Glucosamine hydrochloride functionalized tetraphenylethylene: A novel fluorescent probe for alkaline phosphatase based on the aggregation-induced emission

Article abstract of DOI:10.1039/c002894k

Grafting of glucosamine hydrochloride moieties to tetraphenylethylene (TPE) motif furnished a novel cationic water-soluble tetraphenylethylene derivative (GH-TPE). With aggregation-induced emission properties, GH-TPE was used for fluorometric detection to alkaline phosphatase through enzyme-triggered de-aggregation of the ensemble of GH-TPE and substrate.

Full text of DOI:10.1039/c002894k

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Glucosamine hydrochloride functionalized tetraphenylethylene: A novel
fluorescent probe for alkaline phosphatase based on the
aggregation-induced emissionw
Qi Chen,a Ning Bian,ab Chun Cao,a Xi-Long Qiu,ab Ai-Di Qib and Bao-Hang Han*a
Received 11th February 2010, Accepted 10th April 2010
First published as an Advance Article on the web 10th May 2010
DOI: 10.1039/c002894k
Grafting of glucosamine hydrochloride moieties to tetraphenyl-
ethylene (TPE) motif furnished a novel cationic water-soluble
tetraphenylethylene derivative (GH-TPE). With aggregation-
induced emission properties, GH-TPE was used for fluorometric
detection to alkaline phosphatase through enzyme-triggered
de-aggregation of the ensemble of GH-TPE and substrate.
sulfonate group or ammonium group was used in the
establishment of a fluorometric assay method for acetyl-
cholinesterase13c or studies of label-free DNA assay systems.13b
To extend the potential applications of the TPE-based AIE
active materials in biomacromolecule detection, we designed a
glucosamine hydrochloride functionalized TPE, considering
introduction of glucosamine hydrochloride moieties into a
fluorescent dye molecule can not only improve its water-
solubility and bio-compatibility, but also provide many positive-
charged ammonium binding sites for the electrostatic interaction
between the dyes and other artificial or natural polyanions.14
The design rationale for the ALP assay is schematically
illustrated in Scheme 1. GH-TPE, possessing two units of
glucosamine hydrochloride, is expected to display a weak
photoluminescence (PL) in aqueous media. According to
previous studies,13c monododecylphosphate, as an amphiphilic
compound, can form a heteroaggregation complex with
positively charged GH-TPEs based on the electrostatic binding
when the concentration of monododecylphosphate is much
lower than its critical micelle concentration.15 As a result, the
fluorescence of the ensemble would increase significantly,
which is induced by the aggregation of GH-TPE. When
treated with ALP, the aggregated complex would disassemble
after loss of phosphate groups, resulting in a significant
emission reduction of the system. Thus, the purpose to use
GH-TPE as a fluorescent probe for alkaline phosphatase
detection would be achieved.
In clinical practice, enzymatic reactions are usually used in the
diagnosis of disease. Alkaline phosphatase (ALP, EC 3.1.3.1),
as one of the most commonly assayed enzymes, is capable
of catalyzing hydrolysis of a wide variety of phosphate
compounds and has broad substrate specificity in vitro.1 Several
diseases, such as bone disease,2 liver dysfunction,3 breast and
prostatic cancer,4 and diabetes,5 can be preliminarily diagnosed
in light of an abnormal level of alkaline phosphatase in
serum.6
Among various methods for alkaline phosphatase assays,
fluorescence-based assays have drawn much attention, because
they are high sensitive, convenient, cost-effective, and easy to
scale-up to a high-throughput screening format.7 However,
aggregation-caused fluorescence quenching of traditional dyes
often takes place when dispersed in aqueous media or inter-
acted with biomacromolecules, resulting in drastically negative
effects on efficiencies and sensitivities of biosensors or
bioprobes.8 To overcome this problem, in this work, a novel
cationic water-soluble tetraphenylethylene (TPE) derivative,
GH-TPE, was prepared as a fluorescent probe for alkaline
phosphatase assay based on the aggregation-induced emission
effect.
The synthetic route to GH-TPE is shown in Scheme 2.
