Design and synthesis of peptide-MCA substrates for a novel assay of histone methyltransferases and their inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.01.011

Histone methyltransferases (HMTs) play an important role in controlling gene expression through site-specific methylation of lysines in core and linker histones within chromatin. As the typical HMTs, G9a and Set7/9 have been intensively studied that G9a is specific to the methylation at H3K9 and H3K27 and represses transcription, while Set7/9 methylates at H3K4. In this report we prepared various peptide-MCAs (4-methylcoumaryl-7-amides) related to histone tail and protein-substrates such as p53 and estrogen receptor-α. The fluorogenic substrates are applied for the assay of HMTs and an inhibitor, for example. The most sensitive and specific MCA-substrates to G9a and Set7/9 are discovered. The peptide-MCAs corresponding to the methylation sequences are promising for screening of HMT inhibitors.

Full text of DOI:10.1016/j.bmc.2014.01.011

Bioorganic & Medicinal Chemistry 22 (2014) 1268–1275
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of peptide-MCA substrates for a novel assay
of histone methyltransferases and their inhibitors
a
Hongfang Chi a, Yasushi Takemoto b, Tienabe K. Nsiama a, Tamaki Kato a, , Norikazu Nishino ,
⇑
Akihiro Ito b, Minoru Yoshida b
a Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Wakamatsu, Kitakyushu 808-0196, Japan
b Chemical Genomics Research Group, RIKEN Center for Sustainable Resource Science, Saitama 351-0198, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Histone methyltransferases (HMTs) play an important role in controlling gene expression through site-
specific methylation of lysines in core and linker histones within chromatin. As the typical HMTs, G9a
and Set7/9 have been intensively studied that G9a is specific to the methylation at H3K9 and H3K27
and represses transcription, while Set7/9 methylates at H3K4. In this report we prepared various pep-
tide-MCAs (4-methylcoumaryl-7-amides) related to histone tail and protein-substrates such as p53
Received 20 November 2013
Revised 5 January 2014
Accepted 6 January 2014
Available online 21 January 2014
and estrogen receptor-a. The fluorogenic substrates are applied for the assay of HMTs and an inhibitor,
Keywords:
for example. The most sensitive and specific MCA-substrates to G9a and Set7/9 are discovered. The pep-
tide-MCAs corresponding to the methylation sequences are promising for screening of HMT inhibitors.
Ó 2014 Elsevier Ltd. All rights reserved.
Peptide-MCA substrates
4-Methylcoumaryl-7-amides
Histone methyltransferases
Histone methyltransferase inhibitors
1. Introduction
transcription. Methylation is mediated by various histone methyl-
transferases (HMTs). Also it is believed that these enzymes are sig-
Chromatin, the complexes between eukaryotic DNA and pro-
teins, distributes in the nucleus that condenses to form chromo-
somes during cell division. Histones are the major protein of
chromatin which contains a high proportion of basic amino acids
that facilitate binding to the DNA molecule. Five types of histones
are known which are H1, H2A, H2B, H3, and H4 consisting of a
globular carboxyl terminus and linear amino terminus that is
called histone tails.1–3 These tails are controlled by the post-trans-
lational modifications such as acetylation, methylation, phosphor-
ylation, ubiquitination, sumoylation and so on4,5 (Fig. 1). The
functional characterization of histone modifications are believed
to affect cellular processes like gene transcription or expression,
apoptosis, DNA repair, and the regulation of cell cycle.6–10
nificant to unrestricted expression that associates with diseases
such as cancer.10–13 By recent studies, the relation between meth-
ylation marks and etiological role in human disorders had been
known.14–21 As the typical HMTs, G9a (specific to H3K9 and
H3K27) and Set7/9 (specific to H3K4) have been extensively stud-
ied so far.22–26 Thus, the HMT inhibitor is expected as a therapeutic
agent for diseases and to be applied to regenerative medicine using
iPS cells. Accordingly, the convenient assay method of HMTs and
their inhibitors is of an urgent need.
Based on the above understanding, the screening of HMT inhib-
itor has been extensively challenged, though the assay method is
limited by the conventional methods such as radioisotope (RI)
detection and ELISA.27,28 Recently, the Immuno-Bead based assays
were developed, which used AlhpaLISA technology for measuring
the catalytic activity of epigenetic enzymes and their inhibi-
tors.29,30 However, the RI method is limited in safety due to the
use of radioisotope and requiring many experimental procedures
including the absorption of reaction products to filter paper. The
immunoassay methods have the drawbacks such as a number of
experimental procedures like washing procedure, which takes
much time. Since the AlhpaLISA assay depends on the methylation
antibody, the assay system is hard to work well without good anti-
body. Also, it is costly and it needs special device for measure-
ments. In the present study, we attempted to solve above
problems and construct a simple and sensitive evaluation system
The histone methylation of lysine side chain on histone
tails has important roles in regulating chromatin dynamics and
Abbreviations: AcOH, acetic acid; Ac2O, acetic anhydride; AcOEt, ethyl acetate;
AMC, 7-amino-4-methylcoumarin; DCM, dichloromethane; DCC, dicyclohexylcar-
bodiimide; DIEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide;
Fmoc, fluorenylmethyloxycarbonyl; HOBtꢀH2O, 1-hydroxybenzotriazole hydrate;
HBTU, O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate;
MCA, 4-methylcoumaryl-7-amide; HPLC, high performance liquid chromategra-
phy; HRMS (FAB), high resolution fast atom bombardment mass spectroscopy;
TFA, trifluoroacetic acid; TLC, thin-layer chromatography; SAM, S-adenosyl-
methionine.
