Intramolecular long-distance nucleophilic reactions as a rapid fluorogenic switch applicable to the detection of enzymatic activity

Article abstract of DOI:10.1002/chem.201406093

Long-distance intramolecular nucleophilic reactions are promising strategies for the design of fluorogenic probes to detect enzymatic activity involved in lysine modifications. However, such reactions have been challenging and hence have not been established. In this study, we have prepared fluorogenic peptides that induce intramolecular reactions between lysine nucleophiles and electrophiles in distal positions. These peptides contain a lysine and fluor-escence-quenched fluorophore with a carbonate ester, which triggers nucleophilic transesterification resulting in fluorogenic response. Transesterification occurred under mild aqueous conditions despite the presence of a long nine-amino-acid spacer between the lysine and fluorophore. In addition, one of the peptides showed the fastest reaction kinetics with a half-life time of 3.7 min. Furthermore, the incorporation of this fluorogenic switch into the probes allowed rapid fluorogenic detection of histone deacetylase (HDAC) activity. These results indicate that the transesterification reaction has great potential for use as a general fluorogenic switch to monitor the activity of lysine-targeting enzymes.

Full text of DOI:10.1002/chem.201406093

DOI: 10.1002/chem.201406093
Full Paper
&
Fluorescent Probes
Intramolecular Long-Distance Nucleophilic Reactions as a Rapid
Fluorogenic Switch Applicable to the Detection of Enzymatic
Activity
[a]
[a, b, c]
[a, b]
Reisuke Baba, Yuichiro Hori,
and Kazuya Kikuchi*
Abstract: Long-distance intramolecular nucleophilic reac-
tions are promising strategies for the design of fluorogenic
probes to detect enzymatic activity involved in lysine modifi-
cations. However, such reactions have been challenging and
hence have not been established. In this study, we have
prepared fluorogenic peptides that induce intramolecular
reactions between lysine nucleophiles and electrophiles in
distal positions. These peptides contain a lysine and fluor-
mild aqueous conditions despite the presence of a long
nine-amino-acid spacer between the lysine and fluorophore.
In addition, one of the peptides showed the fastest reaction
kinetics with a half-life time of 3.7 min. Furthermore, the in-
corporation of this fluorogenic switch into the probes
allowed rapid fluorogenic detection of histone deacetylase
(HDAC) activity. These results indicate that the transesterifi-
cation reaction has great potential for use as a general
fluorogenic switch to monitor the activity of lysine-targeting
enzymes.
escence-quenched fluorophore with
a carbonate ester,
which triggers nucleophilic transesterification resulting in
fluorogenic response. Transesterification occurred under
[
4]
Introduction
macrocyclization. Thus, it is a great challenge to design the
probes that induce a fluorogenic switch following long-
distance nucleophilic reactions.
Intramolecular nucleophilic reactions have been adopted to
design fluorogenic probes for the detection of enzymatic reac-
tions. In these probes, nucleophiles generated after the reac-
tion with the target enzymes attack internal electrophiles to
Lysine is an important target of post-translational modi-
[5]
fications such as acetylation, methylation, and succinylation.
These reverse reactions are catalyzed by various “eraser”
[
1]
[6]
form five- or six-membered cyclic intermediates. Subsequent
elimination of the cyclic structure in the parent probe results
in the production of a fluorescent molecule, whereas the initial
state of the probe is nonfluorescent. By using this self-immola-
enzymes including histone deacetylases (HDACs), lysine de-
[7]
[8]
succinylases, and lysine demethylases, which play important
roles in diverse pathological processes such as tumorigenesis,
[9,10]
metabolic diseases, and neurodegenerative disorders.
Thus,
[2]
tive reaction, the fluorogenic detection of enzyme activity
fluorogenic probes for the detection of these enzymatic
activities are valuable tools in biological and medical studies.
