Fluorescence quenching detection of peanut agglutinin based on photoluminescent silole-core carbosilane dendrimer peripherally functionalized with lactose

Article abstract of DOI:10.1016/j.tetlet.2009.07.153

A glycocluster peripherally functionalized with a lactose (Lac: Galβ1→4Glcβ1-) derivative possessing a silole moiety as a luminophore was synthesized. The photoluminescence spectrum of the glycocluster showed extremely strong emission at 474 nm and the ab

Full text of DOI:10.1016/j.tetlet.2009.07.153

Tetrahedron Letters 50 (2009) 5816–5819
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Fluorescence quenching detection of peanut agglutinin based
on photoluminescent silole-core carbosilane dendrimer peripherally
functionalized with lactose
*
Ken Hatano , Hitoshi Saeki, Hiroo Yokota, Hiroaki Aizawa, Tetsuo Koyama, Koji Matsuoka, Daiyo Terunuma
Division of Material Science, Graduate School of Science and Technology, Saitama University, 255 Shimo-Ohkubo, Sakura-ku, Saitama 338-8570, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A glycocluster peripherally functionalized with a lactose (Lac: Galb1?4Glcb1–) derivative possessing a
silole moiety as a luminophore was synthesized. The photoluminescence spectrum of the glycocluster
showed extremely strong emission at 474 nm and the absolute quantum yield was estimated to be
92% in distilled water. The emission intensity was decreased by increasing the amount of peanut agglu-
tinin (PNA), a lactose-binding lectin, and plots of the relative fluorescence intensity revealed a decline of
95% in emission intensity. Fluorescence quenching of the glycocluster upon mixing with PNA could be
easily observed by the naked eye under UV irradiation, whereas no distinct change in fluorescence
properties of the glycocluster was observed when wheat germ agglutinin (WGA) was employed.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 10 June 2009
Revised 17 July 2009
Accepted 31 July 2009
Available online 6 August 2009
Keywords:
Aggregation-induced emission
Biosensor
Carbohydrate
Fluorescence quenching
Glycocluster
Lectin
Silole
Recent advances in glycoscience have revealed that glycoconju-
gates, such as glycoprotein and glycolipid, generally located on the
cell surface play a critical role in the process of cell adhesion with
proteins of pathogens.1 It is known that the early stage of cell adhe-
sion involves carbohydrate-mediated specific recognition of patho-
gens. Lee et al. discovered that the clustering of carbohydrates
leads to the enhancement of individual interactions between car-
bohydrates and proteins.2 A combination of the specific recogni-
tion and the carbohydrate cluster effect has been applied for the
molecular design of artificial inhibitors and neutralizing agents of
pathogens, such as toxins, bacteria, and viruses, and several forms
of glycoclusters have been developed so far.3 We have reported the
syntheses and biological activities of some glycoclusters in carbos-
ilane dendrimers employed as carbohydrate scaffolds.4
On the other hand, glycoclusters possessing luminophores have
recently attracted much attention in both clinical and chemical
biology fields from the viewpoint of a fluorescent marker that ad-
heres to the target analyte.5 However, there have been few reports
on the application of luminescent glycoclusters for a biosensor
whose emission can be enhanced/quenched or blue/red-shifted
in the presence of the target analyte.6 We have recently reported
the synthesis of not only the first glycocluster possessing siloles
(silacyclopentadienes) but also the first hydrophilic silole deriva-
tive, and we have shown that the emission intensity was enhanced
by aggregate formation of the silole moiety.7 This unique photolu-
minescence property motivated us to explore the usefulness of the
silole-glycocluster as a biosensor of pathogens. In this letter, we
present a convenient fluorescence quenching sensor for lectin as
an alternate analyte instead of pathogens based upon the aggrega-
tion-induced enhanced emission (AIE) feature of silole derivatives.
A glycocluster peripherally functionalized with lactose possess-
ing a silole moiety was prepared according to the same route as
that described in our previous report (Scheme 1).7 Coupling reac-
tion between a silole-core dendrimer 1 and a peracetylated lactose
derivative 2 was achieved by nucleophilic substitution of the ter-
minal bromide on the dendrimer 1 with a thiolate anion generated
from 2 by treatment with sodium methoxide in methanol. The
silole-core glycodendrimer 3 fully substituted by lactose was
obtained in 71% yield after re-acetylation followed by recycling
GPC purification.8 De-O-acetylation of the glycodendrimer 3 by a
combination of Zemplén’s condition and saponification afforded
the related dendrimer 4, combining silole-luminophore and lactose
moieties having PNA recognition ability, in 90% yield.9
A photoluminescence (PL) spectrum of 4 measured in distilled
water showed an emission band at around 474 nm from the silole
moiety and the absolute quantum yield (UFL) was estimated to be
92% in water.10 Silole luminophore must have some molecular
vibrational modes when it is molecularly dissolved in a solvent,
and rotation of the phenyl groups linked to the silole nucleus
* Corresponding author. Tel./fax: +81 48 858 3535.
E-mail address: khatano@fms.saitama-u.ac.jp (K. Hatano).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.07.153

