Tailored glycopolymers as anticoagulant heparin mimetics

Article abstract of DOI:10.1002/anie.201306968

Not to clot: Heparin and its low-molecular-weight derivatives are clinical therapeutics used to treat and prevent blood clots. The synthesis of heparin-based glycopolymers that are potent and potentially safer mimetics of heparin is described. The mimetic

Full text of DOI:10.1002/anie.201306968

.
Angewandte
Communications
DOI: 10.1002/anie.201306968
Biomimetic Polymers Very Important Paper
Tailored Glycopolymers as Anticoagulant Heparin Mimetics**
Young In Oh, Gloria J. Sheng, Shuh-Kuen Chang, and Linda C. Hsieh-Wilson*
Heparin, a heterogeneously sulfated glycosaminoglycan, has
been used as an anticoagulant drug for over 80 years.
Although it is a highly effective and inexpensive agent,
heparin in its unfractionated form has several limitations as
a therapeutic. First, it is extracted from porcine or bovine
mucosa and tissue, and carries the potential risk of contam-
ination. The global distribution of contaminated heparin in
2
007, for example, resulted in over 100 deaths and hundreds
[1]
of additional cases reporting adverse clinical effects.
Scheme 1. Chemical structures of Arixtra, Org31550, and synthetic glyco-
polymers 1 and 2.
Second, heparin displays a variable dose–response relation-
ship among patients, in part owing to its structural hetero-
geneity, and thus often requires active monitoring to fine-tune
predictable pharmacokinetics, longer half-lives, and reduced
[2]
[3]
the dosages. Third, approximately 3% of patients under-
going prolonged heparin therapy experience heparin-induced
risk of side effects. However, animal-derived LMWH still
has considerable structural heterogeneity and is susceptible to
[
3]
[1b]
thrombocytopenia (HIT), a severe autoimmune response
triggered by formation of a complex between heparin and
platelet factor 4 (PF4). Altogether, these factors underscore
the critical need to develop safer alternatives to animal-
derived heparin that have more predictable bioactivity and
reduced side effects.
external contamination. ULMWH is notoriously difficult
and expensive to synthesize (ca. 50 chemical steps, ca. 0.1%
[
5a,b]
overall yield in the case of Arixtra)
and exhibits limited
inhibitory activity toward thrombin (also known as factor IIa
[
5a]
(FIIa)) owing to its shorter length.
Thus, despite the
notable advantages of LMW and ULMW heparins, new
methods are greatly needed for the efficient preparation of
defined heparin-like molecules with broad anticoagulation
profiles and improved safety.
In recent decades, two forms of heparin-based antico-
agulants have emerged as alternatives to unfractionated
heparin. Low-molecular-weight heparin (LMWH) is pro-
duced by the chemical or enzymatic degradation of heparin,
yielding fragments with an average molecular weight of
[
8]
Synthetic glycopolymers bearing short, well-defined
heparin epitopes may offer an attractive alternative to natural
[
4]
6
(
000 Da.
Shorter ultralow-molecular-weight heparin
heparins and Arixtra. Here, we utilize ring-opening meta-
[5]
[9]
ULMWH; ca.1500 Da) is prepared through the synthesis
thesis polymerization (ROMP)
chemistry to generate
of a specific heparin pentasaccharide that represents the
minimal epitope required for antithrombin III (ATIII) acti-
a series of heparin-based glycopolymers and demonstrate
that their structures can be tailored to recapitulate the potent
activity of anticoagulant drugs. Previously, we showed that
[
6]
vation. The commercial drug Arixtra (GlaxoSmithKline;
Scheme 1) is a methylated derivative of this pentasaccharide.
