Tetraphenylethylene-based glycoconjugate as a fluorescence "turn-on" sensor for cholera toxin

Article abstract of DOI:10.1002/asia.201100141

Tetraphenylethylene (TPE)-based glycoconjugates were easily synthesized by copper(I)-catalyzed "click reactions" between propargyl-attached TPE and azido-functionalized sugars. The TPE compound bearing lactosyl moieties (Lac-TPE) was found to be a fluores

Full text of DOI:10.1002/asia.201100141

FULL PAPERS
DOI: 10.1002/asia.201100141
Tetraphenylethylene-based Glycoconjugate as a Fluorescence “Turn-On”
Sensor for Cholera Toxin
Xin-Ming Hu,[a, b] Qi Chen,[a] Jin-Xiang Wang,[a, c] Qian-Yi Cheng,[a] Chao-Guo Yan,[c]
Jie Cao,*[d] Yu-Jian He,*[b] and Bao-Hang Han*[a]
Abstract: Tetraphenylethylene (TPE)-
based glycoconjugates were easily syn-
thesized by copper(I)-catalyzed “click
reactions” between propargyl-attached
TPE and azido-functionalized sugars.
The TPE compound bearing lactosyl
moieties (Lac-TPE) was found to be a
fluorescence “turn-on” sensor for chol-
era toxin by virtue of aggregation-in-
duced emission characteristics of the
TPE motif owing to the specific inter-
action of lactose with the cholera toxin
B subunit, whilst a cellobiose-function-
alized TPE derivative did not show any
response to the toxin. Therefore, Lac-
TPE shows promising applications in
the detection of cholera toxin, as well
as in the investigation of carbohydrate–
protein interaction.
Keywords: aggregation-induced
emission · cholera toxin · lactose ·
tetraphenylethylene
Introduction
architecture. The five identical B-subunits of such toxin bind
selectively to the glycolipid ganglioside GM1 that contains a
pentasaccharide. It is the recognition of this pentasaccharide
that facilitates the initial attachment of cholera toxin B-sub-
unit (CTB) to the intestinal cells, which is the first step to-
wards contracting the disease cholera. CTB is known to
bind to lactose through the recognition of the terminal gal-
actose portion of the molecule,[2] which is also the terminal
sugar in the pentasaccharide headgroup of GM1 ganglio-
side.[3]
Cholera continues to represent a major threat to both
human and animals, especially in developing countries.
Cholera is an acute intestinal infection, characterized by
profuse watery diarrhea and vomiting, which can lead to
severe dehydration, electrolyte imbalance, and even death if
treatment is not given appropriately.[1] Cholera toxin (CT),
the primary virulence factor of cholera, is secreted by the
bacterium Vibrio cholerae and possesses an AB5 hexameric
It is well-known that carbohydrate-mediated multivalent
interactions play important roles in numerous biological
processes, such as cell-growth and -recognition, bacterial
and viral infections, inflammation, and cancer metastasis.[4]
The specificity and affinity of these interactions depend
strongly on multivalency, owing to the well-known “cluster
glycoside effect”.[5] Because most sugar ligands weakly bind
to their protein acceptors, multivalency in carbohydrate–
protein interactions becomes a prevalent principle to im-
prove the specificity and affinity.[6] Lactosyl-bearing multiva-
lent systems, such as dendrimers[2b] and nanoparticles,[2c]
have been shown to possess specific binding to CTB, in
which the specific interactions were assayed by fluores-
cence-quenching and red-shift of the adsorption, respective-
ly.
[a] X.-M. Hu, Dr. Q. Chen, J.-X. Wang, Q.-Y. Cheng, Prof. B.-H. Han
National Center for Nanoscience and Technology
Beijing 100190 (China)
Fax : (+86)10-8254-5576
E-mail: hanbh@nanoctr.cn
[b] X.-M. Hu, Prof. Y.-J. He
College of Chemistry and Chemical Engineering
Graduate University of Chinese Academy of Sciences
Beijing 100049 (China)
E-mail: heyujian@gucas.ac.cn
[c] J.-X. Wang, Prof. C.-G. Yan
College of Chemistry and Chemical Engineering
Yangzhou University
Yangzhou 225002 (China)
[d] Prof. J. Cao
MOE Key Laboratory of Cluster Science
Department of Chemistry
Beijing Institute of Technology
Beijing 100081 (China)
Recently, molecules with aggregation-induced emission
(AIE) characteristics have provided a promising platform in
applications ranging from optical materials to sensors, owing
to their enhanced emission in their aggregate or solid-state
forms.[7] The AIE effect can significantly improve the fluo-
E-mail: jcao@bit.edu.cn
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201100141.