L-
⇑
Corresponding author. Tel./fax: +81 093 695 6063.
E-mail address: tmkato@life.kyutech.ac.jp (T. Kato).
0968-0896/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2014.01.011

H. Chi et al. / Bioorg. Med. Chem. 22 (2014) 1268–1275
1269
(preparation will be reported elsewhere). They were subjected to
the action of trypsin and LEP (Fig. 2). We used excess amount of
trypsin and LEP to digest for 15 min as described in Section 4.3.1.
These results showed that the addition of trypsin or LEP to
Boc-Lys-MCA increased the fluorescence intensity of AMC
(kex 390 nm/kem 460 nm) in a manner dependent on the concentration
of Boc-Lys-MCA. In details, when trypsin was applied to 1000 lM
Boc-Lys(Me)n-MCA, the fluorescence intensities of AMC released
from Boc-Lys(Me)-MCA, Boc-Lys(Me)2-MCA, and Boc-Lys(Me)3-
MCA (when the fluorescence intensity from Boc-Lys-MCA was set
to 100%) were only 2.7%, 0.12%, and 0.24%, respectively (Fig. 2A).
Figure 1. Post-translational modifications of histone tails. Ac: Acetylation; Me:
Methylation and P: Phosphorylation.
When LEP was used to 1000 lM Boc-Lys(Me)n-MCA, the fluores-
cence intensities of AMC released from Boc-Lys(Me)-MCA,
Boc-Lys(Me)2-MCA, and Boc-Lys(Me)3-MCA (when the fluores-
cence intensity from Boc-Lys-MCA was set to 100%) were only
0.096%, 0.053%, and 0.25%, respectively (Fig. 2B).
Furthermore, we successfully detected the fluorescence from
Boc-Lys(Me)n-MCA (n = 0, 1, 2, and 3) by kex 330 nm/kem 380 nm
without interruption by fluorescence of AMC as shown in
Figure 3A1 and A2. In addition, Boc-Lys(Me)n-MCA and AMC also
could be discriminated (Fig. 3B1 and B2) (Section 4.3.2 gives the
details).
that is different from the past. We designed and synthesized a
number of peptide-4-methylcoumaryl-7-amides (peptide-MCAs)
with lysine at the C-terminal as an index to measure the activities
of HMTs and their inhibitors. Not only that the peptide-Lys-MCA
releases fluorescent 7-amino-4-methylcoumarin (AMC) upon the
action of peptidases such as trypsin and lysyl endopeptidase
(LEP), but also Lys residue could be the methylation substrate of
HMTs. Therefore, we attempted to utilize these actions of enzymes
by combination into a system for convenient assay of HMTs and
their inhibitors.
According to the facts above, though we at first focused on the
decrease of fluorescence intensity of AMC (kex 390 nm/kem 460 nm)
as an index of measuring HMT activity, we found out that the
increase of fluorescence intensity of methylated peptide-MCA
(kex 330 nm/kem 380 nm) can be also measured as HMT activity.
Thus, we designed our assay strategy as shown in Figure 4.
2. Results and discussion
At our earliest stage, in order to confirm the difference of
methylated and unmethylated peptide-MCAs. We prepared
Boc-Lys(Me)n-MCA, where n = 0, 1, 2, and 3 as model compounds
20000
20000
Boc-Lys-MCA
Boc-Lys(Me)-MCA
Boc-Lys(Me)2-MCA
Boc-Lys(Me)3-MCA
Boc-Lys-MCA
Boc-Lys(Me)-MCA
Boc-Lys(Me)2-MCA
Boc-Lys(Me)3-MCA
15000
10000
5000
15000
10000
5000
0
0
0
1
3
10 30 100 300 1000
0
1
3
10
30 100 300 1000
Boc-Lys(Me)n-MCA (μM)
B: LEP
Boc-Lys(Me)n-MCA (μM)
A: Trypsin
Figure 2. Susceptibilities of Boc-Lys-MCA and methylated Boc-Lys-MCA to trypsin and LEP. (A) Trypsin. (B) LEP.
Boc-Lys-MCA
Boc-Lys-MCA
Boc-Lys-MCA40 μM
Boc-Lys-MCA20 μM + AMC 20 μM
AMC 40 μM
Boc-Lys(Me)-MCA
Boc-Lys-MCA40 μM
Boc-Lys-MCA20 μM + AMC 20 μM
AMC 40 μM
Boc-Lys(Me)-MCA
Boc-Lys(Me)2-MCA
Boc-Lys(Me)3-MCA
AMC
Boc-Lys(Me)2-MCA
Boc-Lys(Me)3-MCA
AMC
60000
16000
14000
12000
10000
8000
6000
4000
2000
0
60000
460 nm
16000
14000
12000
10000
8000
6000
4000
2000
0
λex 330 nm
λex 390 nm
380 nm
40000
20000
0
40000
20000
0
λex 330 nm
λex 390 nm
300 350 400 450 500 550 600
350 400 450 500 550 600 650
300 350 400 450 500 550 600
350 400 450 500 550 600 650
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
A1
A2
B1
B2
Figure 3. Fluorescence spectra of Boc-Lys(Me)n-MCA (n = 0, 1, 2, 3) and AMC. (A1 and B1) exited at 330 nm. (A2 and B2) Exited at 390 nm.

