Real-time colorimetric screening of endopeptidase inhibitors using adenosine triphosphate (ATP)-stabilized gold nanoparticles

Article abstract of DOI:10.1016/j.tetlet.2010.02.061

The gold nanoparticles (AuNPs) that were stabilized with adenosine triphosphate (ATP) were stable over a wide range of pHs for the buffer, even in the presence of high concentrations of salt and protein. However, these stabilized AuNPs immediately aggrega

Full text of DOI:10.1016/j.tetlet.2010.02.061

Tetrahedron Letters 51 (2010) 2228–2231
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Tetrahedron Letters
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Real-time colorimetric screening of endopeptidase inhibitors using
adenosine triphosphate (ATP)-stabilized gold nanoparticles
Mi Hee Kim a, Soo Suk Lee b, Sang J. Chung c, Hyun Hye Jang a, Sujung Yi a, Sudeok Kim a, Suk-Kyu Chang a,
Min Su Han a,
*
a Department of Chemistry, Chung-Ang University, Seoul 156-756, Republic of Korea
b Bio & Health Group, Emerging Center, Samsung Advanced Institute of Technology, Mt. 14-1, Nongseo-dong, Giheung-gu, Yongin-si, Gyeonggi-do 446-712, Republic of Korea
c BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology(KRIBB), Yuseong, Daejeon 305-333, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
The gold nanoparticles (AuNPs) that were stabilized with adenosine triphosphate (ATP) were stable over
a wide range of pHs for the buffer, even in the presence of high concentrations of salt and protein.
However, these stabilized AuNPs immediately aggregated when they were exposed to thiol-containing
compounds, such as thiophenol. Endoprotease hydrolyzed the thioester bond in the CBZ–Phe–S–Ph sub-
strate, and the hydrolyzed product (thiophenol) reacted with the AuNPs that were stabilized with ATP,
causing them to aggregate, which in turn resulted in a visible color change in the AuNPs solution. This
method enabled the real-time monitoring of the inhibition potencies of various endopeptidase inhibitors
and the activity of endoprotease. This assay discriminated between the inhibition activities of various
protease inhibitors for endoprotease on the basis of the color change of the assay solution.
Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
Received 24 November 2009
Revised 8 February 2010
Accepted 12 February 2010
Available online 20 February 2010
The plasmon absorption bands of gold nanoparticles (AuNPs)
depend on their shape and size.1 Typically, discrete AuNPs exhibit
an absorption band with a high extinction coefficient, which is 3–5
orders higher than the absorption band of organic dye molecules,
at around 520 nm. However, the typical absorption band corre-
sponding to the aggregated AuNPs appears at longer wavelengths
because of the intense color (blue-purple) of the NP solution.
AuNPs have widely been used for the colorimetric detection of
DNA sequences, proteins, and metal ions because of their high
extinction coefficients and distance-dependent optical properties.2
Colorimetric enzyme assays using AuNPs have gained consider-
able attention in bioassay studies because of their simplicity, high
sensitivity, and low cost. However, few colorimetric systems have
beendevelopedfortheevaluationof theenzymaticactivities.3 A pre-
vious study showed that the colorimetric bioassays can be divided
into two types corresponding to modified and unmodified AuNPs.
In the first method, the aggregates of the AuNPs that are modified
using the enzyme substrates are used to monitor the enzymatic
activity. The AuNP substrate bond can be cleaved using a suitable en-
zyme in order to disperse the AuNPs in the solution.3a–c This process
brings about a change in the color of the AuNPs solution, and there-
fore, the real-time monitoring of the enzymatic activities is possible.
In the second method, the enzyme substrate and the unmodified
AuNPs are used to monitor the enzymatic activities.3d–f The sub-
strate is pretreated with the target enzyme and then exposed to
the unmodified AuNPs. The activity of the target enzyme is moni-
tored on the basis of the color change of the AuNPs that results from
the two-stage process. Although the colorimetric assays that use
unmodified AuNPs are simpler than the assays that use modified
AuNPs, they cannot be used for thereal-time monitoringof the activ-
ity of the target enzyme because the AuNPs are highly sensitive to
various factors such as the electrolyte concentration, the pH of the
buffer, and the protein concentration.1,2a Hence, a change in any of
theabove-mentioned parametersresultsin the irreversible aggrega-
tion of the unmodified AuNPs and a significant red shift in the absor-
bance spectrum. Therefore, the unmodified AuNPs are not easily
used for the real-time monitoring of the activity of the target
enzyme.
Recently, Zhao et al. reported that a real-time enzyme assay
method using the ATP-stabilized AuNPs could be prepared by sim-
ply mixing the AuNPs with adenosine triphosphate. The stabilized
AuNPs are stable in the buffer solution in the presence of high con-
centrations of salt and protein.4 However, the stabilized AuNPs
aggregate immediately when they are exposed to the enzyme that
hydrolyzes the phosphate bond in ATP because of the change in the
solubility of the stabilized AuNPs in buffer solution at higher salt
concentrations. With this information on the properties of the
ATP-stabilized AuNPs, an operationally simple colorimetric assay
was examined for the simultaneous real-time monitoring of the
endopeptidase activity and the estimation of the potency of the
endopeptidase inhibitors. The ATP-stabilized AuNPs (henceforth
referred to as the ‘stabilized AuNPs (sAuNPs)’) and an endoprotease
substrate (1) were used in this assay. Endoprotease hydrolyzed the
* Corresponding author. Tel.: +82 2 820 5198; fax: +82 2 825 4736.
E-mail address: mshan@cau.ac.kr (M.S. Han).
0040-4039/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.02.061

