Tunable heparan sulfate mimetics for modulating chemokine activity

Article abstract of DOI:10.1021/ja4027727

Heparan sulfate (HS) glycosaminoglycans participate in critical biological processes by modulating the activity of a diverse set of protein binding partners. Such proteins include all known members of the chemokine superfamily, which are thought to guide the migration of immune cells through their interactions with HS. Here, we describe an expedient, divergent synthesis to prepare defined HS glycomimetics that recapitulate the overall structure and activity of HS glycosaminoglycans. Our approach uses a core disaccharide precursor to produce a variety of differentially sulfated glycopolymers. We demonstrate that a specific trisulfated mimetic antagonizes the chemotactic activity of the proinflammatory chemokine RANTES with potency similar to that of heparin, without inhibiting serine proteases in the blood coagulation cascade. Our work provides a general strategy for modulating chemokine activity and dissecting the pleiotropic functions of HS/heparin through the presentation of defined sulfation motifs within polymeric scaffolds.

Full text of DOI:10.1021/ja4027727

Communication
pubs.acs.org/JACS
Tunable Heparan Sulfate Mimetics for Modulating Chemokine
Activity
Gloria J. Sheng,† Young In Oh,† Shuh-Kuen Chang,§ and Linda C. Hsieh-Wilson*,†
†Division of Chemistry and Chemical Engineering, California Institute of Technology and Howard Hughes Medical Institute, 1200
East California Boulevard, Pasadena, California 91125, United States
§Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, United States
S
* Supporting Information
have demonstrated that a unique sulfated sequence found within
heparin is primarily responsible for its anti-coagulant activity.6
ABSTRACT: Heparan sulfate (HS) glycosaminoglycans
participate in critical biological processes by modulating
the activity of a diverse set of protein binding partners.
Such proteins include all known members of the
chemokine superfamily, which are thought to guide the
migration of immune cells through their interactions with
HS. Here, we describe an expedient, divergent synthesis to
prepare defined HS glycomimetics that recapitulate the
overall structure and activity of HS glycosaminoglycans.
Our approach uses a core disaccharide precursor to
produce a variety of differentially sulfated glycopolymers.
We demonstrate that a specific trisulfated mimetic
antagonizes the chemotactic activity of the proinflamma-
tory chemokine RANTES with potency similar to that of
heparin, without inhibiting serine proteases in the blood
coagulation cascade. Our work provides a general strategy
for modulating chemokine activity and dissecting the
pleiotropic functions of HS/heparin through the presenta-
tion of defined sulfation motifs within polymeric scaffolds.
Heparin has also been shown to have potent anti-inflammatory
activity in models of asthma, chronic dermatitis, and ulcerative
colitis, but it is not recommended as an anti-inflammatory agent
in clinical practice due to its anti-coagulant activity.7 Moreover,
heparin isolated from natural sources can induce other
undesirable physiological effects due to its structural hetero-
geneity.6a The development of chemically defined HS/heparin
mimetics that lack anti-coagulant activity offers a potential
solution to these challenges. Although a few studies have
modified natural polysaccharides or undertaken the semi-
synthesis of glucan sulfate mimetics,8 such approaches lack the
precision of chemical synthesis9 in controlling the sulfation
sequence. To our knowledge, only one study9a exploited
chemical synthesis to generate complex, HS-based oligosacchar-
ides that modulate pro-inflammatory chemokines. Here, we
describe a new class of simplified HS/heparin glycomimetics that
have a highly tunable structure, controllable lengths, and defined
sulfation motifs. Importantly, these molecules possess the ability
to inhibit pro-inflammatory chemokines with similar efficiency to
natural heparin, yet they do not interact with key factors in the
coagulation cascade.
eparan sulfate (HS) glycosaminoglycans are a ubiquitous
Hclass of sulfated polysaccharides that are involved in
diverse physiological processes, such as development, wound
healing, angiogenesis, and inflammation.1 Assembled from
disaccharide subunits, HS polysaccharides exhibit subtle
variations in stereochemistry, length, and patterns of sulfation.