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H. Chi et al. / Bioorg. Med. Chem. 22 (2014) 1268–1275
Table 1
Peptide-Lys-MCA
HMTs
Peptide-MCA substrates of HMTs based on histone tail13
Peptide-MCA
Amino acid sequence
Ac-histone H3 (1–4)-MCA
Ac-histone H3 (5–9)-MCA
Ac-histone H3 (1–9)-MCA
Ac-histone H3 (7–9/25–27)-MCA
Ac-histone H3 (23–27)-MCA
Ac-histone H3 (19–27)-MCA
Ac-ARTK-MCA
Ac-QTARK-MCA
Ac-ARTKQTARK-MCA
Ac-ARK-MCA
Ac-KAARK-MCA
Ac-QLATKAARK-MCA
Peptide-Lys(Me)n-MCA
Peptidase
No reaction
Peptide-Lys-MCA
Peptidase
Peptide + AMC
Table 2
Peptide-MCA substrates of HMTs based on methylation sequence in proteins32–35
λex 390 nm
em 460 nm
λex 330 nm
em 380 nm
λ
λ
Peptide-MCA
Amino acid sequence
Ac-p53 (369–372)-MCA
Ac-p53 (367–372)-MCA
Ac-LKSK-MCA
Figure 4. Strategy of HMTs assay using peptide-MCA containing Lys. Note that the
fluorescence of AMC and MCA could be detected by different sets of kex /kem
Ac-SHLKSK-MCA
Ac-KRSK-MCA
Ac-MIKRSK-MCA
Ac-RKLK-MCA
Ac-GARKLK-MCA
Ac-RKTK-MCA
Ac-EARKTK-MCA
.
Ac-ER
Ac-ER
a
a
(299–302)-MCA
(297–302)-MCA
Ac-AR (630–633)-MCA
Ac-AR (628–633)-MCA
Ac-GR (491–494)-MCA
Ac-GR (489–494)-MCA
In the next stage, we applied Boc-Lys-MCA for HMT assay using
GST-mG9a and trypsin. S-Adenosyl-
-methionine (SAM)31 was also
L
employed (Fig. 5A). Figure 5B shows that the fluorescence intensi-
ties of AMC released by trypsin decreased in order of the
GST-mG9a concentrations. Though the increasing concentration
of Boc-Lys-MCA, from 0.009 to 0.09 mM, reflected fluorescence
intensities, there observed was almost no difference in relative
intensities according to the GST-mG9a concentrations. The concen-
tration of SAM was examined for the effect in the methylation
reaction. 1 mM concentration gave the lowest intensity, which
means the highest activity of GST-mG9a (Fig. 5C).
ER
a
, estrogen receptor
a
; AR, androgen receptor; GR, glucocorticoid receptor.
In the third stage, we designed and synthesized a series of pep-
tide-MCAs containing Lys at the C-terminal that were chosen from
the amino acid sequences of histone tail and some methylated pro-
teins such as p53 (Tables 1 and 2). We adopted G9a and Set7/9 as
HMTs (Figs. 6 and 7). The results given in Figure 6A show that most
Boc-Lys-MCA
SAM
GST-mG9a
SAH
Boc-Lys(Me)-MCA
Trypsin
Boc-Lys(Me)2-MCA
Boc-Lys-MCA
(remained)
Trypsin
Boc-Lys + AMC
Trypsin
No reaction
No reaction
A
Boc-Lys-MCA
0.09 mM
Boc-Lys-MCA
0.03 mM
Boc-Lys-MCA
0.009 mM
-G9a
+G9a
1500
3000
2000
1000
0
1000
500
0
GST-mG9a
Relative
intensity of
fluorescence
(% of control)
100 61 52 27 100 66 53 29 100 70 60
32
0
0.01 0.03
0.1
0.3
1
GST-mG9a 0, 0.015, 0.05, 0.15 μg/μL
SAM (mM)
B
C
Figure 5. Measurements of G9a activity using Boc-Lys-MCA. (A) Outline of measuring G9a activity. (B) Dependences of concentrations of Boc-Lys-MCA and G9a.
(C) Dependence of SAM concentration.

H. Chi et al. / Bioorg. Med. Chem. 22 (2014) 1268–1275
1271
of peptide-MCA based on histone tail except Ac-histone H3 (1-4)-
MCA are susceptible to G9a. On the other hand, Set7/9 methylates
Ac-histone H3 (1–4)-MCA selectively (Fig. 6 gives data of Ac-p53
(369–372)-MCA for comparison). Figure 7 shows the results of
action of G9a and Set7/9 to peptide-MCA based on sequences
of methylated proteins. The G9a exhibited no methylation of
120
100
80
60
40
20
0
140
120
100
80
60
40
20
0
0
0.05
0.1
0.15
0
0.5
1
1.5
His-Set7/9 (μg/μL)
GST-mG9a (μg/μL)
A
B
Figure 6. Susceptibilities of peptide-MCAs based on histone tail sequences to HMTs. (A) G9a. (B) Set7/9.
