Sugar-bearing tetraphenylethylene: Novel fluorescent probe for studies of carbohydrate-protein interaction based on aggregation-induced emission

Article abstract of DOI:10.1039/c0ob00680g

Neutral sugar-bearing tetraphenylethenes (TPE) are designed and prepared as "turn-on" luminescent sensors for lectins and glycosidases based on aggregation-induced emission. Through aggregation derived from carbohydrate-lectin binding, multivalent mannosy

Full text of DOI:10.1039/c0ob00680g

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PAPER
Sugar-bearing tetraphenylethylene: novel fluorescent probe for studies of
carbohydrate–protein interaction based on aggregation-induced emission†
Jin-Xiang Wang,a,b Qi Chen,a Ning Bian,a,c Fen Yang,a,b Jing Sun,b Ai-Di Qi,c Chao-Guo Yan*b and
Bao-Hang Han*a
Received 6th September 2010, Accepted 15th December 2010
DOI: 10.1039/c0ob00680g
Neutral sugar-bearing tetraphenylethenes (TPE) are designed and prepared as “turn-on” luminescent
sensors for lectins and glycosidases based on aggregation-induced emission. Through aggregation
derived from carbohydrate–lectin binding, multivalent mannosyl-bearing TPE shows a good selectivity
and sensitivity to Con A by switching on the fluorescence of water-soluble tetraphenylethylene-based
glyco-conjugates in aqueous solution. Meanwhile, cellobiosyl-bearing TPE can be used to investigate
enzymatic hydrolysis based on emission enhancing by glycosidase-induced aggregation.
(AIE) effect can greatly improve the fluorescence quantum yields
Introduction
of the molecules by up to three orders of magnitude, enhancing
Carbohydrate-mediated biological interactions play a crucial role
the photoluminescence (PL) intensity from faint luminophores
into strong emitters.5 Especially, since Tang’s group reported the
AIE feature of tetraphenylethylene (TPE)-based luminophors,6
TPE-based AIE active materials have already shown practical
applications in OLEDs,6 chem-sensors,5,7 and bio-probes,8 due to
their efficient preparation and facile functionalization.
in numerous biological processes such as cell growth, recognition
and differentiation, cancer metastasis, inflammation, and bacterial
and viral infection.1 As a model system of natural carbohydrates,
synthetic glyco-conjugates have demonstrated to be an important
well-defined tool for investigating carbohydrate-based biological
events. In particular, fluorescent glyco-conjugates which possess
both fluorescent dye and reporting carbohydrate ligands are
attractively used in carbohydrate–protein interaction studies and
biosensor applications because of their intrinsic optical properties,
high sensitivities to minor stimuli, and good biocompatibilities.2
Furthermore, fluorescence-based assays are convenient, cost-
effective, and easily scaled-up to a high-throughput screening
format.
However, when traditional dyes are dispersed in aqueous media
or interacted with biomacromolecules, aggregation-induced fluo-
rescence quenching often takes place, which results in drastically
negative effects on efficiencies and sensitivities of biosensors or
bioprobes.3 To overcome this problem, recently, molecules with
aggregation-induced emission characteristics have been developed
and drawn much attention, due to their enhanced emission in
aggregate form or solid-state.4 The aggregation-induced emission
Various functional groups were introduced to the
tetraphenylethylene scaffold for potential sensing purposes. For
example, TPE motif containing charged groups such as a sulfonate
group or an ammonium group was used in the establishment of a
fluorometric assay method for acetylcholinesterase or studies of
a label-free DNA assay system.8b,8c Glucosamine hydrochloride-
functionalized TPE developed by our group was used for
fluorometric detection of alkaline phosphatase.8e However, the
presence of ionic groups in TPE might cause some interfering
responses due to nonspecific electrostatic interactions in a
complicated biological environment, resulting in negative effects
on sensitivity and selectivity.9 Therefore, it is important to explore
neutral TPE-based bio-probes or bio-sensors with well-defined
structures to investigate biological interactions. Recently, neutral
sugar-substituted tetraphenylethenes were designed as “turn-on”
luminescent sensors for lectins and influenza virus.10 To extend
the potential applications of the TPE-based AIE active probes
in biomacromolecule detections, herein, neutral water-soluble
TPE-based artificial glycoclusters were designed and employed
for studies of carbohydrate–protein interaction. In addition, a
novel method to detect glycosidases based on the AIE effect
was also developed. Integration of sugar moieties with TPE can
not only improve water-solubility and bio-compatibility of the
dyes, but also provide neutral ligands to bind specific proteins.
As illustrated schematically in Scheme 1, carbohydrate-bearing
TPE nearly shows no luminescence in diluted aqueous solution
aNational Center for Nanoscience and Technology, Beijing 100190, China.
