strategy described here is
intended to enable the generation of sulfated oligosaccharide
libraries derived from a common intermediate. This includes
access to sulfation patterns not observed in isolated HS
fragments, such as those featuring a 3-O-sulfate on the uronic
acid moiety.7
Thioglycoside 1 (available in multigram quantities from
D-glucosamine)14 was converted to orthogonally protected
derivative 3 by reductive cleavage of the p-anisylidene acetal
to the 4-O-p-methoxybenzyl (PMB) ether using borane and
Bu2BOTf,15 followed by protection of the C6 hydroxyl as a
tert-butyldiphenylsilyl (TBDPS) ether, replacement of the
a Selected abbreviations: BSP ) benzenesulfinylpiperidine;
DTBMP ) di-t-Bu-4-methylpyridine; en ) ethylenediamine; im
) imidazole; PMP ) p-methoxyphenyl.
(8) Spillmann, D.; Witt, D.; Lindahl, U. J. Biol. Chem. 1998, 273,
15487-15493.
Methyl D-glucoside derivative 2 was transformed into a
bicyclic lactone via reductive cleavage to the 4-O-PMB ether,
followed by tetramethyl-1-piperidineoxy (TEMPO)-mediated
oxidation17 and lactonization to the [3.2.1] isomer of 3,6-
glucuronolactone.18 Removal of the PMB group by ceric
ammonium nitrate (CAN) produced glycosyl acceptor 4,
which was coupled with thioglycoside 3 using benzenesulfi-
(9) (a) Venkataraman, G.; Shriver, Z.; Raman, R.; Sasisekharan, R.
Science 1999, 286, 537-542. (b) Keiser, N.; Venkataraman, G.; Shriver,
Z.; Sasisekharan, R. Nature Med. 2001, 7, 123-128. (c) Liu, J.; Shriver,
Z.; Pope, R. M.; Throp, S. C.; Duncan, M. B.; Copeland, R. J.; Raska, C.
S.; Yoshida, K.; Eisenberg, R. J.; Cohen, G.; Linhardt, R. J.; Sasisekharan,
R. J. Biol. Chem. 2002, 277, 33456-33467.
(10) For a recent review, see: Poletti, L.; Lay, L. Eur. J. Org. Chem.
2003, 2999-3024.
(11) Recent examples of HS syntheses: (a) Tabeur, C.; Mallet, J. M.;
Bono, F.; Herbert, J. M.; Petitou, M.; Sinay, P. Bioorg. Med. Chem. 1999,
7, 2003-2012. (b) Ojeda, R.; de Paz, J. L.; Mart´ın-Lomas, M. Chem.
Commun. 2003, 2486-2487. (c) Ojeda, R.; Terenti, O.; de Paz, J.-L.; Mart´ın-
Lomas, M. Glycoconjugate J. 2004, 21, 179-195. (d) Lubineau, A.; Lortat-
Jacob, H.; Gavard, O.; Sarrazin, S.; Bonnaffe´, D. Chem. Eur. J. 2004, 10,
4265-4282. (e) Code´e, J. D. C.; Stubba, B.; Schiattarella, M.; Overkleeft,
H. S.; van Boeckel, C. A. A.; van Boom, J. H.; van der Marel, G. A. J.
Am. Chem. Soc. 2005, 127, 3767-3773.
(15) (a) Jiang, L.; Chan, T.-H. Tetrahedron Lett. 1998, 39, 355-358.
(b) Herna´ndez-Torres, J. M.; Achkar, J.; Wei, A. J. Org. Chem. 2004, 69,
7206-7211.
(16) (a) Alper, P. B.; Hung, S.-C.; Wong, C.-H. Tetrahedron Lett. 1996,
37, 6029-6033. (b) Liew, S.-T.; Wei, A. Carbohydr. Res. 2002, 337, 1319-
1324.
(17) (a) Boulineau, F. P.; Wei, A. Org. Lett. 2002, 4, 2281-2283. (b)
Boulineau, F. P.; Wei, A. Org. Lett. 2004, 6, 119-121.
(18) (a) Kornilov, A. V.; Sherman, A. A.; Kononov, L. O.; Shashkov,
A. S.; Nifantiev, N. E. Carbohydr. Res. 2000, 329, 717-730. (b) Kornilov,
A. V.; Sukhova, E. V.; Nifantiev, N. E. Carbohydr. Res. 2001, 336, 309-
313.
