Synthesis and biological evaluation of a unique heparin mimetic hexasaccharide for structure-activity relationship studies

Article abstract of DOI:10.1021/jm4016069

To date, the structure-activity relationship studies of heparin/heparan sulfate with their diverse binding partners such as growth factors, cytokines, chemokines, and extracellular matrix proteins have been limited yet provide early insight that specific sequences contribute to this manifold biological role. This has led to an impetus for the chemical synthesis of oligosaccharide fragments of these complex polysaccharides, which can provide an effective tool for this goal. The synthesis of three heparin mimetic hexasaccharides with distinct structural patterns is described herein, and the influence of the targeted substitution on their bioactivity profiles is studied using in vitro affinity and/or inhibition toward different growth factors and proteins. Additionally, the particularly challenging synthesis of an irregular hexasaccharide is reported, which, interestingly, in spite of being considerably structurally similar with its two counterparts, displayed a unique and remarkably distinct profile in the test assays.

Full text of DOI:10.1021/jm4016069

Article
pubs.acs.org/jmc
Synthesis and Biological Evaluation of a Unique Heparin Mimetic
Hexasaccharide for Structure−Activity Relationship Studies
Sucharita Roy,† Ahmed El Hadri,‡,§ Sebastien Richard,‡ Fanny Denis,‡ Kimberly Holte,† Jay Duffner,†
Fei Yu,† Zoya Galcheva-Gargova,† Ishan Capila,† Birgit Schultes,† Maurice Petitou,‡
and Ganesh V. Kaundinya*,†
†Momenta Pharmaceuticals Inc., 675 West Kendall Street, Cambridge, Massachusetts 02142, United States
‡Endotis Pharma, Biocitech Park, 102 Avenue Gaston Roussel, 93230 Romainville, France
§CarboMimetics, 2 rue Franco
̧ is Mouthon, 91380 Chilly-Mazarin, France
S
* Supporting Information
ABSTRACT: To date, the structure−activity relationship
studies of heparin/heparan sulfate with their diverse binding
partners such as growth factors, cytokines, chemokines, and
extracellular matrix proteins have been limited yet provide
early insight that specific sequences contribute to this manifold biological role. This has led to an impetus for the chemical
synthesis of oligosaccharide fragments of these complex polysaccharides, which can provide an effective tool for this goal. The
synthesis of three heparin mimetic hexasaccharides with distinct structural patterns is described herein, and the influence of the
targeted substitution on their bioactivity profiles is studied using in vitro affinity and/or inhibition toward different growth factors
and proteins. Additionally, the particularly challenging synthesis of an irregular hexasaccharide is reported, which, interestingly, in
spite of being considerably structurally similar with its two counterparts, displayed a unique and remarkably distinct profile in the
test assays.
oligosaccharide library as a tool required to effectively probe the
SAR.
INTRODUCTION
■
Glycosaminoglycans such as heparin (H) and heparan sulfate
(HS) are considered attractive therapeutic agents because they
modulate many biological processes and have been implicated
in numerous pathologies, including cardiovascular,1 cancer,2
inflammation,3 metabolic, and neurodegenerative diseases4 and
viral infections.5 These biological functions are believed to be
dependent on the interaction of these linear polysaccharides
with key proteins such as growth factors, cytokines, proteases,
adhesion proteins, lipid binding proteins, etc., which have a
heparin binding domain in common and are termed heparin
binding proteins (HBPs).6,7 However, their inherent structural
diversity and microheterogeneity coupled with the challenging
chemical synthesis of defined HS sequences have hindered the
development of a detailed understanding of the specific
structural motifs that mediate their biological activity and
their development as therapeutic agents. We have previously
reported8 the generation of a large library of modified H/HS
derivatives by selective manipulation of the sulfation pattern
and its consequent effect on biological activity. This effort gave
significant preliminary insight into the structure−activity
relationship (SAR) of these polysaccharides. However, the
presence of numerous protein binding sites across chains as
well as within each chain of the heterogeneous mixture, in
addition to the inability to selectively manipulate a specific
residue in the HS domain chemically, allowed only constrained
assessments of a particular interaction. This clearly defined the
need for the generation of a distinct homogeneous synthetic
Although a large number of HBPs have been identified over
the years, there have been only a few reports of crystallographic
and molecular modeling studies to identify definite heparin
binding motifs on proteins that might be responsible for
bioactivity. These have been limited to certain target proteins
such as bFGF,9 aFGF,10 VEGF,11 P-selectin,12 HSV,13 foot and
mouth disease virus,14 etc. One important development has
been the complete elucidation of the heparin−antithrombin
interaction, allowing the generation of the first synthetic
anticoagulant Fondaparinux,15 albeit through a lengthy
chemical process involving ∼50 steps. Thus, a fundamental
opportunity to synthesize numerous targeted synthetic
oligosaccharides exhibiting bioactivity against a myriad of
protein targets still exists.