E-mail: hanbh@nanoctr.cn; Fax: +86-10-82545576
bCollege of Chemistry & Chemical Engineering, Yangzhou University,
Yangzhou 225002, China
cCollege of Traditional Chinese Medicine, Tianjin University of Traditional
Chinese Medicine, Tianjin 300193, China
† Electronic supplementary information (ESI) available: 1H NMR, 13C
NMR and MS spectra of the key compounds; fluorescence spectra of
CTPE-1 in the absence and presence of different proteins; fluorescence
spectra of CTPE-4 in the absence and presence of b-glucosidase. See DOI:
10.1039/c0ob00680g
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propargyl glycosides (10 or 11) through “click” ligation and
Zemple´n deacetylation to detect specific glycosidase based on
the AIE effect. All the synthetic glyco-conjugates are soluble in
aqueous solution. All the synthetic TPE-based glyco-conjugates
are soluble in aqueous solution and well characterized by 1H
NMR, 13C NMR, IR, and MS (in the ESI†).
Aggregation derived from carbohydrate–lectin binding
In order to study carbohydrate–protein interaction using the syn-
thetic water-soluble TPE-based glyco-conjugates, Concanavalin
A (Con A), a member of the lectin family, was chosen as a
target protein since it is a well-known a-mannose- or a-glucose-
binding protein, which exists predominantly as a tetramer of four
identical subunits and possesses four binding sites to interact with
four a-mannose or a-glucose units simultaneously.13 In dilute
aqueous solution, as expected, CTPE-1 nearly does not show any
luminescence. After treatment with Con A (30 mM), the aqueous
mixture is highly emissive, due to the formation of the CTPE-
1–Con A ensemble. From the molecular solution in water to the
aggregating state with Con A, the fluorescence intensity of CTPE-
1 at 469 nm increases by 11-fold (Fig. 1). The non-emissive nature
of the molecular state and emissive nature of the aggregates were
clearly indicated in the photographs given in the insets in Fig. 1. To
evaluate the influence of the glycoside cluster effect, CTPE-2 was
also treated with Con A (30 mM) under the same conditions. In
presence of Con A (30 mM), the fluorescence intensity of CTPE-2
at 469 nm only increased by 3-fold compared to that in the absence
of Con A, which means the TPE motif containing the glycocluster
residue possesses a higher affinity to lectin. Fluorescence spectra of
CTPE-1 [20 mM in PBS (10 mM) buffer solution, pH = 7.6] in the
presence of Con A at different concentrations were shown in Fig. 2,
indicating that the emission enhancement of CTPE-1 is observed
at a concentration of Con A as low as 1.0 mM. To investigate
the selectivity of fluorometric detection to Con A, CTPE-1 was
also treated by BSA protein under the same conditions and no
significant change in photoluminescence intensity was observed
(Fig. S1 in the ESI†).
Scheme 1 Illustration of studies of carbohydrate–protein interaction
using TPE-based artificial glyco-conjugates as fluorescent probes based
on the AIE feature.
when existing in the molecular state. However, aggregation
derived from specific carbohydrate–lectin binding or selective
glycosidase-induced
hydrolysis
can
“turn
on”
the
photoluminescence due to the TPE moiety with the AIE feature,
which can be used to study carbohydrate–protein interactions.
Based on the rationale aforementioned, a multivalent mannosyl-
bearing TPE was designed, which is expected to show high
photoluminescence in the presence of Con A through aggregation
derived from selective mannose–Con A binding. Meanwhile,
cellobiosyl-bearing TPE, as a substrate for b-glucosidase, can
be hydrolyzed by the enzyme in aqueous buffer solution and
lose glucose units to give a fluorescent suspension due to the
formation of a water-insoluble aggregate. Thus, it can be used to
investigate enzymatic hydrolysis based on emission enhancing by
glycosidase-induced aggregation.
Results and discussion
Design and synthesis of TPE-based glyco-cojugates
It’s well-known that the affinity and specificity of carbohydrate-
mediated biological interactions strongly depend on multiva-
lency due to the so-called glycoside cluster effect.11 Therefore,
TPE-based multivalent glyco-conjugate, CTPE-1, was synthe-
sized according to the synthetic route shown in Scheme 2.
Treatment of known TPE derivative 1 with excess NaN3 in
DMF gave azido-functionalized TPE 2 quantitatively. Cu(I)-
catalyzed “click” ligation12 between azido-attached manno-
side 4 derived from mannose derivative 3 and the known
tetrakis(2-propynyloxymethyl)methane 5 furnished propargyl-
bearing mannopyranoside cluster 6, which was reacted with
2 through cycloaddition reaction again to afford peracetylated
TPE-based multivalent glyco-conjugate 7. After global deacetyla-
tion under Zemple´n condition in NaOMe/MeOH, the desired
glycocluster CTPE-1 was obtained in an excellent yield. To
investigate the significance of multivalency, as a control study,
TPE derivative CTPE-2 containing two mannopyranosyl moieties
was also obtained according to a similar method from propargyl-
attached TPE 8. Glycosyl-bearing luminophores are often used to
assess glycosidase capability. Therefore, cellobiosyl- or lactosyl-
attached TPEs (CTPE-3 or CTPE-4) were synthesized from
Fig. 1 Fluorescence spectra of CTPE-1 and CTPE-2 [20 mM in PBS
(10 mM) buffer solution, pH = 7.6] in the absence and presence of Con A
(30 mM); the insets display the photos of the corresponding buffer solutions
of CTPE-1 (20 mM) in the absence (A) and presence (B) of Con A (30 mM)
under UV light (365 nm) illumination.