However, the development of such probes has been
[
3]
and reductive environment has been achieved, showing the
benefit of this mechanism. However, the application of this flu-
orogenic switch to probe design is limited mostly to molecules
with chemical structures that can form the above-mentioned
cyclic intermediates after biological reactions, whereas the use
of such a switch has been scarcely reported for intramolecular
[11]
considered unfeasible because the lysine side chain contains
a simple saturated aliphatic amine and does not allow p-
conjugation of the amino group to a fluorophore for direct
modulation of its electronic state. As a solution to this prob-
lem, a fluorogenic switch based on an intramolecular nucleo-
[12]
philic reaction can be utilized for the probe design, since the
nucleophilic amine in lysine is generated as a result of the
[
a] R. Baba, Dr. Y. Hori, Prof. K. Kikuchi
Graduate School of Engineering
Osaka University, Suita
“
eraser” enzymatic reactions. In this study, we have synthesized
Osaka, 565-0871 (Japan)
Fax: (+81)6-6879-7875
E-mail: kkikuchi@mls.eng.osaka-u.ac.jp
peptides that can induce rapid long-distance intramolecular
reactions between lysine nucleophiles and their distal target
electrophiles and trigger a fluorogenic response.
[
b] Dr. Y. Hori, Prof. K. Kikuchi
Immunology Frontier Research Center
Osaka University, Suita
We have previously applied the intramolecular nucleophilic
reaction to design a fluorogenic probe K4(Ac)-CCB for the
Osaka, 565-0871 (Japan)
[12]
detection of HDAC activity. K4(Ac)-CCB consists of the his-
[c] Dr. Y. Hori
tone H3 (1–9) peptide containing acetyl-lysine as an HDAC
substrate and a fluorescence-quenched coumarin fluorophore
with a carbonate ester (Figure 1a). HDAC activity is detected
as a fluorescence signal produced by an intramolecular trans-
JST, PRESTO
Suita, Osaka, 565-0871 (Japan)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201406093.
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Figure 1. a) A previously developed probe K4(Ac)-CCB and the principle of the fluorogenic switch. The trimethylated lysine at position 9 is marked with an
asterisk; b,c) Structures of the peptides inducing intramolecular transesterification reactions; d) The histone H3 peptide (amino acids 1–17); lysine at position
9
is colored red.
esterification between the HDAC-deacetylated lysine and the
carbonate ester (Figure 1a). However, the peptide sequence in-
ducing the transesterification reaction is currently limited to
K4(Ac)-CCB. This limitation hinders the development of probes
for the detection of enzymes with distinct substrate specificity,
because the activity of most lysine-targeting enzymes depends
on the recognition of long and diverse lysine-neighboring se-
the kinetics of intramolecular transesterification critically affect
the fluorogenic response of a probe, its improvement would
lead to a rapid detection of enzymatic activity. Therefore, the
development of peptide probes with structures that can pro-
vide rapid transesterification is critical for monitoring enzymat-
ic activity. To overcome the limitations and demonstrate
a broad applicability of the fluorogenic switch, we designed
and synthesized a series of lysine-containing peptides that ex-
hibited transesterification, and investigated the effect of the
peptide length on the kinetics of intramolecular transesterifica-
tion. The results indicate that intramolecular reaction occurred
in all the synthetic peptides under mild aqueous conditions
despite the long distance between the lysine residue and fluo-
rophore. Moreover, the optimization of the peptide structure
[
7,13]
quences.
It is therefore essential to clarify whether the pep-
tides containing different or longer spacers between the target
lysine and fluorophore can also induce transesterification. The
variability in spacer amino acid composition would expand the
applicability of the transesterification switch to probe design.
Another problem for K4(Ac)-CCB is that the transesterification
reaction is slow, requiring a long incubation time (2 h). Because
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remarkably improved transesterification kinetics. By using the
optimized peptides, we successfully designed new fluorogenic
probes with rapid fluorogenic response for the detection of
HDAC activity, and applied these probes to evaluate the
potency of an HDAC inhibitor. Overall, our data indicate that
the probes based on the intramolecular transesterification
could be widely used as a general fluorogenic switch for the
detection of various lysine-targeting enzymes.