The commercial syntheses of the pentasaccharides Fonda-
parinux and Idraparinux have spurred the efforts of different
research groups toward the generation of diverse synthetic
oligosaccharides as biologically active GAG analogues.16,17 We
report here our ongoing attempts to more deeply comprehend
the SAR to identify unique HS mimetic oligosaccharides with
inhibitory activity against several growth factors and proteins
(VEGF-A, Shh, PDGF-B, SDF-1α, heparanase, etc.) involved in
tumor growth and metastasis. We present herein the synthetic
strategies used to generate three hexasaccharides that were
Received: October 16, 2013
Published: May 1, 2014
© 2014 American Chemical Society
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Figure 1. Structures of hexasaccharides 1−3.
Scheme 1. Retrosynthetic Route for the Synthesis of Hexasaccharides 2 and 3
further evaluated in vitro for their affinity for these protein
targets, as well as heparanase inhibition.
glycosidic bond formation, the different functional makeup of
the amino groups (NH2, NHSO3Na, and NHAc), followed by
the introduction of sulfate ester groups at specific positions.
More so, the synthesis of irregular structures such as 3 presents
a difficult task, with the successful synthesis of an irregular
octasaccharide being reported only recently.25 Despite the
similarity of the three hexasaccharide templates, minor changes
in the sulfation/acetylation pattern proved to have a significant
impact on the synthesis route. It was observed that simply
introducing one nonsulfated uronic acid accompanied by an N-
sulfo glucosamine residue in 3 proved to be a formidable
synthesis exercise compared to the synthesis of 1 or 2. Herein,
we have proposed efficient solutions for the backbone
construction and the control of stereochemistry in each case
and attempted to study the influence of the targeted
substitution pattern on their bioactivity profiles.
The hexasaccharides contain the same (L-iduronic acid-D-
glucosamine)3 carbohydrate backbone but varying substitution
patterns. The synthesis of compound 1 via another route has
been reported by Petitou and co-workers.18 We present here a
new synthesis of 2, the β isomer having been described
previously by Seeberger and co-workers,17 and a concise
synthesis of the irregular α-pentyl hexasaccharide 3.
RESULTS AND DISCUSSION
■
Although the chemical synthesis of the regular sequences
appearing in H/HS, particularly the IdoA2S-GlcNS,6S disacchar-
ide (GlcNS,6S represents N- and 6-O-sulfonated glucosamine),
has been well-documented,19−24 the preparation of such
structures is often tedious and challenging.
Synthesis of 1. Preparation of N-acetylated hexasaccharide
The major concerns in the preparation of 1−3 involve the
backbone assembly: regio- and stereochemical control in
1 is today a classic exercise26 after the description of several
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a
Scheme 2. Synthesis of Hexasaccharide 2
a
Reagents and conditions: (a) TBDPSCl, Et3N, DMAP, CH2Cl2, RT, 88%; (b) NIS, TfOH, CH2Cl2, 4 Å molecular sieves, 0 °C, 80%; (c) CH3ONa,
CH3OH/THF, RT; (d) (CH3O)2C(CH3)2, CSA, DMF, RT; (e) Ac2O, Et3N, DMAP, CH2Cl2, RT; (f) 60% AcOH, THF, 80 °C; (g) TEMPO,
bromodan, CH3CN/aqueous NaHCO3, RT; (h) CH3I, NaHCO3, DMF, RT overnight, 23% overall yield for steps c−h; (i) TBDMSOTf, toluene, 4
Å molecular sieves, −20 to 0 °C, 91%; (j) NH2NH2/AcOH, pyridine/AcOH (75/25), RT, 75%; (k) TBDMSOTf, toluene, 4 Å molecular sieves,
−20 to 0 °C, 80%; (l) K2CO3, MeOH, RT; (m) Py·SO3, pyridine, 55 °C; (n) (1) NH4F, MeOH, 50 °C, (2) 0.7 M LiOH, MeOH, RT, 64% overall
yield for steps l−n; (o) propanedithiol, Et3N, MeOH, RT, 92% ; (p) Py·SO3, Na2CO3, aqueous NaHCO3, 0 °C, 75% ; (q) H2, 10% Pd/C and
Pd(OH)2, H2O, RT, 87%.
routes toward N-sulfated analogues, the so-called “regular
region of heparin”. Thus, 1 was obtained after selective
acetylation of its triamino precursor resulting from a classical
disaccharide building-block approach.21,27 Several similar N-
acetylated oligosaccharides that differ from 1 by the nature of
the aglycon have been previously reported.17,28 In the case
presented here, the pentenyl moiety was originally introduced
at the anomeric position of the monosaccharide unit to allow
further functionalization. Fortuitously, the pentenyl aglycon
afforded easier separation of the α and β isomers during the
preparation of the reducing end monosaccharide.
Synthesis of 2. The route to hexasaccharide 2 is outlined in
Schemes 1 and 2. The difficulty in the synthesis of this
compound was caused by the presence of the free amine that
was introduced in the aglycon to allow further modification.
This required the use of an amino protecting group orthogonal
to the protecting group used at the 2N-position of the
glucosamine units. An efficient solution was obtained by using
the benzyloxycarbonylated protected amine 8 as described in
previous reports on heparin oligosaccharide synthesis.17,29
Conversely, azido groups were used as protecting groups for
later conversion to N-sulfates without affecting the protection
of the other primary amine, -N(Bn)CBz, present on the
aglycon, which was successfully deprotected later by catalytic
hydrogenation. During this synthesis, a TBDPS group was
employed as the permanent protecting group of the glucos-
amine primary alcohols.
