Real-time fluorometric assay for acetylcholinesterase activity and inhibitor screening through the pyrene probe monomer-excimer transition

Article abstract of DOI:10.1021/ol400619t

A choline labeled pyrene probe (Py-Ch) was designed and synthesized. Poly(vinylsulfonate) (PVS) could induce Py-Ch aggregation. The aggregation and deaggregation process could be finely controlled by the acetylcholinesterase (AChE) enzymatic hydrolysis of Py-Ch. The resulting excimer-monomer transition provided a facile way for real-time AChE activity fluorometric assay and inhibitor screening.

Full text of DOI:10.1021/ol400619t

ORGANIC
LETTERS
XXXX
Vol. XX, No. XX
000–000
Real-Time Fluorometric Assay for
Acetylcholinesterase Activity and
Inhibitor Screening through the Pyrene
Probe MonomerÀExcimer Transition
Jian Chen, Dongli Liao, Yan Wang, Huipeng Zhou, Wenying Li, and Cong Yu*
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied
Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China, and
Graduate School of the Chinese Academy of Sciences, Beijing 100039, P. R. China
congyu@ciac.jl.cn
Received March 8, 2013
ABSTRACT
A choline labeled pyrene probe (Py-Ch) was designed and synthesized. Poly(vinylsulfonate) (PVS) could induce Py-Ch aggregation. The
aggregation and deaggregation process could be finely controlled by the acetylcholinesterase (AChE) enzymatic hydrolysis of Py-Ch. The
resulting excimerÀmonomer transition provided a facile way for real-time AChE activity fluorometric assay and inhibitor screening.
Acetylcholinesterase (AChE, EC 3.1.1.7) is a hydrolase
that can catalyze the hydrolysis of acetylcholine to choline
and acetate.1 It is a key enzyme in the central and periph-
eral nervous system.2 AChE inhibitors are currently used
for the treatment of a number of neuromuscular disorders
and Alzheimer’s disease.3 Detection of AChE activity and
the screening for its potential inhibitors are therefore of
great importance.
In recent years, AChE has been detected by colorimetric,4
chemiluminescent,5 electrochemical,6 and fluorescent7À10
methods. Fluorescent methods exhibit higher sensitivity
compared with other methods and have drawn more atten-
tion. However, certain drawbacks exist. For instance, some
methods require a complicated, time-consuming, and expen-
sive synthesis; some fluorescent materials are somewhat
toxic; and some may produce false-positive output detection
signals. Therefore, the development of a simple, fast, sensitive,
(1) Dvir, H.; Silman, I.; Harel, M.; Rosenberry, T. L.; Sussman, J. L.
Chem. Biol. Interact. 2010, 187, 10–22.
(2) Quinn, D. M. Chem. Rev. 1987, 87, 955–979.
(3) (a) Silman, I.; Sussman, J. L. Curr. Opin. Pharmacol. 2005, 5, 293–
302. (b) Selkoe, D. J. Physiol. Rev. 2001, 81, 741–766.
(4) (a) Pavlov, V.; Xiao, Y.; Willner, I. Nano Lett. 2005, 5, 649–653.
(b) Wang, M.; Gu, X. G.; Zhang, G. X.; Zhang, D. Q.; Zhu, D. B.
Langmuir 2009, 25, 2504–2507. (c) Li, Y. G.; Bai, H.; Li, C.; Shi, G. Q.
ACS Appl. Mater. Interfaces 2011, 3, 1306–1310. (d) Xue, W. X.; Zhang,
D. Q.; Zhang, G. X.; Zhu, D. B. Chin. Sci. Bull. 2011, 56, 1877–1883. (e)
Liu, D. B.; Chen, W. W.; Wei, J. H.; Li, X. B.; Wang, Z.; Jiang, X. Y.
Anal. Chem. 2012, 84, 4185–4191.
(7) (a) Peng, L. H.; Zhang, G. X.; Zhang, D. Q.; Xiang, J. F.; Zhao,
R.; Wang, Y. L.; Zhu, D. B. Org. Lett. 2009, 11, 4014–4017. (b) Cui, K.;
Chen, Z. L.; Wang, Z.; Zhang, G. X.; Zhang, D. Q. Analyst 2011, 136,
191–195. (c) Shen, X.; Liang, F. X.; Zhang, G. X.; Zhang, D. Q. Analyst
2012, 137, 2119–2123. (d) Wang, C. I.; Chen, W. T.; Chang, H. T. Anal.
Chem. 2012, 84, 9706–9712.
(8) (a) Feng, F.; Tang, Y. L.; Wang, S.; Li, Y. L.; Zhu, D. B. Angew.
Chem., Int. Ed. 2007, 46, 7882–7886. (b) Zhang, W. J.; Zhu, L. N.; Qin,
J. G.; Yang, C. L. J. Phys. Chem. B 2011, 115, 12059–12064.
(9) (a) Gill, R.; Bahshi, L.; Freeman, R.; Willber, I. Angew. Chem.,
Int. Ed. 2008, 47, 1676–1679. (b) Saa, L.; Virel, A.; Sanchez-Lopez, J.;
Pavlov, V. Chem.;Eur. J. 2010, 16, 6187–6192.
(5) Briman, S. Biochem. J. 1985, 225, 825–828.
(10) (a) Wang, M.; Gu, X. G.; Zhang, G. X.; Zhang, D. Q.; Zhu, D. B.
Anal. Chem. 2009, 81, 4444–4449. (b) Xue, W. X.; Zhang, G. X.; Zhang,
D. Q.; Zhu, D. B. Org. Lett. 2010, 12, 2274–2277. (c) Chen, Q.; Bian, N.;
Cao, C.; Qiu, X. L.; Qi, A. D.; Han, B. H. Chem. Commum. 2010, 46,
4067–4069.
(6) (a) 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, 1882–1186. (b) Du, D.;
Wang, J.; Wang, L. M.; Lu, D. L.; Smith, J. N.; Timchalk, C.; Lin, Y. H.
Anal. Chem. 2011, 83, 3770–3777.
r
10.1021/ol400619t
XXXX American Chemical Society