K. Hatano et al. / Tetrahedron Letters 50 (2009) 5816–5819
5817
Ph
Ph
OAc
OAc
O
OAc
O
i, ii
AcO
O
+
O
SAc
AcO
Ph
Ph
Br
Si
Si
Si
Br
OAc
OAc
3
3
1
2
Ph
Ph
Ph
Ph
iii, iv
Ph
Ph
4 (y. 90%)
Ph
Ph
3 (y. 71%)
Lac
Si
Si
Si
Lac
(AcO)Lac
Si
Si
Si
Lac(OAc)
3
3
3
3
OR
OR
OR
O
RO
O
O
S
RO
O
OR
OR
Lac(OAc) : R = Ac
Lac : R = H
Scheme 1. Reagents and conditions: (i) 28% NaOMe, MeOH, DMF, rt, on; (ii) Ac2O, pyridine, rt, on; (iii) 28% NaOMe, MeOH, rt, 1 h; (iv) 0.1 M NaOH, rt, on.
900
may occur mainly in the vibrational modes. Since a considerable
0 µL
part of photo-excited energy would be spent on the phenyl-rota-
tion, low quantum yield and low emission intensity were eventu-
ally observed in the solvent. In an aggregational state, however,
the phenyl-rotation could be efficiently inhibited by intermolecu-
lar repulsion of the phenyl substituents, ultimately resulting in
high quantum yield and high emission intensity. The intramolecu-
lar restriction of phenyl-rotation may be the main cause for the AIE
800
700
600
500
400
300
200
100
0
10 µL
20 µL
30 µL
40 µL
50 µL
phenomenon in the case of a p-system peripherally functionalized
with phenyl substituents such as silole. Dynamic light scattering
(DLS) measurement strongly suggested the formation of monodi-
spersed aggregates of globotriaose analogue of 4 in distilled water
(9.0–18.1 nm, 1 mg/100 mL).11 Thus, the extremely high lumines-
cent efficiency of 4 observed in distilled water is attributable to
aggregation formation by amphiphilic silole dendrimer 4 in water;
the hydrophobic silole groups are oriented within the aggregate
and the hydrophilic lactose moieties are exposed to surrounding
water.
400
450
500
550
600
Wavelength (nm)
Figure 1. PL spectra of silole-core dendrimers
amounts of PNA (from 0 to 50 L). Concentration of 4: 0.6
(5 mM, pH 7.4). Concentration of PNA: 20
Excitation: 360 nm.
4
in the presence of different
M in HEPES buffer
lM in HEPES buffer (5 mM, pH 7.4).
l
l
The PL spectrum of 4 measured in aqueous buffer solution
[0.6
strong emission at 474 nm. Upon addition of PNA solution
(20 M in HEPES buffer), the emission of 4 became faint light.
lM in HEPES buffer (5 mM, pH 7.4), 3.0 mL] also showed
l
The PL spectra of 4 were obtained with successive addition of ali-
quots of PNA solution (Fig. 1), and the relative fluorescence inten-
sity [(I-I0)/I0], where I0 and I are emission intensities of 4 in the
absence and presence of PNA, respectively, versus PNA concentra-
tions are plotted in Figure 2. The emission intensity decreased pro-
gressively with an increase in the amount of PNA and there was a
[PNA] (µM)
0
0.1
0.2
0.3
0.4
0
-0.1
-0.2
-0.3
-0.4
-0.5
-0.6
-0.7
-0.8
-0.9
-1
decline of 95% in emission intensity with addition of 50 lL of the
PNA solution; however, emission maximums were constantly de-
tected at around 474 nm in all experiments. Fluorescence quench-
ing of 4 upon mixing with PNA can be easily observed by the naked
eye under UV irradiation as shown in Figure 3. Analogous treat-
ments of 4 with WGA, a GlcNAc/Neu5Ac-binding lectin, and with
a blank solution (buffer solution without lectin) were carried out
for comparison. No remarkable fluorescence quenching was de-
tected in both the PL spectra (Fig. 4); additions of 80 lL of each
solution led to a decline of only 2% in emission intensity due to
the diluting effect. Therefore, the variations of emission intensity
from 4 were essentially inert, unlike the case of PNA, clearly
indicating that silole dendrimer 4 specifically recognizes PNA. A
Figure 2. Plots of relative fluorescence intensity [(IꢀI0)/I0] of solution of 4 at
474 nm versus concentration of PNA.