Three building blocks (9, 12, and 13) were required for the
synthesis of 2. For the preparation of imidates 12 and 13,
known monosaccharides 4 and 5a were condensed to give 10,
which was converted to the desired imidate using a classical
series of reactions.18 Reducing end disaccharide 9 was prepared
(Scheme 2) from monosaccharides 7 and 8 (prepared from 19)
as described by Boons.30
The assembly of hexasaccharide 27 was thus accomplished
using disaccharide units 9, 12, and 13 as outlined in Scheme 2.
Coupling of 9 and 12 afforded fully protected tetrasaccharide
25. Removal of the levulinoyl group at C4 and coupling with 13
resulted in hexasaccharide 27. Deacetylation using K2CO3 gave
28 in quantitative yield followed by O-sulfation using the
pyridine−sulfur trioxide complex in pyridine, cleavage of O-silyl
groups at position 6, and hydrolysis of ester groups to afford 30.
The azido groups of 30 were subsequently reduced to amines
using propanedithiol and then subjected to N-sulfation using
the pyridine−sulfur trioxide complex to afford 32. Hydro-
genolysis of 32 using H2 in the presence of Pd catalyst afforded
2.
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a
Scheme 3. Synthesis of Hexasaccharide 3
a
Reagents and conditions: (a) NIS, TfOH, CH2Cl2, 4 Å molecular sieves, 0 °C, 68%; (b) 0.5 M MeONa, MeOH, RT, 1 h, 55%; (c) TBDPSCl, Et3N,
DMAP, CH2Cl2, RT; (d) BnBr, NaH, DMF, RT, 12 h, 84% overall yield for steps c and d; (e) 80% aqueous AcOH/THF (7/3), 80 °C; (f) TEMPO,
BAIB, CH2Cl2, H2O, RT; (g) MeI, NaHCO3, DMF, RT, overnight, 69% overall yield for steps e−g; (h) propanedithiol, Et3N, CH3OH, 45 °C, 83%;
(i) Fmoc-Cl, DIPEA, CH2Cl2, 4 °C, 71%; (j) TBDMSOTf, 4 Å molecular sieves, CH2Cl2, −20 °C, 53%; (k) NH2-NH2, AcOH, pyridine, RT, 83%;
(l) TBDMSOTf, 4 Å molecular sieves, CH2Cl2, −20 °C, 61%; (m) K2CO3, MeOH/THF (7/3), RT, 81%; (n) propanedithiol, MeOH, Et3N, RT,
90%; (o) Fmoc-Cl, CH2Cl2, DIPEA, 4 °C, 76%; (p) BzCl, pyridine, RT, 2 h, 86%; (q) NIS, TfOH, 4 Å molecular sieves, CH2Cl2, −20 °C to RT,
∼22%; (r) piperidine, DMF, Ac2O, RT, 90%; (s) MeONa, MeOH/THF (4/1), RT, 89%; (t) HF·Py, pyridine, TMSOMe, RT, >99%; (u) Py·SO3,
pyridine, MeOH, Et3N, 55 °C, 88%; (v) LiOH, H2O, 40 °C, 33%; (w) Pd(OH)2, H2, H2O, RT, 69%; (x) Py·SO3, saturated aqueous NaHCO3,
Na2CO3, 0 °C, 95%.
Synthesis of 3. As mentioned before, the challenge in the
synthesis of hexasaccharide 3 arose from its “irregular structure”
and the presence of N-acetylated and N-sulfated D-glucos-
amines and one nonsulfated uronic acid unit. We successfully
devised the route outlined in Scheme 3 in which the key
glycosylation reaction connects two trisaccharide building
blocks bearing differential N-protected glucosamine precursors.
Thus, the stereoselectivity of the last created glycosidic bond
was secured by the adjacent benzoyl groups via neighboring
group participation. An additional key reaction was the selective
reduction of the azido group on the hexasaccharide in the
presence of the Fmoc group using propanedithiol and
triethylamine in methanol.
including the reduction of the azido group with propanedithiol
followed by protection as an Fmoc carbamate. Reaction of 14
with imidate 1518,31 led to trisaccharide 41, and subsequent
removal of the levulinoyl group gave glycosyl acceptor 17. The
synthesis of the second trisaccharide required the preparation
of acceptor monosaccharide 16, which was obtained by the
oxidation of the primary alcohol derivative, followed by
esterification of the carboxylic acid, as reported by Tabeur et
al.31 Reaction of 16 with disaccharide imidate 13 gave
trisaccharide 18 after a series of routine protecting group
manipulations.