This structural diversity enables HS to modulate the activity of
>400 different proteins, including ∼50 members of the
chemokine superfamily.2 Chemokines are chemoattractant
proteins that control the activation and migration of specific
leukocyte populations toward sites of injury, inflammation, and
atherosclerosis. Accumulation of pro-inflammatory chemokines
into epithelial spaces contributes to the pathogenesis of allergy,
arthritis, psoriasis, and other inflammatory disorders.3 Although
HS proteoglycans are required for establishing chemokine
gradients in vivo,4 the precise carbohydrate structural determi-
nants involved have yet to be elucidated, limiting the develop-
ment of HS-based strategies for targeting chemokine activity.
A major challenge to understanding the structure−activity
relationships of HS and developing HS-based therapeutic
approaches has been the chemical complexity and heterogeneity
of HS in vivo. Heparin, a close structural relative of HS, displays
less heterogeneity and is used clinically as an anti-coagulant drug
for the prevention and treatment of thrombosis.5 Elegant studies
Most chemokines have a primary HS-binding site occupied by
at least two sugar residues.10 Based on our prior work with
chondroitin sulfate glycosaminoglycans,11 we envisaged that HS
disaccharide epitopes appended onto a multivalent polymer
backbone might be sufficient to target this binding site, provided
that their binding affinity could be enhanced through avidity
(Scheme 1a). A polynorbornene backbone was selected to allow
for maximal control over chain length and low polydispersity
through ring-opening metathesis polymerization chemistry
(Scheme 1b).12 Reduction of the unsaturated backbone was
expected to emulate the conformational plasticity found in native
HS/heparin and facilitate its interaction with proteins.13
Furthermore, by controlling the sulfation pattern prior to
polymerization, we sought to increase the specificity of the
polymers for particular HS-binding proteins. Notably, these
structures would represent the first example of high-molecular-
weight polymers prepared from minimal HS disaccharide units.
A library of differentially sulfated glycopolymers 1−4 was
derived from a single synthetic precursor, 5 (Scheme 1b). The
orthogonal protecting group strategy for 5 was designed to allow
Received: March 19, 2013
© XXXX American Chemical Society
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Journal of the American Chemical Society
Communication
a
Scheme 1. (a) Synthetic Mimetic of Heparin/HS
Scheme 2. Synthesis of Disaccharide 5
Polysaccharides and (b) Retrosynthesis of Glycopolymers 1−
4
a
Reagents and conditions: (a) AcCl, Py, DMAP, CH2Cl2, −40 °C,
quant.; (b) (i) TiBr4, CH2Cl2, (ii) 2,4,6-collidine, MeOH, CH2Cl2,
75%; (c) NaOMe, MeOH, −10 °C, 80%; (d) TBSOTf, Py, 0 °C, 92%;
(e) HOPO(OBu)2, CH2Cl2, 4 Å MS, quant.; (f) TMSOTf, 7, CH2Cl2,
4 Å MS, 93%; (g) (i) TBAF, AcOH, THF, 0 °C, (ii) Cl3CCN, DBU,
CH2Cl2, 0 °C, 89%; (h) BF3·OEt2, 8, CH2Cl2, 4 Å MS, 80%.
multiple sulfation patterns to be accessed using a minimum
number of steps. We chose to install a tert-butyldimethylsilyl
(TBS) group at the C4′-hydroxyl and benzyl ethers (Bn) at the
non-sulfated positions C3′ and C3 to enhance the solubility of
the sulfated monomers during the polymerization step. Acetyl
(Ac) and levulinoyl (Lev) ester groups were selected for sites that
would ultimately carry the O-sulfonate groups (C2′ and C6,
respectively); the former group would also provide anchimeric
assistance in generating the 1,2-trans glycosidic linkage.
