A fluorescence "turn-on" ensemble for acetylcholinesterase activity assay and inhibitor screening

Article abstract of DOI:10.1021/ol9016723

By making use of the aggregation-induced emission feature of compound 1 and the cascade reactions among acetylthiocholine iodide (ATC), AChE, and compound 2, a new fluorescence "turn-on" method is developed for AChE assay and inhibitor-screening.

Full text of DOI:10.1021/ol9016723

ORGANIC
LETTERS
2
009
Vol. 11, No. 17
014-4017
A Fluorescence “Turn-On” Ensemble for
Acetylcholinesterase Activity Assay and
Inhibitor Screening
4
Lihua Peng, Guanxin Zhang, Deqing Zhang,* Junfeng Xiang, Rui Zhao,
Yilin Wang, and Daoben Zhu
Beijing National Laboratory for Molecular Sciences, Organic Solids Laboratory,
Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China
dqzhang@iccas.ac.cn
Received July 22, 2009
ABSTRACT
By making use of the aggregation-induced emission feature of compound 1 and the cascade reactions among acetylthiocholine iodide (ATC),
AChE, and compound 2, a new fluorescence “turn-on” method is developed for AChE assay and inhibitor-screening.
Alzheimer’s disease (AD), a neurodegenerative disease
characterized by a low concentration of acetylcholine (ACh)
in the hippocampus and cortex, is the leading cause of
AChE activity and inhibition are traditionally monitored
with absorption spectroscopy (e.g., with the Ellman’s re-
agent), but such assay methods show low sensitivity.
1
6
dementia among older people. Hydrolysis of acetylcholine
Meanwhile, fluorometric approaches with good sensitivity
have been reported for screening of AChE inhibitors.
7
,8
(
ACh) by acetylcholinesterase (AChE) is a key process for
the regulation of the neutral response system. The “cholin-
ergic hypothesis of AD” has became the neurobiologic
incentive for AD treatment. Clinical treatment of AD is
mainly based on the AChE inhibitors. Therefore, the
development of a reliable assay method for AChE activity
and its inhibitor screening is of great importance. Addition-
ally, nerve gases and pesticides of organophosphorus and
Chemiluminescent probes have also been reported for AChE
activity assay and inhibitor screening. AChE activity assay
9
and the corresponding inhibitor-screening have been suc-
cessfully achieved by making use of enzyme-involved
cascade reactions in which AChE assay can be converted to
1
0,11
the detection of H
2 2
O .
However, drawbacks are found
for these assay methods. For instance, the use of Ellman’s
reagent may result in a false-positive effect, and some of
these assay methods are discontinuous and time-consuming
2
,3
carbamate types are inhibitors of AChE; thus, an efficient
assay method for AChE activity will be useful for detection
4
,5
of these nerve gases and pesticides.
(
6) Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone, R. M.
Biochem. Pharmacol. 1961, 7, 88–90.
(
1) Whitehouse, P. J.; Price, D. L.; Struble, R. G.; Clark, A. W.; Coyle,
J. T.; Delon, M. R. Science 1982, 215, 1237–1239.
2) Zejli, H.; Hidalgo-Hidalgo de Cisneros, J. L.; Naranjo-Rodriguez,
I.; Liu, B; Temsamani, K. R.; Marty, J.-L. Talanta 2008, 77, 217–221
3) Chen, H. D.; Zuo, X. L.; Su, S.; Tang, Z. Z.; Wu, A. B.; Song,
S. P.; Zhang, D. B.; Fan, C. H. Analyst 2008, 133, 1182–1186
4) Su, S.; He, Y.; Zhang, M.; Yang, K.; Song, S.; Zhang, X.; Fan, C.
(7) Maeda, H.; Mastuno, H.; Ushida, M.; Katayama, K.; Saeki, K.; Itoh,
N. Angew. Chem., Int. Ed. 2005, 44, 2922–2925
(8) Feng, F. D.; Tang, Y. L.; Wang, S.; Li, Y. L.; Zhu, D. B. Angew.
Chem., Int. Ed. 2007, 46, 7882–7886
.
(
.
.