120
100
80
60
40
20
0
140
120
100
80
60
40
20
0
0
0.1
0.2
0.3
0.4
0.5
0.6
0
0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16
His-Set7/9 (μg/μL)
GST-mG9a (μg/μL)
A
B
Figure 7. Susceptibilities of peptide-MCAs based on methylation sequence in proteins to HMTs. (A) G9a. (B) Set7/9.
1200
5000
2000
1500
6000
4000
2000
800
400
4000
2000
1000
500
0
0
0
0
0
0.005 0.015 0.05 0.15
0
0.005 0.015 0.05 0.15
0
0.005 0.015 0.05 0.15
0
0.005 0.015 0.05 0.15
His-Set7/9 (μg/μL)
GST-mG9a (μg/μL)
GST-mG9a (μg/μL)
His-Set7/9 (μg/μL)
A1
A2
B1
B2
Figure 8. Measurements of HMTs activities in different sets of kex/kem. (A1 and A2) G9a activity (Ac-ARTKQTARK-MCA) by measuring AMC (kex/kem: 390 nm/460 nm) and
methylated peptide-MCA (kex/kem: 330 nm/380 nm), respectively. (B1 and B2) Set7/9 activity (Ac-KRSK-MCA) by measuring AMC (kex/kem: 390 nm/460 nm) and methylated
peptide-MCA (kex/kem: 330 nm/380 nm), respectively.

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H. Chi et al. / Bioorg. Med. Chem. 22 (2014) 1268–1275
-
+GST-mG9a
-
+His-Set7/9
1200
1000
800
2000
1500
1000
500
100
80
60
40
20
Set7/9
G9a
600
400
200
0
0
0
1
10
100
0
0
1
3
10 30 100
0
0
1
10
100
-20
Gliotoxin (μM)
Gliotoxin (μM)
Gliotoxin (μM)
A
B
C
Figure 9. Evaluation of an HMT inhibitor (gliotoxin) with peptide-MCAs. (A) G9a with Ac-ARTKQTARK-MCA. (B) Set7/9 with Ac-LKSK-MCA. (C) The rates (%) of methylation
inhibition by gliotoxin.
peptide-MCA based on proteins. The data of Ac-histone H3 (1–9)-
MCA is shown as reference in Figure 7A. On the contrary, most of
peptide-MCA from proteins are susceptible to Set7/9 (Fig. 7B).
demonstrated by the measurement of inhibitory activity of gliotox-
in. These substrates are convenient indicators for screening of
HMTs inhibitors.
Among them, the most sensitive one is Ac-ER
According to the above results, the peptide-MCA derived from his-
tone H3 (1–9) is specific for G9a and ER (299–302) is somewhat
a (299–302)-MCA.
4. Experimental
a
selective for Set7/9. Thus, we could figure out the substrate speci-
ficities by using peptide-MCAs. (Section 4.3.4 gives the details.)
As the strategy described previously in Figure 4, the decrease of
fluorescence intensity of AMC could be an index at kex 390 nm/kem
460 nm, and the increase of fluorescence intensity of methylated
peptide-MCA could be reference at kex 330 nm/kem 380 nm. Actu-
ally Figure 8 shows the results of the assays of G9a and Set7/9
4.1. General methods
Unless otherwise noted, all solvents and reagents were reagent
grade obtained from WAKO, Japan. Flash chromatography was per-
formed using silica gel 60 (230–400 mesh) eluting with solvents as
indicated. All compounds were routinely checked by thin layer
chromatography (TLC) and/or high performance liquid chromatog-
raphy (HPLC). TLC was performed on aluminum-backed silica gel
plates (Merck DC-Alufolien Kieselgel 60 F254) with spots visualized
by UV light or charring.
using peptide-MCAs (Ac-histone H3 (1–9)-MCA and Ac-ER
a
(299–302)-MCA) at kex 390 nm/kem 460 nm to measure AMC, kex
330 nm/kem 380 nm to measure peptide-MCA, respectively. (The
experimental and evaluation details were not shown.) This could
eliminate some misunderstandings, such as the unclearly distin-
guish of fluorescence signal whether HMTs works well or the fluo-
rescence just quenched accidentally.
In summary, we confirmed the substrate specificities of HMTs
regarding the amino acid sequences. We discovered that
Ac-ARTKQTARK-MCA (Ac-histone H3 (1–9)-MCA), the longest pep-
tide is the most specific and susceptible substrate of G9a. However,
Set7/9 methylated moderately histone tail-related peptide-MCA,
Analytical HPLC was performed on a Hitachi L-7100 instrument
equipped with
a chromolith performance RP-18e column
(4.6 ꢁ 100 mm, Merck). The mobile phases used were A: H2O with
0.1% TFA, B: CH3CN with 0.1% TFA using a solvent gradient of A–B
over 15 min with a flow rate of 2 mL/min, with detection at
220 nm. HRMS (FAB) were measured on a JEOL JMS-SX 102A
instrument.