The addition of another 3-O-sulfate group has been shown to
confer even greater specificity for ATIII activation
[10]
glycopolymers containing defined chondroitin sulfate (CS)
[
11]
and heparan sulfate (HS)
disaccharide epitopes could
mimic the activity of natural glycosaminoglycans in neurite
[
7]
[10]
[11]
(
Org31550; Scheme 1). For the prophylaxis and treatment
outgrowth
and immune cell migration
assays, respec-
of thromboembolic diseases, LMW and ULMW heparins are
preferred over unfractionated heparin because of their more
tively. However, the application of ROMP to heparin, the
most highly charged and synthetically challenging glycosami-
noglycan, has not been investigated. Moreover, synthetic
glycopolymers with medicinally significant anticoagulant
activity have not been extensively explored as potential
heparin mimetics. Our approach overcomes the challenges of
heterogeneous sulfation and complex oligosaccharide syn-
thesis and shows promise for the development of a new class
of medicinal agents.
[
*] Y. I. Oh, G. J. Sheng, Prof. L. C. Hsieh-Wilson
Division of Chemistry and Chemical Engineering and
Howard Hughes Medical Institute
California Institute of Technology
1200 E. California Blvd, Pasadena, CA 91125 (USA)
E-mail: lhw@caltech.edu
S.-K. Chang
Department of Chemistry and Biochemistry
The Ohio State University
The first challenge in designing an anticoagulant glyco-
polymer was to select a minimal unit that encapsulated the
key determinants of the ATIII-binding pentasaccharide.
Although previous studies had suggested that a pentasacchar-
281 W. Lane Ave, Columbus, OH 43210 (USA)
[
**] We thank Prof. Robert H. Grubbs and Dr. Jean Li for their generous
donation of the ruthenium catalyst and for helpful discussions. This
research was supported by National Institutes of Health grant R01
GM093627 (L.H.W.).
[
5,6]
ide was the minimum active motif,
we reasoned that
a disaccharide epitope might be sufficient when presented on
a multivalent polymeric scaffold, which might bring about an
[
12]
enhancement of binding affinity through avidity. Thus, we
chose two monosaccharide units (G and H, Scheme 1) from
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201306968.
1
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Angew. Chem. Int. Ed. 2013, 52, 11796 –11799

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Chemie
the reducing end of the pentasaccharide, because of their
well-characterized interactions with the A-helix (R46, R47)
[13]
and P-helix (K114, D113) of ATIII. The inclusion of unit G
allowed us to exploit the conformational flexibility of l-
iduronic acid, which substantially improves the affinity of
[14]
heparin for ATIII. We chose unit H of Org31550 because it
contains a rare 3-O-sulfate modification that significantly
[7]
enhances the interaction of heparin with ATIII.
The tetrasulfated glycopolymer 1, comprised of units G
and H, can be readily derived from disaccharide 3 (Scheme 2).
We devised a protecting group strategy for 3 that would allow
Scheme 3. Synthesis of tetrasulfated heparin glycopolymer 1. Condi-
tions: a) TMSOTf, CH Cl , À308C, 85%. b) LiOH (0.7m), H O , 258C;
2
2
2
2
MeOH, NaOH (4m), 258C, 82% over two steps. c) SO ·TMA, DMF,
3
608C, 71%. d) PMe , THF, 258C, quant. e) SO ·pyr, pyr/Et N, 608C,
3 3 3
5
4% over two steps. f) MeOH/(CH Cl) , 558C. g) Pd(OH) /C, H
2
2
2
2
(1 atm), MeOH, phosphate buffer (pH 7.4), 63–92%. TMSOTf=tri-
methylsilyl trifluoromethanesulfonate, TMA=trimethylamine,
DMF=N,N-dimethylformamide, pyr=pyridine.