M. H. Kim et al. / Tetrahedron Letters 51 (2010) 2228–2231
2229
thioester bond in the CBZ–Phe–S–Ph substrate (1).5 The hydrolyzed
product (thiophenol) reacted with the sAuNPs, causing them to
aggregate because of the change in the solubility of the sAuNPs,
which in turn resulted in a visible color change in the AuNP solu-
tion (Scheme 1). The color change was used to screen the endopep-
tidase inhibitors and the endoprotease activity.
The AuNP substrates (sAuNPs + 1) were used to evaluate the
enzymatic activity of endopeptidase. In a typical experiment,
known concentrations of a-chymotrypsin were added to a solution
containing the AuNP substrates. The color of the AuNP solution
gradually changed from red to purple upon the hydrolysis of the
thioester in 1 with
a-chymotrypsin. The quantitative analysis of
The stability of sAuNPs to 1 was dependent on the concentra-
the -chymotrypsin-catalyzed hydrolysis of 1 was carried out by
a
tion of ATP and without
a
-chymotrypsin, the thioester reacted
measuring the absorbance of the solution at 680 nm (Fig. 2). The
reaction rate (Kobs) increased with the enzyme concentration, and
this increase was monitored in real time. From these results, the
detection limit for the method with endoprotease was estimated
to be 0.38 nM (see Supplementary data), which was 13-fold better
than the recently reported the AuNP-based colorimetric detection
of endoprotease.3f
with the sAuNPs, causing them to aggregate because the gold sur-
face catalyzed the thioester hydrolysis and then the hydrolysis
product can attach to AuNPs.3f The stability of the sAuNPs to 1
was evaluated at various ATP concentrations in order to avoid
the color change that was induced by 1 in the absence of
trypsin.3f When AuNPs were stabilized at ATP concentrations that
were lower than 10 M in the presence with 1, the surface plas-
a-chymo-
l
In addition to the screening of the enzyme activities, the pro-
posed assay was used to evaluate the inhibitory potency of the
mon resonance (SPR) band of the sAuNPs changed in 30 min. How-
ever, for the AuNPs that were stabilized with ATP concentrations of
a
-chymotrypsin inhibitors. This method has been demonstrated
using chymostatin, a selective and strong -chymotrypsin inhibi-
tor.6 The stock solution of
-chymotrypsin was added to the assay
mixture containing the sAuNPs (3 nM), the substrate (1, 50 M),
more than 20
lM, the SPR band did not change after 30 min
a
(Fig. 1A). Therefore, the AuNP substrates were prepared for the
endoprotease assay by combining 1 (hydrolyzable substrate) with
a
l
the sAuNPs that were stabilized by mixing ATP (30
lM) with the
and various concentrations of chymostatin in a pH 7.0 buffer solu-
tion (10 mM phosphate buffered saline (PBS) + 0.1 M NaCl), with a
final concentration of 0.48 nM. Figure 3 shows the enzymatic activ-
AuNPs (diameter: 13 nm).4 The dispersion of the sAuNPs was con-
firmed using transmission electron microscopy (TEM; Fig. 1B) and
the appearance of the SPR band at 520 nm in the UV–vis spectrum.
ity of a-chymotrypsin in the presence of various concentrations of
However, the sAuNPs aggregated upon the addition of
a
-chymo-
chymostatin. In this figure, the enzymatic activity loss that was
caused by chymostatin was monitored using the change in the
UV–vis absorbance of the solution. From Figure 3, IC50 was about
trypsin to the solution (Fig. 1C), and the SPR band red shifted from
520 to 680 nm (see Supplementary data). Therefore, the thioester
was hydrolyzed by the endopeptidase to produce the correspond-
ing thiol, which then reacted with the sAuNPs.
This reaction caused the aggregation of the sAuNPs, causing the
color change to purple and the red shift in the SPR band. This red
shift is a well-known phenomenon and is used to confirm the for-
mation of the nanoparticle aggregates.1,2 The color change in the
AuNPs was detected by the naked eye, and the extinction coeffi-
cients were measured from the UV–vis spectrum at 680 nm.
26 nM for the inhibition of
a-chymotrypsin by chymostatin (see
Supplementary data). This value was similar to the previously re-
ported value (IC50 = 10 nM).6b
The proposed assay was used to evaluate the relative activities
of various protease inhibitors. In a typical assay,
was added to the control solution and the AuNP solutions contain-
ing one of the following protease inhibitors: chymostatin (1 M,
a-chymotrypsin
l
chymotrypsin-selective inhibitor), N-acetyl-Trp-Glu-His-Asp-al
0.5 µM
1 µM
a)
0.6
3 µM
5 µM
7 µM
10 µM
20 µM
30 µM
50 µM
100 µM
0.5
0.4
0.3
0.2
0.1
0.0
0
200 400 600 800 1000 1200 1400 1600 1800
Time (sec)
Figure 1. (a) Stabilities of the sAuNPs to 1 in the presence of various concentrations of ATP; (b) TEM image of the mixture of the 13 nm ATP-stabilized AuNPs and 1 at pH 7.0;
(c) TEM image of the mixture after the addition of -chymotrypsin. The scale bar represents 200 nm.
a