Results and Discussion
Design of peptides with a fluorogenic switch
To analyze the peptide structures that trigger spontaneous in-
tramolecular transesterification under mild aqueous conditions,
we synthesized 34 lysine-containing peptides conjugated to
coumarin derivatives (Figure 1b,c). In these peptides, coumarin
fluorescence is quenched by the incorporation of a carbonate
ester into its 7-hydroxy group and is expected to be recovered
by transesterification between the carbonate ester and lysine,
as described earlier. A series of target peptides distinct from
K4(Ac)-CCB were designed based on the histone H3 tail se-
quence containing lysine at position 9 (H3K9) and surrounding
regions of different lengths. H3K9 is an important substrate of
enzymatic acetylation/deacetylation or methylation/demethyla-
Figure 2. Reversed-phase HPLC analyses of the intramolecular transesterifica-
tion reactions in LN1 (a,b) and LC8 (c,d). Each peptide (5 mm) was incubated
for 180 min. The absorption wavelength was 305 nm (a,c), and the excita-
tion and emission wavelengths were 338 and 458 nm (b,d), respectively.
were injected into a reversed-phase HPLC column. The chro-
matograms for LN1 and LC8 are shown in Figure 2 and those
for the other peptides in Figure S2 (see the Supporting Infor-
mation). For all the peptides, with the exception of C0, LC0,
N0, and LN0, the initial peaks were diminished within a 3 h in-
cubation; instead, single peaks appeared at different retention
times (Figure 2 and the Supporting Information, Figure S2).
The eluted compounds represented by the newly generated
peaks showed mass values of the transferred products, which
were also the same as those of the intact peptides (the
Supporting Information, Figure S2). The fluorescence chroma-
tograms verified that the peaks observed after the peptide
incubation exhibited fluorescence, whereas those without the
incubation did not. The HPLC-purified compounds obtained
after the incubation with LN6 and LC9, which contained the
longest sequences among the N- and C-type peptides, were
further analyzed using matrix-assisted laser-desorption/ioniza-
tion time-of-flight/time-of-flight mass spectrometry (MALDI-
[
14]
tion for epigenetic regulation of gene expression. In addi-
tion, an enzyme that catalyzes desuccinylation or demalonyla-
[
7]
tion of H3K9 has been recently identified. H3K9 is therefore
a suitable test substrate to monitor transesterification during
the design of fluorogenic probes for the detection of lysine-
targeting enzymatic activity. The peptides with C-terminal cou-
marin, C-type peptides, were designed to contain 0 to 9 amino
acids between H3K9 and coumarin, and were designated as C0
to C9, respectively. Other C-type peptides named LC0–LC9 con-
tained 4 additional residues at the N-terminal side of H3K9 to
examine the influence of the neighboring sequence on trans-
esterification. In all the C-type peptides except C0 and LC0,
glutamine was the C-terminal residue that allowed the intro-
duction of coumarin into the peptides by using a solid-phase
procedure (see the Supporting Information). Similarly, the
peptides with the N-terminal coumarin, N-type peptides, were
designed by introducing 0 to 6 amino acids between H3K9
and coumarin, and LN0–LN6 were also obtained by adding
four extra residues at the C-terminus of H3K9. Lysines other
than K9 were trimethylated to prevent the possibility of
undesirable transesterification reactions. All the peptides
were synthesized using Fmoc solid-phase chemistry (the
Supporting Information, Schemes S2–S5).
[15]
LIFT-TOF/TOF MS). It was demonstrated that transesterifica-
tion in the peptides occurred at lysine 9 (the Supporting Infor-
mation, Figures S3 and S4). It is notable that transesterification
was detected even in LC9 despite the long distance between
lysine 9 and coumarin. C0, LC0, N0, and LN0 also showed trans-
esterification as evidenced by the results of the HPLC and ESI-
MS experiments, but the reaction took nearly 10 h to complete
(
the Supporting Information, Figures S2 and S6). A significant
Intramolecular transesterification in the peptides
difference in the reaction rate suggests that in the peptides
lacking spacer amino acids between lysine9 and coumarin,
steric constraints may influence lysine attack of the carbonate
ester. To investigate the occurrence of intermolecular reactions,
LN1 was mixed with LN1(Ac), an acetylated form of LN1, and
subjected to HPLC analysis. The intermolecular reaction prod-
uct, in which both Lys and coumarin of LN1 were acylated,
The intramolecular transesterification reactions were investi-
gated by high-pressure liquid chromatography (HPLC) and
electrospray ionization mass spectrometry (ESI-MS; see
Figure 2 and the Supporting Information, Figure S2 and Table
S1). After each peptide was incubated at 378C in HEPES·NaOH
+
buffer (20 mm, pH 8.0) containing NAD (300 mm), aliquots
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was not detected (Figure S5 and the discussion in the Support-
ing Information). In addition, whereas the hydrolysis of the
coumarin carbonate ester in LN1(Ac) was observed (the Sup-
porting Information, Figure S5), such a reaction in LN1 was not
detected. Similarly, no hydrolysis product was detected in the
other peptides except C0, LC0, N0, and LN0 (Figure 2, and the
Supporting Information, Figures S2 and S5). These results indi-
cate that rapid intramolecular transesterification precedes car-
bonate ester hydrolysis, which is thus not detectable during
transesterification. Taken together, these results clearly demon-
strate that the intramolecular transesterification occurred in all
the peptides under mild aqueous conditions regardless of the
relative positions of lysine and coumarin. The data also provide
evidence that peptide sequences other than that of K4(Ac)-
CCB can induce transesterification, indicating the versatility of
the lysine-based fluorogenic switch.
