Development of tetraphenylethylene-based fluorescent oligosaccharide probes for detection of influenza virus

Article abstract of DOI:10.1016/j.bbrc.2010.02.155

Tetraphenylethylene (TPE) derivatives have strong fluorescence in aggregated state. We designed and synthesized a tetraphenylethylene derivative bearing alkyne groups which were used for combination by click chemistry. The new TPE compound bearing alkyne groups was used to synthesize fluorescence oligosaccharide probes which have lactosyl and 6′-sialyllactosyl moieties as ligands. We found that the TPE compounds bearing lactosyl and 6′-sialyllactosyl moieties were useful for detection of RCA120 and SSA lectins, respectively. Moreover, we have shown that TPE-based fluorescent oligosaccharide probe bearing 6′-sialyllactose moiety can be utilized as a "turn-on" fluorescent sensor for detection of influenza virus.

Full text of DOI:10.1016/j.bbrc.2010.02.155

Biochemical and Biophysical Research Communications 394 (2010) 200–204
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Development of tetraphenylethylene-based fluorescent oligosaccharide probes
for detection of influenza virus
c
c
d
Tomohisa Kato a,b, , Atsushi Kawaguchi , Kyosuke Nagata , Kenichi Hatanaka
*
a Japan Chemical Innovation Institute, 1-3-5 Jimbocho, Chiyoda-ku, Tokyo 101-0051, Japan
b Frontier Biochemical & Medical Research Laboratories, Kaneka Corporation, 1-8 Miyamae-machi, Takasago-cho, Takasago, Hyogo 676-8688, Japan
c Department of Infection Biology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
d Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 February 2010
Available online 25 February 2010
Tetraphenylethylene (TPE) derivatives have strong fluorescence in aggregated state. We designed and
synthesized a tetraphenylethylene derivative bearing alkyne groups which were used for combination
by click chemistry. The new TPE compound bearing alkyne groups was used to synthesize fluorescence
oligosaccharide probes which have lactosyl and 60-sialyllactosyl moieties as ligands. We found that the
TPE compounds bearing lactosyl and 60-sialyllactosyl moieties were useful for detection of RCA120 and
SSA lectins, respectively. Moreover, we have shown that TPE-based fluorescent oligosaccharide probe
bearing 60-sialyllactose moiety can be utilized as a ‘‘turn-on” fluorescent sensor for detection of influenza
virus.
Keywords:
Biosensor
Influenza virus
Oligosaccharide probe
Fluorescent
Ó 2010 Elsevier Inc. All rights reserved.
Click chemistry
1. Introduction
paramyxovirus, parvovirus, adenovirus-associated virus, herpesvi-
rus, flavivirus, and norovirus are known as viruses binding to oligo-
A biosensor is a device for detection of an analyte using the
molecular recognition mechanism of organisms. Since the first bio-
sensor was developed by Updike and Hicks [1], a variety of biosen-
sors have been developed [2]. Recently, Tong et al. have discovered
the novel aggregation-induced emission (AIE) phenomenon [3] and
applied it to biosensor technology. Among the AIE dyes, tetraphen-
ylethylene (TPE) is synthesized by simple reactions and emits very
efficiently in aggregated or crystal states. The water-soluble cat-
ionic TPE derivatives can be used as bioprobes for detection of pro-
teins, DNA, and RNA [4]. The TPE compounds bearing adenine and
saccharides [9].
Influenza is one of the most common existing infectious dis-
eases to both human and animal in the world. Recently, an influ-
enza virus causes a global outbreak that becomes a menace to
humanity. The receptor binding specificity of hemagglutinin,
which is a viral membrane protein to attach cell surface, varies dif-
ferently depending on the relation between hosts and viruses. Hu-
man influenza virus binds to the Neu5Ac2,6Gal sequence, while
equine and avian viruses specifically bind to Neu5Ac2,3Gal. Por-
cine virus, which is called ‘‘New Influenza A (H1N1)”, bind both
to both Neu5Ac2,6Gal and Neu5Ac2,3Gal [10–13].
thymine moieties can be used as chemosensors for Ag+ and Hg2+
,
respectively [5]. Moreover, sugar-phosphole oxide conjugates were
synthesized as ‘‘turn-on” fluorescent sensors for lectins [6].