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Chem. Asian J. 2011, 6, 2376 – 2381

rescence quantum yields of the molecules by up to two
orders of magnitude, and enhance the photoluminescence
intensity from faint luminophores into strong emitters.[8] Of
these AIE molecules, TPE can be readily produced from a
facile synthesis and shows an enhanced emission in both its
aggregated form and the solid state.[9] In contrast to aggrega-
tion-caused fluorescence-quenching of conventional dyes,
TPE-based AIE-active materials improve efficiency and sen-
sitivity as chemosensors[10] or bio-probes[11] and have already
shown practical applications in these fields. For example,
Zhang and co-workers demonstrated that the TPE com-
pounds bearing adenine and thymine moieties can be used
as chemosensors for silver(I) and mercury(II) ions, respecti-
vely.[10b] In addition, Tang and co-workers synthesized diphe-
nylated TPE dyes and found that the emission of such dyes
could be switched off and on reversibly by wetting and de-
wetting with solvent vapor (e.g. CHCl3), thereby manifesting
their ability to optically sense volatile organic compound-
s.[10a] They also developed various types of TPE motifs con-
taining charged groups, such as ammonium or sulfonate
groups, which were used as turn-on luminescent probes for
biomacromolecules, such as proteins and DNA.[9,11a,b] Kato
et al. prepared a TPE-based fluorescent oligosaccharide
probe bearing a 6’-sialyllactose moiety and utilized the
probe as a “turn-on” fluorescent sensor for the detection of
the influenza virus.[11e] Sanji and co-workers synthesized a
series of sugar-modified TPEs, through which carbohydrate–
protein and protein–protein interactions were investiga-
ted.[11f,g] Considering the specificity and affinity of carbohy-
drate–protein interactions, the grafting of carbohydrates
onto TPE can give rise to new properties and potential ap-
plications as biosensors and in investigating carbohydrate–
protein interaction.
sensor provides a unique platform to investigate carbohy-
drate–protein interactions.
Results and Discussion
Synthesis of Sugar–TPE Derivatives
Sugar–TPE derivatives were prepared as shown in
Scheme 1. TPE derivative 2 was first prepared using the
McMurry reaction[12] from 4,4’-dihydroxybenzophenone.
Herein, we report the synthesis of a new AIE-active TPE
derivative bearing lactose moieties and its potential applica-
tion as a fluorescence sensor for CT. Four lactose moieties
were readily introduced onto the TPE-derivative by a cop-
per(I)-catalyzed “click reaction” between propargyl-at-
tached TPE and azido-functionalized lactose. The introduc-
tion of four lactose moieties not only improves the water-
solubility and biocompatibility, but also provides multiple
binding sites towards CTB and enhances the binding
through multivalent interactions. Lac-TPE becomes signifi-
cantly luminescent when CTB is added, whilst cellobiose-
functionalized TPE derivative (Cel-TPE) does not show the
same phenomenon. Furthermore, we believe that the use of
Scheme 1. Synthetic route to Lac-TPE and Cel-TPE.
carbohydrate-bearing TPE as
a fluorescence “turn-on”
However, the yield of this reaction was poor (only 34%),
which is consistent with the result reported by Kato et al.[11e]
Further investigation showed that McMurry coupling of 4,4’-
dimethoxybenzophenone to produce compound 1 and then
demethylation with BBr3 can give 2 smoothly. Compared
with the straightforward coupling of 4,4’-dihydroxybenzo-
phenone, the yield of the two-step preparation was much
higher (up to 84%). The poor yield of the one-step prepara-
tion may result from some side-reactions induced by the
active hydroxyl groups during the McMurry reaction. Be-
Abstract in Chinese:
Chem. Asian J. 2011, 6, 2376 – 2381
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J. Cao, Y.-J. He, B.-H. Han et al.