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Scheme 2 Synthetic route to TPE-based glyco-conjugates.
Aggregation derived from glycosidase-induced hydrolysis
the fluorescence intensity of the TPE moiety with AIE property
will significantly increase due to glycosidase-induced aggregation
and the purpose to detect the glycosidase can be achieved. As
shown in Fig. 3, CTPE-3 (20 mM) displays a weak fluorescence
in aqueous buffer solution. After being treated with b-glucosidase
(1.5 U), the resulting mixture became turbid (Fig. S2 in the ESI†),
which indicates the formation of the aggregate. Fluorescence
intensity of the resulting suspension at 460 nm increases by 9-fold
compared to CTPE-3 in absence of b-glucosidase. Photographs
of solutions of CTPE-3 and the resulting suspension in buffer
b-Glucosidase with specificity for a variety of b-D-glycoside sub-
strates catalyzes the hydrolysis of terminal non-reducing residues
in b-D-glucosides with release of glucose.14 b-Cellobiosyl-carrying
TPE (CTPE-3), as a certain substrate for b-glucosidase, can be
hydrolyzed by the enzyme in aqueous buffer solution and lose
glucose units one by one. Therefore, it can be expected that the
water-soluble CTPE-3 will be transformed gradually into water-
insoluble TPE derivatives after enzymatic hydrolysis. In that case,
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Conclusions
In summary, four TPE-based glyco-conjugates were prepared
through Cu(I)-catalyzed “click” ligation and used for studies
of carbohydrate–protein interaction and related biosensing. Sig-
nificant fluorescence change of water-soluble glyco-conjugates
can be observed based on aggregation derived from specific
carbohydrate–lectin binding or selective glycosidase-induced hy-
drolysis in aqueous solution, due to the TPE moiety with an AIE
feature. We believe the method inspired by this novel concept
to investigate carbohydrate-mediated biological interaction shows
promising applications in biomacromolecule detections and gly-
cobiology studies.
Experimental
Fig. 2 Fluorescence spectra of CTPE-1 [20 mM in PBS (10 mM) buffer
solution, pH = 7.6] in the presence of Con A at different concentrations:
Con A concentrations (mM) from the bottom upwards are 0, 5, 10, 15, 20,
25, and 30.
Materials and characterization
All chemical reagents were commercially available and used as
received unless otherwise stated. Ultra-pure water was purified
with a Millipore purification system (Milli-Q water). Con A
and b-glucosidase were purchased from Sigma-Aldrich Co. TPE
derivatives (1, 8)3c,8e and sugar derivatives (3, 10, 11)15 were
prepared according to reported methods.
The 1H and 13C NMR spectra were recorded on a Bruker
DMX400 NMR spectrometer. Mass spectra were recorded with
a Thermo LCQ Deca XP MAX mass spectrometer using the
ESI(+) technique. The optical rotations were measured with a
JASCO DIP-1000 digital polarimeter. Infrared (IR) spectra were
recorded in KBr pellets using a Spectrum One Fourier transform
IR spectrometer (Perkin Elmer Instruments Co. Ltd). Ultraviolet-
visible (UV-vis) spectra were measured using a Perkin-Elmer
Lambda 950 UV-vis-NIR spectrophotometer and quartz cells with
1 cm path length. The fluorescence spectra were measured in a
conventional cell with 1 cm path length using a Perkin-Elmer
LS55 spectrophotometer.
taken under illumination of a UV lamp are displayed in the
insets in Fig. 3. The enzymatic hydrolysis process can be further
confirmed by ESI-MS. Fig. 4 shows the mass spectrum of a CTPE-
3 sample digested by b-glucosidase for 1 h. The ion at m/z 1264.64
corresponds to [CTPE+H]+. The signal peaks at m/z 1102.82,
939.33, 775.85, and 614.54 indicate the glucose losses, which
clearly shows that the glucose residues in CTPE-3 are released one
by one. To investigate the selectivity of fluorometric detection to
b-glucosidase, lactosyl-carrying TPE (CTPE-4) was also treated
by b-glucosidase under the same conditions and no significant
change of photoluminescence intensity was observed (Fig. S3 in
the ESI†). The terminal non-reducing sugar residue in CTPE-4 is
b-galactose, which can not be easily hydrolyzed by b-glucosidase. It
can be inferred from these data that functionalization of TPE with
certain glycosyl residues provided a unique platform for selective
glycosidase detection.