Table 1. Transesterification kinetics in the peptides.
2
2
Peptide
kꢁ10
t
1/2
Peptide
kꢁ10
[min
t
1/2
[min]
ꢀ
1
[a]
[b]
ꢀ1 [a]
[b]
[
min
]
[min]
]
[
c]
[d]
[e]
DP
DP
C0
C1
C2
C3
C4
C5
C6
C7
C8
C9
2.13
4.43
n.d.
33
N0
N1
N2
N3
N4
N5
N6
LN0
LN1
LN2
LN3
LN4
LN5
LN6
n.d.
17.4
10.4
6.24
6.47
7.91
5.92
>80
16
>100
39
4.0
6.7
[
d]
[e]
1.78
2.21
2.67
2.23
3.11
4.77
2.38
1.95
3.58
11
11
8.8
12
>80
31
26
31
22
15
29
36
19
[
d]
[e]
n.d.
18.6
12.3
3.7
5.6
6.81
6.75
7.87
6.50
10
10
8.8
11
[
d]
[e]
LC0
LC1
LC2
LC3
LC4
LC5
LC6
LC7
LC8
LC9
n.d.
>100
17
4.20
3.49
3.71
3.32
4.65
4.92
5.26
7.36
4.74
20
19
21
15
14
13
9.4
15
Effect of salt concentration on the transesterification
reaction rate
The analysis of the peptide properties demonstrated that salt
concentration in the reaction buffer affected the kinetics of
transesterification. The reaction rate was evaluated by using
two different reaction buffers: High salt (HEPES·NaOH (20 mm),
[a] All data were obtained in triplicated experiments. k=first-order rate
constant. [b] t1/2 =transesterification half-life time calculated using k value
(
except for C0, LC0, N0, and LN0). [c] k was determined using high-salt
pH 8.0, containing NaCl (150 mm), KCl (2.7 mm), MgCl (1 mm),
2
buffer (HEPES·NaOH (20 mm), pH 8.0, containing NaCl (150 mm), KCl
+
and NAD (300 mm)), which is frequently used in the assays of
+
(
2
2.7 mm), MgCl (1 mm), and NAD (300 mm)). [d] Not determined. [e] t1/2
+
NAD -dependent HDACs, and low salt (HEPES·NaOH (20 mm),
was estimated from the data shown in Figure S6 (the Supporting
Information).
+
pH 8.0, containing NAD (300 mm)). For all the peptides except
C0, LC0, N0, and LN0, intramolecular transesterification was
monitored by fluorescence intensity. The enhancement of the
peptide fluorescence under low-salt conditions was markedly
faster than that under high-salt conditions, although in several
peptides the kinetic differences were slight (the Supporting
Information, Figure S7). These results show that the decrease
in salt concentration in the reaction buffer leads to an increase
in the rate of intramolecular transesterification. Therefore, the
following experiments were conducted under low-salt
conditions, unless otherwise noted.