Since AIE system makes it possible to detect intermolecular
bindings by fluorescence signals, we envisioned that this new bio-
sensor using AIE systems may be useful for determination of the
recognition specificity of xenobiotics (bacteria, virus, and protein)
for cell surface receptors. The cell surface oligosaccharides act as
receptors for bacteria, virus, toxin, and other cells (a white blood
corpuscle, a cancer cell). The sialyllacto/sialylneolacto-series sugar
chains in glycoproteins and glycolipids are the functional receptor
sugar chains for influenza A virus of humans and animals [7,8]. In
addition, orthomyxovirus, polyomavirus, reovirus, coronavirus,
It is known that bacterial enterotoxins bind to the receptor mol-
ecules on the host cell surface. The cell surface receptors are glyco-
conjugates, especially glycosphingolipids. For example, chorela
toxin to ganglioside GM1, Escherchia coli heat-labile enterotoxin
to ganglioside GD1b, and Shiga toxins to Gb3Cer are known. Oligo-
saccharides are key molecules for development of the technology
to analyze toxin–ligand interaction [14–17].
In this paper, we developed a simple method for the prepara-
tion of fluorescence oligosaccharide probes, which is a ‘‘turn-on”
type sensor using tetraphenylethylene derivative, for detection of
virus, toxin, and lectin. The merit of method for preparation of
these probes is to be able to introduce ligands to probe compounds
by ‘‘click chemistry”, a Cu(I)-catalyzed azide-alkyne cycloaddition
method, and can facilitate the manufacture of various biosensors.
This reaction is very suitable for the preparation of various kinds
* Corresponding author. Fax: +81 3 5452 6806.
E-mail address: tkato@iis.u-tokyo.ac.jp (T. Kato).
0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2010.02.155

T. Kato et al. / Biochemical and Biophysical Research Communications 394 (2010) 200–204
201
of probes because it has high efficiency and proceeds in a variety of
solvents, including aqueous alcohol or organic co-solvent and
water [18]. Furthermore, using the fluorescent oligosaccharide
probes bearing 60-sialyllactose moiety, we could detect human
influenza A virus.
and dried over anhydrous Na2SO4. After evaporation of the sol-
vents, the crude product was purified by a silica gel column using
hexane-dichloromethane (1:1, v/v) as eluent. Compound 2 was
obtained in 66% yield (0.09 g). 1H NMR (CDCl3, 600 MHz): d 6.93
(d, 8H), 6.70 (d, 8H), 4.62 (s, 8H), 2.51 (s, 4H); 13C NMR (CDCl3,
600 MHz): d 156.1, 138.7, 137.5, 132.6, 114.1, 78.7, 75.5, 55.9;
ESI-MS: m/z 548.2 ([M+H]+: 549.207).
2. Materials and methods
2.5. Preparation of 12-azidododecyl -lactoside and 12-azidododecyl
60-sialyllactoside
2.1. Reagents
Lectins were purchased from Funakoshi (Tokyo, Japan) or Sei-
kagaku Biobusiness (Tokyo, Japan). All chemical reagents were pur-
chased from Sigma (St. Louis, MO), Wako Pure Chemical Industries
(Osaka, Japan) or Nacalai Tesque (Kyoto, Japan). Nitrocellulose
12-Azidododecyl b-lactoside was prepared as described in liter-
ature [20]. The addition of
a2,6-linked sialic acid to 12-azidodode-
cyl b-lactoside was performed using
a
2,6-sialyltransferase from
Photobacterium damselae JT160 (ST6, Japan Tobacco, Tokyo, Japan).
Fifty microliters of 100 mM 12-azidododecyl b-lactoside in DMSO
and 1.95 ml reaction buffer (20 mM Bis-Tris, pH 6.0, containing
membrane (pore size = 0.2 lm) was purchased from Bio-Rad
(Hercules, CA). Virus suspension was prepared from influenza
virus-infected Madin–Darby canine kidney (MDCK) cells [19].