and inexpensive fluorometric assay for AChE activity and its
inhibitors is highly desirable.
Scheme 1. (a) Schematic Illustration of the Fluorometric
Detection of Ache Activity through the ExcimerÀMonomer
Transition; (b) Hydrolysis of Py-Ch by AChE; (c) Structure of PVS
Controlled self-assembly has been widely used by all
forms of life for the construction of sophisticated func-
tional units. And in recent years it has also been used for
the development of a number of novel biosensing techn-
iquesfortheselective sensing ofenzymeactivities, proteins,
and other biomolecules.11
Pyrene is a planar aromatic compound. The aggregation
of pyrene monomer molecules (emission at 370À420 nm)
could produce excimer emission. As a result, a red-shifted
and broad emission band at 450À550 nm could be
observed.12 Monomer emission could be restored through
the deaggregation process.13 This particular property has
been employed for the design of a number of fluorescent
sensors.14 In this work, a choline labeled cationic pyrene
probe (Py-Ch) was designed and synthesized. It shows
considerable water solubility (>3 mM) and exhibits blue
fluorescence in an aqueous solution. Using Py-Ch as an
AChE substrate, we herein report a simple, fast, inexpen-
sive, and sensitive fluorescent method for real time AChE
activity assay.
The principle of the assay is shown in Scheme 1. (1) In an
aqueous solution, Py-Ch mainly exists in the monomeric
form, because of the positive charge electrostatic repulsive
interactions. Strong pyrene probe monomer fluorescence
is detected. (2) Poly(vinylsulfonate) (PVS) is a polyanion.
When PVS is added to the assay solution, strong attractive
electrostatic and hydrophobic interactions between PVS
and Py-Ch result in the aggregation of Py-Ch. Both the
increase of the pyrene excimer emission and the decrease of
the monomer emission could be observed. (3) AChE can
catalyze the hydrolysis of the cationic Py-Ch to the anionic
1-pyrenebutyrate and choline. Upon the addition of AChE to
the assay solution, the pyrene probe is hydrolyzed and
repulsed from PVS becasue of the charge reversal. The pyrene
excimer returns to the monomeric state as a result of the
deaggregation. An excimerÀmonomer transition is detected,
and a convenient fluorometric assay for AChE activity is
therefore established. (4) In the presence of an AChE in-
hibitor, the activity of AChE is reduced. A reduced degree of
excimerÀmonomer transition is detected, which could be
used for the screening for potential AChE inhibitors.
Figure 1 shows that, in the absence of PVS, Py-Ch
exhibited characteristic monomer emission with peaks at
375 and 396 nm. A new and broad emission band with a
peak at 486 nm appeared, and the intensity gradually
increased upon the gradual increase of the PVS concentra-
tion. Meanwhile, a gradual decrease of the monomer emis-
sion was also observed. The emission band at 486 nm was
attributed to the formation of a pyrene excimer as a result of
the PVS-induced Py-Ch aggregation. The changes in assay
solution emission color from blue to green could be easily
observed by the naked eye (inset I, Figure 1). When 300 μM
PVS was introduced, the intensity ratio (the IE/IM value) of
the excimer (at 486 nm) to monomer emission (at 375 nm)
reached its maximum (inset II of Figure 1). A further
increase of the PVS concentration caused no further increase
of the IE/IM value, indicating that a saturation point was
reached where most of the monomic pyrene probe had been
transformed to the aggregated forms.
(11) (a) Law, B.; Weissleder, R.; Tung, C.-H. Bioconjugate Chem 2007,
ꢀ
18, 1701–1704. (b) Gonzalez, D. C.; Savariar, E. N.; Thayumanavan, S.
J. Am. Chem. Soc. 2009, 131, 7708–7716. (c) Mizusawa, K.; Ishida, Y.;
Takaoka, Y.; Miyagawa, M.; Tsukiji, S.; Hamachi, I. J. Am. Chem. Soc.
2010, 132, 7291–7293. (d) Wang, B.; Yu, C. Angew. Chem., Int. Ed. 2010, 49,
1485–1488.
(12) Zhang, R. X.; Tang, D.; Lu, P.; Yang, X. Y.; Liao, D. L.; Zhang,
Y. J.; Zhang, M. J.; Yu, C.; Yam, V. W. W. Org. Lett. 2009, 11, 4302–
4305.
Figure 1. Changes in emission spectra of Py-Ch (100 μM) upon
the addition of increasing concentrations of PVS. Inset: (I)
photograph of the Py-Ch sample solution in the absence (left)
and presence (right) of PVS (300 μM) under UV light illumina-
tion at 365 nm; (II) Changes in the IE/IM value upon the addition
of increasing concentrations of PVS.
(13) Ma, B. L.; Zeng, F.; Li, X. Z.; Wu, S. Z. Chem. Commun. 2012,
48, 6007–6009.
(14) (a) Liu, L.; Zhang, D. Q.; Zhang, G. X.; Xiang, J. F.; Zhu, D. B.
Org. Lett. 2008, 10, 2271–2274. (b) Yu, C.; Yam, V. W. W. Chem.
Commun. 2009, 45, 1347–1349. (c) Li, Y.; Li, H.-W.; Ma, L.-J.; Dang,
Y.-Q.; Wu, Y. Q. Chem. Commun. 2010, 46, 3768–3770. (d) Yang, X. Y.;
Liu, D.; Lu, P.; Zhang, Y. J.; Yu, C. Analyst 2010, 135, 2074–2078. (e)
Dai, Q.; Liu, W. M.; Zhuang, X. Q.; Wu, J. S.; Zhang, H. Y.; Wang, P. F.
Anal. Chem. 2011, 83, 6559–6564.
AChE could catalyze the hydrolysis of the cationic Py-Ch
to the anionic 1-pyrenebutyrate and choline. 1-Pyrenebuty-
rate was repulsed from the polyanion (PVS) as a result of the
B
Org. Lett., Vol. XX, No. XX, XXXX