Azido-functionalized TPE 2
To a mixture of compound 1 (90 mg, 0.16 mmol) and NH4Cl
(17 mg, 0.31 mmol) in DMF solution (5 mL) was added NaN3
(60 mg, 0.93 mmol). The resulting mixture was stirred at 80 ◦C for
12 h. The reaction mixture was then diluted with water (20 mL)
and was further extracted with ethyl acetate (2 ¥ 25 mL). The
combined organic phase was washed with water (2 ¥ 20 mL) and
dried over anhydrous Na2SO4. After evaporation at diminished
pressure, the desired product 2 was obtained as an off-yellow oil
(72 mg, 95%). 1H NMR (400 MHz, CDCl3): d 7.12–7.07 (m, 6H),
7.04–6.99 (m, 4H), 6.96–6.90 (m, 4H), 6.67–6.62 (m, 4H), 4.25–
4.19 (m, 4H), 3.62–3.57 (m, 4H). 13C NMR (100 MHz, CDCl3): d
156.6, 144.1, 139.9, 137.2, 132.7, 131.5, 127.8, 126.4, 113.9, 67.8,
58.6. ESI(+)-MS: calcd. for C30H26N6O2: 502.21 [M]; found 503.29
[M+H]+. IR (KBr): 2930, 2855, 2105, 1640, 1464, 1403, 1256, 800
cm-1.
Fig. 3 Fluorescence spectra of CTPE-3 [20 mM in citric acid–Na2HPO4
buffer solutions, pH = 5.8] in the absence and presence of b-glucosidase:
b-glucosidase from the bottom upwards are 0, 0.5, 1.0, 1.2, and 1.5 U; the
inset displays the photos of the corresponding buffer solutions of CTPE-3
(20 mM) in the absence (A) and presence (B) of b-glucosidase (1.5 U) under
UV light (365 nm) illumination.
Thio-mannopyranosyl derivative 4
A mixture of 3 (2.12 g, 5.82 mmol) and potassium carbonate
(930 mg, 6.73 mmol) in acetone–water (2 : 1, 24 mL) was stirred
under nitrogen. Then, 1,2-dibromoethane (6.3 mL, 72.7 mmol)
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Fig. 4 Mass spectrum of CTPE-3 solution digested by b-glucosidase for 1 h.
was added and the reaction was stirred at room temperature for
3 h. The mixture was extracted twice with CH2Cl2 (50 mL). The
combined organic layer was dried with anhydrous Na2SO4 and
concentrated to give a yellow oil. The residue was purified by a
silica gel chromatography using petroleum ether and ethyl acetate
(3 : 1) as eluent to give the bromide intermediate as a yellow oily
45 mL) were added ascorbate sodium (30 mg) and CuSO4 (20 mg)
as catalyst. The mixture was refluxed for 1.5 h. The reaction
mixture was cooled to room temperature and then extracted with
ethyl acetate (2 ¥ 30 mL). The organic layer was combined and
concentrated at diminished pressure. The residue was purified by
silica gel chromatography (ethyl acetate) to furnish 6 (166.6 mg,
30%). [a]2D5 +140 (c 0.5, CHCl3); 1H NMR (400 MHz, CDCl3): d
7.67 (s, 3H), 5.28–5.20 (m, 9H), 5.14 (dd, J = 2.8, 10.0 Hz, 3H),
4.60–4.57 (m, 7H), 4.55 (s, 6H), 4.29 (d, J = 8.4 Hz, 3H), 4.25 (d,
J = 6.4 Hz, 3H), 4.08 (t, J = 8.4 Hz, 6H), 4.03 (d, J = 6.0 Hz, 6H),
3.18–3.08 (m, 6H), 2.42 (s, 1H), 2.12 (s, 9H), 2.02 (s, 9H), 2.00 (s,
9H), 1.94 (s, 9H). 13C NMR (100 MHz, CDCl3): d 170.6, 169.9,
169.7, 167.7, 145.6, 123.0, 82.9, 82.0, 74.4, 71.8, 70.8, 69.5, 69.3,
69.1, 66.1, 65.9, 64.9, 62.4, 60.4, 58.7, 58.2, 49.5, 21.1, 20.9, 20.6,
19.2. ESI(+)-MS: calcd. for C65H89N9O31S3: 1588.64 [M]; found
1611.70 [M+Na]+. IR (KBr): 2928, 2855, 2100, 1750, 1464, 1403,
1240, 1080, 650 cm-1.