Peptide-MCAs were synthesized by conventional solution
method or automated solid phase peptide synthesis (SPPS). Some
protected peptide fragments were assembled on Barlos resin (sub-
stitution 0.7 mmol/g) by peptide synthesizer (Applied Biosystems
433A) using Fmoc/piperidine strategies in FastMoc Chemistry at
0.25 mmol scale. HBTU and HOBtꢀH2O were used as activating
agent. After capping the N-terminal with acetic anhydride, the
protected peptides were cleaved from the resin in DCM containing
TFE and AcOH for 2 h. The protected peptides were condensed with
H-Lys(Boc)-MCA. All protecting groups were removed by the
treatment with TFA containing phenol (5%), thioanisole (5%), eth-
anedithiol (2.5%) and water (5%). The peptides were precipitated
by tert-butyl methyl ether. Crude peptides were purified by size
exclusion chromatography performed on a column of Sephadex
G-25 with 10% AcOH. The purified peptide-MCAs were analyzed
by HRMS (FAB). HPLC profiles and HRMS (FAB) data are provided
in Supplementary data.
but showed higher activity to p53 and estrogen receptor
sequences; Ac-KRSK-MCA (Ac-ER (299–302)-MCA) from ER
was the best substrate of Set7/9.
At the final stage, we carried out the evaluation of gliotoxin,36
known G9a inhibitor by this new assay method using Ac-ARTKQT-
a
a
a
a
ARK-MCA and obtained IC50 as 2.8 lM, close to the reported
value37 (Fig. 9). When Ac-LKSK-MCA was used to examine the
Set7/9 inhibition by gliotoxin, this compound showed no inhibi-
tory activity (>100 lM) as expected to be specific to G9a.
3. Conclusion
We have revisited the action of trypsin and LEP to
Boc-Lys(Me)n-MCA, where n = 0, 1, 2, and 3, and confirmed that
methylated Boc-Lys(Me)n-MCA (n >1) show very poor to null sus-
ceptibility to the key hydrolytic enzymes. As sensitive fluorogenic
substrates of HMTs we have designed a series of peptide-MCAs
whose sequences are selected from the known methylation points
in histone and other proteins. The most sensitive and specific sub-
strates to G9a (Ac-ARTKQTARK-MCA) and Set7/9 (Ac-KRSK-MCA)
are discovered. The usefulness of the new assay method was
4.2. Synthesis of peptide-MCAs
4.2.1. Ac-ARTK-MCA
To a chilled solution of Fmoc-Lys(Boc)-OH (1.86 g, 4 mmol) in
DCM (10 mL) was added DCC (0.42 g, 2 mmol). After 1 h on ice

H. Chi et al. / Bioorg. Med. Chem. 22 (2014) 1268–1275
1273
bath, AMC (0.36 g, 2 mmol) was added to the mixture. The suspen-
sion was stirred at room temperature overnight, then the product
was filtered and washed with methanol. Fmoc-Lys(Boc)-MCA was
obtained as crystalline solid (1.13 g, 90%). Fmoc-Lys(Boc)-MCA
(1.13 g, 1.8 mmol) was dissolved in 20% piperidine/DMF (5 mL) at
room temperature. After 30 min the reagent and solvent were re-
moved by evaporation. The residues were extracted into AcOEt,
washed with sat. Na2CO3, and dried over anhydrous Na2CO3. The
filtrate was concentrated to yield H-Lys(Boc)-MCA as free amine
(0.73 g, 100%). Fmoc-Thr(tBu)-OH (119 mg, 0.3 mmol) and H-Lys(-
Boc)-MCA (121 mg, 0.3 mmol) were condensed by the aid of HBTU
(125 mg, 0.33 mmol), HOBtꢀH2O (50 mg, 0.33 mmol) and DIEA
(0.06 mL, 0.33 mmol) in DMF for 2 h. The product was extracted
into AcOEt, washed with 10% citric acid, 4% NaHCO3, and brine.
After dried over MgSO4, the AcOEt solution was filtrated and con-
centrated to give Fmoc-Thr(tBu)-Lys(Boc)-MCA as white solid
(384 mg, 87%). The protected dipeptide-MCA was treated with
20% piperidine/DMF as described above. The free amine H-Thr
(tBu)-Lys(Boc)-MCA was obtained (140 mg, 100%). Fmoc-
Arg(Pmc)-OH (165 mg, 0.25 mmol) and H-Thr(tBu)-Lys(Boc)-MCA
(140 mg, 0.25 mmol) were condensed by the same procedure
Sephadex G-25 column with 10% AcOH, and lyophilization gave
20 mg of Ac-ARTKQTARK-MCA_(AcOH)4. HRMS (FAB) m/z found:
1258.6858, calcd for [M+H]+ C55H92N19O15: 1258.7020.
4.2.4. Ac-ARK-MCA
This compound was synthesized starting from Fmoc-Lys
(Boc)-MCA by solution method as described above. Deprotection
of Ac-Ala-Arg(Pmc)-Lys(Boc)-MCA (66 mg), purification, and
lyophilization gave 32 mg of Ac-ARK-MCA_(AcOH)2. HRMS (FAB)
m/z found: 573.3113, calcd for [M+H]+ C27H41N8O6: 573.3149.
4.2.5. Ac-KAARK-MCA
The protected peptide Ac-Lys(Boc)-Ala-Ala-Arg(Pmc)-OH was
prepared by assembling on Barlos resin, capping with Ac2O, and
cleavage as described above. This fragment was condensed
with H-Lys(Boc)-MCA to obtain Ac-Lys(Boc)-Ala-Ala-Arg(Pmc)-Lys
(Boc)-MCA. Deprotection of fully protected peptide (60 mg), purifi-
cation, and lyophilization gave 15 mg of Ac-KAARK-MCA_(AcOH)3.