(
(9) Sabelle, S.; Renard, P. Y.; Pecorella, K.; de Suzzoni-Dezard, S.;
Creminon, C.; Grassi, J.; Mioskowski, C. J. Am. Chem. Soc. 2002, 124,
4874–4880.
.
(
Appl. Phys. Lett. 2008, 93, 023113
.
(10) Pavlov, V.; Xiao, Y.; Willner, I. Nano Lett. 2005, 5, 649–653.
(
5) Pohanka, M.; Jun, D.; Kalasz, H.; Kuca, K. Protein Pept. Lett. 2008,
(11) Gill, R.; Bahshi, L.; Freeman, B.; Willner, I. Angew. Chem., Int.
Ed. 2008, 47, 1676–1679
1
5, 795–798
.
.
10.1021/ol9016723 CCC: $40.75
Published on Web 08/10/2009
 2009 American Chemical Society

9
,12
for AChE activity and inhibition studies.
Therefore,
in Scheme 1 and explained as follows: (1) compound 2 is a
hydrophobic compound with a maleimide group; (2) acetylth-
iocholine iodide (ATC) is a good substrate of AChE; i.e.,
ATC can be hydrolyzed into thiocholine in the presence of
AChE. Michael reaction of thiocholine with the maleimide
group in 2 would lead to the formation of an amphiphilic
compound 3; (3) such amphiphilic compound would induce
the aggregation of compound 1, and accordingly the fluo-
rescence of the ensemble would increase significantly; (4)
in the presence of the corresponding inhibitor the hydrolysis
of acetylthiocholine would be retarded, and as a result, a
smaller amount of the amphiphilic compound would be
generated. Consequently, the fluorescence enhancement for
the ensemble would be reduced. Therefore, the ensemble of
compounds 1 and 2 can be used for the AChE activity assay
and the inhibitor-screening by making use of the AIE feature
of TPE compounds.
convenient and continuous methods for AChE activity assay
and the inhibitor screening are still highly desired.
We have very recently described a convenient and
continuous fluorometric assay method for AChE and inhibitor
screening based on the ensemble of compound 1 (Scheme
) units and myristoylcholine
by making use of the aggregation-induced emission (AIE)
feature of tetraphenyl ethylene (TPE). This assay method
is based on the following facts: (1) in the presence of
myristoylcholine, an amphiphilic compound with one am-
monium group, aggregation of compound 1 occurred leading
to strong fluorescence; (2) myristoylcholine can easily be
hydrolyzed into myristic acid and choline in the presence of
AChE. Accordingly, the aggregation complex of compound
-
1
) with two sulfonate (-SO
3
1
3
1
and myristoylcholine formed in aqueous solution would
be disassembled because of the Coulombic repulsive interac-
tion between 1 and the hydrolysis product of myristoylcho-
line. As a result, the fluorescence of the ensemble decreases.
Compound 1 was prepared according to a previous
1
4
report. Compound 2 was obtained simply by the reaction
of maleic anhydride and dodectylamine, and compound 3
was obtained by further reaction of compound 2 with
thiocholine (see the Supporting Information). First, we
discuss the fluorescence enhancement of compound 1 in
the presence of compound 3 (Scheme 1), the Michael
adduct of compound 2 with thiocholine. As anticipated,
compound 1 [20 µM in HEPES (10 mM) buffer solution,
pH ) 7.35] showed rather weak fluorescence. After
addition of compound 3, the fluorescence of compound 1
increased greatly. For instance, the fluorescence intensity
at 490 nm for the aqueous solution of compound 1 (20
µM) was enhanced by 45.5 times when the concentration
of compound 3 in the ensemble reached 24 µM. In fact,
the fluorescence quantum yield of the solution of 1 (20
µM) increased from 0.0090 to 0.064 (by reference to
quinine hemisulfate monohydrate) after introduction of
compound 3 (24 µM) to the solution. Interestingly, the
fluorescence intensity of the ensemble solution of com-
pounds 1 and 3 increased almost linearly with the
concentration of compound 3 in the range of 0-20 µM
as displayed in the inset of Figure 1.