5818
K. Hatano et al. / Tetrahedron Letters 50 (2009) 5816–5819
plausible mechanism for the fluorescence quenching of 4 upon
mixing with PNA is agglomerate dispersion of 4 formed in the ab-
sence of the lectin due to carbohydrate–lectin specific recognition,
that is, PNA addition converts a highlyemissive ‘AIE active’ situa-
tion of silole 4 into a non-emissive ‘AIE inactive’ situation. Silole-
core dendrimers peripherally functionalized with carbohydrates
such as 4 are potentially useful for detection of a target lectin by
means of the high affinity and specificity of the glycocluster and
the AIE effect of the silole derivative. Further investigations of sil-
ole-core dendrimers functionalized with bioactive oligosaccha-
rides and their applications to detection of pathogens as a novel
biosensor are currently in progress.
Acknowledgments
This work was supported by a Grant-in-Aid for Scientific Re-
search (C) (No. 21550152) from the Japan Society for the Promo-
tion of Science. We thank Professor K. Tamao, Dr. T. Matsuo and
Dr. T. Fukawa [The Institute of Physical and Chemical Research (RI-
KEN)] for measurements of absolute quantum yield of the silole
dendrimers.
Supplementary data
Figure 3. Pictures of photoluminescence of the HEPES buffer solution of 4 in the
(A) absence and (B) presence of PNA. Excitation: 360 nm.
Supplementary data (analytical data for new compounds) asso-
ciated with this article can be found, in the online version, at
doi:10.1016/j.tetlet.2009.07.153.
800
A
0 µL
700
20 µL
References and notes
40 µL
600
60 µL
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Kolusheva, S. Chem. Rev. 2004, 104, 5987–6015.
80 µL
500
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M.; Lönn, H. J. Biol. Chem. 1983, 258, 199–202; (b) Lee, Y. C. FASEB J. 1992, 6,
3193–3200.
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0
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J. Chem. Rev. 2002, 102, 555–578; (b) Chabre, Y. M.; Roy, R. Curr. Top. Med. Chem.
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3617–3620; (e) Mori, T.; Hatano, K.; Matsuoka, K.; Esumi, Y.; Toone, E. J.;
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Koyama, T.; Matsuoka, K.; Esumi, Y.; Terunuma, D. Carbohydr. Res. 2006, 341,
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T.; Matsuoka, K.; Terunuma, D. Tetrahedron Lett. 2007, 48, 4365–4368.
400
450
500
550
600
Wavelength (nm)
800
700
600
500
400
300
200
100
0
B
0 µL
20 µL
40 µL
60 µL
80 µL
400
450
500
550
600
8. Compound 3: ½a D39
ꢁ
ꢀ8.7 (c 1.0, CHCl3). 1H NMR: (CDCl3, 400 MHz) d 6.73–7.12
Wavelength (nm)
(m, 20H, aromatic), 5.34 (d, 6H, J = 2.8 Hz, H-40), 5.18 (t, 6H, J = 9.6 Hz, H-3),
5.10 (t, 6H, J = 8.0 Hz, H-20), 4.95 (dd, 6H, J3 ,4 = 2.8 Hz, J2 ,3 = 10.4 Hz, H-30),
4.87 (t, 6H, J = 8.4 Hz, H-2), 4.43–4.49 (m, 18H, H-1, H-10, H-6b), 4.04–4.15 (m,
18H, H-6a, H-60ab), 3.87 (t, 6H, J = 6.4 Hz, H-50), 3.76–3.85 (m, 12H, H-4,
OCH2b), 3.56–3.60 (m, 6H, H-5), 3.40–3.46 (m, 6H, OCH2a), 2.42 (br s, 24H,
SCH2), 1.96–2.15 (m, 126H, OAc), 1.51–1.61 (m, 24H, OCH2CH2CH2), 1.37–1.49
(m, 28H, SCH2CH2, SiCH2CH2), 1.05–1.07 (m, 4H, SiCH2CH2CH2SiCH2), 0.47–0.56
0
0 0
0
00
Figure 4. PL spectra of silole-core dendrimers 4. (A): Presence of different amounts
of WGA (from 0 to 80 L). Concentration of 4: 0.6 M in HEPES buffer (5 mM, pH
7.4). Concentration of WGA: 40 M in HEPES buffer (5 mM, pH 7.4). Excitation:
360 nm. (B): Addition of different amounts of blank solution (from 0 to 80
Excitation: 360 nm.
l
l
l
lL).