(12) Recent examples of orthogonally protected HS fragments: (a) Haller,
M. F.; Boons, G.-J. Eur. J. Org. Chem. 2002, 2033-2038. (b) Prabhu, A.;
Venot, A.; Boons, G.-J. Org. Lett. 2003, 5, 4975-4978.
(13) Lubineau, A.; Bonnaffe´, D. Eur. J. Org. Chem. 1999, 2523, 3-2532.
(14) Herna´ndez-Torres, J. M.; Liew, S.-T.; Achkar, J.; Wei, A. Synthesis
2002, 487-490.
5096
Org. Lett., Vol. 7, No. 22, 2005

nyl piperidine (BSP) and Tf2O as activating agents.11e,19
Employing a Lev group at the C3 position of the glycosyl
donor 3 created some obstacles in glycosidic coupling, due
to its reactivity with Tf2O and its proximity to the activated
anomeric center.20,21 Nevertheless, the desired R-linked
disaccharide could be obtained in good yield by inverting
the order of reagent addition (activating BSP with Tf2O prior
to the addition of glycosyl donor) and by maintaining the
reaction temperature below -55 °C. Methanolysis of the
disaccharide lactone produced the corresponding methyl ester
in 64% isolated yield over two steps. The free C3 hydroxyl
was subsequently protected as the 2-trimethylsilylethoxy-
methyl (SEM) ether, resulting in orthogonally protected
heparan disaccharide 5.
Scheme 2
The use of bicyclic lactone 4 as glycosyl acceptor is
1
noteworthy in several respects. First, the C4 conformation
forces the C4 hydroxyl into an axial configuration with
relatively low steric encumbrance, a geometry known to be
favorable for R-glycosidic couplings in related systems.22
Performing the coupling of 3 with GlcA derivative 6 under
identical conditions resulted in low yields of 5 (ca. 25%)
and required a tedious separation from unreacted glycosyl
acceptor. Second, the lactone ring opening after glycosidic
coupling provides greater flexibility in the choice of O3
protecting group at a late stage. Third, the carboxyl group
can be readily converted into a variety of esters or other acyl
derivatives, introducing a seventh protecting group. For
example, SiO2-mediated hydrolysis of the disaccharide
lactone yielded the free acid 7 in 60% yield, followed by
alkylation under biphasic conditions to produce benzyl ester
8 in 42% overall yield from 3 (See Scheme 2).23 For the
purposes of our study, we opted to constrain our investiga-
tions to the six hydroxyl and amine protecting groups
featured in methyl ester 5.
Table 1. Reagents and Conditions for Orthogonal Deprotection
of 5
group
deprotection conditions
TBDPS TBAF (30 equiv) in THF, adjusted to pH 10, 1 day, rt
PMB
Lev
SEM
Ac
CAN in 90% aq CH3CN (3 equiv), 12 h, 0 °C
N2H4‚H2O (10 equiv) in 3:2 pyridine/AcOH, 6 h, rt
MgBr2‚Et2O (10 equiv), CH3NO2 (20 equiv), Et2O, 6 h, rt
Mg(OMe)2 (15 equiv) in 1:1 MeOH/CH2Cl2, 12 h, rt
(to -NHAc) AcSH (40 equiv), pyridine, 44 h, rt
(to -NH2) Bu3P (1.5 equiv) in CH2Cl2, 5 h then
1:1 H2O/CH2Cl2, 12 h, rt
N3
the generation of sulfated oligosaccharide libraries im-
mobilized on solid-phase supports. Second, the â-GlcA
linkage in HS suggests the placement of an acyl protecting
group for the C2 hydroxyl, as their utility to assist â-gly-
cosidation is well-known. Third, we wished to take advantage
of orthogonal cleavage conditions which had already been
developed for protecting groups with similar reactivities.25,26
However, it must be noted that their relative stabilities are
also dependent on their position, a frequent observation in
the regioselective formation and cleavage of carbohydrate
protecting groups.27 For example, switching the positions of
Lev (C3′) and Ac (C2) in 5 resulted in a loss of chemose-
lectivity during methanolysis due to the greater lability of
acyl groups at the C2 position.
Orthogonal deprotection conditions were developed for
converting disaccharide 5 to mono-O-sulfate esters 9-13 (see
Tables 1 and 2).24 In addition to the obvious need for
chemoselectivity, several other factors were considered in
the placement and cleavage of each protecting group. First,
the deprotection conditions should also be applicable toward
(19) Crich, D.; Smith, M. J. Am. Chem. Soc. 2001, 123, 9015-9020.
(20) Boons and co-workers have shown that the Lev groups at the C3
position on glycosyl acceptors or at the C2 position of L-Ido donors are
compatible with glycosidic coupling. See ref 12.
