Novel linear polymers bearing thiosialosides as pendant-type epitopes for influenza neuraminidase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.05.016

A conventional synthesis of α-thioglycoside of sialic acid as a glycomonomer was accomplished. Radical copolymerization of the glycomonomer with vinyl acetate proceeded smoothly to afford a new class of glycopolymers having thiosialoside residues, in which all protection was removed by a combination of transesterification and saponification to provide a water-soluble thiosialoside cluster. The results of a preliminary study on biological responses against influenza virus neuraminidases using the thiosialoside polymer as a candidate for a neuraminidase inhibitor showed that the glycopolymer has potent inhibitory activity against the neuraminidases.

Full text of DOI:10.1016/j.bmcl.2007.05.016

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3826–3830
Novel linear polymers bearing thiosialosides
as pendant-type epitopes for influenza neuraminidase inhibitors
Koji Matsuoka,a,* Chiharu Takita,a Tetsuo Koyama,a Daisei Miyamoto,b,c
Sangchai Yingsakmongkon,d Kazuya I. P. J. Hidari,b,c Wipawee Jampangern,e
Takashi Suzuki,b,c Yasuo Suzuki,c,f Ken Hatanoa and Daiyo Terunumaa
aArea for Molecular Function, Division of Material Science, Graduate School of Science and Engineering,
Saitama University, Sakura, Saitama 338-8570, Japan
bDepartment of Biochemistry, University of Shizuoka, School of Pharmaceutical Sciences,
and COE Program in the 21st Century, Suruga, Shizuoka 422-8526, Japan
cCore Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency,
Kawaguchi, Saitama 332-0012, Japan
dDepartment of Microbiology and Immunology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand
eDepartment of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand
fDepartment of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Kasugai, Aichi 487-8501, Japan
Received 13 March 2007; revised 11 April 2007; accepted 8 May 2007
Available online 13 May 2007
Abstract—A conventional synthesis of a-thioglycoside of sialic acid as a glycomonomer was accomplished. Radical copolymeriza-
tion of the glycomonomer with vinyl acetate proceeded smoothly to afford a new class of glycopolymers having thiosialoside res-
idues, in which all protection was removed by a combination of transesterification and saponification to provide a water-soluble
thiosialoside cluster. The results of a preliminary study on biological responses against influenza virus neuraminidases using the thi-
osialoside polymer as a candidate for a neuraminidase inhibitor showed that the glycopolymer has potent inhibitory activity against
the neuraminidases.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Synthetic glycoclusters are of great interest in biochemical
as well as biomedical fields because the multivalent com-
pounds have various advantages such as (1) simplicity
of the synthetic process, (2) obvious epitope structures
in the molecule, and (3) chemical and biological stabili-
ties.1 Among the various synthetic glycoclusters, glyco-
polymers are attractive candidate for manufacturing
therapeutic reagents.2 We have previously reported glyco-
polymers having trisaccharidic epitopes of globotriaosyl
ceramide (Gb3; Gala1 ! 4Galb1 ! 4Glcb1 ! Cer)3
and their biological evaluations.4 The results of a study
on biological evaluations showed that the activity of the
glycopolymers was highly enhanced by the sugar-cluster
effect, and the therapeutic treatment for mice was also
demonstrated.4 A sialic acid (Neu5Ac) is a unique mono-
saccharide and is specifically recognized by various carbo-
hydrate-related proteins.5 Several proteins in influenza
virus are the carbohydrate-related proteins. In general,
influenza viruses have different types of carbohydrate-
related proteins on their viral particle surfaces,6 such as
hemagglutinin (HA), which is a kind of lectin and specif-
ically binds to sialyl oligosaccharides on host cells,7 and
glycosidase, which is referred to as either sialidase or neur-
aminidase (NA) and cleaves sialic acid residues from
sialoglycoproteins as well as gangliosides on the surfaces
of the host cells.8 Neuraminidase inhibitors, such as
zanamivir9 and oseltamivir,10 have been synthesized and
widely used as therapeutic agents in the clinical treatment
of influenza A and B viruses.11 Although these drugs have
extremely high inhibitory potencies to the release of influ-
enza virions from infected cells, NA inhibitor-resistant
viruses have already been generated.12 Oseltamivir-resis-
tant avian influenza virus has been isolated, and the death
of a human from influenza A virus (H5N1) infection has
recently been reported.13 Since the inhibitors are not
naturally obtained and are designed as transition-state
analogues after cleavage of sialic acid residues by an
Keywords: Thioglycosides; Enzyme inhibitors; Sialic acid; Influenza
virus; Sialidases; Glycopolymers.
*
Corresponding author. Tel./fax: +81 48 858 3099; e-mail: koji@fms.
saitama-u.ac.jp
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.05.016