Kinetic analyses of the intramolecular transesterification
reactions
The detailed comparison of the intramolecular transesterifi-
cation kinetics was performed based on the fluorescence data
shown in Figure S7 (the Supporting Information). Because all
the peptides except C0, LC0, N0, and LN0 induced transesterifi-
cation without any side reaction, the fluorescence data were
fitted to a first-order reaction equation (Table 1, Figure 3, and
the Supporting Information, Figure S8). The transesterification
kinetic constant for DP, the deacetylated product of
K4(Ac)-CCB, was also determined (Table 1, Figure 3, and the
Supporting Information, Figure S9). Among all the peptides,
Figure 3. Changes in the first-order rate constants of intramolecular
transesterification with the number of amino-acid residues between lysine
(
H3K9) and terminal coumarin in the tested peptides. Blue and red bars
represent k for the C- and N-type peptides, respectively. Closed and hatched
bars represent k for the peptides with and without, respectively, 4-amino
acid sequences before or after H3K9. The green bar shows k for DP deter-
mined using low-salt buffer.
showed k values higher than that of DP. In the C-type peptides,
ꢀ
2
ꢀ1
LC8 showed the highest value (k=7.36ꢁ10 min ), even
though the spacer between lysine9 and coumarin contained
as many as eight amino acids. This result contrasts with the
tendency observed for the N-type peptides, when the species
with 1- and 2-amino acid spacers showed better transesterifica-
tion kinetics than those with longer spacers. However, in both
ꢀ
2
ꢀ1
LN1 showed the highest value (k=18.6ꢁ10 min ), which
ꢀ
2
ꢀ1
was 4.2-fold higher than that of DP (k=4.43ꢁ10 min );
under high-salt conditions, the value was 8.7-fold higher than
ꢀ
2
ꢀ1
that of DP (k=2.13ꢁ10 min ; Table 1 and the Supporting In-
formation, Figure S10). Interestingly, all the N-type peptides
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peptide types, spacer elongation did not cause significant de-
crease in the reaction kinetics (Figure 3), which is surprising be-
cause the accessibility of the nucleophile for the intramolecular
electrophile is considered to depend on their mutual distance.
Furthermore, the extra 4-amino acid sequence in the side op-
posite to coumarin tended to accelerate the reaction kinetics:
ꢀ
2
ꢀ1
For example, the rate for LC8 (k=7.36ꢁ10 min ) was 3.8-
ꢀ
2
ꢀ1
fold higher than that for C8 (k=1.95ꢁ10 min ) (Table 1 and
Figure 3). These kinetic data demonstrate that the optimization
of the peptide structures improved the transesterification ki-
netics compared to DP. In addition, these results also indicate
that long amino acid sequences with a lysine in the middle
can be used for fluorogenic switching. These structures can be
applied to the design of fluorogenic probes for the detection
of the enzymes such as histone deacetylases, desuccinylases,
and demethylases that target modified lysines and require
specific
lysine-neighboring
sequences
for
substrate
recognition. Moreover, long spacers may be important when
one needs to determine the amino acid sequence specific for
substrate recognition by the target enzyme.
Design of fluorogenic probes for the detection of HDAC
activity
Fluorogenic probes for the detection of HDAC activity were
designed by applying the transesterification switch of the pep-
tides described above. LN1(Ac) and LC8(Ac), which contain
acetylated Lys, were selected as the substrate regions in the
probes based on the transesterification kinetics (Figure 4): LN1
and LC8 showed the highest k values among the N- and
C-type peptides, respectively. The reactivity of LN1(Ac) and
Figure 5. Reversed-phase HPLC analyses of the enzymatic reaction of
LN1(Ac) and LC8(Ac) with Sirt1. Sirt1 (100 nm) was incubated with 5 mm
LN1(Ac) (a,b) or LC8(Ac) (c,d) in HEPES·NaOH buffer (20 mm, pH 8.0) contain-
ing NAD (300 mm) at 378C for the designated times. The absorption wave-
length was 305 nm (a,c); the excitation and emission wavelengths were 338
and 458 nm, respectively (b,d).
+
[
16]
LC8(Ac) with class III HDAC Sirt1
was investigated using
porting Information, Figures S15 and S16). Thus, these results
indicate that acetyl-lysine in LN1(Ac) was deacetylated by Sirt1.
This peak was diminished after 20 min incubation. HPLC analy-
sis also showed that, similar to LN1 (Figure 2), the peak at the
retention time of 17.8 min increased with incubation and was
fluorescent (Figure 5b). The eluate corresponding to this peak
had the same mass as LN1 and its transferred product (the
Supporting Information, Figure S11). Moreover, MALDI-LIFT-
HPLC and ESI-MS. Following the 10 min incubation of LN1(Ac)
with the enzyme, the peak corresponding to the intact probe
(
retention time=20.0 min) completely disappeared (Figure 5a).
