Synthesis of a cluster-forming sialylthio-d-galactose fullerene conjugate and evaluation of its interaction with influenza virus hemagglutinin and neuraminidase

Article abstract of DOI:10.1016/j.bmcl.2014.04.032

In order to obtain self assembling, multivalent ligand for influenza virus hemagglutinin α-N-acetylneuraminyl-(2-6)-d-galactopyranose has been synthesized and bonded to a water soluble fullerene derivative using 1,3-dipolar cycloaddition click reaction. The aggregating amphiphilic compound did not inhibit the influenza virus hemagglutinin, but it proved to be an inhibitor of its neuraminidase with a 50% inhibitory concentration of 81 μM.

Full text of DOI:10.1016/j.bmcl.2014.04.032

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2420–2423
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of a cluster-forming sialylthio-D-galactose fullerene
conjugate and evaluation of its interaction with influenza virus
hemagglutinin and neuraminidase
Szilvia Tollas a, Ilona Bereczki a, Anikó Borbás a, Gyula Batta b, Evelien Vanderlinden c, Lieve Naesens c,
,
⇑
Pál Herczegh a,
⇑
a Department of Pharmaceutical Chemistry, University of Debrecen, H-4010 Debrecen, Hungary
b Department of Organic Chemistry, University of Debrecen, H-4010 Debrecen, Hungary
c Rega Institute for Medical Research, KU Leuven, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to obtain self assembling, multivalent ligand for influenza virus hemagglutinin
raminyl-(2-6)-D-galactopyranose has been synthesized and bonded to a water soluble fullerene deriva-
tive using 1,3-dipolar cycloaddition click reaction. The aggregating amphiphilic compound did not
inhibit the influenza virus hemagglutinin, but it proved to be an inhibitor of its neuraminidase with a
50% inhibitory concentration of 81 lM.
a-N-acetylneu-
Received 3 March 2014
Revised 8 April 2014
Accepted 9 April 2014
Available online 18 April 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Sialic acid
Thiodisaccharide
Fullerene
Aggregation
Multivalent ligand
Antiviral activity
Human influenza A and B viruses are highly contagious respira-
tory pathogens causing considerable morbidity and mortality,
particularly in aged individuals.1 Current influenza vaccines
require annual updating and provide only partial protection in
elderly people.2 Currently available anti-influenza drugs include
the M2 blockers amantadine and rimantadine, which nowadays
are rarely used in clinical praxis because of widespread viral
resistance to these agents, and the neuraminidase inhibitors
oseltamivir and zanamivir.3 Influenza viruses carry two surface
glycoproteins with a sialic acid recognition site: the hemagglutinin
(HA), that is responsible for initial virus attachment to host cell
receptors, and the neuraminidase (NA) which releases the newly
formed virions at the end of the virus life cycle.
all influenza virus (sub)types,4 interfering with the HA–sialic acid
interaction is a relevant antiviral strategy. According to the
O
O
O
HO
4
O
O
OH
4
N
Due to its crucial role in viral entry, the influenza virus HA rep-
resents4,5 a suitable target for antiviral intervention. The HAs of
human-adapted influenza viruses bind mainly to host cell surface
O
O 4
glycans having sialyl-
influenza viruses preferentially bind to glycans with sialyl-
(2-3)-galactosyl termini.6,7 Since sialic acid binding is similar in
a(2-6)-galactosyl termini, while avian
HO
a
⇑
Corresponding authors. Tel.: +36 52 512 900 22895; fax: +36 52 512 914 (P.H.).
E-mail addresses: lieve.naesens@rega.kuleuven.be (L. Naesens), herczeghp@
1
gmail.com (P. Herczegh).
Figure 1. Structure of the fullerene–pyrrolydine carrier 1.
http://dx.doi.org/10.1016/j.bmcl.2014.04.032
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