1
liquid in 68% yield (1.86 g). [a]2D5 +65 (c 1, CHCl3); H NMR
(400 MHz, CDCl3): d 5.19 (s, 1H), 5.16 (s, 1H), 5.09 (t, J = 6.8 Hz,
1H), 5.03 (dd, J = 2.0, 6.4 Hz, 1H), 4.23 (t, J = 4.8 Hz, 1H), 4.11 (dd,
J = 4.4, 8.0 Hz, 1H), 3.95 (d, J = 3.6 Hz, 1H), 3.50–3.46 (m, 1H),
3.39–3.34 (m, 1H), 2.99–2.78 (m, 2H), 1.99 (s, 3H), 1.94 (s, 3H),
1.90 (s, 3H), 1.82 (s, 3H). To a mixture of the bromide intermediate
(470 mg, 1.00 mmol) and ammonium chloride (55 mg, 1.0 mmol)
in dried DMF was added sodium azide (200 mg, 3.0 mmol). The
resulting mixture was stirred at 80 ◦C for 7 h. The reaction mixture
was poured into water (20 mL) and extracted twice with ethyl
acetate (50 mL). The combined organic layer was washed once
by water (20 mL). After separation, the organic layer was dried
over Na2SO4 and concentrated to give a yellow oil. The residue
was purified by a silica gel chromatography using petroleum ether
and ethyl acetate (3 : 1) as eluent to give thio-mannopyranosyl
derivative 4 as a yellow oily liquid (424.76 mg, 98%). [a]2D5 +56 (c
1, CHCl3);1H NMR (400 MHz, CDCl3): d 5.23–5.20 (m, 2H), 5.17
(t, J = 10.0 Hz, 1H), 5.09 (dd, J = 2.8, 10.0 Hz, 1H), 4.28–4.23 (m,
1H), 4.17 (dd, J = 6.0, 12.4 Hz, 1H), 3.98 (dd, J = 2.0, 12.0 Hz, 1H),
3.49–3.36 (m, 2H), 2.81–2.74 (m, 1H), 2.72–2.65 (m, 1H), 2.04 (s,
3H), 1.97, (s, 3H), 1.93 (s, 3H), 1.86 (s, 3H). 13C NMR (100 MHz,
CDCl3): d 170.2, 169.6, 169.5, 169.4, 82.7, 70.5, 69.0, 65.9, 62.2,
50.6, 30.5, 20.6, 20.4, 20.3. ESI(+)-MS: calcd. for C16H23N3O9S:
433.12 [M]; found 456.08 [M+Na]+. IR (KBr): 2935, 2860, 2100,
1750, 1450, 1250, 1080, 610 cm-1.
Peracetylated TPE-based mannopyranosyl cluster 7
Glyco-cluster 6 (88.9 mg, 55.95 mmol) and compounds 2 (15.3 mg,
30.4 mmol) in THF–water (2 : 1, 15 mL) was added a freshly
prepared aqueous ascorbate sodium (30 mg) and CuSO4 (20 mg).
The mixture was stirred at 80 ◦C for 9 h. The resulting mixture was
extracted twice with ethyl acetate (50 mL). The combined organic
layer was dried over Na2SO4 and concentrated to give a yellow oil.
Silica gel chromatography (ethyl acetate : methanol = 20 : 1) gives
compound 7 in a yield of 52% (53.2 mg). [a]2D5 +185 (c 0.2, CHCl3);
1H NMR (400 MHz, CDCl3): d 7.71 (br, 8H), 7.09–7.04 (m, 6H),
6.99–6.94 (m, 4H), 6.92–6.87 (m, 4H), 6.62–6.57 (m, 4H), 5.31 (s,
10H), 5.28 (d, J = 9.6 Hz, 6H), 5.18 (dd, J = 2.4, 10.0 Hz, 6H), 4.71
(br, 6H), 4.62–4.58 (m, 12H), 4.55 (br, 18H), 4.35–4.26 (m, 18H),
4.13–4.06 (m, 18H), 3.22–3.10 (m, 12H), 2.14 (s, 18H), 2.05 (s,
18H), 2.04 (s, 18H), 1.97 (s, 18H). 13C NMR (100 MHz, CDCl3): d
170.6, 169.9, 169.8, 167.8, 156.4, 145.6, 145.2, 139.8, 137.3, 132.7,
Mannosyl cluster 6
To the mixture of thio-mannopyranosyl derivative 4 (454.5 mg,
1.05 mol) and 5 (145.3 mg, 0.50 mol) in THF–water (2 : 1,
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132.5, 131.4, 131.0, 128.9, 127.8, 124.0, 123.1, 113.9, 113.7, 82.9,
71.9, 70.8, 69.5, 69.4, 69.2, 66.2, 65.0, 62.5, 58.5, 49.8, 45.3, 21.0,
20.8, 20.7, 19.3. ESI(+)-MS: calcd. for C160H204N24O64S6: 3679.18
[M]; found 3702.31 [M+Na]+. IR (KBr): 2930, 2850, 1640, 1465,
1403, 1250, 1080, 650 cm-1.
4H), 6.83 (d, J = 8.8 Hz, 4H), 5.25 (s, 2H), 5.06 (s, 4H), 5.04 (d,
J = 9.3 Hz, 2H), 4.87 (br, 2H), 4.77(br, 2H), 4.70–4.63 (m, 4H),
4.61–3.56 (m, 2H), 3.72 (d, J = 11.6 Hz, 2H), 3.69 (s, 2H), 3.65 (d,
J = 6.8 Hz, 2H), 3.47 (dd, J = 7.2, 11.6 Hz, 2H), 3.41–3.37 (m, 4H),
3.09 (t, J = 6.8 Hz, 4H). 13C NMR (100 MHz, d6-DMSO): d 156.6,
143.7, 142.4, 139.3, 136.0, 132.0, 130.8, 128.7, 127.8, 124.8, 114.0,
85.4, 74.8, 71.7, 71.5, 67.3, 61.2, 56.0, 52.6, 49.2. ESI(+)-MS: calcd.
for C48H54N6O12S2: 971.11 [M]; found 994.75 [M+Na]+. IR (KBr):
3400, 2934, 2843, 1640, 1464, 1400, 1075, 810 cm-1. Elem. anal.
calcd: C, 59.37; H, 5.60; found: C, 59.30; H, 5.64.