A new peak transiently detected at the retention time of
5.0 min, corresponded to LN1 (Figure 2 and the Supporting
1
Information, Figure S11); this peak was not observed in the
chromatogram of the probe incubated without Sirt1 (the Sup-
Figure 4. Detection of HDAC activity using LN1(Ac) and LC8(Ac).
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TOF/TOF MS clearly detected acylation of the substrate lysine
in the probe (the Supporting Information, Figure S12). Based
on these analyses, this peak can be assigned to the transferred
product of LN1.
The fluorescence intensity of LN1(Ac) increased during incuba-
tion, reaching saturation after 20 min period, which was signifi-
[12]
cantly shorter than that for K4(Ac)-CCB (120 min) (Figure 6a).
For LC8(Ac), the increase in fluorescence intensity was ob-
served up to 60 min after the probe addition (Figure 6b). As
expected from the transesterification kinetics of LN1 and LC8
(Table 1 and Figure 3), saturation for LN1(Ac) was reached
faster than for LC8(Ac). Nevertheless, the incubation time re-
quired for the saturation of fluorescence intensity was much
shorter for LC8(Ac) than for K4(Ac)-CCB. A slight increase in the
fluorescence of each probe in the absence of the enzyme was
due to some hydrolysis of the carbonate ester (Figure 6). To
suppress the hydrolysis, a more stable functional group such
as carbamate ester or imide ester can be utilized instead of
carbonate ester. These structural optimizations are now under
investigation. However, the hydrolysis did not prevent the
clear detection of HDAC activity because the increase in fluo-
rescence due to the enzymatic reaction was considerably
greater than that resulting from the probe hydrolysis. To
confirm that the enzymatic deacetylation of the probes was
caused by Sirt1, the probes were incubated with Sirt1 dena-
tured at 958C for 5 min, which reduced the fluorescence to the
background level (the Supporting Information, Figure S17).
HPLC analyses of these reaction mixtures demonstrated that
no compounds corresponding to the deacetylation products
of the probes were generated (the Supporting Information,
Figure S18). These results demonstrate that the probes can
specifically and rapidly detect Sirt1 activity.
Similar results were obtained for LC8(Ac) (Figure 5c,d). After
the disappearance of the probe peak (retention time=16.8
min), a new peak corresponding to the deacetylated product
(LC8) was transiently generated (retention time=14.0 min).
Subsequently, a fluorescent peak representing the transferred
product of LC8 was detected at the retention time of 14.7 min.
For the Sirt1-free LC8(Ac), these peaks were not observed (the
Supporting Information, Figure S15). Similar to LN1(Ac), each
of the peaks was analyzed by ESI-MS (the Supporting Informa-
tion, Figures S13 and S16) and MALDI-LIFT-TOF/TOF MS (the
Supporting Information, Figure S14). In Figure 5b and 5d, sev-
eral small fluorescent peaks, which most likely corresponded
to reaction side products, were observed. However, these
products were generated at the level below MS detection limit
and therefore were not identified. These results clearly demon-
strate that Sirt1 catalyzes the deacetylation of both LN1(Ac)
and LC8(Ac), triggering subsequent intramolecular transesterifi-
cation that switches on the fluorescence of the probe.
Rapid fluorogenic detection of HDAC activity
The time course for HDAC activity with LN1(Ac) and LC8(Ac) as
substrates was monitored by fluorescence intensity. After
adding LN1(Ac) or LC8(Ac) to Sirt1 solution, fluorescence inten-
sity was recorded at the indicated incubation times (Figure 6).
We also examined the reactivity of the probes with HDAC
isoforms other than Sirt1: HDACs of class I (HDAC1, 2, 3/
NCOR1, and 8), class II (HDAC4, 5, 6, 7, 9, and 10), class III (Sirt2,
[17]
3
, 4, 5, 6, and 7), and class IV (HDAC11). For both probes,
a significant fluorescence enhancement was observed in the
reactions with HDAC3/NCOR1 and Sirt3 (the Supporting
Information, Figures S19 and S20).