(23) Bocchi, V.; Casnati, G.; Dossena, A.; Marchelli, R. Synthesis 1979,
957-960.
(24) O-Sulfate esters were prepared by treating partially deprotected
disaccharides with SO3‚Me3N in pyridine for 20 h at 55 °C. See the
Supporting Information for details.
(21) Recent evidence by Woerpel and co-workers suggests that electro-
negative substituents on tetrahydropyrylium ions prefer to adopt pseudoaxial
orientations: (a) Ayala, L.; Lucero, C. G.; Romero, J. A. C.; Tabacco, S.
A.; Woerpel, K. A. J. Am. Chem. Soc. 2003, 125, 15521-15528. (b)
Chamberland, S.; Ziller, J. W.; Woerpel, K. A. J. Am. Chem. Soc. 2005,
127, 5322-5323.
(25) (a) Greene, T. W.; Wuts, P. G. M. Protecting Groups in Organic
Synthesis, 3rd ed.; John Wiley: New York, 1999. (b) Kocienski, P. J.
Protecting Groups, 3rd ed.; Thieme: Stuttgart, 2004.
(26) Xu, Y.-C.; Bizuneh, A.; Walker, C. Tetrahedron Lett. 1996, 37,
455-458.
(27) Selected reviews: (a) David, S.; Hanessian, S. Tetrahedron 1985,
41, 643-663. (b) Stanek, J. Top. Curr. Chem. 1990, 154, 209-256.
(22) Orgueira, H. A.; Bartolozzi, A.; Schell, P.; Seeberger, P. H. Angew.
Chem., Int. Ed. 2002, 41, 2128-2131.
Org. Lett., Vol. 7, No. 22, 2005
5097

of our knowledge the stability of sulfate esters under multiple
deprotection conditions has not been studied methodically.
We chose to address this issue by removing protecting groups
adjacent to the O-sulfate esters in compounds 9-13 using
the conditions listed in Table 1. These cleavage reactions
proceeded smoothly to afford the disaccharide monosulfates
14-18 in high yields (see Table 2). In the case of 3′-O-
sulfate 11, the azide was converted directly to N-acetyl
disaccharide 16 by addition of thioacetic acid.12a,28 Last,
N-sulfate disaccharide 19 was prepared from 5 by first
removing the Lev group followed by Bu3P reduction of the
azide and hydrolysis and then selective N-sulfation using
PhOSO2Cl in CH2Cl2.29 It is worth mentioning that com-
pound 19 could be purified by silica gel chromatography in
75% isolated yield. Chemoselective N-sulfation is typically
used as the final step in HS oligosaccharide synthesis because
of the product’s sensitivity to aqueous acid, but the stability
of N-sulfates in organic solvents may be significantly higher
and warrants further study.
Table 2. Heparan Disaccharide Monosulfates Derived from 5a
The orthogonal deprotection-sulfation strategy presented
here demonstrates that this approach is capable of generating
diverse sulfation profiles from a common synthetic heparan
precursor. Further implementation will require its adaptation
to oligosaccharides on solid-phase supports, so that fully
deprotected HS ligands can be prepared with minimal
attrition.
Acknowledgment. We gratefully acknowledge financial
support from the National Insititutes of Health (GM-06982-
01), the American Cancer Society (CDD-106244), the
American Heart Association (30399Z), and the Purdue
Cancer Center. J.A. thanks the American Heart Association
Midwest Affiliates for support in the form of a predoctoral
fellowship (0110248Z).
Supporting Information Available: Experimental details
on the synthesis and spectroscopic characterization of
compounds 3-5 and 8-19 and 1H and 13C NMR spectra of
all compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
a Deprotection and sulfation conditons are described in Table 1 and ref
24. b Isolated yields over two steps. c Isolated yields after second depro-
tection. d Isolated yield over three steps from 5.
OL052130O
To achieve full orthogonality, each deprotection condition
must also be compatible with O-sulfate esters already present
on the carbohydrate. While several cleavage reactions have
been shown to be compatible with O-sulfates,13 to the best
(28) (a) Jacquinet, J.-C. Carbohydr. Res. 1990, 199, 153-181. (b)
Shangguan, N.; Katukojvala, S.; Greenberg, R.; Williams, L. J. J. Am. Chem.
Soc. 2003, 125, 7754-7755.
(29) Kerns, R. J.; Linhardt, R. J. Synth. Commun. 1996, 26, 2671-2680.
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Org. Lett., Vol. 7, No. 22, 2005