CTPE-1
To a solution of 7 (36 mg, 9.78 mmol) in methanol (5 mL) was
added a 1 M solution of NaOMe in methanol until the pH value
reached 11. The reaction was stirred for 24 h at room temperature,
and then, the resulting mixture was neutralized by acidic resin.
After filtration, the filtrate was concentrated to furnish CTPE-1
as a white solid (24.56 mg, 95%). [a]2D5 +163 (c 0.2, H2O); 1H-NMR
(400 MHz, d6-DMSO): d 8.21 (br, 8H), 7.42–7.38 (m, 6H), 7.25–
7.18 (m, 8H), 6.72–6.69 (m, 4H), 5.78 (s, 16H), 5.22 (s, 6H), 5.02
(s, 16H), 4.70–4.60 (m, 12H), 4.59–4.49 (m, 12H), 3.78–3.60 (m,
20H), 3.48–3.40 (m, 10H), 3.39–3.37 (m, 14H), 3.34–3.30 (m, 4H),
3.20–3.07 (m, 20H). 13C-NMR (100 MHz, d6-DMSO): d 156.3,
145.8, 145.1, 139.7, 137.5, 132.6, 132.4, 131.5, 131.0, 128.8, 127.8,
124.1, 123.1, 113.8, 113.6, 82.9, 71.6, 70.8, 69.7, 69.5, 69.1, 62.5,
60.9, 59.1, 53.5, 49.8, 45.2. ESI(+)-MS: calcd. for C112H156N24O40S6:
2670.96 [M]; found 2694.79 [M+Na]+. IR (KBr): 3400, 2928, 2857,
1634, 1464, 1403, 1085, 799 cm-1. Elem. anal. calcd: C, 50.36; H,
5.89; found: C, 50.42; H, 5.92.
TPE-based peracetylated b-cellobioside 12
To a solution of peracetylated b-cellobiose (0.68 g, 1.0 mmol) in
5 mL of anhydrous dichloromethane were added boron trifluoride
diethyl etherate (0.85 g, 3.0 mmol) and propargyl alcohol (0.07 g,
1.2 mmol). The mixture was stirred for 2 h at room temperature,
and then diluted with dichloromethane (30 mL) and the organic
phase was washed with brine (60 mL) and dried over anhydrous
Na2SO4. The solvent was removed to give peracetylated propargyl
b-cellobioside 10 as a white powder (0.54 g, 80%). The crude
product was used without further purification. To a mixture of
2 (53 mg, 0.11 mmol) and 10 (178 mg, 0.26 mmol) in H2O–
THF (1 : 1, 10 mL) were added freshly prepared aqueous 1.0 M
sodium ascorbate (150 mL, 0.15 mmol) and CuSO4 (12 mg,
0.075 mmol). The heterogeneous mixture was stirred vigorously
in a dark room at 50–60 ◦C until the complete consumption of the
reactants based on TLC analyses. After removal of THF under
reduced pressure, water (20 mL) was added and the product was
extracted with EtOAc (3 ¥ 25 mL). The combined organic layer
was dried over anhydrous Na2SO4 and evaporated in vacuo. The
crude product was subjected to column chromatography (ethyl
acetate : petroleum ether = 5 : 1) to give 12 as a foamy solid (162 mg,
Peracetylated TPE-based mannopyranosyl derivative 9
To the mixture of 8 (95 mg, 0.21 mol) and mannosyl derivative
4 (202 mg, 0.46 mol) in THF–water (2 : 1, 10 mL) were added
ascorbate sodium (15 mg) and CuSO4 (10 mg) as catalyst. After
refluxing for 8 h, the reaction mixture was cooled to room
temperature and then extracted with ethyl acetate (2 ¥ 35 mL).
The combined organic layer was concentrated and then purified
by silica gel chromatography (petroleum ether : ethyl acetate = 1 : 2)
to yield 9 (270.8 mg, 96%) as a foamy solid. [a]2D5 +110 (c 1, CHCl3);
1H NMR (400 MHz, CDCl3): d 7.68 (s, 2H), 7.08–6.97 (m, 10H),
6.91 (d, J = 8.4 Hz, 4H), 6.70 (d, J = 8.8 Hz, 4H), 5.29 (d, J =
3.2 Hz, 2H), 5.24 (s, 2H), 5.17 (dd, J = 3.6, 10.0 Hz, 2H), 5.10 (d, J =
11.6 Hz, 4H), 4.65–4.53 (m, 4H), 4.32–4.26 (m, 4H), 4.12–4.06 (m,
4H), 3.21–3.07 (m, 4H), 2.14 (s, 6H), 2.03 (s, 6H), 2.01 (s, 6H), 1.96
(s, 4H). 13C NMR (100 MHz, CDCl3): d 170.5, 170.0, 169.8, 169.7,
156.7, 144.0, 139.8, 137.0, 132.6, 131.4, 127.7, 127.6, 126.3, 113.9,
82.9, 70.7, 69.5, 69.2, 66.1, 62.5, 61.8, 60.4, 49.7, 20.9, 20.7, 20.6,
18.5. ESI(+)-MS: calcd. for C64H70N6O20S2: 1307.40 [M]; found
1308.55 [M+H]+. IR (KBr): 2935, 2850, 1643, 1465, 1400, 1240,
1080, 660 cm-1.