Compound 17 was condensed with glycosyl donor 18 to
obtain the desired hexasaccharide template 46 (Scheme 3),
which was subsequently subjected to a series of functional
group transformations to obtain fully functionalized hexasac-
charide 3. Replacement of the Fmoc protecting group in 46
with an acetyl group using acetic anhydride in a DMF/
Having tested the key steps of the process, we embarked on
the synthesis of 46, the fully protected hexasaccharide precursor
of 3. Disaccharide 33 obtained by coupling 5b18 and 618 was
converted into 14 following a classical series of reactions,
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Figure 2. Heat map depicting the affinities of 1−3 compared to those of the enoxaparin hexamer mixture for a panel of 22 selected HBPs as
measured by KD (nanograms per milliliter). Inhibition of PSEL/PSGL1 interaction by the test compounds is expressed as Ki values. Darker colors
indicate lower KD/Ki values with higher affinities for the tested protein, while lighter shades indicate lower affinities with higher KD/Ki values. The
gray sections indicate values not measured.
piperidine mixture yielded 47. The benzoate groups were
removed using sodium methoxide in a MeOH/THF mixture,
and deprotection of primary alcohols was conducted in a HF·
Py/pyridine/TMSOMe mixture to yield 49. The free hydroxyls
of 49 were sulfated by treatment with the pyridine−sulfur
trioxide complex, and the resulting compound was saponified
with LiOH in water to yield 51. The azido and benzyl groups
were subsequently reduced using Pd(OH)2 followed by
sulfation of the amino groups using the pyridine−sulfur trioxide
complex in the presence of aqueous NaHCO3/Na2CO3. Target
molecule 3 was finally obtained after gel filtration and Sephadex
LH-20 column purification.
We selected a group of seven growth factors (FGF1, FGF2,
FGF4, FGF7, HBEGF, VEGF, and PDGFBB), one adhesion
protein (PSEL), two cytokines (IL23 and IFNγ), six chemo-
kines (CCL2, CCL5, CCL11, CXCL2, CXCL4, and CXCL12),
and six differentiation proteins (BMP2, BMP4, BMP6, sFRP1,
Shh, and WNT3A) for screening in the SPR assay. The results
from this analysis are depicted in Figure 2. The affinity
constants (KD values) for each saccharide−protein interaction
are listed in Table 1. The selection of our chosen protein panel
was driven by the theorized involvement of the individual
groups in cancer biology, including tumor angiogenesis (growth
factors), tumor metastasis and invasion (PSEL), immunomo-
dulation (cytokines), inflammation/leukocyte migration (che-
mokines), and oncogenesis (differentiation proteins).
The binding ability of the synthetic hexamers was compared
to controls such as unfractionated heparin (UFH), enoxaparin,
a hexamer fraction pool isolated from enoxaparin, and
Fondaparinux, a synthetic pentasaccharide. As expected, all
the hexamers that were tested typically exhibited affinities for
HBPs 10−1000-fold lower than that of the larger enoxaparin
and UFH but similar or slightly higher than that of
Fondaparinux (data not reported). Over the course of this
study, we chose to compare the binding data of the synthetic
oligomers with those of the heterogeneous enoxaparin hexamer
pool that was observed to exhibit either higher or equivalent
affinity values, rather than Fondaparinux in the assays tested. In
general, the synthetic hexasaccharides exhibited a range of
The structures of 1−3 were analyzed and supported by one-
and two-dimensional NMR experiments together with high
resolution ESI-MS measurements (see the Supporting In-
formation).
Heparin Binding Protein Affinity Results. The effect of
the chemical pattern imprinted on these hexamers on their
affinity for various HBPs was assessed by surface plasmon
resonance (SPR) assays. Affinity-in-solution assays were
designed to monitor the formation and interactions of
oligosaccharide−protein complexes and determine the affinity
of the interaction between the hexasaccharides and different
heparin binding proteins. In addition, a specific inhibition
format assay was used to determine the ability of molecules to
disrupt the interaction between platelet selectin (PSEL) and
platelet selectin glycoprotein ligand 1 (PSGL1).
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which has a backbone and charge density similar to 1 (with the
exception of the internal disaccharide possessing an N-sulfate
and 2-OH instead of a -NAc/2-OS moiety), exhibited fairly
higher affinities (∼2−6-fold increase) for the tested proteins, as
compared to the latter, suggesting that molecular recognition
definitely plays a significant role.
Table 1. In Vitro Affinity and/or Inhibition of the
Hexasaccharide−HBP Interactions Tested
a
KD (μM)
HBP
enoxaparin hexamer
14
1
2
3
VEGF165
PDGFBB
HBEGF
FGF7
66
86
14
b
b
b
b
25000
25000
25000
25000
25000
25000
25000
25000
25000
25000
25000
25000
25000
25000
25000
25000
25000
A closer examination of the synthetic hexamer binding
relative to the size-fractionated enoxaparin hexamer yielded
additional insights and supported the hypothesis that specific
heparin sequences exhibit affinities for certain heparin binding
proteins higher than that of the overall mixture. Specific
synthetic heparins exhibited affinities for proteins BMP2,
CXCL12, Shh, and CXCL4 higher than that of the enoxaparin
hexamer fraction (Table 1). All three compounds (1−3)
exhibited an affinity for BMP2 higher than that of the hexamer
fraction, even though there was no significant preference
observed within the group. In contrast, both 1 and 3 displayed
∼4-fold increased affinity for Shh compared to that of 2 and the
enoxaparin hexamer pool. However, only 3 exhibited an affinity
for CXCL4 and CXCL12 substantially higher than those of the
others, as shown in Figure 2. This clearly indicates that the
structural pattern plays a role in in vitro potency, although no
clear trend emerged from the analogues studied.