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sides characterization by NMR spectroscopy, compound 1
was further identified by single-crystal X-ray diffraction
analysis. We found that the four phenyl rings of TPE were
non-planar (Figure 1), owing to the steric effect of the pe-
Figure 1. Crystal structure of TPE derivative 1.[16]
ripheral phenyl rings. Efficient transformation from com-
pound 1 into 2 was confirmed by the absence of resonance
peaks of methoxy protons in the 1H NMR spectrum after
the reaction had been completed. Further treatment of 2
with 3-bromo-1-propyne in the presence of K2CO3 afforded
propargyl-attached TPE derivative 3. Subsequently, lactosyl
moieties were introduced onto TPE via copper(I)-catalyzed
“click reactions”[13] between 3 and azido-functionalized lac-
tose derivative 4a, which was prepared according to previ-
ous procedures.[14] The formation of the triazole ring was
confirmed by a chemical shift at 7.78 ppm (single peak, 4H)
Figure 2. a) UV/Vis and b) fluorescence spectra for aqueous Lac-TPE so-
lution of different concentrations (from 5 to 40 mm).
sorption intensity increases with the concentration and the
red-shift of up to 12 nm of the adsorption near 250 nm was
attributed to the aggregation of the Lac-TPE molecules.
Addition of tetrahydrofuran or acetonitrile (even up to
99% in volume fraction) into the dilute aqueous solution of
Lac-TPE did not dramatically improve its emission efficien-
cy (see the Supporting Information, Figure S1). This phe-
nomenon should be attributed to the amphiphilic properties
of Lac-TPE, whose lactosyl groups are hydrophilic and TPE
moiety is lipophilic. The Tang group has also obtained simi-
lar results with a TPE derivative containing amino groups
that is hydrophilic.[11b] Therefore, a co-solvent system of di-
chloromethane (a poor solvent) and dimethyl sulfoxide
(DMSO, a good solvent) was used to investigate the AIE
effect of Lac-TPE. As shown in the Supporting Information,
Figure S2, in the dilute dimethyl sulfoxide solution, Lac-
TPE is nonemissive, regardless of addition of dichlorome-
thane up to 80% (v/v). When the volume fraction of di-
chloromethane reached 85% or higher (the final concentra-
tion of Lac-TPE was kept constant at 3.7 mm), the fluores-
cence intensity increased significantly under the same mea-
surement conditions owing to the aggregation of Lac-TPE
in the dimethyl sulfoxide/dichloromethane solution, which
leads to restricted intramolecular rotations and decreased
nonradiative decay.[7c,d] The inset photos in the Supporting
1
in H NMR spectrum and two peaks at 121.3 and 144.9 ppm
in 13C NMR spectrum (see the Supporting Information).
After the removal of all of the acetyl groups with CH3ONa/
CH3OH, the desired water-soluble Lac-TPE was obtained in
an excellent yield (97%). Compared with the protected pre-
cursor 5a, the peaks of acetyl groups (2.14–1.95 ppm) in the
1H NMR spectrum disappeared and those for the hydroxy
groups (5.16–4.57 ppm) of Lac-TPE appeared after efficient
deprotection (see the Supporting Information). Analysis of
the ESI-ion-trap mass spectrum of Lac-TPE revealed a
peak at m/z 2075.04 ([M+H2O+K]+). Similarly, Cel-TPE
was also prepared and characterized for the control study.
AIE effect of Lac-TPE
With Lac-TPE in hand, its AIE effect was studied firstly
based on solvent-assistant aggregation. When dissolved in
soluble solvents, such as water, Lac-TPE was nonlumines-
cent in a relatively dilute solution. The fluorescence emis-
sion enhanced significantly with an increase in concentration
of Lac-TPE in aqueous solution (Figure 2b). There may be
two reasons for this enhanced fluorescence intensity, that is,
the increased adsorption and the aggregation of Lac-TPE in
water at a higher concentration. Figure 2a confirms the ad-
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Chem. Asian J. 2011, 6, 2376 – 2381

A Fluorescence “Turn-On” Sensor for Cholera Toxin
Information, Figure S2, show the enhanced photolumines-
cence intensity of Lac-TPE solution from a faint lumino-
phore into a strong emitter when Lac-TPE aggregates. In
addition, Cel-TPE showed a similar phenomena (see the
Supporting Information, Figure S3).
Fluorescence “Turn-On” Assay for Cholera Toxin
CT is a pentamer composed of five identical monomers,
each with a binding site for the GM1 ganglioside.[3a] These
binding sites, matching with the galactose moieties of the
GM1 ganglioside, make it possible for CTB to bind to vari-
ous types of molecules that have the same terminal galac-
tose moiety. Lactose, as a disaccharide consisting of glucose
and terminal-galactose units, was thus used to design CT
sensors. The design rationale for the CT assay is illustrated
in Scheme 2. Lac-TPE, possessing four lactosyl units, is ex-
Figure 3. a) Fluorescence spectra of Lac-TPE (2.0 mm in 50 mm PBS solu-
tion, pH 7.3) in the presence of different amounts of CTB (0, 1.0, 2.0,
and 5.0 mm); left inset: photos of the corresponding solutions of Lac-TPE
in the absence (A) and presence of CTB (B; 5.0 mm) under UV light
(365 nm) illumination; right inset: plot of the fluorescence intensity
(I475 nm) versus the concentration of CTB. b) UV/Vis spectra of Lac-TPE
PBS solution in the absence and presence of CTB (5.0 mm).