K. Hatano et al. / Tetrahedron Letters 50 (2009) 5816–5819
5819
(m, 16H, SCH2CH2CH2SiCH2). 13C NMR (CDCl3, 100 MHz) d 170.17, 170.14,
169.96, 169.86, 169.62, 169.39, 168.90, 154.96, 140.37, 139.98, 138.66, 129.75,
128.71, 127.81, 127.28, 126.05, 125.34, 100.86, 100.37, 76.11, 72.66, 72.42,
71.51, 70.80, 70.46, 69.72, 68.94, 66.45, 61.87, 60.63, 35.88, 31.90, 29.18, 28.89,
24.95, 24.05, 20.69, 20.63, 20.54, 20.44, 20.32, 18.24, 16.68, 16.41, 11.81. IR
(KBr) 2938 (w, sh), 1781 (s, sh), 1437 (w, sh), 1369 (m, sh), 1233 (s, sh), 1171
100 MHz) d 140.19, 137.18, 125.50–129.69 (br m), 102.77, 102.18, 78.29, 75.07,
74.44, 74.30, 73.01, 72.56, 72.38, 70.70, 70.35, 69.92, 69.69, 68.29, 67.80, 60.77,
60.09, 35.44, 32.12, 31.51, 29.23, 28.75, 28.32, 24.81, 23.86, 18.32, 16.91, 11.46.
IR (KBr) 3383 (s, br), 2922 (m, sh), 1643 (w, br), 1416 (w, br), 1074 (s, br) cmꢀ1
.
UV–vis (water) kmax 360 nm
(e = 6700). MALD-TOF calcd for C154H254O66
S6Si3Na [M+Na]+ 3460.409; found 3458.414.
(w, sh), 1132 (w, sh), 1051 (m, sh) cmꢀ1
.
UV–vis (CHCl3) kmax 361 nm
10. The quantum yield was determined by means of the absolute PL quantum
measurement system C9920-03 (Hamamatsu Photonics K. K.). The UFL is the
second highest efficiency in silole derivatives, following 1,2-bis(1-methyl-
2,3,4,5-tetraphenylsilacyclopentadienyl)ethane reported by Murata et al.
Murata, H.; Kafafi, Z. H.; Uchida, M. Appl. Phys. Lett. 2002, 80, 189–191.
11. More details, including DLS measurement in distilled water, will be published
elsewhere.
(e
= 8300). MALD-TOF calcd for C238H338O108S6Si3Na [M+Na]+ 5225.855;
found 5226.436.
9. Compound 4: ½a D37
ꢁ
45.0 (c 1.0, H2O). 1H NMR (D2O, 400 MHz) d 6.41–7.15 (br m,
20 H, aromatic), 4.29–4.51 (br m, 12H, H-1, H-10), 3.21–4.10 (br m, 78H, CH2O,
H-2, H-3, H-4, H-5, H-6ab, H-20, H-30, H-40, H-50, H-60ab), 2.22–2.65 (br s, 24H,
CH2SCH2), 1.18–1.84 (m, 52H, OCH2CH2CH2CH2SCH2CH2CH2SiCH2CH2), 0.85–
1.13 (br s, 4H, SiCH2CH2CH2SiCH2), 0.20–0.75 (br, 16H, SiCH2). 13C NMR (D2O,