In general, the data obtained showed that the synthetic
oligosaccharides could interact with several growth factors and
chemokine sets implicated in different disease biologies.
Although no obvious trend emerged from the three
hexasaccharides tested, it was clear that the sulfation pattern
influenced their affinities and potentially inhibitory activities
significantly. Despite all three hexasaccharides possessing six
overall sulfates, the variation in the placement of the sulfate
groups was observed to undeniably influence the binding
profiles of three hexasaccharides. For example, the interesting
binding profile of 3, which possesses two N-acetyl glucosamines
at the terminal end and an internal iduronic acid-N-sulfated
glucosamine disaccharide and can be considered a hybrid of 1
(having N-acetyl glucosamines) and 2 (having N-sulfated
glucosamines), may be attributed to its atypical conformational
orientation as compared to those of the other two hexamers.
This study also suggests that apart from the typically studied
oligosaccharides with repeating disaccharide structures, syn-
thetic approaches afford the ability to make transition or
unusual domains that may elicit a varied ability to engage with
different subsets of target heparin binding proteins. It was
inferred that comparing the hexamers to different heparin-like
molecules other than unfractionated heparin might identify
specific structural requirements that affect binding between
HBPs and synthetic heparin-like molecules. This may allow
further identification of potential oligosaccharide probes (or
drugs) that could have selective activity in certain specific
biological areas.
b
b
b
b
b
b
b
b
b
b
b
b
b
2
b
25000
b
FGF4
25000
FGF2
0.1
0.3
97
1
8
FGF1
PSEL/PSGL1
IL23
250
358
7
96
4
b
b
25000
25000
b
IFNγ
12
25000
77
10
b
7
b
CXCL12
CXCL4
CXCL2
CCL11
CCL5
250000
250000
24
b
b
b
b
b
b
b
b
25000
25000
25000
250000
25000
3
b
b
b
b
b
b
b
250000
250000
25000
250000
b
25000
11
b
b
25000
25000
25000
25000
CCL2
39
250000
250000
250000
250000
61
18
c
WNT3A
Shh
NT
111
27
b
122
23
b
b
sFRP1
BMP6
250000
250000
9
b
250000
c
b
b
NT
250000
250000
250000
9
b
b
b
BMP4
250000
250000
46
250000
34
b
BMP2
250000
24
a
It should be noted that the measurements shown were derived from
b
only one set of binding curves. Above the maximum for the given
assay. Not tested.
c
affinities similar to that of the enoxaparin hexamer pool for
most of the HBPs tested, the exceptions being FGF1, FGF2,
and HBEGF (Table 1). Among the 22 proteins tested, eight
(BMP4, CCL5, CCL11, CXCL2, FGF4, FGF7, HBEGF, and
PDGFBB) exhibited very low affinities for all synthetic
hexamers, which could not be assessed by the SPR assay.
Binding preferences for individual proteins differed between
the synthetic hexamers. Compound 1 exhibited the lowest
affinity for any of the heparin-binding proteins (with the
exception of Shh), consistent with prevalent literature
suggesting that the N-sulfate groups are critical for these
interactions. In the case of Shh, it was observed that 1 exhibited
an affinity similar to that of 3 but an affinity increase of 4-fold
compared to that of 2. This lower affinity of 2 was attributed to
the absence of 6-O-sulfates, which is in accordance with
literature reports.32
Compound 2 exhibited an affinity higher than those of the
other hexamers for FGF1, FGF2, and sFRP1. This compound
was noticeably different from the other two compounds,
because it completely lacked 6-O-sulfate groups but possessed
three N-sulfated glucosamine residues. This is in concordance
with previously reported data17 that the presence of N-sulfates
was requisite as compared to 6-O-sulfates in the interaction of
heparins with FGF2. Little about the interactions between
sFRP1 and heparin has been published other than a report33
postulating that 2-O-sulfation in heparin was critical for the
interaction, which is consistent with our results.
STD NMR. Saturation transfer difference (STD) NMR has
recently emerged as a powerful tool for SAR studies, for
screening synthetic ligand compound libraries for their binding
to proteins and for determining the binding epitopes of the
ligands.34 In STD experiments, the magnetic saturation is
initiated in the target protein and is transferred to the ligand
protons in the proximity of the binding sites. The binding
epitope of the ligands can therefore be mapped by accessing
those saturations.
Among the three hexasaccharides tested, irregular α-pentyl
hexasaccharide 3 exhibited the highest affinity for BMP6,
CCL2, CXCL12, CXCL4, IFNγ, IL23, PSEL/PSGL1, VEGF,
and WNT3A. This set largely consists of proteins in the
immunomodulatory/inflammation category. Interestingly, 3,
The three hexasaccharides were analyzed by STD NMR in an
attempt to map or study the binding activity of the saccharides
against FGF2. FGF2 was chosen as the target protein because
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Figure 3. STD spectrum for hexasaccharide 2 complexed with FGF2.
of extensive studies published previously on FGF2−heparin
oligosaccharide interactions17,35 and reported along with the X-
ray crystallography36 of the heparin−FGF2 complex.