resulting compounds 16 and 17 were regioselectively N-sulfated,
O-sulfated, and N-acetylated at the desired positions. Con-
veniently, we found that simultaneous sulfation at the O- and N-
positions could be achieved using SO3·Py and mild heating
conditions (55 °C). Polymerization of the sulfated monomers
with 5 mol % of Grubbs’ fast-initiating catalyst [(H2IMes)-
(Py)2(Cl)2RuCHPh]17 led to rapid conversion within
minutes to the desired glycopolymers (22−25). Late-stage
deprotection conditions were first established using monomer 18
to prepare a monovalent trisulfated disaccharide bearing the
norbornyl linker at the reducing end (26, see SI) and then
applied to 22−25. We were pleased to find that TMSOK-
mediated saponification of the methyl ester also deprotected the
TBS group at C4′, likely due to its proximity to the resulting C5′
carboxylate anion. Finally, catalytic hydrogenation using Pd-
(OH)2/charcoal in a co-solvent of phosphate buffer and
methanol9b delivered 26 and glycopolymers 1−4, which were
Disaccharide 5 was generated from iduronic acid (IdoA)
phosphate donor 6, glucosamine (GlcN) acceptor 7,14a and
norbornyl linker 8.12c An efficient route to donor 6 and its
subsequent transformation to disaccharide 5 is shown in Scheme
2. First, the 1,2,4-triol 9 was synthesized from commercially
available diacetone glucose in six steps14b and treated with AcCl
and DMAP at −40 °C15a to afford the triacetylated β-isomer 10
in quantitative yield. Conditions using Ac2O or higher
temperatures15b were avoided as they led to a mixture of α/β-
pyranose and α/β-furanose isomers. Anomeric bromination of
15a
10 using TiBr4 formed a glycosyl halide intermediate, which
was directly transformed into 1,2-orthoester 11 using 2,4,6-
collidine as the base. The 1,2-orthoester moiety locked the
pyranose ring into the 4C1 conformation and allowed for
selective cleavage of the remaining acetyl group under Zemplen
́
1
characterized by H NMR spectroscopy and size exclusion
conditions. After several unsuccessful attempts using standard
conditions, we found that TBS protection of the C4′ hydroxyl of
12 required excess TBSOTf in neat pyridine to compensate for
its poor nucleophilicity.
chromatography multi-angle light scattering. Comparison of the
1H−1H gCOSY NMR spectrum of trisulfated monomer 26 to
that of glycopolymer 1 confirmed that the sulfation pattern
remained intact during the post-polymerization deprotection
steps. By varying the amount of catalyst, we could predictably
control the glycopolymer lengths within a relatively narrow
polydispersity range, affording polymers with lengths compara-
ble to the length of commercially available heparin (Table S1).
With the library of glycopolymers in hand, we characterized
their binding specificities for RANTES, a basic chemokine that
induces the migration of T cells, monocytes, natural killer cells,
and other classes of leukocytes.18 Although RANTES preferen-
tially recognizes trisulfated heparin (Kd = 32.1 nM) over other
classes of glycosaminoglycans,19 the effects of HS sulfation on
RANTES binding have not been examined using homogeneously
sulfated molecules. We compared the relative ability of each
glycopolymer to block RANTES binding to heparin (EC50 = 12.2
nM; Figure S1) using a competitive enzyme-linked immuno-
sorbent assay. Trisulfated glycopolymer 1 bound strongly to
RANTES (IC50 = 9.3 1.1 μg/mL (334 39 nM)), albeit with
reduced affinity compared to heparin of similar chain length
(IC50 = 0.90 0.03 μg/mL (45.0 1.5 nM); Figure 1 and Table
Direct reaction of orthoester 13 with acceptor 7 gave low
yields of the desired IdoA-GlcN disaccharide 14. We therefore
chose to explore the reactivity of the corresponding IdoA
phosphate donor, which could be accessed from 13 using
16
HOPO(OBu)2 in a single quantitative step. Fortuitously,
phosphate donor 6 reacted with acceptor 7 and stoichiometric
TMSOTf16 to deliver the α-linked disaccharide 14 in 93% yield.
To tether the HS disaccharide to norbornyl linker 8, we
converted 14 into glycosyl imidate 15 by deprotecting the
anomeric TBS and treating the resulting hemiacetal with
trichloroacetonitrile and DBU. Glycosidic coupling of 15 and 8
with catalytic TMSOTf resulted in a 1:1 mixture of α/β isomers;
however, treatment with BF3·OEt2 (0.34 equiv) gave exclusively
the β-isomer in 80% yield (δ = 4.34 ppm, J12 = 8 Hz).
Disaccharide 5 was then diversified into four differentially
sulfated polymers (1−4; Scheme 3). The azide was reduced
using 1,3-propanedithiol,9b and the resulting intermediate was
treated either with K2CO3 to deprotect both ester groups or with
hydrazine acetate9c to selectively cleave the Lev ester. The
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a
Scheme 3. Synthesis of Glycopolymers 1−4
a
Reagents and conditions: (a) 1,3-propanedithiol, DIPEA, MeOH, 87%; (b) K2CO3, MeOH, 78%; (c) H2NNH2·H2O, AcOH, Py, quant.; (d)
SO3·Py, NEt3, Py, 55 °C, 78−85%; (e) Ac2O, NEt3, MeOH, quant.; (f) (H2IMes)(Py)2(Cl)2RuCHPh, DCE, MeOH, quant.; (g) (i) TMSOK,
TBAI, THF, 80−88%, (ii) Pd(OH)2/charcoal, H2 (1 atm), phosphate buffer (pH 7.4), MeOH, 37−55%.
in a multivalent framework and underscore the importance of
avidity in the recognition of glycosaminoglycan structures.