2230
M. H. Kim et al. / Tetrahedron Letters 51 (2010) 2228–2231
Control
0 nM
0.6
0.5
0.4
0.3
0.2
0.1
0.0
N-Acetyl-Trp-Glu-His-Asp-al
Leupeptin
0.16 nM
0.48 nM
0.64 nM
0.80 nM
0.96 nM
1.28 nM
1.60 nM
0.25
0.20
0.15
0.10
0.05
Pepstatin A
Phosphoramidon
Chymostatin
0
200
400
Time (sec)
600
800
1000
0
200
400
600
800
1000
Time (sec)
Figure 2.
7.0 (10 mM PBS + 0.1 M NaCl; ATP: 30
was monitored at an extinction wavelength of 680 nm. Inset: replot of the initial
a
-Chymotrypsin-catalyzed hydrolysis of 1 (50
lM) to thiophenol at pH
lM) using the AuNPs (3 nM). The hydrolysis
Figure 4. Inhibition of
UV absorbance at 680 nm against time. The increase in the absorbance intensity
was caused by the addition of the endoprotease inhibitor (1 M) to the pH 7.0
M) containing 1 (50 M),
a-chymotrypsin by the endoprotease inhibitors. Plot of the
rate against the
and 1.6 nM).
a-chymotrypsin concentrations (0, 0.16, 0.48, 0.64, 0.8, 0.96, 1.28,
l
buffer solution (10 mM PBS + 0.1 M NaCl; ATP 30
chymotrypsin (0.48 nM), and AuNPs (3 nM).
l
l
a-
0.0 nM
5.0 nM
15 nM
0.25
25 nM
30 nM
50 nM
100 nM
250 nM
500 nM
1000 nM
5000 nM
20000 nM
0.20
0.15
0.10
0.05
0
200
400
600
800
1000
Time (sec)
Figure 5. Color change of the AuNP substrates (1 + sAuNPs) in the absence and
presence of the endoprotease inhibitors (1 M) at specific time intervals after the
addition of -chymotrypsin. (From left to right): control, chymostatin, N-acetyl-
Figure 3. Inhibition of
inhibitor. Plot of the UV absorbance at 680 nm against time. The absorbance
intensity increased after the addition of chymostatin (final concentration of the
a
-chymotrypsin by chymostatin, a selective
a
-chymotrypsin
l
a
assay mixture: 0–20
lM) to the pH 7.0 buffer solution (10 mM PBS + 0.1 M NaCl;
Trp-Glu-His-Asp-al, leupeptin trifluoroacetate salt, pepstatin A, and phosphorami-
ATP: 30 M) containing 1 (50
l
l
M), -chymotrypsin (0.48 nM), and AuNPs (3 nM).
a
don disodium salt.
Scheme 1. Schematic illustration of the aggregation of the AuNP substrates in the colorimetric assay of the endopeptidase activity.