bell-shaped inhibition curves, which to the best of our
knowledge have not been reported for heparins. It was unclear
whether this unusual profile curve reflected a different
mechanism of action or could be attributed to substrate
competition at higher concentrations. As shown in Figure 4,
compound 3 showed an inhibitory activity profile stronger than
that of 1, exhibiting ∼47% maximal inhibition at 4.2 mg/mL
and ∼31% maximal inhibition at 8.3 mg/mL. Both 1 and 2
showed similar inhibition profiles at concentrations ranging
from 0.03 to 8 mg/mL, but their profiles diverged at higher
concentrations. Both of these hexasaccharides possess similar 2-
O-sulfations but differ in their N-substitution patterns, with 1
having only N-acetylated glucosamines as compared to all N-
sulfated building blocks in 2. This indicates that the N-sulfates
seem to contribute significantly to heparanase inhibition at
higher concentrations. Interestingly, 3 showed more potent
heparanase inhibition than 1 and 2 up to a concentration of 4
mg/mL, followed by decreased inhibitory activity with
increasing concentrations. The data suggested that weaker 2-
O-sulfation may facilitate heparanase inhibition, but that strong
N-sulfation was essential for continued heparanase inhibition at
higher concentrations. It may be possible that more sulfated
compounds exhibit an inhibition mechanism different from that
of their less sulfated counterparts, or there might be a size
dependence that needs to be explored further. Though all three
hexasaccharides tested show weak inhibition of heparanase
Although weak STD signals were observed for both
hexasaccharides 1 and 3, the STD spectrum shows no
1
preferentially enhanced signals compared to the reference H
spectrum, which suggested the manifestation of nonspecific
interactions between the hexamers and FGF2. On the other
hand, the STD spectrum for 2 showed noticeable preferential
enhancement of residue D, especially the H-2 resonance,
indicating the 2-O-sulfate of residue D plays an essential role in
its binding to FGF2 (Figure 3). Enhancement was also
observed for glucosamine residue C adjacent to residue D as
shown in Figure 3. This reasonably established the internal
disaccharide sequence “C-D” in hexasaccharide 2 as the binding
epitope in the oligomer−FGF2 interaction. STD NMR thus
provides an elegant tool for gleaning information about the
SAR of heparin oligosaccharides by probing the specific epitope
mapping and/or interactions of oligosaccharides with different
proteins.37
Heparanase Inhibition Results. All three hexasaccharides
were assessed for heparanase inhibition using an HTR-FRET
assay. Remarkably, only 2 showed a typical heparanase
inhibition profile with a calculated IC50 value of 66.4 mg/mL,
consistent with other LMWHs and Fondaparinux (data not
shown). However, hexasaccharides 1 and 3 showed atypical
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EXPERIMENTAL SECTION
■
All reactions were conducted under an atmosphere of nitrogen unless
stated otherwise. All compounds were homogeneous as determined by
thin layer chromatography (TLC) and had spectral properties
consistent with their assigned structures. Purifications in organic
solvents were performed by flash column chromatography on a Merck
cartridge (GX0171511110LK [554-3646]; EVFD17, Si60, 15−40 μm;
10 g) using the Isolera flash purification system (Biotage). Gel
permeation chromatography with aqueous eluents was performed
using Sephadex G-25 (GE Healthcare). Gel filtration chromatography
with DMF or aqueous methanol as the eluent was performed using
Sephadex LH-20. The compound purity was checked by TLC on silica
gel 60 F254 (E. Merck) with detection by charring with sulfuric acid.
The chemical purity of all compounds was determined by HPLC and
1
ESI-TOF or LC−MS and confirmed to be ≥95%. H NMR spectra
were recorded on a Bruker 400 MHz instrument; chemical shifts were
expressed relative to an internal tetramethylsilane (TMS; spectra
recorded in organic solvents) or trimethylsilyl propionate (TSP;
spectra recorded in D2O) standard unless stated otherwise. MS
analyses were performed on a Q-TOF QSTAR instrument (Applied
Biosystems Inc.; R = 10000) and an FT-ICR instrument (7T Apex
Bruker; R = 1000000). Before analysis in D2O, samples were passed
through a Chelex (Bio-Rad) ion exchange column and lyophilized
three times from D2O.
Figure 4. Dose-dependent heparanase inhibition by hexasaccharides
1−3.
General Method for O-Glycosylation. In a dry round-bottom
flask, the saccharide donor (1.3 equiv) and the saccharide acceptor (1
equiv) were dissolved in anhydrous toluene (0.2−0.4 M per acceptor)
under a nitrogen atmosphere containing 4 Å molecular sieves (1
weight equivalent) previously activated at 400 °C. After being stirred
for 30 min at room temperature, the solution was cooled to −20 °C,
and a freshly prepared 0.1 M solution of tert-butyldimethylsilyl
trifluoromethanesulfonate in toluene (0.2 equiv vs donor) was added
dropwise. The reaction mixture was warmed from −20 to 0 °C over 30
min and stirred at this temperature for 1 h. The reaction mixture was
neutralized with Et3N until the pH reached 7, filtered through a pad of
Celite, and concentrated to dryness under reduced pressure. The
residue was purified by chromatography on a silica gel column to
afford the glycosylated compound.