Next, we tested whether site-defined modifications to the
sulfation pattern of 1 would alter its affinity for RANTES.
Removal of either the N-sulfate group of GlcN (2) or the 2-O-
sulfate group of IdoA (4) decreased binding to RANTES (IC50 =
31.1 6.2 μg/mL (852 170 nM) and 58.0 5.7 μg/mL (1760
170 nM), respectively), and unsulfated 3 had no appreciable
activity (Figure 1a). The observation that removal of 2-O-
sulfation has a greater effect than removal of N-sulfation suggests
that precise positioning of the sulfate groups (in addition to
overall charge) is important for determining the affinity of the
glycopolymers for RANTES. Importantly, none of the
glycopolymers possessed anti-coagulant activity, as demonstra-
ted by their inability to potentiate the inhibition of factor Xa and
thrombin substrates by antithrombin III (Figures 1b and S2).
Thus, controlling the positioning of sulfate groups within the
glycopolymer enables the anti-inflammatory function of HS/
heparin to be dissected from its anti-coagulant function.
Furthermore, modifications to the sulfation pattern can be
exploited to adjust the affinity of the glycopolymers for different
HS-binding proteins and may facilitate the development of
glycosaminoglycan-based therapeutic agents with fewer off-
target side effects.
Figure 1. (a) Comparison of the ability of 1−4 to compete with heparin
for binding to RANTES. P < 0.05 for IC50 values of 2 and 4 compared to
1; P < 0.05 for IC50 value of 2 compared to 4. IC50 values corrected for
ligand valence are reported in Table S2. (b) 1−4 do not potentiate the
anti-coagulant activity of antithrombin III, as determined by their ability
to inhibit factor Xa substrates in a chromogenic assay. Data represent the
mean standard error for quadruplicate assays in (a) and (b).
The chemotactic activity of RANTES, which is essential to the
pathogenesis of allergic inflammatory responses such as asthma,
is mediated in part by the G-protein coupled receptor CCR3.20
To test whether 1 can interfere with RANTES-induced
chemotaxis via CCR3, we probed the migration of murine L1.2
pre-B cells that were stably transfected with the CCR3
receptor.20a Using a modified Boyden chamber, we observed
that the directional migration of L1.2-CCR3 cells (but not wild-
type L1.2 cells lacking CCR3) was dependent on the RANTES
concentration and elicited a maximal response at 10 nM (Figure
S3). Pre-incubation of RANTES (10 nM) with either 1 or
heparin diminished the chemotactic activity of RANTES in a
dose-dependent manner (Figure 2). Further corroborating these
results, lower levels of RANTES were detected on the surface of
CCR3-expressing cells after the chemokine (100 nM) was
pretreated with 1 or heparin, as determined by flow cytometry
S2). However, the glycopolymer competed more effectively for
RANTES binding compared to heparin at its maximum
inhibitory concentration. Whereas heparin exhibited a maximum
inhibition of 58.4%, 1 inhibited RANTES binding by up to 90.8%
under the same assay conditions, which could be attributed to
differences in their interactions with oligomeric forms of
RANTES.4 As expected, monovalent disaccharide 26 failed to
compete with heparin for RANTES binding (Figure 1). These
results provide the first demonstration that an HS disaccharide
epitope can be sufficient for chemokine binding when presented
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AUTHOR INFORMATION
Corresponding Author
lhw@caltech.edu
■
Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank Rochelle Diamond at the Flow Cytometry Cell Sorting
Facility, Dr. Mona Shahgholi in the Mass Spectrometry Facility,
and Dr. David VanderVelde at the High Resolution NMR
Facility at Caltech. We also thank Dr. Osamu Yoshie (Kinki
University, School of Medicine, Japan) for generously providing
murine L1.2 cells stably expressing CCR3 and CCR5. This work
was supported by the NIH (R01 GM093627).
D
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