Determination of the IC50 value for an HDAC inhibitor
Finally, we applied LN1(Ac), which showed the fastest fluoro-
genic response to enzymatic deacetylation, for a quantitative
evaluation of the potency of a Sirt1 selective inhibitor EX-
[18]
5
27. In this assay, Sirt1 was mixed with the probe in the
presence (1 nm to 20 mm) or absence of EX-527. EX-527 inhibit-
ed Sirt1 activity in a concentration-dependent manner (the
Supporting Information, Figure S21). Using the fluorescence
data, 50% inhibitory concentration (IC50) was calculated as
[19]
0.52 mm, which is within the reported range of 0.1–1 mm.
Thus, LN1(Ac) is demonstrated to be a practical tool to investi-
gate the potency of HDAC inhibitors by a simple procedure.
Conclusion
Figure 6. Time course of a) LN1(Ac) and b) LC8(Ac) fluorescence intensity in
the presence (*) or absence (*) of Sirt1. The reaction was performed with
LN1(Ac) or LC8(Ac) (5 mm) and Sirt1 (100 nm) in HEPES·NaOH buffer (20 mm,
We developed 34 peptides that exhibit a fluorogenic switch
due to intramolecular transesterification. The reaction was trig-
gered by the nucleophilic amine of a lysine residue, leading to
a fluorogenic response. In all the synthesized peptides, the
transesterification proceeded under mild aqueous conditions.
+
pH 8.0) containing NAD (300 mm). Fluorescence was measured at 378C.
The excitation and emission wavelengths were (350ꢁ20) and (450ꢁ20) nm,
respectively.
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The reaction kinetics of the new peptides were greatly
improved compared with the case of a previously developed
peptide DP: One of the peptides showed the highest reaction
rate, with a transesterification half-life time of 3.7 min. Impor-
tantly, the reaction also occurred in the peptides containing
a long 9-amino acid spacer between the lysine residue and flu-
orophore. Although nucleophilic amino acids such as cysteine,
histidine, and tyrosine might affect the intramolecular trans-
esterification, this would not be a serious problem because the
histone tails contain no cysteine or tyrosine, and only one
histidine residue exists at the end of the H4 tail. Based on the
structure of the peptides inducing rapid transesterification, we
designed fluorogenic probes LN1(Ac) and LC8(Ac) for
monitoring HADC activity. The fluorogenic responses of the
new probes after enzymatic deacetylation were much faster
than that of a previously reported probe, K4(Ac)-CCB. We also
demonstrated that LN1(Ac) can be used to evaluate the
potency of an HDAC inhibitor.
and Sirt1-free LC8 (Ac), the probes (5 mm) were incubated in
HEPES·NaOH buffer (20 mm, pH 8.0) containing NAD (300 mm) at
+
3
78C. Aliquots were taken at 0, 20, and 60 min, treated with TFA to
minimize the hydrolysis of the carbonate ester in the probe, and
analyzed by HPLC. Heat-denatured Sirt1 (100 nm) was prepared by
heating at 958C for 5 min and mixed with each of the probes
+
(
(
5 mm) in HEPES·NaOH buffer (20 mm, pH 8.0) containing NAD
300 mm) at 378C. After 90 min of incubation, the mixture was
treated with TFA, and analyzed by HPLC.
Fluorescence spectroscopy
The fluorescence intensity of the peptides and probes was mea-
sured every 5 min using the excitation and emission wavelengths
of (350ꢁ20) and (450ꢁ20) nm, respectively. To investigate the
effect of salt concentration, C1–C9, LC1–LC9, N1–N6, LN1–LN6, and
DP were incubated in high-salt buffer (HEPES·NaOH (20 mm),
+
pH 8.0, NaCl (150 mm), KCl (2.7 mm), MgCl2 (1 mm), and NAD
(300 mm)) or in low-salt buffer (HEPES·NaOH (20 mm), pH 8.0,
+
NAD (300 mm)) at 378C, and the fluorescence intensity was
measured. The fluorescence data were normalized according to
Equation (1):
Currently, two-step enzyme-coupled methods for the
[20]
detection of HDAC activity are commercially available.