13a
Propargylation of the known TPE derivative 1
afforded
Recently, molecules with aggregation-induced emission
(AIE) characteristics provide a unique platform for exploiting
9
novel optical materials and sensors,10 due to their enhanced
emission in aggregate form or solid state.11 Especially, since
Tang’s group reported the facile preparation of TPE-based
luminophors by a one-step reaction in a one-pot procedure,9a
TPE-based AIE active materials have already shown practical
applications in OLEDs,9a chem-sensors,12 and bio-probes.8b,13
For example, TPE motif containing charged groups such as a
a National Center for Nanoscience and Technology, Beijing 100190,
China. E-mail: hanbh@nanoctr.cn; Fax: +86-10-82545576
b College of Traditional Chinese Medicine, Tianjin University of
Traditional Chinese Medicine, Tianjin 300193, China
w Electronic supplementary information (ESI) available: Preparation
and characterization details for compounds 2, 5, 6, Glu-TPE, and
GH-TPE; absorption and emission spectra of the aggregate suspensions
and complexes containing GH-TPE; 1H and 13C NMR spectra of
compounds 2, 5, 6, Glu-TPE, and GH-TPE. See DOI: 10.1039/c002894k
Scheme 1 Illustration of fluorometric assay for alkaline phosphatase
using GH-TPE as a fluorescent probe based on AIE feature.
ꢀc
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 4067–4069 | 4067

View Article Online
structure and amphiphilic property for SDS and mono-
dodecylphosphate. In dilute aqueous solution, GH-TPE is
non-luminescent to the aforementioned. After treatment with
SDS, the aqueous mixture is highly emmissive, due to formation
of the GH-TPE/SDS ensemble. From the free state in aqueous
solution to the aggregating state with SDS, the fluorescence
intensity of GH-TPE at 469 nm increased by 9 fold (Fig. S3
in the ESIw). The non-emissive nature of free state and
emissive nature of the aggregates were clearly indicated in the
photographs given in Fig. S3 (ESIw), respectively.
Heteroaggregate complexation of the water-soluble
GH-TPE with monododecylphosphate was studied by spectro-
metric titrations in aqueous phosphate buffer solution (PBS,
10 mM, pH = 8.0) at 25 1C. With addition of the mono-
dodecylphosphate [the concentration of monododecylphosphate
(0–30 mM) is much lower than its critical micelle concentration,
nearly 2.0 mM], the fluorescence intensity of GH-TPE gradually
increased by 11 fold (Fig. 1). Meanwhile, a red shift as large as
90 nm (from 377 nm to 467 nm) of its emission maximum was
observed. The absorption band around 312 nm also red-
shifted by 16 nm in the presence of monododecylphosphate
(30 mM) as shown in Fig. S4 (ESIw). These results are in
agreement with the formation of the heteroaggregate complex.
Photographs of GH-TPE and the ensemble GH-TPE/
monododecylphosphate in PBS taken under illumination of
a UV lamp are displayed in Fig. 1. To prove the NH3+ group
of glucosamine is critical for the fluorescence change caused by
the electrostatic interaction, Glu-TPE was also treated identically
with monododecylphosphate (30 mM) and no significant
fluorescence change was observed (Fig. S5 in the ESIw).
As aforementioned, ALP is able to catalyze the hydrolysis of
monododecylphosphate into dodecyl alcohol and phosphic
acid. Therefore, it is expected that the fluorescence of the
ensemble of GH-TPE/monododecylphosphate would reduce
after addition of ALP, because the interaction between
GH-TPE and monododecylphosphate was weakened after loss
of phosphate group, leading to disassembly of GH-TPE/
monododecylphosphate heteroaggregate complexes and fluore-
scence reduction. Fig. 2 shows the fluorescence spectra of the
ensemble of GH-TPE [20 mM in PBS (10 mM), pH = 8.0] and
Scheme 2 Synthetic route to GH-TPE and Glu-TPE.
propargyl-attached TPE 2 under a basic condition. Cu(I)-
catalyzed ‘‘click’’ ligation16 between 2 and azido-functionalized
glucosamine derivative 314 furnished a sugar-bearing TPE 5
smoothly in 96% yield. Formation of the triazole ring is
confirmed by the chemical shift at 7.93 ppm (single peak) on
the 1H NMR spectrum and two peaks at 122.2 and 144.3 ppm
on the 13C NMR spectrum (Fig. S1 in the ESIw). After
removing all the acetyl groups with MeONa/MeOH and
deprotecting the N-Boc groups in aqueous HCl-THF solution
(4 M), the desired water-soluble GH-TPE was obtained in an
excellent yield (90% in two steps) by a one-pot procedure.
Similarly, a glucopyranosyl-bearing TPE (Glu-TPE) was also
prepared for the control studies. Compared with the 1H NMR
spectrum of protected precursor 5, we can find that the peak
intensities of Boc (d = 1.24 ppm), Ac (d = 2.05 ppm), and
NH-Boc (dNÀH = 5.98 ppm) groups in 5 disappear after final
+
efficient global deprotection and the peak of NH3 group
(d = 8.61 ppm) in GH-TPE appears clearly (Fig. S1 in the
ESIw), which confirms the successful preparation of the hydro-
chloride salt.