Scheme 1. (A) Chemical Structures of Compounds 1-3. (B)
Cascade Reactions among ATC, AChE, and Compound 2. (C)
Illustration of the Aggregation of Compound 1 in the Presence
of Compound 3
In this paper, we want to report an alternative fluorescence
turn-on assay method for AChE activity and the inhibitor-
screening based on the ensemble of compound 1 and
compound 2 (Scheme 1). The design rationale is illustrated
(
12) Rhee, I. K.; Van Rijn, R. M.; Verpoorte, R. Phytochem. Anal. 2003,
4, 127–131
13) Wang, M.; Gu, X. G.; Zhang, G. X.; Zhang, D. Q.; Zhu, D. B.
1
.
(
Anal. Chem. 2009, 81, 4444–4449. It should be noted that several new
chemo-/biosensors based on AIE features of silole and TPE derivatives have
been described recently: (a) Hong, Y.; Lam, J. W. Y.; Tang, B Chem.
Commun. 2009, 4332–4353, and references cited therein. (b) Liu, J. Z.;
Lam, J. W. Y.; Tang, B. Z. J. Inorg. Organomet. Polym. Mater. 2009, 19,
2
49–285. (c) Peng, L.; Wang, M.; Zhang, G.; Zhang, D.; Zhu, D. Org.
Figure 1. Fluorescence spectra of the ensemble of compound 1
Lett. 2009, 11, 1943–1946. (d) Liu, L.; Zhang, G. X.; Xiang, J. F.; Zhang,
D. Q.; Zhu, D. B Org. Lett. 2008, 10, 458–461. (e) Wang, M.; Zhang,
D. Q.; Zhang, G. X.; Tang, Y. L.; Wang, S.; Zhu, D. B. Anal. Chem. 2008,
[20 µM in HEPES (10 mM) buffer solution, pH ) 7.35] in the
presence of different concentrations of compound 3 (from 0 to 1.2
equiv); inset shows the plot of fluorescence intensity of compound
8
0, 6443–6448. (f) Zhao, M. C.; Wang, M.; Liu, H. J.; Liu, D. S.; Zhang,
G. X.; Zhang, D. Q.; Zhu, D. B. Langmuir 2009, 25, 676–678. (g) Ning,
Z.; Chen, Z.; Zhang, Q.; Yan, Y; Qian, S; Cao, Y.; Tian, H. AdV. Funct.
Mater. 2007, 17, 3799–3807.
1
at 490 nm vs the concentration of compound 3.
Org. Lett., Vol. 11, No. 17, 2009
4015

According to previous studies, such fluorescence enhance-
ment observed for compound 1 should be ascribed to the
aggregation of compound 1 in the presence of compound 3.
The dynamic light scattering (DLS) and surface tension
experimental results support this conclusion. The DLS signal
shown in Figure S1 (Supporting Information) indicates the
formation of aggregates with sizes around 460 nm in the
15
ensemble solution of compounds 1 (20 µM) and 3 (30 µM).
The variation of the surface tension vs the concentrations of
compounds 1 and 3 (see Figure S2, Supporting Information)
implies that there is a phase-transition when the concentra-
tions of 1 and 3 are 15 and 22 µM, respectively. These results
clearly indicate the aggregation of compound 1 in the
presence of compound 3. The aggregation of compound 1
in the presence of compound 3 may occur via the following
mechanism: molecules of 1 and 3 may form comicelle, in
which the hydrophobic moieties of 1 and 3 (TPE framework
and alkyl chain) may stack to generate the core and their
hydrophilic parts (sulfonate and ammonium) may form the
corona. These comicelles may be further interconnected by
molecules of compound 1 due to electrostatic interaction to
form large aggregates as illustrated in Scheme 1.
Figure 2
.
Fluorescence spectra of the ensemble of compound 1
[
2
20 µM in HEPES (10 mM) buffer solution, pH ) 7.35], compound
(30 µM), and ATC (30 µM) in the presence of AChE (0.1 U/mL)
incubated at room temperature for different periods; inset shows
the photos of the corresponding mixed buffer solution in the absence
(
1
A) and presence (B) of AChE (0.1 U/mL) after incubation for
5.0 min under UV light (365 nm) illumination.
