A fluorescent probe for detection of histone deacetylase activity based on aggregation-induced emission

Article abstract of DOI:10.1039/c2cc36591j

A tetraphenylethylene-derivative fluorescent probe for the one-step detection of histone deacetylases (HDAC) was developed. The deacetylation of the probe triggers electrostatic interaction between the molecules and automatically leads to fluorescence enhancement based on aggregation-induced emission (AIE). The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2cc36591j

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COMMUNICATION
A fluorescent probe for detection of histone deacetylase activity based on
aggregation-induced emissionw
Koushik Dhara,a Yuichiro Hori,a Reisuke Babaa and Kazuya Kikuchi*ab
Received 11th September 2012, Accepted 11th October 2012
DOI: 10.1039/c2cc36591j
A tetraphenylethylene-derivative fluorescent probe for the one-step
detection of histone deacetylases (HDAC) was developed. The
deacetylation of the probe triggers electrostatic interaction
between the molecules and automatically leads to fluorescence
enhancement based on aggregation-induced emission (AIE).
assay, enzyme deacetylation of the peptides is initiated. In the
second step, the HDAC reaction is detected via trypsin cleavage
of the fluorophore from the peptides. The main limitation of this
assay is its inability to permit continuous monitoring of the
enzyme activity.
In this research, to overcome these problems, a new fluorescent
probe, K(Ac)PS-TPE, was developed, which enables the detection
of the deacetylation activity by simple mixing with HDAC. As a
probe scaffold, tetraphenylethylene (TPE) was employed with a
switching mechanism that is based on aggregation-induced
emission (AIE). A recent breakthrough in the synthesis of
fluorescent organic dyes, which induce AIE phenomena, has
attracted special attention.11 While TPE derivatives are AIE-active
and weakly fluorescent in solution, these dyes become highly
fluorescent upon aggregation.12 In the dilute solution of TPE, fast
rotation of the phenyl rings and partial twisting of the CQC bond
quench its fluorescence.13 On the other hand, in TPE aggregates,
close intermolecular interactions obstruct the rotation of the
phenyl groups resulting in fluorescence enhancement. The unique
luminescence behavior of TPE has been harnessed for the
development of biological sensors,14 solid-state lighting materials,15
and luminescent polymers.16
Epigenetic regulation of gene expression plays an essential role in
development, differentiation, and prevention of diseases.1 The
level of gene expression is modulated by the modification of DNA
or histone proteins without any change of genetic information
encoded in DNA sequences. The reversible acetylation of lysine
residues near the N-terminus of nucleosomal histones by histone
deacetylases (HDACs) and histone acetyltransferases (HATs)
regulates chromatin structure and transcriptional activity.1,2
Histone acetylation catalyzed by HATs generally leads to
transcriptional activation, whereas deacetylation activity executed
by HDACs results in transcriptional repression.3 The activities of
both HATs and HDACs affect various biological phenomena
including angiogenesis,4 apoptosis,5 and the pathogenesis of
malignant diseases.6 In particular, HDACs are attracting
attention, because these enzymes are the major targets of drug
development for diseases such as cancer, neurological diseases,
and metabolic diseases.7 Therefore, the detection of HDAC
activities is key to medical sciences as well as basic biology.
Although tremendous efforts have been made to develop methods
for investigating the deacetylation of HDAC substrates, all of
these methods require multiple laborious processes for the detection
of enzyme activity. Classical assays for the determination of
HDAC activity were based on the incubation of the enzyme
with [3H]acetylated histones8 and peptide substrates.9 The
radio-active assays, however, require the separation of the
product from the substrate, and this process limits the assay
throughput. A non-isotopic HDAC assay utilizing a fluoro-
phore-conjugated peptide was reported in the literature and also
requires a coupled two-step procedure.10 In the first step of the
Considering this fluorescence property of TPE, the design
rationale for the HDAC probe was devised, as illustrated in
Scheme 1. K(Ac)PS-TPE contains N-a-t-butoxycarbonyl-
N-e-acetyl-L-lysine and propane sulphonic acid, which were
attached to TPE. In this design strategy, the sulphonate
moiety was chosen to increase the water solubility and to
serve as a negative terminal charge. In this acetylated state, it
was expected that the probe shows weak fluorescence due to
the lack of aggregation of the probe. The deacetylation of the
probe with HDAC generates KPS-TPE, which possesses
primary aliphatic e-amine of lysine. The amine was protonated
under physiological conditions, as the pKa value was close to
10.5. As a result, the newly generated cationic unit would
trigger the aggregation of the probe (Scheme 1b) owing to the
probable electrostatic interaction among the sulphonate unit
and the cationic N-e-ammonium ion. This phenomenon
prevents the free rotor motions of the phenyl moiety because
of the physical restraints in the aggregated state, and thus,
KPS-TPE becomes highly fluorescent.