Briefly, MDCK cells were infected with A/WSN/33 strain at the
multiplicity of infection of 0.1 in minimal essential medium con-
0.02% Triton-X100) were mixed thoroughly. Then, 100 ll of
50 mg/ml CMP-NeuAc solution in reaction buffer and 0.1 unit
ST6 was added to the solution, and the solution was incubated at
30 °C for 16 h. The crude product was purified by the SAX cartride
(GL Science, Tokyo, Japan) as described previously [21]. The eluate
was desalted using a C18 Sep-Pak cartridge (Waters, Milford, MA)
and the solvents were removed in vacuo.
taining 0.1% BSA, 1ꢀ MEM vitamin solution (Gibco), and 1
lg/ml
trypsin. After 48 h post infection, the culture fluid was collected
and stored at ꢁ80 °C until use.
2.2. Apparatus
1H- and 13C NMR spectra were measured using a 600-MHz JEOL
spectrometer (JEOL, Tokyo, Japan). Electrospray ionization mass
spectrometry (ESI-MS) used was a HCTultraTM (high-capacity ion
trap mass spectrometer, Bruker Daltonics, Bremen, Germany). Ma-
trix-assisted laser desorption/ionization time-of-flight mass spec-
tra (MALDI-TOF/MS) were obtained using an AutoFlex MALDI-
TOF MS (Bruker Daltonics, Bremen, Germany). The photographs
under UV illumination were taken by using a FAS-III UV-image
analyzer (Toyobo, Osaka, Japan). Fluorescence intensity and spec-
trum were measured on a Varioskan plate reader (Thermo Fisher
Scientific, Waltham, MA).
2.6. Introduction of oligosaccharides to TPE derivative
A reaction solution containing10 mM 12-azidododecyl b-lacto-
side (or 12-azidododecyl 60-sialyllactoside), 2.5 mM compound 2,
10 mM CuSO4ꢂ5H2O and 50 mM sodium ascorbate in H2O–
DMSO(1:1, v/v) was stirred at room temperature for 24 h. The
crude product was purified by silica gel column chromatography
(Purif-pack SI-15, Moritex, Tokyo, Japan) using chloroform–metha-
nol–H2O (5:4:1, v/v/v) as eluent. As mentioned above, two kinds of
fluorescence oligosaccharide probes bearing lactosyl (Lac-TPE) and
60-sialyllactosyl (
a2,6SL-TPE) moieties were manufactured.
2.7. Analysis of reactivity against lectins by dot blotting
2.3. Synthesis of TPE derivative compound 1
Lectins (10
Rad Laboratories, Richmond, CA) and dried up. Then, the mem-
branes were soaked in 20 M Lac-TPE/ 2,6SL-TPE solution in
10 mM Tris–HCl, pH 7.6. After 10 min of exposure to the probe
solutions the membranes were washed with 10 mM Tris–HCl, pH
7.6 and detected under UV illumination.
lg) were spotted on nitrocellulose membrane (Bio-
This reaction was performed following the procedures de-
scribed in the literature [5]. A suspension of TiCl4 (0.78 ml,
7.0 mmol) and Zn dust (0.92 g, 14.0 mmol) in 35 ml of dry THF
was refluxed under N2 atmosphere for 2 h. A solution of 4,40-dihy-
droxybenzophenone (1.50 g, 7.0 mmol) in dry THF (15 ml) was
added to the suspension of the titanium reagent, and the reaction
was allowed to proceed at reflux for 4 h. The reaction mixture was
cooled to 25 °C, poured into a 10% aqueous K2CO3 solution (50 ml),
and after vigorous stirring for 5 min, the dispersed insoluble mate-
rial was removed by vacuum filtration using Celite pad. The organ-
ic layer was separated, and the aqueous layer was extracted three
times with ethyl acetate (25 ml). The combined organic fractions
were washed with water and dried over MgSO4.The solvents were
removed in vacuo to afford the compound 1. The crude product
was purified by a silica gel column using hexane-ethyl acetate
(1:1, v/v) as eluent. Compound 1 was obtained in 29% yield
(0.40 g). 1H NMR (CD3OD, 600 MHz): d 6.79 (d, 8H), 6.78 (d, 8H);
13C NMR (CD3OD, 600 MHz): d 155.3, 138.3, 136.1, 132.4, 114.0.
l
a
2.8. Fluorescence measurement
Fluorescence intensity was measured on a Varioskan plate read-
er with kex/kem set at 319/460 nm. Solutions of influenza virus and
fluorescence probe were mixed in a 96-well microtiter plate. After
incubation at room temperature for 10 min, fluorescence intensity
and spectrum were measured.