The protected dye 5 is soluble in common organic solvents
such as acetonitrile, chloroform, and THF, but insoluble in
water. GH-TPE, on the other hand, is soluble in polar solvents
including water, DMF, and DMSO. In dilute aqueous solution,
GH-TPE, as expected, does show a very limited luminescence.
However, when methanol, acetonitrile, THF, or dioxane is
added to its aqueous solution, the emission of GH-TPE in the
mixture still remains as faint as it is in the aqueous solution,
which means these organic solvents can not induce aggregation
of the salt. Similar phenomenon was observed for studies of
TPE derivatives containing other ammonium groups by
Tang’s group and was ascribed possibly to the amphiphilic
nature of those TPE derivatives.13b Further investigation
demonstrated that GH-TPE in the DCM/DMSO mixture with
high DCM fractions was highly emissive, implying that GH-TPE
is AIE-active (Fig. S2 in the ESIw).
Fig. 1 Fluorescence spectra of GH-TPE [20 mM in PBS (10 mM)
buffer solution, pH = 8.0] in the presence of different amounts of
monododecylphosphate (from 0 to 30 mM); the inset displays the
photos of the corresponding buffer solutions of GH-TPE (20 mM) in
the absence (A) and presence (B) of monododecylphosphate (30 mM)
under UV light (365 nm) illumination.
With GH-TPE in hand, the investigation into electrostatic
interactions between sodium dodecyl sulfonate (SDS) and
TPE salt was first performed as model studies for GH-TPE/
monododecylphosphate interactions, due to the similar chemical
ꢀc
This journal is The Royal Society of Chemistry 2010
4068 | Chem. Commun., 2010, 46, 4067–4069

View Article Online
Grant 2007CB808000) and the National Science Foundation
of China (Grant 20972035).
Notes and references
1 H. N. Fernley, Enzyme, 1971, 4, 417.
2 D. Goltzman and D. Miao, Encyclopedia Endocr. Dis., 2004, 1, 164.
3 K. Ooi, K. Shiraki, Y. Morishita and T. Nobori, J. Clin. Lab.
Anal., 2007, 21, 133.
4 G. Ramaswamy, V. R. Rao, L. Krishnamoorthy, G. Ramesh,
R. Gomathy and D. Renukadevi, Indian J. Clin. Biochem., 2000,
15, 110.
5 G. M. M. Rao and L. O. Morghom, Enzyme, 1986, 35, 57.
6 Y. Liu and K. S. Schanze, Anal. Chem., 2008, 80, 8605.
7 H. Sahoo, A. Hennig, M. Florea, D. Roth, T. Enderle and
W. M. Nau, J. Am. Chem. Soc., 2007, 129, 15927.
8 (a) J. B. Birks, Photophysics of Aromatic Molecules, Wiley,
London, 1970; (b) H. Tong, Y. N. Hong, Y. Q. Dong,
M. Haussler, J. W. Y. Lam, Z. Li, Z. F. Guo, Z. H. Guo and
B. Z. Tang, Chem. Commun., 2006, 3705.
Fig. 2 Fluorescence spectra of the ensemble of GH-TPE [20 mM in
PBS (10 mM), pH = 8.0] and monododecylphosphate (30 mM) in the
presence of ALP (1.5 U/mL) incubated at 25 1C for different periods;
the inset displays the photos of the corresponding solutions of
GH-TPE and monododecylphosphate in the absence (A) and presence
(B) of ALP (1.5 U/mL) after 30 min of incubation at 25 1C under UV
light (365 nm) illumination.
9 (a) Y. Dong, J. W. Y. Lam, A. Qin, J. Liu, Z. Li, B. Z. Tang, J. Sun
and H. S. Kwok, Appl. Phys. Lett., 2007, 91, 11111;
(b) K. H. Cheng, Y. Zhong, B. Y. Xie, Y. Q. Dong, Y. Hong,
J. Z. Sun, B. Z. Tang and K. S. Wong, J. Phys. Chem. C, 2008, 112,
17507; (c) S.-J. Lim, B.-K. An and S. Y. Park, Macromolecules,
2005, 38, 6236; (d) Y. Liu, X. Tao, F. Wang, X. Dang, D. Zou,
Y. Ren and M. Jiang, J. Phys. Chem. C, 2008, 112, 3975.
10 (a) T. Sanji, K. Shiraishi and M. Tanaka, ACS Appl. Mater.