K. Matsuoka et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3826–3830
3827
i)
OH
OMs
1
2
OAc
OAc
OAc
OAc
COOMe
S
COOMe
SAc
ii)
AcO
AcHN
AcO
AcHN
O
O
OAc
OAc
3
4
Scheme 1. Reagents and conditions: (i) MsCl, Pyr, 0 ꢁC, 3 h; (ii) 2, K2CO3, MeOH, 0 ꢁC ! rt, overnight, then Ac2O–Pyr, rt, overnight.
OR1
COOR2
S
OR1
O
R1O
AcHN
i)
OR1
OAc
+
4
m
n
5 : R1 = Ac, R2 = Me
6 : R1 = R2 = H
ii)
R1O
Scheme 2. Reagents and conditions: (i) AIBN, reflux temp; (ii) NaOMe, MeOH, rt, 3 h, then 0.5 M aqueous NaOH, rt, 2 days.
30
½aꢀ +28.7ꢁ (c 1.0, CHCl3), J7,8 = 8.2 Hz, Dd |H-9a-H9b| =
NA, a therapeutic treatment of influenza using oseltami-
vir would result in the oseltamivir-resistant virus.12a It
seems that an epitope of the neuraminidase of influenza
virus is a natural sialic acid, and the resistant virus
therefore appeared during treatment of the infection.
Therefore, we planned to synthesize a new polymeric
thiosialoside cluster as an additional antiviral agent
because thioglycosidic linkage is usually not hydrolyzed
by glycosidases such as NA.14 However, since monomeric
sialoside does not have any potent inhibitory activity
for NA, we utilized the sugar-cluster effect for enhance-
ment of the binding activities.15 In addition, a thiosialo-
side polymer is expected to show enhancement of the
binding activity, since NAs display tetrameric structures
on the virion surface.16 In this paper, we describe
syntheses of thiosialoside polymers and preliminary bio-
logical evaluations of the activity of the polymers against
influenza virus NAs.
D
0.20 ppm.19
Since the preparation of glycomonomer 4 was accom-
plished, we directed our attention to utilization of the
monomer for radical polymerization. We first investi-
gated a radical polymerization of deprotected 4 with
an acrylamide in water by a previously reported meth-
od.3 However, a glycopolymer having sialic acid resi-
dues was not obtained in the present study. Therefore,
a direct copolymerization of glycomonomer 4 having
protecting groups with vinyl acetate (VA) was exam-
ined. The synthetic scheme of the reaction is shown in
Scheme 2. A polymerization of 4 with VA was initially
carried out in the presence of AIBN as a radical initiator
in MeOH as a solvent, but a polymeric product was
unfortunately not obtained. Consequently our synthetic
plan had to be changed. Bulk polymerization was one of
the suitable procedures for preparation of a polymer.
Thus, the copolymerization of 4 with VA as both a
comonomer and a solvent was investigated. The results
of the radical polymerization, in which the monomer
ratio of the polymerization reaction was fixed at 1:10
and the reaction temperature as well as reaction time
were changed, are summarized in Table 1. The results
of polymerization showed that the reaction temperature
of 90 ꢁC and the reaction time of 3 h gave the best result.
The effects of different amounts of AIBN as an initiator
were then examined, and the results are summarized in
Table 2. The yields of the polymerization were gradually
increased until around 40% according to addition of
AIBN. The weight–average molecular weights ðMwÞ of
the glycopolymers were around 20 kDa as estimated
by the results of size exclusion chromatography (SEC),
and SEC experiments also gave information on the
degree of polydispersion ðMw=MnÞ of the polymers,
which was estimated to be around 1.7. 1H NMR spectra
The synthetic scheme of a convenient pathway for prepa-
ration of carbohydrate monomer 4 is shown in Scheme 1.
Commercially available x-undecylenyl alcohol (10-unde-
cen-1-ol) 1 was selected as a precursor of aglycon because
the alkenyl groups have a C@C double bond at an end
terminal, which has polymerizable potency with other
monomers.17 In addition, the alcohol has a long aliphatic
chain, which can increase the degree of freedom of a
carbohydrate moiety bearing from linear backbone
after polymerization and has an adequate hydrophobicity
as a lipid-like component. Thus, O-mesylate 2 having
a leaving group was prepared from 1 in quantitative
yield. Known thioacetate of sialic acid 318 was treated
with mesylate 2 in the presence of potassium carbonate
togive a-thioglycoside 4 in 92% yield afterre-acetylation,
All new compounds with specific rotation data gave satisfactory
results of elemental analyses.