3. Results and discussion
The TPE derivatives were prepared as shown in Scheme 1A.
Compound 1 was synthesized by a McMurry coupling reaction
[22] between two molecules of 4,40-dihydroxybenzophenone.
Compound 1 was converted to tetrapropargyl compound 2 by reac-
tion with 3-bromo-1-propyne in the presence of K2CO3 in accept-
able yield. The chemical structures and molecular masses of
compounds 1 and 2 were confirmed from their NMR and mass
spectra. Lactosyl and 60-sialyllactosyl moieties were introduced
into compound 2 by click chemistry between the propargyl group
2.4. Synthesis of TPE derivative compound 2
The mixture of compound 1 (0.1 g, 0.25 mmol), 3-bromo-1-pro-
pyne (0.15 g, 1.25 mmol) and K2CO3 (0.35 g, 2.5 mmol) in anhy-
drous DMF (5 ml) was stirred vigorously under N2 atmosphere at
room temperature for 24 h. Then the solution was extracted with
chloroform. The organic layer was washed successively with water

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T. Kato et al. / Biochemical and Biophysical Research Communications 394 (2010) 200–204
Scheme 1. Synthesis of fluorescence oligosaccharide probes. (A) Synthesis of compounds 1 and 2. (B) Copper(I)-catalyzed synthesis of Lac-TPE and a2,6SL-TPE.
of TPE derivative and the azide group of aglycon of oligosaccharide
compounds (12-azidododecyl b-lactoside and 12-azidododecyl
60-sialyllactoside) using CuSO4 and sodium ascorbate as shown in
Scheme 1B. 12-Azidododecyl b-60-sialyllactoside was obtained by
enzymatic reaction from 12-azidododecyl b-lactoside using ST6
in 70% yield. Because the solubility of TPE derivative and12-azi-
dododecyl b-lactoside to water was low, they were dissolved in
DMSO and added to the reaction solution. TLC analysis (chloro-
form–methanol–H2O, 5:4:1) of these mixtures showed no sign of
the starting compound 2. Analysis of the MALDI-TOF mass spec-
trum of Lac-TPE and
a2,6SL-TPE revealed a peak at m/z 2777.6
([M+Na]+) and 4030.9 ([M-4H+5Na]+), respectively.
By dot blot method using lectin we confirmed the binding spec-
ificity of two kinds of fluorescent oligosaccharide probes (Lac-TPE
and
cifically to lactose and sialyl(
quence, respectively. Lac-TPE and
a
2,6SL-TPE). Lectins RCA120 and SSA are known to bind spe-
2,6)Gal/sialyl( 2,6)GalNAc se-
2,6SL-TPE bound to RCA120
a
a
a
and SSA, respectively (Fig. S1). These fluorescent oligosaccharide
probes were confirmed to be useful tools for detection by dot blot
method. It is usually necessary for the analytes to have plural bind-
Fig. 1. Fluorescence intensity at 460 nm of Lac-TPE and a2,6SL-TPE (2.5 lM) in the
presence of different concentrations of influenza virus A/WSN/33 in 10 mM Tris–
HCl, pH 7.6.

T. Kato et al. / Biochemical and Biophysical Research Communications 394 (2010) 200–204
203
ing sites to form aggregate states in the solution. However, the ana-
lytes immobilized on the membranes were detectable even if they
have only one binding site.
aggregates to induce such fluorescence enhancement by AIE effect.