HRMS (FAB) m/z found: 772.4436, calcd for [M+H]+ C36H58N11O8:
772.7740.
described above. After purification with
a
silica gel column
4.2.6. Ac-QLATKAARK-MCA
Fmoc-Arg(Pmc)-Thr(tBu)-Lys(Boc)-MCA was obtained as white
solid (238 mg, 82%). The protected tripeptide-MCA was
treated with 20% piperidine/DMF as described above. The free
amine H-Arg(Pmc)-Thr(tBu)-Lys(Boc)-MCA was obtained (197 mg,
100%). Fmoc-Ala-OH (62 mg, 0.2 mmol) and H-Arg(Pmc)-Thr
(tBu)-Lys(Boc)-MCA (197 mg, 0.2 mmol) were condensed by the
same procedure described above. After silica gel column purifica-
tion, Fmoc-Ala-Arg(Pmc)-Thr(tBu)-Lys(Boc)-MCA was obtained as
white solid (168 mg, 70%). Fmoc-Ala-Arg(Pmc)-Thr(tBu)-Lys(Boc)-
MCA (84 mg, 0.07 mmol) was treated with 20% piperidine/DMF
as described above. The free amine H-Ala-Arg(Pmc)-Thr(tBu)-Lys(-
Boc)-MCA was obtained (75 mg, 100%), which was reacted with
Ac2O (0.08 mmol) and NEt3 (0.08 mmol) in DMF for 2 h. In the
work-up step, the product was crystallized in AcOEt. Finally the
deprotection of Ac-Ala-Arg(Pmc)-Thr(tBu)-Lys(Boc)-MCA (76 mg)
was carried out with TFA reagent containing phenol, thioanisole,
ethanedithiol, and water as described before for 2 h. Concentration
and addition of tert-butyl methyl ether (20 mL) gave white
precipitate, which was collected by centrifugation and washed
with same solvent. The crude material was passed through a col-
umn of Sephadex G-25 with 10% AcOH. Lyophilization gave
42 mg of Ac-ARTK-MCA_(AcOH)2. HRMS (FAB) m/z found:
674.3607, calcd for [M+H]+ C31H48N9O8: 674.3626.
The protected peptide Ac-Gln(Trt)-Leu-Ala-Thr(tBu)-Lys(Boc)-
Ala-Ala-Arg(Pmc)-OH was prepared by assembling on Barlos resin,
capping with Ac2O, and cleavage as described above. This fragment
was condensed with H-Lys(Boc)-MCA to obtain Ac-Gln(Trt)-Leu-
Ala-Thr(tBu)-Lys(Boc)-Ala-Ala-Arg(Pmc)-Lys(Boc)-MCA. Deprotec-
tion of fully protected peptide (136 mg), purification, and
lyophilization gave 28 mg of Ac-QLATKAARK-MCA_(AcOH)3. HRMS
(FAB) m/z found: 1185.6716, calcd for [M+H]+ C54H89N16O14
1185.6744.
:
4.2.7. Ac-LKSK-MCA
This compound was synthesized starting from Fmoc-Lys(Boc)-
MCA by solution method as described above. Deprotection of Ac-
Leu-Lys(Boc)-Ser(tBu)-Lys(Boc)-MCA (87 mg), purification, and
lyophilization gave 25 mg of Ac-LKSK-MCA_(AcOH)2. HRMS (FAB)
m/z found: 674.3658, calcd for [M+H]+ C31H48N9O8: 674.3626.
4.2.8. Ac-SHLKSK-MCA
The protected peptide Ac-Ser(tBu)-His-Leu-Lys(Boc)-Ser(tBu)-
OH was prepared by assembling on Barlos resin, capping with
Ac2O, and cleavage as described above. This fragment was con-
densed with H-Lys(Boc)-MCA to obtain Ac-Ser(tBu)-His-Leu-
Lys(Boc)-Ser(tBu)-Lys(Boc)-MCA. Deprotection of fully protected
peptide (120 mg), purification, and lyophilization gave 32 mg of
Ac-SHLKSK-MCA_(AcOH)2. HRMS (FAB) m/z found: 898.4748, calcd
for [M+H]+ C42H64N11O11: 898.4709.
4.2.2. Ac-QTARK-MCA
This compound was synthesized starting from Fmoc-Lys(Boc)-
MCA by solution method as described above. Deprotection of
Ac-Gln(Trt)-Thr(tBu)-Ala-Arg(Pmc)-Lys(Boc)-MCA (57 mg), purifi-
cation, and lyophilization gave 26 mg of Ac-QTARK-MCA_(AcOH)2.
4.2.9. Ac-KRSK-MCA
This compound was synthesized starting from Fmoc-Lys(Boc)-
MCA by solution method as described above. Deprotection of Ac-
Lys(Boc)-Arg(Pmc)-Ser(tBu)-Lys(Boc)-MCA (930 mg), purification,
and lyophilization gave 500 mg of Ac-KRSK-MCA_(AcOH)3. HRMS
(FAB) m/z found: 717.4039, calcd for [M+H]+ C33H53N10O8:
717.3770.
HRMS (FAB) m/z found: 802.4196, calcd for [M+H]+ C36H56N11O10
:
802.4212.