(see Scheme 1); accordingly, the fluorescence of the en-
semble is enhanced. In fact, the formation of compound 3
within the ensemble of compounds 1 and 2, ATC, and AChE
was confirmed by HPLC analysis as shown in Figure S4
We now demonstrate the application of the ensemble of
compounds 1, 2, and acetylthiocholine (ATC) for AChE
activity assay and the inhibitor screening. A clear solution
of 1 (20 µM), 2 (30 µM), and ATC (30 µM) in mixture of
HEPES buffer (10 mM) and THF (v/v, 1000/3) was prepared
for the following spectral studies. Initially, the ensemble
solution of compounds 1, 2, and acetylthiocholine (ATC)
exhibited rather weak fluorescence as shown in Figure 2.
After addition of AChE (0.1 U/mL), the fluorescence
intensity of the ensemble started to increase; moreover, the
fluorescence intensity increased gradually by prolonging the
incubation time (see Figure 2). For instance, the fluorescence
intensity at 490 nm of the ensemble was enhanced 43 times
after AChE (0.1 U/mL) was introduced and the solution was
incubated for 15 min. Actually, the fluorescence enhance-
ment, observed for the ensemble solution after addition of
AChE, can be distinguished by naked eye when the buffer
solution is illuminated with UV light (365 nm) as indicated
in the inset of Figure 2.
(
Supporting Information).
The fluorescence spectra of the ensemble of 1 (20 µM), 2
(30 µM), and ATC (30 µM) containing different concentrations
of AChE (0, 0.005, 0.01, 0.02, 0.05, 0.1U/mL) were measured
after incubation for different times. As shown in Figure 3, the
fluorescence intensity at 490 nm remained almost unchanged
in the absence of AChE, but the fluorescence intensity increased
gradually in the presence of AChE by prolonging the reaction
time at room temperature. Obviously, a large degree of
fluorescence enhancement was detected for the ensemble
containing a high concentration of AChE. AChE with concen-
trations as low as 0.005 U/mL can be analyzed. This is
understandable by considering the fact that the presence of more
AChE would induce the formation of more amount of com-
pound 3, which would trigger the aggregation of compound 1
leading to fluorescence enhancement.
The control experiments confirm that the fluorescence
enhancement observed for compound 1 requires the presence
of compound 2, ATC, and AChE. Neither the ensemble of
compound 1/AChE or that of compound 1/compound
2/AChE or that of compound 1/ATC/AChE exhibits obvious
fluorescence enhancement as displayed in Figure S3 (Sup-
porting Information). The fact that the fluorescence of the
ensemble of 1, 2, and ATC increases after addition of AChE
can be understood as follows: ATC is hydrolyzed to generate
thiocholine, which in turn reacts with compound 2 to form
the amphiphilic compound 3. As discussed above, aggrega-
tion of compound 1 occurs in the presence of compound 3
Figure 3. Variation of the fluorescence intensity at 490 nm vs the
(
14) Tong, H.; Hong, Y. N.; Dong, Y. Q.; Haussler, M.; Li, Z.; Lam,
reaction time for the ensemble of compounds 1 [20 µM in HEPES
(10 mM) buffer solution, pH ) 7.35], 2 (30 µM), and ATC (30 µM)
in the presence of different concentrations of AChE (0, 0.005, 0.01,
J. W. Y.; Dong, Y. P.; Sung, H. H. Y.; Williams, I. D.; Tang, B. Z. J.
Phys. Chem. B 2007, 111, 11817.
(
15) Because of the amphiphilic feature, compound 3 can also self-
0.02, 0.05, and 0.1 U/mL); the excitation wavelength was 340 nm.
assemble into aggregates with sizes around 44 nm in aqueous solution as
indicated by the DLS result (see Figure S1, Supporting Information).
4016
Org. Lett., Vol. 11, No. 17, 2009

It is known that the hydrolysis of ATC catalyzed by AChE
will be retarded in the presence of corresponding AChE
inhibitors. Thus, it is anticipated that the degree of fluores-
cence enhancement for the ensemble of compounds 1, 2,
ATC, and AChE will become small after addition of the
inhibitors of AChE. Accordingly, the ensemble of com-
pounds 1, 2, ATC, and AChE can be employed for screening
donepezil, which are also the inhibitors of AChE. Figures
S6 and S8 (Supporting Information) show the plots of the
fluorescence intensity at 490 nm of the ensemble vs the
reaction time in the presence of different amounts of tacrine
and donepezil, respectively. Similarly, the IC50 values were
estimated to be 22.5 and 7.3 nM for tacrine and donepezil,
respectively, based on the plots of the inhibition efficiency
vs the respective concentrations of tacrine and donepezil (see
Figures S7 and S9, Supporting Information). These IC50
values are close to those obtained with other AChE assay
1
6
the inhibitors of AChE. Neostigmine (see Figure 4),
a
typical inhibitor of AChE, was selected to demonstrate the
application of the ensemble to screen the AChE inhibitors.