a Graduate School of Engineering, Osaka University,
Osaka 565-0871, Japan. E-mail: kkikuchi@mls.eng.osaka-u.ac.jp;
Web: http://www-molpro.mls.eng.osaka-u.ac.jp/;
Fax: +81 6-6879-7875
b Immunology Frontier Research Center, Osaka University,
Osaka 565-0871, Japan
The synthetic route for K(Ac)PS-TPE is shown in Scheme
S1 (ESIw). It involves cross McMurry coupling of benzophenone
and 4,40-diaminobenzophenone resulting in compound 1.
w Electronic supplementary information (ESI) available: Detailed
experimental description, Scheme S1 and Fig. S1–S9. See DOI:
10.1039/c2cc36591j
c
11534 Chem. Commun., 2012, 48, 11534–11536
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20-O-acetyl-ADP-ribose. This protein is implicated in diverse
biological phenomena, including chromatin silencing,22 axonal
degeneration, and cell death.23 After incubation of the probe
with Sirt1 for 3 h, a new peak appeared in the chromatogram
(Fig. 1a). Time-course experiments showed that the enzymatic
reaction was almost completed within 3 h and the reaction
yield was 42% (Fig. 1 and Fig. S3, ESIw). The elutant
corresponding to the peak at the retention time of 13.9 min
was collected and examined by ESI mass spectrometry. The
presence of the expected deacetylated compound KPS-TPE
(m/z, [M + H+]: found 713.28; calculated 713.33) was
confirmed. These results clearly indicated that the probe was
deacetylated by Sirt1. The influence of acetate on the enzyme
reaction was investigated to check whether the back reaction
of KPS-TPE occurred in the presence of acetate, since the
reaction yield did not increase after 3 h. Enzyme reaction was
conducted in the presence of acetate and the reversed-phase
HPLC showed that the acetate had no significant inhibitory
effect on the enzyme activity (Fig. S4, ESIw). The possibility of
enzyme inactivation during the assay was also examined.
Fresh Sirt1 was added to the enzyme reaction mixture, which
has been already incubated for 3 h, and then the enzyme
reaction was checked with reversed-phase HPLC (Fig. S5, ESIw).
No significant progress of the enzyme reaction was observed
after the addition of fresh enzyme. A possible reason for the
imperfect reaction is that the probe, K(Ac)PS-TPE, interacted
with the product, KPS-TPE, and participated in the aggregate
formation and thereby hindered further interaction of the
probe with the enzyme.
Scheme 1 (a) Proposed enzymatic reaction of K(Ac)PS-TPE with
HDAC. (b) Schematic representation of the aggregation-induced
fluorescence enhancement of K(Ac)PS-TPE by HDAC reaction.
N-a-t-Butoxycarbonyl-N-e-acetyl-L-lysine was attached to one
of the amino groups of 1 and 1,3-propanesultone was reacted
with the other amino group to form K(Ac)PS-TPE. First, the
stability of the probe in buffer (pH 8.0) at 37 1C was examined.