Interfaces, 2009, 1, 270; (b) M. C. Zhao, M. Wang, H. J. Liu,
D. S. Liu, G. X. Zhang, D. Q. Zhang and D. B. Zhu, Langmuir,
2009, 25, 676; (c) M. Wang, D. Q. Zhang, G. X. Zhang,
Y. L. Tang, S. Wang and D. B. Zhu, Anal. Chem., 2008, 80,
6443; (d) M. Wang, D. Q. Zhang, G. X. Zhang and D. B. Zhu,
Chem. Commun., 2008, 4469.
11 (a) J. Luo, Z. Xie, J. W. Y. Lam, L. Cheng, H. Chen, C. Qiu,
H. S. Kwok, X. Zhan, Y. Liu, D. Zhu and B. Z. Tang, Chem.
Commun., 2001, 1740; (b) J. Chen, C. C. W. Law, J. W. Y. Lam,
Y. Dong, S. M. F. Lo, I. D. Williams, D. Zhu and B. Z. Tang,
Chem. Mater., 2003, 15, 1535; (c) J. Xu, L. Wen, W. Zhou, J. Lv,
Y. Guo, M. Zhu, H. Liu, Y. Li and L. Jiang, J. Phys. Chem. C,
2009, 113, 5924; (d) J. He, B. Xu, F. Chen, H. Xia, K. Li, L. Ye and
W. Tian, J. Phys. Chem. C, 2009, 113, 9892; (e) S. Dong, Z. Li and
J. Qin, J. Phys. Chem. B, 2009, 113, 434.
12 (a) L. Liu, G. Zhang, J. Xiang, D. Zhang and D. Zhu, Org. Lett.,
2008, 10, 4581; (b) A. Qin, J. W. Y. Lam, L. Tang, C. K. W. Jim,
H. Zhao, J. Sun and B. Z. Tang, Macromolecules, 2009, 42, 1412.
13 (a) H. Tong, Y. N. Hong, Y. Q. Dong, M. Haeussler, Z. Li, J. W.
Y. Lam, Y. P. Dong, H. H. Y. Sung, I. D. Williams and
B. Z. Tang, J. Phys. Chem. B, 2007, 111, 11817; (b) Y. N. Hong,
M. Haussler, J. W. Y. Lam, Z. Li, K. K. Sin, Y. Q. Dong, H. Tong,
J. Z. Liu, A. J. Qin, R. Renneberg and B. Z. Tang, Chem.–Eur. J.,
2008, 14, 6428; (c) M. Wang, X. Gu, G. Z. D. Zhang and D. Zhu,
Anal. Chem., 2009, 81, 4444.
monododecylphosphate (30 mM) in the presence of ALP
(1.5 U/mL) after a different reaction time. Apparently, the
fluorescence intensity of the ensemble started to decrease
gradually upon treatment with ALP. By prolonging the
hydrolysis time, significant reduction of the fluorescence intensity
was observed. The fluorescence variation of the ensemble
solution and the disassemble solution upon addition of ALP
can be distinguished with the naked eye under UV illumination as
shown in the inset of Fig. 2. Therefore, GH-TPE could be a
potentially useful fluorescent probe for detection of ALP.
In summary, a novel cationic water-soluble tetraphenyl-
ethylene derivative, GH-TPE, was designed and efficiently
synthesized. Grafting of glucosamine hydrochloride moieties
into TPE can not only improve its water-solubility and bio-
compatibility, but also provide positive-charged ammonium
binding sites towards other artificial or natural polyanions
through electrostatic interaction. By taking advantage of the
aggregation-induced enhanced emission feature, a convenient
fluorescence-based assay for alkaline phosphatase has been
developed. Heteroaggregate complexation of the GH-TPE
with monododecylphosphate can ‘‘turn on’’ the photo-
luminescence. After addition of alkaline phosphatase, the
fluorescence gradually reduced through enzyme-triggered
de-aggregation of the ensemble of GH-TPE/monododecyl-
phosphate. These results reveal that GH-TPE possesses
potential applications in biomacromolecule detections and
related inhibitor screening.
14 Q. Chen, Q.-Y. Cheng, Y.-C. Zhao and B.-H. Han, Macromol.
Rapid Commun., 2009, 30, 1651.
15 (a) N. C. Maiti, S. Mazumdar and N. Periasamy, J. Phys. Chem. B,
1998, 102, 1528; (b) Z. EI-Hachemi, G. Mancini, J. M. Ribo
A. Sorrenti, J. Am. Chem. Soc., 2008, 130, 15176.
´
and
16 (a) H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem.,
Int. Ed., 2001, 40, 2004; (b) V. V. Rostovtsev, L. G. Green,
V. V. Fokin and K. B. Sharpless, Angew. Chem., Int. Ed., 2002,
41, 2596.
This work was supported by the Ministry of Science and
Technology of China (National Basic Research Program,
ꢀc
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 4067–4069 | 4069