M. H. Kim et al. / Tetrahedron Letters 51 (2010) 2228–2231
2231
(1
(1
l
l
M, cysteine protease inhibitor),7 leupeptin trifluoroacetate salt
M, serine protease inhibitor),8 pepstatin A (1
M, aspartic pro-
M, metal-
grant that was provided by the Korean Ministry of Education Sci-
ence & Technology(MEST) in 2009 (2008-00656).
l
tease inhibitor),9 and phosphoramidon disodium salt (1
l
loprotease inhibitor).10 The inhibitor activity was monitored at
680 nm as a function of time (sample scan rate: 30 sÀ1). The slope
of the plot (rate) was used to measure the inhibition. Figure 4
Supplementary data
Supplementary data (the experimental details for the colori-
metric screening assay, the synthesis of CBZ–Phe–S–Ph (1), the
UV–vis spectrum of the SAuNPs versus time in the presence of a-
shows that only chymostatin effectively inhibited a-chymotrypsin.
High throughput screening (HTS) is widely used to identify the
hit compounds from combinatorial libraries.11 The proposed assay
chymotrypsin, the detection limit of the assay method for
a-chy-
was examined for the HTS of the
catalytic hydrolysis of the substrate (1) in the assay solution with
-chymotrypsin brought about a color change from red to purple
for the solution. Therefore, if -chymotrypsin was successfully
inhibited, the color of the solution remained unchanged. In Figure
5, the inhibition activities of various protease inhibitors for -chy-
a-chymotrypsin inhibitors. The
motrypsin, and the IC50 value of chymostatin for the -chymotryp-
a
sin EDX data of the SAuNPs) associated with this article can be
found, in the online version, at doi:10.1016/j.tetlet.2010.02.061.
a
a
References and notes
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motrypsin were differentiated on the basis of the color change of
the assay solution.
In conclusion, a new colorimetric assay was developed for
screening endoprotease activities and determining the relative
inhibitory potencies of the endoprotease inhibitors by monitoring
the kinetics of the sAuNP aggregation. This screening method
was simpler than the other assays based on the AuNPs and was
used for the easy real-time monitoring of the inhibition potencies
of various endopeptidase inhibitors. Furthermore, this assay was
used for the qualitative and quantitative estimation of the inhibi-
tion. This assay can also be adapted to the HTS of potential drug
candidates from large combinatorial libraries and important bio-
logical endoproteases by simply changing the thiol-bearing
substrate.
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This work was supported by the Korea Research Foundation
Grant funded by the Korean Government (MOEHRD, Basic Research
Promotion Fund) (KRF-2008-331-C00171), the Priority Research
Centers Program through the National Research Foundation of
Korea(NRF) funded by the Ministry of Education, Science and Tech-
nology (2009-0093817), and the Korea Foundation for Interna-
tional Cooperation of Science & Technology(KICOS) through a