B. Wang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 552–555
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Scheme 1. The mechanism of acetylcholinesterase activity screening based on
compound 1.
acetylthiocholine in the presence of AChE, the fluorescein-based
probe can rapidly induce 1,4-addition of the hydrolysis product
thiocholine to a,b-unsaturated ketone in the compound 1 to form
thioether, resulting in strong fluorescence enhancement and
absorption change. However, in the presence of the corresponding
inhibitor, the fluorescence enhancement or the absorption change
would be inhibited in that the formation of thiocholine was hin-
dered. Based on this strategy, this compound was successfully ap-
plied to the detection of the AChE activity and its inhibitors
screening.
Figure 2. UV/vis absorption spectra of the HEPES buffer (10 mM, pH = 7.4)
containing compound 1 (10 lM), acetylthiocholine (10 lM), and in the presence
of different concentrations of AChE (0, 0.001, 0.003, 0.006, 0.0125, 0.025 U/mL) after
incubation for 5 min. Inset: the plot of the absorption changes at 499 nm with
various concentration of AChE.
To further investigate the corresponding AChE inhibitor, neo-
stigmine, the typical inhibitor of AChE, was used to carry out the
inhibitive activity assay. The ensemble solution containing com-
pound 1 (10 lM), acetylthiocholine (10 lM) and AChE (0.0125 U/
Compound
1 was synthesized according to our previous
mL) was treated with various concentrations of neostigmine (0.5,
1, 2.5, 5 nM) after incubation for 5 min. After neostigmine was
added to the ensemble solution, the fluorescence intensity at
525 nm decreased comparing to that in the absence of inhibitor
after incubation for the same time (Fig. 3a inset). Similarly, when
incubated with neostigmine for 5 min, the absorption intensity at
460 nm of the ensemble solution decreased while the absorption
intensity at 499 nm increased. When more AChE inhibitor was
added to the ensemble solution, the hydrolysis of acetylthiocholine
would be discouraged and less thiocholine would be generated,
resulting in the less changes both in fluorescence and absorption.
On the basis of the plot of the inhibition efficiency dependent on
the concentration of neostigmine, the IC50 value was examined to
be 1.4 nM using the fluorometric method (method A) while
3.2 nM using the colorimetric method (method B) (Fig. 3). Further-
more, tacrine was the first centrally-acting cholinesterase inhibitor
approved for the treatment of Alzheimer’s disease and also used to
carry out the inhibiting experiment. Similarly, on the basis of the
plot of the inhibition efficiency dependent on the concentration
of tacrine, the IC50 value was examined to be 11 nM and 12.8 nM
using the fluorometric and colorimetric methods, respectively
(Fig. 4). Thus, using the fluorescein-based sensor, the colorimetric
and fluorometric assays for acetylcholinesterase and inhibitor
screening were carried out successfully.
report.25 In order to demonstrate that the compound is efficient
for the activity assay and the inhibitor screening, we choose acet-
ylthiocholine as substrate of AChE in that acetylthiocholine could
be hydrolyzed into thiocholine by AChE. Initially, compound 1 is
non-fluorescent in HEPES buffer (10 mM, pH 7.4). Upon addition
of AChE (0.05 U/mL) to aqueous solution containing compound 1
(10 lM) and acetylthiocholine (10 lM), the fluorescence intensity
at 525 nm increased. Time-dependent fluorescence intensity as-
says show the reactions including the hydrolysis induced by AChE
and addition reaction between compound 1 and thiocholine were
almost complete within 5 min (Fig. S1).
After incubation for different time, the fluorescence enhance-
ment of compound
1 in the presence of acetylthiocholine
(10 M) and various concentrations of AChE were shown in Figure
l
1. The fluorescence intensity at 525 nm induced little change in the
absence of AChE. However, upon the addition of as low as 0.001 U/
mL of AChE, the fluorescence intensity displayed an obvious
enhancement by extending the reaction time. Incubation for a
same time, increase of the concentration of AChE led to the
enhancement of the fluorescence intensity at 525 nm since the
hydrolyzing rate of acetylthiocholine into thiocholine is faster. As
shown in Figure S2, after the addition of AChE (0.025 U/mL), the
fluorescence intensity at 525 nm increased with the reaction time.
Similarly, upon the addition of AChE (0.025 U/mL), the absorption
intensity at 499 nm of the assemble solution increased while the
absorption peak at 460 nm decreased (Fig. 2). Using fluorometric
or colorimetric method, the activity of AChE upon a concentration
as low as 0.001 U/mL could be detected. It further proved that
AChE is highly active for the formation of thiocholine. Through
In contrast with fluorescein-based sensor, Ellman’s reagent
(DTNB) was used to substitute compound 1 to carry out the inhib-
itive activity assay, and other condition remain unchanged. As
shown in Figure 5, after the addition of AChE, with the increasing
of the concentration of AChE (0, 0.003, 0.006, 0.0125 and
0.025 U/mL), the absorption peak at 416 nm of the assemble solu-
tion increased while the absorption intensity at 328 nm decreased.
monitoring the reaction kinetics of compound 1 (10
ocholine (10 M), the reaction was completed within 20 s. In con-
trast, it will take more than 300 s for getting to the end of the
reaction of (10 M), acetylthiocholine (10 M) and AChE
lM) and thi-
l
When treated the ensemble of Ellman’s reagent (DTNB) [10
lM in
HEPES (10 mM) buffer solution, pH 7.4], acetylthiocholine (10
l
M)
1
l
l
and AChE (0.0125 U/mL) with different concentrations of neostig-
mine (1, 2.5, 5, 10 nM) and tacrine (5, 10, 20, 50 nM) after incuba-
tion for 5 min, the inhibition efficiencies were obtained. On the
basis of the plot of the inhibition efficiency dependent on the con-
(0.0125 U/mL) (Fig. S3), which showed the thiol trapping reaction
is much faster than the enzyme hydrolysis and the fluorescence
generated is a real time reflection of the enzyme action.
Wang, Bihui