Scheme 2. Retrosynthesis of glycopolymer 1 from orthogonally pro-
tected disaccharide 3.
expedient sulfation at four different sites. A benzoyl (Bz)
group was used to protect C3, and acetyl (Ac) groups were
selected to protect the C2’ and C6 positions. These groups can
be removed under the same conditions required for C5’
using the fast initiating catalyst [(H IMes)(Py) (Cl) Ru=
2
2
2
[20]
CHPh] (9) produced a similar undesired outcome. Ulti-
mately, we found that the polymerization of 3 was fully
compatible with a MeOH/(CH Cl) cosolvent system at
2
2
[
15]
[10]
saponification
to simultaneously expose the three sites
558C,
which resulted in high conversions and good
desired for O-sulfation. In addition, the C2’ Ac group was
expected to facilitate the formation of the 1,2-trans glycosidic
linkage through neighboring group participation. Owing to
the highly charged nature of the sulfated monomer required
for polymerization, we elected to use benzyl (Bn) groups at
the nonsulfated positions (C3’ and C4’) to enhance monomer
solubility during ROMP.
glycopolymer polydispersities. Using this cosolvent system,
we then explored the relationship between catalyst loading
and polymer length. By tuning the amount of catalyst and the
MeOH/(CH Cl) cosolvent ratio, we were able to synthesize
2
2
a series of glycopolymers with controlled, predictable chain
lengths (10-n, where n = degree of polymerization (DP);
Table 1). Under the tested conditions, polymer lengths were
limited to 45 units as a result of premature precipitation of
polymer 10 during ROMP. All glycopolymers were charac-
The orthogonally protected disaccharide 4 was prepared
[
16]
from the norbornene-conjugated acceptor 5 and glycosyl
phosphate donor 6 (Scheme 3 and Scheme S1 in the Support-
ing Information). The coupling reaction utilized stoichiomet-
ric amounts of TMSOTf at À308C, and exclusively produced
the a-linked disaccharide 4 in 85% yield. Late-stage manip-
ulations to the resulting disaccharide were carried out with
1
terized at this stage by H NMR spectroscopy and size-
exclusion chromatography–multiangle light scattering (SEC–
MALS). Hydrogenation of the remaining Bn groups using
Pd(OH) /C in a mixture of MeOH and neutral phosphate
2
21]
[
buffer afforded the desired glycopolymers (1-n) in 63–92%
yield.
[15,17]
modifications to reported procedures.
Briefly, 4 was
subjected to the sequential addition of LiOOH and NaOH to
saponify the C5’ methyl ester and concomitantly remove the
Ac and Bz protecting groups. Global O-sulfation of disac-
We next assessed the anticoagulant activities of the
glycopolymers in comparison to heparin, LMW heparin,
charide 7 using SO ·NMe3 in DMF furnished trisulfated
intermediate 8 in 71% yield. Finally, the azide group was
3
Table 1: Heparin glycopolymer properties.
Entry Polymer
9
MeOH/(CH Cl) n (DP) Mn
PDI
2
2
reduced under Staudinger conditions, followed by regiospe-
À1
[
18]
[Mol%]
[gmol ]
cific N-sulfation with SO ·pyr to give tetrasulfated 3 in 54%
3
[
[
a]
a]
yield over two steps.
1
2
3
4
5
6
7
10-4
10-6
30
17
1:4
1:4
1:3
1:3
1:2.5
1:2.5
1:10
4
6
4373
6167
2.03
1.25
1.29
1.32
1.41
1.25
1.62
With monomer 3 in hand, we evaluated a series of
polymerization conditions. Although polar solvents were
required for complete solubility of 3, unfortunately, such
conditions compromised the overall efficiency of the ROMP
catalyst. Attempts at aqueous ROMP using the water-soluble
[
[
a]
a]
10-10
10-15
10-30
10-45
2-155
10.7
6.5
5.2
2.0
4.0
10
15
30
45
155
11207
15452
32721
42970
164345
[a]
[a]
[
b,c]
catalyst
iPrOC H )] led to incomplete conversion and low-molec-
[{H IMes-poly(ethyleneglycol)}(Cl) Ru=CH(o-
2
2
Degree of polymerization (DP), number average molecular weight (M ),
and polydispersity index (PDI) were determined by SEC-MALS in [a] LiBr
n
[19]
4
4
ular-weight polymers (H IMes = 1,3-dimesityl-4,5-dihydro-
2
(0.2m) in DMF or [b] NaNO (100mm), NaN (200 ppm) in H O. [c] See
3 3 2
imidazol-2-ylidene). Polymerization reactions in MeOH
Ref. [11] for the synthesis of 2.