4.2.3. Ac-ARTKQTARK-MCA
The protected peptide Ac-Ala-Arg(Pmc)-Thr(tBu)-Lys(Boc)-
Gln(Trt)-Thr(tBu)-Ala-Arg(Pmc)-OH was prepared by assembling
on Barlos resin by peptide synthesizer, acetylation at the N-termi-
nal with Ac2O, and subsequent cleavage from the resin. This frag-
ment (428 mg, 0.22 mmol) and H-Lys(Boc)-MCA (88 mg, 0.22
mmol) were condensed by the aid of HBTU (90 mg, 0.24 mmol),
HOBtꢀH2O (36 mg, 0.24 mmol) and DIEA (0.04 mL, 0.24 mmol) in
DMF for 2 h. The product was extracted into AcOEt. The work-up
4.2.10. Ac-MIKRSK-MCA
The protected peptide Ac-Met-Ile-Lys(Boc)-Arg(Pmc)-Ser(tBu)-
OH was prepared by assembling on Barlos resin, capping with
Ac2O, and cleavage as described above. This fragment was con-
densed with H-Lys(Boc)-MCA to obtain Ac-Met-Ile-Lys(Boc)-
Arg(Pmc)-Ser(tBu)-Lys(Boc)-MCA. Deprotection of fully protected
peptide (97 mg), purification, and lyophilization gave 28 mg of
Ac-MIKRSK-MCA_(AcOH)3. HRMS (FAB) m/z found: 961.5269, calcd
for [M+H]+ C44H73N12O10S: 961.5215.
afforded
Ac-Ala-Arg(Pmc)-Thr(tBu)-Lys(Boc)-Gln(Trt)-Thr(tBu)-
Ala-Arg(Pmc)-Lys(Boc)-MCA as white solid (470 mg, 90%).
Deprotection of this final precursor (50 mg) and purification by

1274
H. Chi et al. / Bioorg. Med. Chem. 22 (2014) 1268–1275
4.2.11. Ac-RKLK-MCA
4.3.3. Measurement of G9a activity with Boc-Lys-MCA
This compound was synthesized starting from Fmoc-Lys(Boc)-
MCA by solution method as described above. Deprotection of
Ac-Arg(Pmc)-Lys(Boc)-Leu-Lys(Boc)-MCA (80 mg), purification,
and lyophilization gave 30 mg of Ac-RKLK-MCA_(AcOH)3. HRMS
(FAB) m/z found: 744.4380, calcd for [M+H]+ C36H58N9O8:
744.4408.
GST-fused mG9a (706-stop a.a.) and (His)6-fused Set7/9 were
obtained as previously described.37 10
l
L of 2 ꢁ HMT buffer, 2
g/mL), and a predetermined concentration
(final concentration: 0, 0.015, 0.05, or 0.15 g/ L) of the G9a solu-
tion (total volume to 16 L) to adjust the total volume to 16 L.
Then the resultant solution was incubated at room temperature
for 1 h. Thereafter, 2 L of SAM (10 mM) and 2 L of Boc-Lys-
lL
of a BSA solution (30
l
l
l
l
l
l
l
4.2.12. Ac-GARKLK-MCA
MCA (concentration: 0.009 mM, 0.03 mM, or 0.09 mM) were added
The protected peptide Ac-Gly-Ala-Arg(Pmc)-Lys(Boc)-Leu-OH
was prepared by assembling on Barlos resin, capping with Ac2O,
and cleavage as described above. This fragment was condensed
with H-Lys(Boc)-MCA to obtain Ac-Gly-Ala-Arg(Pmc)-Lys(Boc)-
Leu-Lys(Boc)-MCA. Deprotection of fully protected peptide
(107 mg), purification, and lyophilization gave 42 mg of Ac-GAR-
KLK-MCA_(AcOH)3. HRMS (FAB) m/z found: 871.5152, calcd for
[M+H]+ C41H67N12O9: 871.5076.
thereto, which was mixed and then incubated at 37 °C for 15 min.
Subsequently, 30 lL of a trypsin solution (20 mg/mL) was added
thereto, followed by incubation at 37 °C for 15 min. The fluores-
cence intensity of the solution was measured at kex 390 nm/kem
460 nm.
4.3.4. Substrate specificity of HMTs
Susceptibilities of peptide-MCAs to G9a were examined in the
following conditions. 10
tion (150 g/mL), 4.5 L of GST-mG9a (0, 0.022–0.66
1.1 L of distilled water were mixed and then incubated at room
temperature for 1 h. Then, 2 L of SAM (10 mM) and 2 L of a pep-
tide-MCA solution (0.6 mM) were added thereto, which was then
incubated at 37 °C for 15 min. Thereafter, 30 L of a trypsin solu-
tion (20 mg/mL) was added thereto, followed by incubation at
37 °C for 15 min. Then, the fluorescence intensity of the solution
was measured at kex 390 nm/kem 460 nm. The final concentration
of GST-mG9a during the measurement of fluorescence intensity
l
L of 2 ꢁ HMT buffer, 0.4
l
L of a BSA solu-
4.2.13. Ac-RKTK-MCA
l
l
lg/lL), and
The protected peptide Ac-Arg(Pmc)-Lys(Boc)-Thr(tBu)-OH was
prepared by assembling on Barlos resin, capping with Ac2O, and
cleavage as described above. This fragment was condensed with
H-Lys(Boc)-MCA to obtain Ac-Arg(Pmc)-Lys(Boc)-Thr(tBu)-Lys
(Boc)-MCA. Deprotection of fully protected peptide (100 mg), puri-
fication, and lyophilization gave 44 mg of Ac-RKTK-MCA_(AcOH)3.