1
80%). [a]2D5 -31 (c 0.5, CHCl3); H NMR (400 MHz, CDCl3): d
7.69 (s, 2H), 7.07–7.03 (m, 6H), 6.96–6.93 (m, 4H), 6.90 (d, J =
8.4 Hz, 4H), 6.59 (d, J = 8.8 Hz, 4H), 5.17–5.10 (m, 4H), 5.05 (t,
J = 8.8 Hz, 2H), 4.92–4.87 (m, 6H), 4.77 (d, J = 12.8 Hz, 2H), 4.71
(t, J = 10.0 Hz, 4H), 4.62 (d, J = 8.0 Hz, 2H), 4.54–4.50 (m, 4H),
4.35 (dd, J = 4.4, 8.4 Hz, 2H), 4.27 (t, J = 6.0 Hz, 4H), 4.13–4.07
(m, 2H), 4.02 (dd, J = 2.0, 10.4 Hz, 2H), 3.78 (t, J = 19.2 Hz, 2H),
3.70–3.60 (m, 4H), 2.10 (s, 6H), 2.06 (s, 6H), 2.01 (s, 6H), 1.99 (s,
12H), 1.96 (s, 6H), 1.89 (s, 6H). 13C NMR (100 MHz, CDCl3): d
170.6, 170.4, 170.3, 169.8, 169.7, 169.4, 169.1, 156.2, 144.2, 143.8,
137.4, 132.7, 131.3, 127.7, 126.4, 124.0, 113.8, 100.8, 99.7, 76.4,
73.0, 72.9, 72.5, 72.0, 71.6, 71.5, 67.8, 66.2, 62.9, 61.8, 61.6, 58.4,
49.9, 20.9, 20.7, 20.6, 18.5. ESI(+)-MS: calcd. for C88H102N6O38:
1850.62 [M]; found 1873.50 [M+Na]+. IR (KBr): 2925, 2850, 1644,
1465, 1408, 1250, 1080, 660 cm-1.
CTPE-2
To a solution of compound 9 (100 mg, 76 mmol) in methanol
(15 mL), 1 M sodium methoxide in methanol was added drop-
wise until pH = 11. The reaction mixture was stirred at room
temperature overnight. After neutralization with Amberlite IR120
acidic resin and filtration, the solvent was removed under reduced
pressure to give the desired product CTPE-2 (65 mg, 90%). [a]D25
TPE-based peracetylated b-lactoside 13
TPE-based peracetylated b-lactoside 13 was obtained as a
foamy solid (158 mg, 78%) from peracetylated b-lactose (0.68
g, 1.0 mmol) according to the same synthetic method for 12.
1
[a]2D5 -52 (c 0.5, CHCl3); H NMR (400 MHz, CDCl3): d 7.67
1
+105 (c 0.5, DMF); H NMR (400 MHz, d6-DMSO): d 8.25 (s,
2H), 7.18–7.05 (m, 6H), 6.99–6.94 (m, 4H), 6.90 (d, J = 8.8 Hz,
(s, 2H), 7.10–7.02 (m 6H), 7.00–6.97 (m, 4H), 6.89 (d, J = 8.8 Hz,
4H), 6.57 (d, J = 8.8 Hz, 4H), 5.33 (d, J = 2.8 Hz, 2H), 5.18 (t,
2224 | Org. Biomol. Chem., 2011, 9, 2219–2226
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J = 18.8 Hz, 4H), 5.09 (dd, J = 8.0, 10.4 Hz, 2H), 4.94 (dd, J =
3.2, 10.4 Hz, 2H), 4.88 (d, J = 10.8 Hz, 4H), 4.77 (d, J = 12.8 Hz,
2H), 4.69 (t, J = 5.6 Hz, 4H), 4.62 (d, J = 8.0 Hz, 2H), 4.47 (d,
J = 8.0 Hz, 2H), 4.25 (t, J = 4.4 Hz, 4H), 4.11–4.06 (m, 8H), 3.86
(t, J = 13.6 Hz, 2H), 3.79 (t, J = 18.8 Hz, 2H), 3.64–3.60 (m, 2H),
2.14 (s, 6H), 2.10 (s, 6H), 2.03 (bs, 18H), 1.95 (s, 6H), 1.89 (s, 6H).
13C NMR (100 MHz, CDCl3): d 170.5, 170.3, 170.2, 169.8, 169.7,
169.2, 156.2, 144.2, 143.9, 139.7, 137.4, 132.7, 131.3, 131.0, 128.9,
127.8, 126.5, 124.0, 113.7, 101.1, 99.7, 76.2, 72.9, 72.8, 71.9, 71.6,
71.5, 71.1, 69.1, 66.7, 66.2, 62.9, 61.9, 60.8, 58.4, 49.9, 21.0, 20.9,
20.7, 20.6, 19.2, 18.5. ESI(+)-MS: calcd. for C88H102N6O38: 1850.62
[M]; found 1873.71 [M+Na]+. IR (KBr): 2926, 2850, 1645, 1463,
1405, 1250, 1080, 660 cm-1.