negative charge electrostatic repulsive interactions. Deag-
gregation took place. Therefore, the intensity of the excimer
emission decreased, and the intensity of the monomer emis-
sion increased. Figures 2 and S1 (Supporting Information)
show that, upon the addition of AChE to the sample solution
of Py-Ch and PVS, with the increase of the enzymatic
reaction time, a gradual decrease of the excimer emission
and at the same time a gradual increase of the monomer
emission were observed. Almost complete recovery of the
free Py-Ch monomer emission was obtained after 120 min of
the enzymatic reaction. The emission color of the sample
solution also changed from green back to blue, which could
be easily observed by the naked eye (inset of Figure 2). The
results clearly show that AChE could catalyze the hydrolysis
of Py-Ch, which led to the probe excimer-to-monomer
emission transition.
Control experiments were conducted to verify the principle
of the assay. Py-Ch was incubated with AChE for an ample
amount of time, and the emission spectrum was recorded.
Figure S2 shows clear pyrene monomer emission, and no
excimer emission was observed. The spectrum is quite similar
to that of the free Py-Ch. The UVÀvis absorption spectrum
of Py-Ch also shows minimal changes upon enzymatic
hydrolysis (Figure S3). The results clearly suggest that simple
hydrolysis of Py-Ch could not produce pyrene excimer
emission. Upon the addition of PVS, the absorption bands
of Py-Ch showed clear broadening, along with a significant
decrease in intensity, because of the formation of the probe
aggregates. After the enzymatic hydrolysis by AChE, the
absorption bands of the pyrene probe changed back to that of
the free Py-Ch (Figure S3). The results further support the
conclusion that the enzymatic hydrolysis of the pyrene probe
resulted in deaggregation and restoration of the probe mono-
mer emission. Our results also show that PVS itself shows
little effect on the enzymatic activity of AChE (Figure S4). A
number of related pyrene and choline derivatives were
selected, and their influences on the AChE enzymatic reac-
tion were studied (Figures S5 and S6).
Our method could be used to monitor the AChE activity
in real time (Figure 3). The emission spectra of the sample
mixtureofPy-Ch and PVS containing different amountsof
AChE were recorded at different reation times. The IM/IE
value remained mostly unchanged in the absence of AChE,
but increased gradually with reaction time in the presence
ofAChE. The IM/IE value increased morequickly athigher
AChE concentrations (Figure S7). The results clearly
suggest that the increase in AChE concentration led to
faster substrate (Py-Ch) hydrolysis.
To address the selectivity of our assay, control experi-
ments were conducted. A number of enzymes such as
lysozyme, alkaline phosphatase (ALP), collagenase, and
exonuclease I (Exo I) were tested. These enzymes can
catalyze the hydrolysis of glycoside, phosphate, collagen,
and nucleicacid, respectively. FigureS8 shows thatnoneof
these enzymes had the ability to induce noticeable IM/IE
value changes. The results further confirm that the emission
changes of Py-Ch were due to the AChE-catalyzed Py-Ch
hydrolysis, and the assay is highly selective for AChE.
Figure 3. Real-time IM/IE value changes of Py-Ch (100 μM) at
different AChE concentrations. Assay solutions contained 300 μM
PVS and different concentrations of AChE (0, 0.1, 0.2, 0.5, 1.0, 2.0,
5.0, and 10.0 U/mL, respectively). Inset: Expanded curves with
lower AChE concentrations.
Our AChE fluorometric assay was further tested in com-
plex sample mixtures. The emission spectra of the sample
mixtures containing Py-Ch, PVS, and AChE of various
concentrations in 2% fetal calf serum (or 2% cell lysate)
were measured, and the IM/IE values were obtained. Figures
S9 and S10 show that the more AChE is spiked, the larger the
IM/IE value increase is obtained. The activity of 0.1 U/mL of
AChE could be easily detected. The results clearly show that
our assay could be used in complex sample mixtures.
Figure 2. Changes in emission spectrum of Py-Ch (100 μM) with
the enzymatic reaction time. Emission spectra were recorded at
10 min intervals. The assay solution contained 300 μM PVS and
10 U/mL AChE. Inset: Photograph of the sample solution of
Py-Ch and PVS in the absence (left) and presence (right) of
AChE under UV light illumination at 365 nm.
The assay could also be used for the screening of
potential AChE inhibitors. To demonstrate this, donepezil
Org. Lett., Vol. XX, No. XX, XXXX
C