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Angewandte
Communications
and Arixtra. The binding of heparin to ATIII induces
a conformational change that enables ATIII to inhibit the
O-sulfation and the specificity of the polymer interaction with
ATIII (Figure 1A, Table 2). Thus, controlling the sulfation
sequence and chain length of the glycopolymer allows the fine
modulation of ATIII-mediated inhibition at a critical junction
in the blood coagulation cascade.
[
13]
serine proteases factor Xa (FXa) and FIIa.
Protease
inhibition was measured using chromogenic substrate assays
that monitor the release of p-nitroaniline (lmax 405) from
specific peptide substrates. In a manner consistent with
FIIa is activated downstream of FXa in the coagulation
cascade and facilitates blood clotting by converting soluble
fibrinogen into insoluble fibrin strands. Unlike FXa, which
utilizes a specific pentasaccharide sequence, FIIa requires
a longer, more highly sulfated heparin template to form an
[
5,22]
previous reports,
heparin, LMWH, and Arixtra attenu-
ated FXa activity in the presence of ATIII, with half maximal
inhibitory concentrations (IC ) of (16.5 Æ 1.2), (526 Æ 71),
5
0
and (11.0 Æ 0.1) nm, respectively (Table 2, Figure S1 in the
Supporting Information). To our delight, glycopolymer 1-45
showed greater anti-FXa activity than the clinical anticoagu-
[
5a,13]
inhibitory ternary complex with ATIII.
In agreement
[
5,22]
with this molecular mechanism and previous reports,
heparin displayed strong anti-FIIa activity (Figure 1B,
Table 2; IC = (11.0 Æ 0.1) nm), whereas LMWH and Arixtra
5
0
Table 2: Biological activity of heparin glycopolymers.
showed no appreciable activity (Table 2, Figure S1 in the
Supporting Information). Notably, we found that glycopoly-
mer 1-45 was 100-fold more potent than heparin at inhibiting
FIIa (IC = (114 Æ 1) pm), which is likely due to a greater
[
a]
[a]
Polymer
Anti-FXa
IC50 [nm]
Anti-FIIa
IC50 [nm]
APTT
[s]
PT
[s]
1
1
1
1
1
1
2
-4
-6
>2000
>2000
>2000
>2000
>2000
>2000
>2000
577Æ31
32.5Æ0.3
32.2Æ0.2
59.6Æ0.3
82.9Æ0.6
100.8Æ0.6
119.4Æ0.5
46.1Æ0.4
31.2Æ0.3
>180
13.2Æ0.1
13.2Æ0.3
15.8Æ0.4
23.4Æ1.9
50.8Æ6.3
52.2Æ7.8
12.7Æ0.1
13.3Æ0.1
84.2Æ18.
14.8Æ0.2
15.1Æ0.3
5
0
number of active tetrasulfated motifs and increased polymer
length. These results are exciting because they demonstrate
that synthetic glycopolymers can be designed to surpass the
biological activity of natural glycosaminoglycans. Glycopoly-
-10
-15
-30
-45
-155
1470Æ578
684Æ60
À2
À4
5.76Æ10
0.114Æ10
>2000
mer 1-30 was also active in this assay (IC = (577 Æ 31) nm),
>2000
>2000
50
none
>2000
and as expected, the shorter polymers (1-15, 1-10, 1-6, and
1-4) and the trisulfated glycopolymer (2-155) showed no
significant activity (Figure 1B, Table 2 and Figure S2 in the
Supporting Information).
heparin
LMWH
Arixtra
16.5Æ1.2
526Æ71
11.0Æ0.1
11.0Æ0.1
>2000
117Æ3.0
>2000
78.3Æ0.4
À1
[a] 150 mgmL compound in citrated human plasma, n=3.