This AIE effect is mainly caused by the restriction of intramolecular
rotation of C–C single bond [24]. The a2,6SL-TPE may bind to the
Human influenza viruses bind preferentially to sialic acids
HA molecules on the surface of an influenza virus and freeze its
linked to galactose by an
a2,6 linkage. We examined whether
intramolecular rotation.
influenza virus could be specifically detected by the fluorescence
oligosaccharide probe, because the influenza virus possesses HA
molecules at a high density (about 1000 molecules/virus) on its
surface [23]. Fig. 1 shows the fluorescence spectrum of Lac-TPE
Fig. 2 shows the fluorescence spectrum of
absence and presence of influenza virus A/WSN/33 in 10 mM
Tris–HCl, pH 7.6. 2,6SL-TPE showed very feeble emission at the
concentration of 5 M. The emission of less than 400 nm light de-
a2,6SL-TPE in the
a
l
and
enza virus A/WSN/33 in 10 mM Tris–HCl, pH 7.6. Fluorescence
enhancement of 2,6SL-TPE was observed in the presence of influ-
enza virus. On the other hand, fluorescence of Lac-TPE was not en-
hanced in the presence of influenza virus. These results suggest
that AIE may have been caused by binding of influenza virus to
a
2,6SL-TPE in the presence of different concentrations of influ-
pends on the self-luminosity of the 96-well microtiter plate. How-
ever, after addition of influenza virus A/WSN/33, the emission band
at 460 nm increased. Actually, the difference of fluorescence be-
tween the absence and presence of influenza virus can be distin-
guished by the naked eye under UV illumination as shown in
Fig. 2. Moreover, we examined detectable amounts of influenza
a
6’-sialyllactose moiety ligated to
a
2,6SL-TPE. It is interesting that
virus. The fluorescence intensity of
a2,6SL-TPE at 460 nm in-
whether the TPE derivatives bind to one virus particle or viral
creased significantly as a function of increasing amounts of influ-
enza virus as shown in Fig. 3. The influenza virus with
a
concentration higher than 105 pfu/100
ted.
ll was significantly detec-
It is well-known that influenza viruses bind to sialyl sugar
chain receptors on host cell membranes. Trimeric hemagglutinin
(HA) molecules on the surface of the envelope membrane of the
virus plays an important role in influenza virus infection to hu-
man cells. The binding specificity of HA changes easily by a
mutation of the HA gene (only two sets of amino acid exchange)
[13], so that the detection method of influenza virus using oligo-
saccharide probes is quite useful for diagnosis of the influenza
virus.
In conclusion, we developed a simple method for the prepara-
tion of fluorescence oligosaccharide probes. TPE derivatives with
propargyl residues could be used for ligation to oligosaccharide
compounds with azide residues by click chemistry. Because the
ligation reaction proceeded with CuSO4/ascorbate in water/DMSO
mixtures at room temperature, water-soluble compounds such as
oligosaccharide could be easily ligated to TPE derivatives. By this
technique, we prepared fluorescence oligosaccharide probes and
Fig. 2. Fluorescence spectrum and photographs of
a2,6SL-TPE (5 lM) in the
presence (106 pfu) or absence of influenza virus A/WSN/33 in 10 mM Tris–HCl,
pH 7.6.
demonstrated that
for influenza virus.
a2,6SL-TPE can be used as fluorescent sensor
Fig. 3. Change in (A) fluorescence spectrum and (B) intensity of a2,6SL-TPE (5 lM) after addition of influenza virus A/WSN/33 in10 mM Tris–HCl (pH 7.6). I0 is the value in its
absence.

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T. Kato et al. / Biochemical and Biophysical Research Communications 394 (2010) 200–204
sialyloligosaccharides in membrane-associated gangliosides as its receptor
Acknowledgments
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We would like to acknowledge Dr. Maria Carmelita Z. Kasuya
(The University of Tokyo, Tokyo, Japan) for the helpful discussions
and technical support on this study. We also thank Dr. Koji Mats-
uoka and Dr. Tetsuo Koyama (Saitama University, Saitama, Japan)
for the MALDI-TOF MS analysis. This work was supported by a
grant for ‘‘Development of Novel Diagnostic and Medical Applica-
tions through Elucidation of Sugar Chain Functions” from New
Energy and Industrial Technology Development Organization
(NEDO), the Ministry of Education, Culture, Sports, Science, and
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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bbrc.2010.02.155.
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