In
these methods, peptide sequences in the probe are restricted
because of direct conjugation of the carboxyl group in the
substrate lysine to a fluorophore. In contrast, the fluorogenic
probes developed in this study have a relatively high versatility
in the spacer amino acids between the lysine and fluorophore.
Moreover, the probes detect HDAC activity by using a one-
step procedure, in which the probe is simply mixed with the
enzyme. Thus, the fluorogenic switch based on long-distance
intramolecular transesterification would be useful for the inves-
tigation of the substrate specificity of histone deacetylases,
and will be a valuable tool in the development of HDAC-
targeting drugs.
½
Normalized fluorescence intensityꢂ ¼ ðF ꢀF Þ=ðFmaxꢀF0Þ
ð1Þ
t
0
in which F , F , and F represent observed, maximum, and initial
fluorescence intensity, respectively.
t
max
0
To evaluate the reactivity of LN1(Ac) or LC8(Ac) with class I, II, or IV
HDACs, the probes (5 mm) were incubated with and without
2
00 nm HDAC1 or HDAC2, or 100 nm HDAC3/NCOR1, HDAC4–
HDAC11 in HEPES·NaOH buffer (20 mm, pH 8.0). For class III HDACs
Sirt1–7) each of the probes (5 mm) was incubated with or without
00 nm enzyme in HEPES·NaOH buffer (20 mm, pH 8.0) containing
(
1
+
NAD (300 mm) at 378C, then the fluorescence intensity of the
probe was measured for 90 min. The first-order rate constant, k,
was obtained by fitting the fluorescence data shown in Figure S7
(the Supporting Information) to Equation (2):
Normalized fluorescence intensity ¼ 1ꢀexpðꢀktÞ
ð2Þ
ð3Þ
Experimental Section
The half-life time, t1/2, was calculated using Equation (3):
t1=2 ¼ ln2=k
HPLC analyses
HPLC separation was carried out with an increasing ratio of B
buffer (0.1% HCOOH in acetonitrile) to A buffer (0.1% HCOOH in
H O). All samples were analyzed using a linear gradient of 3–48%
2
B buffer over 30 min. To evaluate the intramolecular transesterifica-
tion, the peptides (5 mm) were incubated in HEPES·NaOH buffer Acknowledgements
+
(
20 mm, pH 8.0) containing NAD (300 mm) at 378C. For C1–C9,
LC1–LC9, N1–N6, and LN1–LN6, aliquots were taken at 0 and
This work was supported by MEXT of Japan (Grants 25220207,
1
0
80 min, mixed with trifluoroacetic acid (TFA; final concentration:
.2%) to stop the reaction, and analyzed by using HPLC. For C0,
26102529, and 25620133 to K.K., 26282215 and 25560403 to
Y.H., and 26·565 to R.B.), by CREST from JST, by JST, PRESTO,
and by Asahi Glass Foundation.
LC0, N0, and LN0, aliquots were taken at 0 min and 18 h
1,080 min), mixed with TFA, and analyzed by HPLC. To investigate
(
the intermolecular reaction, LN1 (5 mm) was incubated with
LN1(Ac) (5 mm) in HEPES·NaOH buffer (20 mm, pH 8.0) containing
NAD (300 mm) at 378C for 60 min. The reaction was stopped by
Keywords: enzymes · fluorescence · fluorescent probes ·
kinetics · peptides
+
TFA, and analyzed by HPLC. The enzymatic reactions were con-
ducted by incubating LN1(Ac) or LC8 (Ac) (5 mm) with Sirt1
+
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Received: November 14, 2014
Published online on && &&, 2015
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&
Fluorescent Probes
R. Baba, Y. Hori, K. Kikuchi*
& – &&
&
Intramolecular Long-Distance
Nucleophilic Reactions as a Rapid
Fluorogenic Switch Applicable to the
Detection of Enzymatic Activity
Fluorogenic switch for lysine: A series
of fluorogenic peptides that contain
a lysine and a fluorescence-quenched
fluorophore were developed. In all the
peptides, intramolecular nucleophilic
transesterification occurred between the
lysine and fluorophore triggering fluo-
rescence (see figure). This fluorogenic
mechanism is useful for detecting the
activity of lysine-targeting enzymes.
Chem. Eur. J. 2015, 21, 1 – 9
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9
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