General Method for Isopropylidene Cleavage. The saccharide
was dissolved in a 1/1 tetrahydrofuran/60% acetic acid mixture in
water (0.16 M) at room temperature. The reaction mixture was stirred
at 80 °C until complete conversion had been achieved. The reaction
mixture was concentrated under reduced pressure and coevaporated
with toluene. The residue was dissolved in dichloromethane and
successively washed with a saturated aqueous solution of NaHCO3 and
a brine solution. The organic layer was dried over MgSO4, filtered, and
concentrated under reduced pressure to afford the crude compound.
General Method for Oxidation. To a solution of 0.015 M
saccharide in an acetonitrile/saturated aqueous NaHCO3 (50/50)
mixture at room temperature were added TEMPO (0.1 equiv) and 1,
3-dibromo-5,5-dimethylhydantoin (2 equiv). The reaction mixture was
stirred for 2 h at room temperature, after which a 1 M aqueous
solution of Na2S2O3 (to neutralize the 1,3-dibromo-5,5-dimethylhy-
dantoin reagent) and ethyl acetate were added. The reaction mixture
was cooled to 0 °C, and an aqueous solution of 1 M H2SO4 was added.
The organic layer was separated, and the aqueous layer was extracted
with ethyl acetate. The organic layers were combined, dried over
MgSO4, filtered, and concentrated under reduced pressure to give the
intermediate carboxylic acid that was directly used in the next step
without any further purification.
activity, the observed patterns are intriguing. Testing of
additional hexasaccharides or higher-order oligosaccharides
with mixed 2-O-sulfation and full N-sulfation patterns would
be a logical next step in further elucidating the mechanism of
action and SAR for heparanase binding and inhibition.
CONCLUSIONS
■
A practical, concise route for the synthesis of three
hexasaccharides, including the challenging synthesis of an
irregular N-differentiated hexasaccharide, has been presented.
Screening the three hexasaccharides through a series of in vitro
protein binding and inhibition assays presented an unexpected
SAR pattern, despite their analogous structures and similar
charge densities. A positive correlation was observed between
the observed 10-fold increase in FGF2 binding affinity for the
hexasaccharide 2 and the distinct signal enhancements observed
in the STD NMR in comparison to those of the other two
hexamers. As a result, the specific binding epitope in 2 involved
in the protein interaction was mapped using this technique.
This has the potential to be used as a valuable tool for mapping
out protein−saccharide interactions for future library screen-
ings. Moreover, we also report here for the first time, to the
best of our knowledge, the unusual bell-shaped heparanase
inhibition curve of the irregular hexasaccharide 3, which calls
for further probing into its mechanisms and implications.
Predictably, 1 showed the lowest affinity for the panel of 22
HBPs tested as well as the weakest heparanase inhibition,
emphasizing the importance of N-sulfates in these specific
interactions.
The ability to synthesize discrete saccharide sequences allows
for an enhanced assessment of the impact of subtle structural
modifications that may not be easily accessible through the
isolation of the more abundant structural motifs in HSGAGs.
Our limited study, therefore, highlights the importance of using
synthetic methodologies to meaningfully probe the structural
space of HS, further leading to an elucidation of specific HS−
protein interactions. Furthermore, the information can be used
to design further compounds with potentially higher affinities
and promising pharmaceutical candidates.
General Method for Esterification. To a solution of carboxylic
acid in anhydrous DMF (0.1 M) under a nitrogen atmosphere was
added iodomethane (10 equiv) followed by solid NaHCO3 (10 equiv).
The reaction mixture was stirred overnight at room temperature. The
reaction mixture was diluted with ethyl acetate and successively
washed with an aqueous solution of Na2S2O3 (1 M), a brine solution,
and water. The organic layer was dried over MgSO4, filtered, and
concentrated under reduced pressure, and the residue was purified by
chromatography on a silica gel column to give the desired compound.
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General Method for Selective Azide Reduction. The azido
compound was dissolved in anhydrous methanol (0.02 M) under a
nitrogen atmosphere. 1,3-Propanedithiol (10 equiv per N3 function)
and Et3N (10 equiv per N3 function) were successively added. The
reaction mixture was protected from light and stirred for 2 days at
room temperature or 40 °C. The reaction mixture was concentrated to
dryness under reduced pressure, and the residue was purified by
chromatography on a silica gel column or by a Sephadex LH20 gel
column to afford the desired product.