The fluorescence spectra of the ensemble of compounds 1
1
1,7
methods (11.8 nM, 6.4 nM) .
(
20 µM), 2 (30 µM), ATC (30 µM), and AChE (0.1U/mL)
containing different concentrations of neostigmine (0, 10,
0, 100, 500 nM) were measured after incubation for
Additionally, cysteine and reduced glutathione (GSH) have
negligible interference toward this fluorometric assay method
for AChE activity and the inhibitor-screening. As shown in
Figure S10 (Supporting Information), the fluorescence in-
tensity of the ensemble of compounds 1 (20 µM) and 2 (30
µM) remained almost unchanged after addition of either
cysteine (up to 10 equiv) or GSH (up to 10 equiv). It is
probable that the corresponding Michael adducts formed
between compound 2 and cysteine/GSH do not possess net
positive charges and as a result aggregation of compound 1
may not take place efficiently. It should be noted that thiol-
containing compounds such as reduced glutathione (GSH)
have interference on the AChE assay with Ellmans’s reagent.
In summary, we successfully demonstrate a new switch-
on fluorometric assay method for AChE and its inhibitor-
screening based on the ensemble of compounds 1, 2, and
ATC. This fluorometric assay is designed by making use of
the following features of the ensemble: (1) cascade reactions
from ATC hydrolysis to the Michael reaction with compound
5
different times at room temperature. As expected, in the
absence of neostigmine the fluorescence intensity increases
by prolonging the reaction time, but in the presence of
neostigmine the degree of the fluorescence enhancement for
the ensemble is reduced gradually (see Figure S5, Supporting
Information). On the basis of the plot of the inhibition
4
efficiency vs the concentration of neostigmine (Figure 4),
the corresponding IC50 value was estimated to be 50.3 nM.
2
to generate the amphiphilic compound 3; (2) aggregation
of compound 1 in the presence of the amphiphilic compound
; (3) the AIE (aggregation-induced emission) feature of
3
tetraphenyl ethylene compounds. Fluorescent spectral inves-
tigations show that this fluorometric assay can be performed
continuously and AChE with concentrations as low as 0.005
U/mL can be analyzed. Moreover, the ensemble of com-
pounds 1, 2, ATC, and AChE can be conveniently used to
screen the inhibitors of AChE. Given its speed, simplicity,
and ease of operation, this fluorescence turn-on assay may
extend to high-throughput screening of AChE inhibitors and
relevant drug discovery.
Figure 4. Plot of the inhibition efficiency of neostigmine to AChE
vs the concentration of neostigmine; the data were obtained with
the ensemble of compounds 1 [20 µM in HEPES (10 mM) buffer
solution, pH ) 7.35], 2 (30 µM), and ATC (30 µM) and AChE
(
0.1 U/mL) in the presence of different concentrations of neostig-
mine (10, 50, 100, 500 nM).
This value of IC50 is close to that obtained with other AChE
Supporting Information Available: Synthesis and char-
acterization data of compounds 2 and 3; DLS and HPLC
data; fluorescence spectra and relevant data for the ensemble.
This material is available free of charge via the Internet at
http://pubs.acs.org.
1
7
assay methods (36 nM). The fluorescence spectra of the
ensemble were recorded in the presence of tacrine and
(
16) Gupta, R. C. Toxicology of organophosphates and carbamate
compounds; Elsevier: Amsterdam, 2006.
17) Kishibayashi, N.; Ishii, A.; Karasawa, A. Jpn. J. Pharmacol. 1994,
6, 397–403.
(
6
OL9016723
Org. Lett., Vol. 11, No. 17, 2009
4017