To assess potential interactions with PF4, we evaluated
the ability of PF4 to neutralize the anti-FIIa activity of
glycopolymers 1-45 and 1-30. PF4 was added to the glyco-
À1
lants; it exhibited an IC50 value of (5.76 Æ 0.04) nm (Fig-
ure 1A, Table 2). As the polymer length decreased, we
observed a significant reduction in anti-FXa activity ((IC =
polymers or heparin (0.5–500 mgmL ) in the presence of
ATIII and excess FIIa, and FIIa activity was measured using
the same chromogenic assay. Both heparin and glycopolymer
1-45 interacted strongly with PF4, a result consistent with
reports that longer polysaccharides are more likely to engage
5
0
6
84 Æ 60) and (1470 Æ 578) nm for 1-30 and 1-15, respectively),
until no activity was detected (1-10, 1-6, and 1-4; Table 2 and
Figure S2 in the Supporting Information). We also assessed
the contribution of the glucosaminyl 3-O-sulfate modification
on unit H by comparing the activity of the glycopolymers to
[
4,23]
PF4.
However, the anti-FIIa activity of glycopolymer 1-30
was only partially neutralized by PF4 (Figure S3 in the
Supporting Information), thus indicating that the PF4 reac-
tivity associated with HIT can be minimized to some extent
by modulating the polymer length.
[
11]
that of the 3-O-desulfated glycopolymer 2-155. Remark-
ably, this single alteration in the sulfation pattern abrogated
the anti-FXa activity, thereby reaffirming the importance of 3-
Finally, we examined the ex vivo clotting times of the
glycopolymers, with comparison to clinical anticoagulants,
using human plasma samples. Since the presence of other
proteins in complex serum can interfere with anticoagulant
activity, this assay represents a more stringent test of anti-
coagulant efficacy. We measured the activated partial throm-
boplastin time (APTT) and prothrombin time (PT) of each
compound to determine its ability to inhibit blood clotting
through the intrinsic and extrinsic pathways of the coagu-
[
24]
lation cascade, respectively.
APTT and PT for clotting compared to the saline control
Table 2), whereas LMWH or Arixtra at the same concen-
Heparin increased both the
(
tration increased only the APTT. Notably, the APTT of the
glycopolymers could be controlled by varying the polymer
length, with a minimum of 10 disaccharide epitopes (1-10)
required to prolong the APTT. A slight increase to 15 units
Figure 1. Tetrasulfated glycopolymers (1-15, 1-30, 1-45) inhibit the
ATIII-mediated activity of A) FXa and B) FIIa in a length- and sulfation-
dependent manner. Chromogenic substrate assays were used to
measure coagulation factor activity in the presence of ATIII and
glycopolymer. Data represent the mean Æstandard deviation for n=3
experiments. See Table 2 for IC50 values.
(1-15) endowed the polymer with APTT properties similar to
Arixtra, whereas the PT was not appreciably altered in either
case. Glycopolymers 1-30 and 1-45 modulated both the APTT
1
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Angewandte
Chemie
and PT, a result consistent with their ability to inhibit FXa and
FIIa in vitro. Remarkably, tuning of the polymer structure
produced anticoagulants with unique hybrid properties.
Whereas the APTTs of 1-30 and 1-45 were comparable to
those of LMWH and Arixtra, their PTs more closely
resembled that of heparin. Lastly, as expected, the trisulfated
glycopolymer 2-155 produced no appreciable change in
APTT or PT, thus reinforcing the importance of the 3-O-
sulfate modification and the potential to direct the selectivity
of the glycopolymers toward specific heparin/HS-binding
proteins by modulating the sulfation pattern. Overall, the
ex vivo profile of the glycopolymers validates not only their
efficacy in human plasma, but also their ability to replicate the
activity profiles of the clinical anticoagulants. Moreover, we
find that fine-tuning of the glycopolymer structure can lead to
agents with novel properties not found in naturally occurring
oligo- and polysaccharides.
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Received: August 8, 2013
Published online: October 9, 2013
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