3828
K. Matsuoka et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3826–3830
Table 1. Results of radical polymerization of 4 with vinyl acetate (VA)
Entry
Monomer ratio sugar:VA
Temp (ꢁC)
Time (h)
Polym. comp.a
Sugar content (wt%)
Total yield (%)
c
1
2
3
4
5
6
7
1:10
1:10
1:10
1:10
1:10
1:10
1:20
60
80
12
6
—
—
—
1:11
N.D.b
N.D.b
1:7
41.1
N.D.b
N.D.b
52.3
56.1
25.0
14.7
6.1
rt ꢂ 80
rt ꢂ 90
90
3
6
6.1
3
31.2
17.4
57.1
90
90
6
1:6
1:23
6
a Polymer compositions of sugar unit:vinyl acetate unit were estimated on the basis of the results of 1H NMR spectra.
b N.D. means ‘not determined’ due to small amount of the polymer.
c Polymerization reaction did not proceed.
Table 2. Effects of the initiator (AIBN) for the radical polymerization in 1:10 of sugar/vinyl acetate (monomer ratio) at 90 ꢁC for 3 h
b
Entry
AIBN (mg)
Total yield (%)
Polym. comp.a
Sugar content (wt%)
Mw ðkDaÞ
Mw=Mn
1
2
3
4
5
6
0.69
1.10
1.57
2.02
2.47
3.02
6.9
26.0
30.4
40.0
37.3
39.0
1:9
1:9
46.0
46.0
46.0
46.0
43.4
46.0
17.4
16.3
18.3
24.3
17.5
16.4
1.6
1.8
1.7
1.9
1.8
1.6
1:9
1:9
1:10
1:9
a Polymer compositions of sugar unit:acrylamide unit were estimated on the basis of the results of 1H NMR spectra.
b The weight–average molecular weights were estimated by size-exclusion chromatography in THF using tandem-bonded Tosoh TSKgel SuperHZM-
M · 2 columns. Calibration curves were obtained using polystyrene standards (0.5, 1.0, 2.5, 5.87, 9.49, 17.1, 37.2, 98.9, 189, 397, 707, and 1110 kDa;
TSK standard, PS-oligomer kit).
Figure 1. 1H NMR spectra of (a) a glycomonomer 4 and (b) a glycopolymer 5.
of the glycomonomer 4 and the glycopolymer 5 in
CDCl3 are shown in Figure 1. Signals due to proton
binding to a C@C double bond of aglycon on 4 ap-
peared at around 6–5 ppm (Fig. 1a), and the signals dis-
appeared after polymerization (Fig. 1b).
filtration to remove low molecular-weight byproducts.
Structural elucidation of 6 was performed by a combina-
tion of spectroscopic analyses, including NMR and IR.
IR spectra of the fully protected glycomonomer 5 and
the deprotected glycopolymer 6 are shown in Figure 2.
Disappearance of a characteristic infrared absorption
band of C@O functional group as stretching vibration
at around 1740 cmꢁ1 and appearance of a characteristic
infrared absorption band of O–H functional group as a
stretching vibration at around 3400 cmꢁ1 in 6 (Fig. 2b)
provided evidence of a completely deprotected structure.
Given the success of preparation of glycopolymers having
thiosialoside residues by radical polymerization, com-
plete removal of the protection in the polymer was our
next objective. Transesterification and subsequent sapon-
ification of 5 gave water-soluble glycopolymer 6 after gel

K. Matsuoka et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3826–3830
3829
Figure 2. IR spectra of (a) a fully protected glycopolymer 5 and (b) a deprotected glycopolymer 6.
Acknowledgment
Table 3. Preliminary results of inhibition assays for influenza virus
sialidases
Compounda
Influenza virus subtype
A/Memphis/1/71 (H3N2) A/PR/8/34 (H1N1)
2.5 10
We are grateful to Snow Brand Milk Products Co., Ltd
for providing the sialic acid used in this study.
6
IC50 values are indicated in millimolar concentration based on a
monomeric sugar unit concentration.
a The structure is shown in Scheme 2.
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Preliminary results of inhibitory activity of a glycopoly-
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showed inhibitory activity potencies not only against
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weak binding efficiency.22 The details of the results pre-
sented here and the further synthetic procedures as well
as biological activities will be reported elsewhere.

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