Scheme 2. Illustration of fluorescence “turn on” assay for CT with Lac-
TPE based on AIE effect.
pected to display weak photoluminescence in aqueous
media. When CT was added to a dilute aqueous solution of
Lac-TPE, multiple binding events occurred between the
toxin and the multivalent lactosyl-attached TPE, leading to
aggregation of Lac-TPE. As a result, the fluorescence of the
ensemble would be increased significantly, which is induced
by the aggregation of Lac-TPE.
Figure 3a shows the fluorescence spectrum of Lac-TPE
and those in the presence of different amounts of CTB in
PBS solution. Lac-TPE showed rather weak emission when
its concentration was 2.0 mm. After the addition of CTB, the
emission band around 475 nm emerged and its intensity in-
creased gradually. The enhanced emission increased signifi-
cantly when the concentration of CTB was below 2.0 mm,
and the enhanced emission got less significant when the con-
centration of CTB was up to 5.0 mm, thereby indicating that
the amount of CTB begins to be in excess. Photographs of
solutions of Lac-TPE and the ensemble Lac-TPE/CTB in
PBS buffer solution taken under illumination of a UV lamp
are given in Figure 3a, left inset. The observed fluorescence
enhancement is mainly caused by restricted intramolecular
rotation of phenyl groups in the aggregated state, which
blocks the nonradiative decay of Lac-TPE and makes it
highly luminescent. The change of fluorescence intensity
became larger with increasing amounts of the toxin owing to
the formation of more and larger aggregates. When the con-
centration of CTB was as low as 1.0 mm, the fluorescence
emission of Lac-TPE was also obviously enhanced, up to
three folds of that of Lac-TPE solution in the absence of
CTB. Compared with previous reported CT sensors,[15] Lac-
TPE has the advantages of a simple synthesis, without the
need for complex instrumentation, though does show a
lower sensitivity. Furthermore, the sensitivity is expected to
increase when more lactosyl moieties are introduced onto
TPE owing to the multivalent interactions.[2b,c]
In order to identify the formation of aggregates, the ab-
sorption spectra of Lac-TPE in the absence and presence of
CTB were investigated. Compared to a Lac-TPE solution
without CTB, the adsorption showed a red-shift of 8 nm
(from 242 nm to 250 nm) when CTB (5.0 mm) was added to
the Lac-TPE solution (Figure 3b). This result is consistent
with both the aforementioned aggregation induced by the
added concentration of Lac-TPE and the reported aggrega-
tion of dimethoxytetraphenyletheylene induced by the in-
creasing poor solvent, which can verify the formation of the
Lac-TPE/CTB ensemble.[11a]
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J. Cao, Y.-J. He, B.-H. Han et al.
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To investigate the selectivity of fluorometric detection for
CT, as well as the specificity of carbohydrate–protein inter-
actions, Lac-TPE was treated with another protein (BSA)
under the same conditions, and no significant change of pho-
toluminescence intensity was observed. Furthermore, when
Lac-TPE was mixed with an equal amount of CTB and
BSA in PBS solution, the mixture displayed an intense emis-
sion, in which the intensity is almost as same as that ob-
served in the case of CTB (Figure 4).
Experimental Section
Materials and Instrumentation
All chemical reagents were commercially available and used as received
unless otherwise stated. CTB and Bovine Serum Albumin (BSA) were
purchased from Sigma–Aldrich Co. Tetrahydrofuran (THF) was purified
by distillation from sodium under nitrogen immediately prior to use. De-
ionized water was obtained with a Millipore purification system (Milli-Q
water).
1H and 13C NMR spectra were recorded on a Bruker DMX400 NMR
spectrometer using the solvent peak as internal reference. Mass spec-
trometry was performed on a Thermo LCQ Deca XP MAX mass spec-
trometer using the ESI(+) technique. Single-crystal X-ray diffraction
analysis of TPE derivative 1 was carried out on a Bruker SMART APEX
II CCD diffractometer. Ultraviolet-visible (UV/Vis) spectra were mea-
sured on a Perkin–Elmer Lamda 950 UV/Vis spectrometer and fluores-
cence spectra were measured on a Perkin–Elmer LS 55 luminescence
spectrometer using a conventional cell with 1 cm path length.