Surface Plasmon Resonance Assays. Surface plasmon reso-
nance (SPR) affinity-in-solution assays were conducted at 25 °C on
either a ProteOn XPR36 instrument (Bio-Rad, Hercules, CA) or a
Biacore T200 instrument (GE Healthcare, Piscataway, NJ). All
recombinant human heparin-binding proteins were purchased from
R&D Systems (Minneapolis, MN) with the exception of CXCL4
(PF4), which was purchased from Peprotech. All affinity-in-solution
assays were conducted using methods adapted from the general format
described by Karlsson.8
Assays conducted on the Biacore T200 chip were prepared in the
same manner described previously.8 For assays on the ProteOn XPR36
instrument, a ProteOn NLC chip was loaded into the instrument,
normalized, and conditioned with four 30 s injections of 50 mM
sodium hydroxide. Neutravidin (Pierce Biotechnology, Rockford, IL)
was immobilized on the reference and sample flow cells, and
biotinylated low-molecular weight heparin was subsequently captured
only on the sample flow cell. The heparin binding proteins at varying
concentrations were mixed with a dilution series of each
hexasaccharide sample and passed over the sensor chip (see
Supporting Information). All data were single reference subtracted
using the sensor chip interspot for each flow channel. A quadratic
curve was used to fit slope versus concentration data for the standard
curve. The free concentration of the protein in each test sample was
calculated from the standard curve. The KD (affinity constant) was
calculated by the following equation:
ASSOCIATED CONTENT
* Supporting Information
Detailed experimental procedures and spectral data for relevant
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*E-mail: ganesh@momentapharma.com. Phone: (617) 491-
9700. Fax: (617) 621-0431.
■
Author Contributions
S. Roy and A.E.H. contributed equally to this work.
Notes
The authors declare the following competing financial
interest(s): A.E.H., S. Richard, F.D., and M.P. conducted the
experimental work as part of a research project funded by
Momenta Pharmaceuticals Inc. S. Roy, K.H., J.D., F.Y., Z.G.-G.,
I.C., and G.V.K. are employees of Momenta Pharmaceuticals
Inc.
ACKNOWLEDGMENTS
■
We thank Dr. John Schaeck and Paul Miller for critical reading
of the manuscript.
ABBREVIATIONS
■
FGF1, fibroblast growth factor 1; FGF2, fibroblast growth
factor 2; FGF4, fibroblast growth factor 4; FGF7, fibroblast
growth factor 71; HBEGF, heparin-binding epidermal growth
factor; VEGF, vascular endothelial growth factor; PDGF,
platelet-derived growth factor; PSEL, P-selectin; IL23, inter-
leukin 23; IFNγ, interferon γ; CCL2, CC chemokine ligand 2;
CCL5, CC chemokine ligand 5; CCL11, CC chemokine ligand
11; CXCL2, CXC chemokine ligand 2; CXCL4, CXC
chemokine ligand 24; CXCL12, CXC chemokine ligand 12;
BMP2, bone morphogenetic protein 2; BMP4, bone
morphogenetic protein 4; BMP6, bone morphogenetic protein
6; sFRP1, secreted frizzled related protein 1; Shh, sonic
hedgehog; WNT3A, wingless-type MMTV integration site 3A;
DMF, N,N-dimethylformamide; Et3N, triethylamine; TEMPO,
2,2,6,6-tetramethylpiperidin-1-oxyl; DMAP, 4-dimethylamino-
pyridine; EDAC, N-(3-dimethylaminopropyl)-N′-ethylcarbodii-
mide hydrochloride; TBDPS, tert-butyldiphenylsilyl; TBDMS,
tert-butyldimethylsilyl; Bn, benzyl; Ph, phenyl; Bz, benzoyl;
PMB, p-methoxybenzyl; Me, methyl; Ac, acetate; Lev,
levulinoyl; Cbz, benzyloxycarbonyl; Fmoc, fluorenylmethox-
ycarbonyl; NIS, N-iodosuccinimide; TfOH, trifluoromethane-
sulfonic acid; CSA, camphorsulfonic acid; Py·SO3, pyridine−
sulfur trioxide; BAIB, β-aminoisobutyric acid
KD or EC50 = ([Pfree][Htotal])/[P
]
bound
where [P] is the protein concentration in molar units and [H] is the
hexasaccharide concentration in mass per volume units. The reported
value is the average KD calculated at three hexasaccharide
concentrations.
Inhibition of the PSEL/PSGL1 interaction by the hexasaccharide
molecules was assessed by an inhibition assay.8 A dilution series of
each hexasaccharide was mixed with 50 nM PSEL (R&D Systems).
Each mixture was passed over the sensor surface coated with the
PSGL1-Fc fusion protein (R&D Systems), and the response at
equilibrium was measured. The IC50 was calculated from the
equilibrium response versus concentration data by nonlinear
regression in GraphPad Prism. The constant of inhibition (Ki) was
calculated from the IC50 using the Cheng−Prusoff equation.
Heparanase Activity. Heparanase activity was measured using
CisBio Bioassays technology based on time-resolved fluorescence
energy transfer (TR-FRET) between europium cryptate and XL665
(allophycocyanin). The recombinant heparanase was preincubated
with different concentrations of the heparin-derived compounds for 15
min at 37 °C prior to being added to the biotin-cryptate-labeled
heparan sulfate substrate and incubated with streptavidin-XL665 for 1
h at 37 °C.
STD NMR. Recombinant human FGF2 was purchased from R&D
Systems. The hexamer−FGF2 sample was prepared as 3.9 μM FGF2
and 780 μM heparin hexamer in 200 μL of PBS in D2O (pH 6.5).
NMR experiments were performed at 25 °C with a Bruker Avance 600
MHz spectrometer equipped with a QCI cryoprobe. STD NMR was
acquired by using the Biospin sequence stddiff.3 with spin lock for
protein signal suppression. The selective saturation was achieved by a
train of Gauss-shaped pulses with a length of 50 ms, and the number of
scans was 1280.
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