S. Tollas et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2420–2423
2421
estimations of Whitesides et al.,8 the association constant for inter-
action of a single sialic acid with monomeric HA is 103 MÀ1. How-
ever, due to polyvalent virus–cell interactions, the binding affinity
between multiple viral HA ligands and sialic acid receptors of an
erythrocyte is estimated 1013 MÀ1. This strong cooperative interac-
tion inspired the pioneering works of the teams of Bovin,9 Roy10
and Whitesides11 to synthesize multivalent sialic acid-containing
polymers and study their effect on HA–sialic acid binding.
In the last decades, many sialoconjugates of polymer carrier
molecules have been synthesized12,13 and evaluated for their
anti-influenza virus activity. However, the polymer backbone of
these sialosides can be immunogenic, insoluble, cytotoxic and
inconsistent. These disadvantages initiated the research of mono-
meric sialosides that can form multivalent aggregates. One of the
other approaches is the incorporation of monosialosides into lipo-
somes.14–16 The Bovin’s group elaborated an ingenious HA-decoy
method: they conjugated sialosides to oligoglycine chains
that can form self-assembled nano-sheets through hydrogen
bonding.17–19
In the framework of our systematic studies of glycopeptide anti-
biotic aglycons, we previously prepared a series of ristocetin and
teicoplanin derivatives bearing lipophilic side-chains,20 several of
which exhibited high antibacterial and anti-influenza virus activ-
ity. We postulated that the unusual/serendipotous antiviral activ-
ity originates from their ability to form nano-aggregates. Since it
is known that fullerene (C60) derivatives can form self-assembled
supramolecular nanostructures in water,21 we designed and pre-
pared the fullerene derivative 1 as one of the lipophilic side-chains.
Compound 1 was provided with tetraethyleneglycole chains for
improving the water solubility and a propargyl ether residue
allowing its functionalization with bioactive compounds via a
1,3-dipolar cycloaddition (click) reaction (Fig. 1).22
We anticipated that conjugation of 1 with an appropriate sia-
lodisaccharide ligand of the HA receptor would result in a sialo-
cluster-forming fullerene derivative with HA-decoy properties.
The thiosialoside analog of N-acetylneuraminyl-a(2,6)-D-galactose
has been chosen for the conjugation, because this is the terminal
structure of human cell surface glycoproteins recognized by the
AcO
AcO
OTs
O
AcO
AcO
RO
RO
SAc
O
SR
O
i
ii
OAc
OAc
O
O
N3
O
O
OAc
OR
iii
OAc
4 R = Ac
R = H
3
2
5
AcO
Cl
OAc
AcO
iv
AcO
O
COOMe
AcO
AcHN
O
COOMe
S
OAc
AcO
O
6
AcHN
AcO
HO
HO
O
O
O
N3
O
OH
7
HO
COOH
OH
HO
v
O
AcHN
S
HO HO
HO
O
O
O
N3
O
O
OH
8
HO
COOH
OH
vi
1
+
HO
O
AcHN
S
HO HO
HO
N
N
N
O
O
O
O
O
O
O
OH
O
HO
4
O
O
OH
4
N
O
O 4
HO
9
Scheme 1. Synthesis of the thiosialoside-functionalized fullerene 9. Reagents and conditions: (i) KSAc, dry DMF, 90 °C, 2 h, 70%; (ii) HO-TEG-N3, BF3ÁEt2O, dry CH2Cl2, rt, 4 h,
33%; (iii) (1) NaOMe, dry MeOH, (2) Serdolite Red (H+); (iv) Na2CO3, H2O, Bu4NHSO4, EtOAc, 40% for 7 over two steps; (v) (1) NaOMe, dry MeOH, 2. Serdolite Red (H+), (3) KOH,
H2O, MeOH, (4) Amberlite IR 120 (H+), 51%; (vi) Et3N, Cu(I)I, Ar, rt, 24 h, dry CH2Cl2, dry MeOH, 60%.

2422
S. Tollas et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2420–2423
100
80
60
40
20
0
In conclusion, compound 9, a fullerene conjugate containing a
thiosialosyl- (2,6)-galactose disaccharide has been prepared, in
a
order to study the multimeric interaction of a sialocluster with
influenza virus HA and NA. This amphiphilic conjugate formed
nano-sized aggregates in water but did not show anti-influenza
virus activity, nor did it inhibit influenza virus-induced hemagglu-
tination. It did show moderate inhibitory activity towards the
influenza virus neuraminidase. We assume that the numerous tet-
raethyleneglycol chains applied in the molecule to improve its
water solubility, probably shielded the active disaccharide part,
preventing its multimeric interaction with viral hemagglutinin.
On the other hand, interestingly, this shielding did not seem to dis-
turb the interaction of 9 with the viral neuraminidase.
Acknowledgments
This study was supported by the TÁMOP 4.2.4.A/2-11-1-2012-
0001 Project (National Excellence Program). The project is
co-financed by the European Union and the European Social Fund.
The study was also supported by the Hungarian Research Fund
(OTKA K 109208), the University of Debrecen (Bridging Fund to
P.H) and
a grant from the Geconcerteerde Onderzoeksacties
(GOA/10/014) from the KU Leuven. The authors wish to thank
Wim van Dam and Stijn Stevens for their dedicated technical assis-
tance. We are indebted to Dr. S. Kéki for the light scattering
measurements.
10
100
1000
d (nm)
Figure 2. Size distribution of aggregates of compound 9 in water.
influenza virus HA. As thioglycosides display significantly lower
susceptibility to enzymatic and acid hydrolysis,23 and thiosialo-
sides are not cleaved by neuraminidase enzyme,24 the planned thio
analog must be stable under physiological conditions.
The synthesis commenced with the conversion of the known
galactose derivative 225 into the 6-S-acetyl-containing compound
3 (Scheme 1). The tosyloxy group of 2 was displaced by potassium
thiolacetate to afford 3. Lewis acid mediated glycosylation of
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
04.032.
References and notes
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l
l
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´
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