for donepezil and 3-hydroxycarbofuran were estimated to be
37.8 and 33.2 nM, respectively (Figure 4).7b,8a The results
clearly demonstrate that our assay could be used not only for
the real-time monitoring of AChE activity but also for the
screening of potential AChE inhibitors.
In summary, a choline labeled pyrene probe (Py-Ch) was
designed and synthesized. Py-Ch was used as an AChE
substrate. Based on the finely controlled aggregation and
deaggregation process, and the resulting excimerÀmonomer
transition, a facile real-time fluorometric assay for AChE
activity has been developed. Our assay has several important
features. First, it is based on the “excimerÀmonomer transi-
tion” mode, which could considerably reduce the likelihood
of false-positive signals associated with other fluorometric
assays. Second, it offers a convenient “mix-and-detect”
approach for the rapid and sensitive detection of AChE
activity and inhibition. Third, the emission spectral changes
could be monitored in real time using a common spectro-
photometer. Fourth, Py-Ch could be easily prepared, and the
polyanion (PVS) is commercially available. All materials
used are fairly inexpensive. The assay is thus fairly cost-
effective. Fifth, the principle of our assay may also be used for
the detection of other enzymes (such as protease and
phosphatase) and the corresponding inhibitors with the
properly designed substrates.
Acknowledgment. This workwas supported by the “100
Talents” program of the Chinese Academy of Sciences, the
National Natural Science Foundation of China (Grants
21075119, 91027036, and 21275139), the National Basic
Research Program of China (973 Program, Grant No.
2011CB911002), and the Pillar Program of Changchun
Municipal Bureau of Science and Technology (Grant No.
2011225).
Figure 4. Plots of the inhibition efficiency of donepezil (a) and
3-hydroxycarbofuran (b) versus the inhibitor concentration.
Assay solutions contained 100 μM Py-Ch, 300 μM PVS, and
10 U/mL AChE.
and 3-hydroxycarbofuran, two known AChE inhibitors,7b,8a
were tested. The emission spectra of the sample mixtures of
Py-Ch, PVS, AChE, and the inhibitors of different concen-
trations were recorded. Figures S11 and S12 show that both
the excimer emission and the monomer emission changed
significantly upon the addition of the inhibitors. And the IM/
IE value decreased with the increase of the inhibitor concen-
tration. The results indicate that the inhibition was more
effective at higher inhibitor concentrations. The IC50 values
Supporting Information Available. Experimental de-
tails, synthesis and characterization of Py-Ch, UVÀvis
and emission studies. This material is available free of
charge via the Internet at http://pubs.acs.org.
The authors declare no competing financial interest.
D
Org. Lett., Vol. XX, No. XX, XXXX