Synthesis of TPE derivative 1
Zn dust (5.31 g, 81.2 mmol) was added to a solution of 4,4’-dimethoxy-
benzophenone (2.01 g, 8.3 mmol) in 40 mL of dry THF. After refluxing
for 20 h, the reaction mixture was cooled to room temperature and fil-
tered. The solvent was evaporated under vacuum and the crude product
was purified by column chromatography on silica gel using dichlorome-
thane/petroleum ether (v/v=1:2) as the eluent. Finally, compound 1 was
obtained as a white solid in 90% yield (1.69 g). 1H NMR (400 MHz,
CDCl3): d=6.92 (d, J=8.6 Hz, 8H), 6.63 (d, J=8.6 Hz, 8H), 3.72 ppm (s,
12H). 13C NMR (100 MHz, CDCl3): d=157.9, 138.5, 137.0, 132.6, 113.1,
55.2 ppm.
Figure 4. Fluorescence spectra of sugar-TPE derivatives (2.0 mm Lac-TPE
or Cel-TPE in 50 mm PBS solution; pH 7.3) in the absence and presence
of different proteins (5.0 mm CTB or BSA in 50 mm PBS solution;
pH 7.3).
Synthesis of TPE Derivative 2
Under nitrogen atmosphere, compound 1 (3.00 g, 6.63 mmol) was dis-
solved and stirred in 40 mL dry dichloromethane. BBr3 (6.0 mL,
63.6 mmol) was quickly added to the solution at À458C. After stirring for
30 min, the solution was allowed to warm to room temperature and was
stirred for a further 10 h. The reaction mixture was poured into cold
water with vigorous stirring until no more precipitate was formed. After
filtration and drying, a purple solid 2 was obtained in 93% yield (2.44 g).
1H NMR (400 MHz, [D6]DMSO): d=6.71 (d, J=8.4 Hz, 8H), 6.49 ppm
(d, J=8.4 Hz, 8H). 13C NMR (100 MHz, [D6]DMSO): d=155.4, 137.7,
135.1, 132.0, 114.5 ppm.
As a control study, Cel-TPE was also used to investigate
the effect of sugar ligand on the selectivity of CT detection.
Compared with high fluorescence intensity induced by Lac-
TPE/CTB ensemble, the CTB cannot “turn on” the fluores-
cence of Cel-TPE, which implies that there is no specific in-
teraction between Cel-TPE and CTB. All the results afore-
mentioned substantiate the specificity of interaction be-
tween Lac-TPE and CTB.
Preparation of TPE Derivative 3
A
mixture of compound 2 (700 mg, 1.77 mmol), propargyl bromide
(80 wt% in toluene, 2.0 mL, 18.0 mmol), K2CO3 (2 g, 14.5 mmol) and
NBu4Br (30 mg) in acetone (30 mL) was refluxed overnight under a ni-
trogen atmosphere. The mixture was then cooled to room temperature
and filtered. After removal of the solvent under reduced pressure, the
residue was purified by column chromatography on silica gel (ethyl ace-
tate/petroleum ether, v/v=1:5) to give the desired product 3 (794 mg,
82%) as a white solid. 1H NMR (400 MHz, CDCl3): d=6.93 (d, J=
8.4 Hz, 8H), 6.70 (d, J=8.8 Hz, 8H), 4.62 (d, J=2.4 Hz, 8H), 2.51 ppm
(t, J=2.4 Hz, 4H). 13C NMR (100 MHz, CDCl3): d=156.1, 138.7, 137.6,
132.6, 114.1, 78.7, 75.6, 55.9 ppm.
Conclusions
In summary, neutral lactose-bearing tetraphenylethenes are
designed and prepared as “turn-on” luminescent sensors for
cholera toxin. Aggregation derived from lactosyl–CTB bind-
ing can switch on the fluorescence of water-soluble tetraphe-
nylethylene-based glycoconjugates in PBS solution, which
can be used for investigation of carbohydrate–protein inter-
actions based on aggregation-induced emission. We believe
that methods inspired by this concept of investigating carbo-
hydrate-mediated biological interactions have promising ap-
plications in biomacromolecule detections and glycobiology
studies.