HRMS (FAB) m/z found: 731.4197, calcd. for [M+H]+ C34H55N10O8:
731.4126.
l
l
l
l
was 0, 0.005, 0.015, 0.05, or 0.15
lg/lL.
4.2.14. Ac-EARKTK-MCA
Susceptibilities of peptide-MCAs to Set7/9 were examined in
The protected peptide Ac-Glu(OtBu)-Ala-Arg(Pmc)-Lys(Boc)-
Thr(Trt)-OH was prepared by assembling on Barlos resin, capping
with Ac2O, and cleavage as described above. This fragment was
condensed with H-Lys(Boc)-MCA to obtain Ac-Glu(OtBu)-Ala-
Arg(Pmc)-Lys(Boc)-Thr(Trt)-Lys(Boc)-MCA. Deprotection of fully
protected peptide (98 mg), purification, and lyophilization gave
37 mg of Ac-EARKTK-MCA_(AcOH)3. HRMS (FAB) m/z found:
931.5050, calcd for [M+H]+ C42H67N12O12: 931.4923.
the following conditions. 10
l
L of 2 ꢁ HMT buffer, 2
l
l
L of a BSA
g/ L), and
solution (30 g/mL), 3 L of His-Set7/9 (0, 0.33–10
l
l
l
1
l
L of distilled water were mixed and then incubated at room
temperature for 1 h. Then, 2 L of SAM (10 mM) and 2 L of a pep-
tide-MCA solution (0.6 mM) were added thereto, which was then
incubated at 37 °C for 15 min. Thereafter, 30 L of a trypsin solu-
l
l
l
tion (20 mg/mL) was added thereto, followed by incubation at
37 °C for 15 min. Then, the fluorescence intensity of the solution
was measured at kex 390 nm/kem 460 nm. The final concentration
of His-Set7/9 during the measurement of fluorescence intensity
4.3. Assays of enzymes
was 0, 0.005, 0.015, 0.05, or 0.15 lg/lL.
4.3.1. Loss of susceptibility to peptidases by methylation of Boc-
Lys-MCA
4.3.5. Inhibition of HMTs by gliotoxin
10 L of a BSA solution (150
L of 2 ꢁ HMT buffer, 0.4
of GST-mG9a (0, 0.5 g/ L) or 4.5 of His-Set7/9
(0, 2.25 g/ L), 1 L of gliotoxin (any various concentrations) and
2.6 L of distilled water were mixed and then incubated at room
temperature for 1 h. Then, 2 L of SAM (10 mM) and 2 L of
Ac-ARTKQTARK-MCA solution (0.6 mM) for G9a or Ac-LKSK-MCA
solution (0.6 mM) for Set7/9 were added thereto, which was then
incubated at 37 °C for 1 h. Thereafter, 30 lL of a trypsin solution
(20 mg/mL) was added thereto, followed by incubation at 37 °C
for 15 min. Then, the fluorescence intensity of the solution was
measured at kex 390 nm/kem 460 nm. The final concentrations of
GST-mG9a and His-Set7/9 during the measurement of fluorescence
1 lL of a Boc-Lys-MCA solution was mixed with 10 lL of 2
l
l
l
lg/mL),
ꢁ HMT buffer (100 mM Tris–HCl (pH 8.5), 20 mM MgCl2, 40 mM
2
l
L
l
lL
KCl, 20 mM 2-mercaptoethanol, 500 mM sucrose), and distilled
l
l
l
water was added to adjust the total volume to 20
lL. To the resul-
l
tant solution was added 30 L of a peptidase solution (20 mg/mL
l
l
l
trypsin or 20 mAU/mL LEP), which was then mixed and incubated
at 37 °C for 15 min. Then, the fluorescence intensity of the solution
was measured at kex 390 nm/kem 460 nm with a SpectraMax M2e
microplate reader (Molecular Devices).
4.3.2. Fluorescence spectra of Boc-Lys-MCA and AMC
2
l
L of Boc-Lys-MCA (2 mM) and 48
mixed to prepare 50 L of a solution (Boc-Lys-MCA 40
of Boc-Lys-MCA (2 mM), 1 L of AMC (2 mM), and 48 L of distilled
water were also mixed to prepare 50 L of a solution (Boc-
Lys-MCA 20 M + AMC 20 M). 2 L of AMC (2 mM) and 48 L of
distilled water were mixed to prepare 50 L of a solution (AMC
40 M). 1 L of Boc-Lys(Me)-MCA (2 mM) and 49 L of distilled
water were mixed to prepare mixed solution. Using
Boc-Lys(Me)2-MCA, Boc-Lys(Me)3-MCA, or AMC in place of Boc-
Lys-MCA, mixed solution was similarly prepared. The
l
L of distilled water were
l
lM). 1
lL
intensity were 0.05 and 0.5 lg/lL, respectively.
l
l
l
Acknowledgments
l
l
l
l
l
This work was supported in part by the Chemical Genomics
Research Project, RIKEN Center for Sustainable Resource Science,
the CREST Research Project, the Japan Science and Technology
Corporation, and by Grants-in-Aid for Innovative Area ‘Cancer’
from the Ministry of Education, Culture, Sports, Science and
Technology of Japan.
l
l
l
a
a
fluorescence spectrum of these solutions was measured with a
SpectraMax M2e microplate reader.

H. Chi et al. / Bioorg. Med. Chem. 22 (2014) 1268–1275
1275
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