Aliquots of lectin in the same buffer were added to the solution.
The final concentration of CTPE-1 is 20 mM. After each addition,
the sample was allowed to equilibrate for 2 h prior to recording a
spectrum. Additions of lectin were continued until no significant
change in the fluorescence signal was observed. The excitation
wavelength was 360 nm and the emission scan ranged from 330 nm
to 650 nm.
Studies of glycosidase-induced hydrolysis based on fluorescence
spectrum
A solution of CTPE-3 was prepared in citric acid–Na2HPO4 buffer
solutions (pH = 5.8). Aliquots of b-glucosidase in the same buffer
were added to the solution. The final concentration of CTPE-3 is
20 mM. After each addition, the sample was allowed to equilibrate
for 3 h prior to recording a spectrum. The excitation wavelength
was 360 nm and the emission scan ranged from 330 nm to 650 nm.
CTPE-3
To a solution of TPE-based peracetylated b-cellobioside 12
(100 mg, 0.054 mmol) in dry CH2Cl2 (5 mL) and MeOH (5 mL)
was added 1.0 M CH3ONa/CH3OH solution dropwise. The pH
was adjusted to 11 and stirred at room temperature for 4 h, and
then, the resulting mixture was neutralized to 7.0 using acidic
resin. After filtration, the filtrate was collected and concentrated
under reduced pressure to give CTPE-3 as a foamy solid (65 mg,
Acknowledgements
This work is supported by the Ministry of Science and Tech-
nology of China (National Basic Research Program, Grant
2007CB808000) and the National Science Foundation of China
(Grants 20972035 and 21002017).
1
95%). [a]2D5 -25 (c 0.2, DMF); H NMR (400 MHz, d6-DMSO):
d 8.19 (s, 2H), 7.11–7.05 (m, 6H), 6.94–6.90 (m, 4H), 6.85–6.80
(m, 4H), 6.72–6.66 (m, 4H), 5.71 (br, 14H), 4.85 (d, J = 12.0 Hz,
2H), 4.71 (br, 4H), 4.63 (d, J = 12.0 Hz, 2H), 4.34–4.26 (m, 8H),
3.79 (d, J = 11.2 Hz, 2H), 3.69–3.62 (m, 4H), 3.45–3.38 (m, 4H),
3.31 (s, 4H), 3.21–3.15 (m, 4H), 3.08–2.97 (m, 6H). 13C NMR
(100 MHz, d6-DMSO): d 156.3, 143.7, 139.4, 136.3, 132.0, 130.8,
127.9, 126.4, 124.9, 114.0, 113.9, 103.3, 101.9, 80.6, 76.9, 76.5,
75.1, 73.4, 73.2, 70.1, 66.0, 61.6, 61.0, 60.5, 56.1, 49.2. ESI(+)-MS:
calcd. for C60H74N6O24: 1263.26 [M]; found 1264.95 [M+H]+. IR
(KBr): 3400, 2930, 2845, 1640, 1463, 1403, 1075, 790 cm-1. Elem.
anal. calcd: C, 57.05; H, 5.90; found: C, 57.10; H, 5.93.
Notes and references
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CTPE-4 was obtained as a foamy solid (44 mg, 65%) from TPE-
based peracetylated b-lactoside 13 (0.68 g, 1.0 mmol) according to
the same synthetic method for CTPE-3. [a]2D5 -46 (c 0.2, DMF); 1H
NMR (400 MHz, d6-DMSO): d 8.18 (s, 2H), 7.13–7.07 (m, 6H),
6.95–6.90 (m, 4H), 6.86–6.80 (m, 4H), 6.70–6.66 (m, 4H), 4.85 (d,
J = 12.0 Hz, 2H), 4.70 (br, 18H), 4.62 (d, J = 12.0 Hz, 2H), 4.34–
4.21 (m, 8H), 3.81 (bs, 4H), 3.64 (s, 4H), 3.49–3.41 (m, 8H), 3.32
(s, 8H), 3.04 (br, 2H). 13C NMR (100 MHz, d6-DMSO): d 156.2,
143.8, 143.7, 139.3, 136.2, 132.0, 131.7, 130.8, 128.8, 127.9, 126.5,
124.8, 113.9, 113.8, 103.9, 101.8, 80.6, 75.6, 75.0, 74.9, 73.3, 73.1,
70.7, 68.0, 66.0, 61.7, 60.5, 60.3, 56.1, 49.1. ESI(+)-MS: calcd.
for C60H74N6O24: 1263.26 [M]; found 1264.91 [M+H]+. IR (KBr):
3400, 2930, 2843, 1640, 1465, 1406, 1070, 790 cm-1. Elem. anal.
calcd: C, 57.05; H, 5.90; found: C, 57.09; H, 5.88.
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Studies of carbohydrate–lectin interaction based on
spectrofluorometric titration
A solution of CTPE-1 was prepared in PBS (10 mM) buffer
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