Synthesis of TPE Derivative 5a
To a suspension of compound 3 (150 mg, 0.27 mmol) and lactose azide
derivative 4a (1.07 g, 1.62 mmol) in THF/H2O (v/v=2:1, 30 mL) were
added sodium ascorbate (30 mg) and CuSO4 (20 mg) as a catalyst. The
mixture was stirred at 708C for 6 h and then extracted three times with
ethyl acetate (50 mL). The combined organic extracts were concentrated
and purified by column chromatography on silica gel (ethyl acetate/pe-
troleum ether, v/v=4:1) to yield
a white solid 5a (751 mg, 86%).
1H NMR (400 MHz, CDCl3): d=7.78 (s, 4H), 6.90 (d, J=8.4 Hz, 8H),
6.69 (d, J=8.8 Hz, 8H), 5.85 (d, J=8.8 Hz, 4H), 5.41–5.38 (m, 8H), 5.34
(d, J=3.2 Hz, 4H), 5.09 (s, 8H), 4.96 (dd, J=3.2, 10.4 Hz, 4H), 4.53 (d,
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A Fluorescence “Turn-On” Sensor for Cholera Toxin
J=8.0 Hz, 4H), 4.46 (d, J=11.6 Hz, 4H), 4.14–4.05 (m, 16H), 3.96–3.89
(m, 12H), 2.14 (s, 12H), 2.07–2.01 (m, 48H), 1.95 (s, 12H), 1.83 ppm (s,
12H). 13C NMR (100 MHz, CDCl3): d=170.4, 170.3, 170.2, 170.1, 169.6,
169.2, 169.2, 156.6, 144.9, 138.6, 137.3, 132.6, 121.3, 114.0, 101.2, 85.6,
76.0, 75.7, 72.7, 71.0, 70.9, 70.6, 69.1, 66.7, 61.9, 60.9, 60.5, 21.1, 20.9, 20.7,
20.6, 20.5, 20.4, 20.3 ppm.
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Synthesis of Lac-TPE
To a solution of compound 5a (410 mg, 0.13 mmol) in dichloromethane/
methanol (v/v=1:3, 20 mL), CH3ONa (1.0m in methanol) was added
dropwise until the pH value of the solution reached 11. After stirring at
room temperature for 5 h, the resultant precipitate was collected by cen-
trifugation at 8000 rpm, washed twice with dichloromethane, dried under
vacuum at 408C, and finally obtained as a yellowish solid (Lac-TPE) in
97% yield (251 mg). 1H NMR (400 MHz, [D6]DMSO): d=8.44 (s, 4H),
6.90 (d, J=8.8 Hz, 8H), 6.84 (d, J=8.8 Hz, 8H), 5.66 (d, J=9.2 Hz, 4H),
5.16–4.57 (m, 36H), 4.26 (d, J=7.2 Hz, 4H), 3.87 (t, J=9.2 Hz, 4H), 3.78
(d, J=10.8 Hz, 4H), 3.67–3.46 (m, 32H), 3.38–3.33 ppm (m, 8H).
13C NMR (100 MHz, D2O): d=157.1, 143.9, 139.7, 138.2, 133.3, 124.7,
115.0, 103.9, 88.6, 78.6, 78.3, 76.3, 75.9, 73.6, 73.3, 71.8, 69.5, 61.9, 60.8,
58.2 ppm. MS (ESI-Ion Trap): m/z calcd for C86H112N12O44: 2017.86 [M];
found: 2075.04 [M+H2O+K]+.
Fluorescence Analysis
A stock solution of Lac-TPE was prepared in aqueous phosphate buffer
solution (PBS, 50 mm, pH 7.3). Aliquots of CTB in the same PBS buffer
were added to the solutions of Lac-TPE. The final concentration of Lac-
TPE was 2.0 mm. After each addition, the sample was allowed to equili-
brate overnight prior to recording a spectrum. The excitation wavelength
was 317 nm and the emission scan ranged from 330–630 nm.
Acknowledgements
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2056–2075; Angew. Chem. Int. Ed. 2001, 40, 2004–2021; b) V. V.
Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, Angew. Chem.
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We thank the Ministry of Science and Technology of China (National
Basic Research Program, Grant 2011CB932500) and the National Science
Foundation of China (Grants 20972035 and 21002017) for financial sup-
port.
[15] a) H. Chen, Y. Zheng, J.-H. Jiang, H.-L. Wu, G.-L. Shen, R.-Q. Yu,
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[16] CCDC 825731 (1) contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
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Received: February 11, 2011
Published online: July 11, 2011
Chem. Asian J. 2011, 6, 2376 – 2381
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
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