The reversed-phase HPLC analyses showed that the probe
displayed sufficient stability in the reaction buffer (pH 8.0) up to
24 h (Fig. S2, ESIw). The progress of the enzymatic reaction
was also monitored by using reversed-phase HPLC (Fig. 1 and
Fig. S3, ESIw). Sirt1 was used as a HDAC for the characterization
of the probe in this research. Sirt1 is a NAD+-dependent
enzyme, which deacetylates various substrates such as
histone,17 p5318 Ku70,19 NF-kB,20 and forkhead proteins.21
For the deacetylation, NAD+ is consumed as a co-substrate to
afford nicotinamide, the deacetylated peptide, and the metabolite
The fluorescence spectra of K(Ac)PS-TPE (10 mM)
were measured at 37 1C in the presence and absence of Sirt1
(500 nM) in HEPES buffer (pH 8.0) containing 500 mM
NAD+ (Fig. 2a). The fluorescence intensity of the probe was
significantly increased in the presence of Sirt1, while only
slight fluorescence enhancement was observed in the absence
of the enzyme. The fluorescence intensity increased after 3 h of
enzyme reaction, though the HPLC indicated the completion
of the reaction. This discrepancy can be explained by the kinetic
difference between the aggregate formation and the enzyme
reaction. It is considered that the fluorescence enhancement was
delayed because the aggregation process was slower than the
enzyme reaction. The influence of NAD+ on the fluorescence
intensity of the probe was also examined. The intensity was
slightly increased after addition of 500 mM of NAD+ due to the
possible non-covalent interaction between the probe and
NAD+ (Fig. S6, ESIw). Basal fluorescence intensity of the
probe in the absence of enzyme was observed due to the
interaction with NAD+. Although NAD+ caused the slight
increase of background fluorescence, the fluorescence intensity
amplified by the enzyme reaction was significantly higher than
the background intensity. Furthermore, the enzymatic reaction
was examined by using inactivated Sirt1 which was achieved by
heating to 100 1C for 5 min. The increase in the fluorescence
intensity of the probe was restrained and almost coincided with
that of the background level (Fig. 2b). These results indicated
that non-specific interaction between the probe and the enzyme
was not observed and that the fluorescence increase in
the enzymatic reaction resulted solely from the formation
of the deacetylated product. From the absorption spectrum
Fig. 1 (a) Reversed-phase HPLC analyses of the reaction progress of
K(Ac)PS-TPE with Sirt1 (500 nM) in reaction buffer (pH 8.0) at 37 1C.
(b) The conversion of K(Ac)PS-TPE to KPS-TPE was plotted against
the progress over time.
c
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Chem. Commun., 2012, 48, 11534–11536 11535

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in inhibitor assays. Thus, this new probe should be valuable to
the field of epigenetics and drug discovery.
This work was supported by MEXT of Japan (Grants
20675004, 24108724, 22 00338 to K.K. and 22685016,
23114710 to Y.H.), by the Funding Program for Word-Leading
Innovative R&D on Science and Tecnology from JSPS, by
CREST from JST, and by Asahi Glass Foundation.
Notes and references
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Fig. 2 (a) Time-dependent fluorescent spectra of the probe (10 mM)
with Sirt1 (500 nM) in the presence of 500 mM of NAD+ in the
reaction buffer (pH 8.0) at 37 1C. The spectra were measured every
30 min after the addition of the enzyme up to 5 h. Excitation
wavelength: 345 nm. (b) Fluorescent intensity of the probe (10 mM)
at 456 nm was plotted against the incubation time. K(Ac)PS-TPE was
incubated in the reaction buffer (pH 8.0) at 37 1C in the presence and
absence of various additives; * corresponds to the Sirt1 inactivated by
heat at 100 1C for 5 min.
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(Fig. S7, ESIw) of the probe after the addition of Sirt1 (500 nM),
it was found that the absorbance at 343 nm was increased. In
contrast, the absorbance remained unchanged in the absence of
Sirt1. The increased absorbance of the enzymatic reaction was
expected to be due to the aggregated state. The fluorescence
excitation maximum (Fig. S8, ESIw) was found at 345 nm,
which coincided with the absorption maximum (343 nm) in this
aggregated state.
The enzymatic reaction in the presence of a potent Sirt1
inhibitor, tenovin-6, was investigated.24 The fluorescent intensity
of the probe with tenovin-6 (1 mM) in the absence and the
presence of Sirt1 (500 nM) was measured. The data showed
that there was no significant difference in the fluorescence
intensity of the probe in both cases (Fig. 2b). Reversed-phase
HPLC analyses (Fig. S9, ESIw) after 5 h of reaction showed
no formation of the deacetylated product. Thus, enzyme
inhibition by tenovin-6 was confirmed. This finding demonstrated
that the probe can be utilized to investigate the inhibitor
activity.
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In conclusion, a fluorescent probe, K(Ac)PS-TPE, for the
detection of Sirt1 activity was designed and synthesized. The
deacetylation of the probe triggers the electrostatic interaction
between the anionic sulphonate and cationic lysine and
automatically leads to fluorescence enhancement based on
AIE. The advantage of this probe is that the enzymatic activity
was distinctly detected by using a one-step procedure, in which
the probe was simply mixed with the enzyme. Since the
fluorescence increase of the probe was restrained in the
presence of an HDAC inhibitor, the probe can also be utilized
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c
11536 Chem. Commun., 2012, 48, 11534–11536
This journal is The Royal Society of Chemistry 2012