Identification of SNAIL1 Peptide-Based Irreversible Lysine-Specific Demethylase 1-Selective Inactivators

Article abstract of DOI:10.1021/acs.jmedchem.5b01323

Inhibition of lysine-specific demethylase 1 (LSD1), a flavin-dependent histone demethylase, has recently emerged as a new strategy for treating cancer and other diseases. LSD1 interacts physically with SNAIL1, a member of the SNAIL/SCRATCH family of transcription factors. This study describes the discovery of SNAIL1 peptide-based inactivators of LSD1. We designed and prepared SNAIL1 peptides bearing a propargyl amine, hydrazine, or phenylcyclopropane moiety. Among them, peptide 3, bearing hydrazine, displayed the most potent LSD1-inhibitory activity in enzyme assays. Kinetic study and mass spectrometric analysis indicated that peptide 3 is a mechanism-based LSD1 inhibitor. Furthermore, peptides 37 and 38, which consist of cell-membrane-permeable oligoarginine conjugated with peptide 3, induced a dose-dependent increase of dimethylated Lys4 of histone H3 in HeLa cells, suggesting that they are likely to exhibit LSD1-inhibitory activity intracellularly. In addition, peptide 37 decreased the viability of HeLa cells. We believe this new approach for targeting LSD1 provides a basis for development of potent selective inhibitors and biological probes for LSD1.

Full text of DOI:10.1021/acs.jmedchem.5b01323

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Article
Identification of SNAIL1 Peptide-Based Irreversible
Lysine Specific Demethylase 1-Selective Inactivators
Yukihiro Itoh, Keisuke Aihara, Paolo Mellini, Toshifumi Tojo, Yosuke Ota, Hiroki Tsumoto,
Viswas Raja Solomon, Peng Zhan, Miki Suzuki, Daisuke Ogasawara, Akira Shigenaga, Tsubasa
Inokuma, Hidehiko Nakagawa, Naoki Miyata, Tamio Mizukami, Akira Otaka, and Takayoshi Suzuki
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01323 • Publication Date (Web): 23 Dec 2015
Downloaded from http://pubs.acs.org on December 29, 2015
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Identification of SNAIL1 PeptideꢀBased Irreversible
Lysine Specific Demethylase 1ꢀSelective
Inactivators
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†
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Yukihiro Itoh, Keisuke Aihara, Paolo Mellini, Toshifumi Tojo, Yosuke Ota, Hiroki Tsumoto,
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‡
Viswas Raja Solomon, Peng Zhan, Miki Suzuki, Daisuke Ogasawara, Akira Shigenaga,
‡
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‡,
Tsubasa Inokuma, Hidehiko Nakagawa, Naoki Miyata, Tamio Mizukami, Akira Otaka, * and
⊥
†, ,
Takayoshi Suzuki
*
†
Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 1ꢀ5
ShimogamohangiꢀCho, SakyoꢀKu, Kyoto, 606ꢀ0823, Japan
‡
Institute of Biomedical Sciences and Graduate School of Pharmaceutical Sciences, Tokushima
University, Shomachi, Tokushima 770ꢀ8505, Japan
§
Graduate School of Pharmaceutical Sciences, Nagoya City University, 3ꢀ1 Tanabeꢀdori,
Mizuhoꢀku, Nagoya, Aichi 467ꢀ8603, Japan
#
Research Team for Mechanism of Aging Tokyo Metropolitan Institute of Gerontology, 35ꢀ2
SakaeꢀCho, Itabashiꢀku, Tokyo 173ꢀ0015, Japan
¶
Graduate School of BioꢀScience, Nagahama Institute of BioꢀScience and Technology, 1266
Tamuraꢀcho, Nagahama, Shiga, 526ꢀ0829, Japan
⊥
CREST, Japan Science and Technology Agency (JST), 4ꢀ1ꢀ8 Honcho Kawaguchi, Saitama 332ꢀ
0012, Japan
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ABSTRACT
Inhibition of lysineꢀspecific demethylase 1 (LSD1), a flavinꢀdependent histone demethylase, has
recently emerged as a new strategy for treating cancer and other diseases. LSD1 interacts
physically with SNAIL1, a member of the SNAIL/SCRATCH family of transcription factors.
This study describes the discovery of SNAIL1 peptideꢀbased inactivators of LSD1. We designed
and prepared SNAIL1 peptides bearing a propargyl amine, hydrazine, or phenylcyclopropane
moiety. Among them, peptide 3 bearing hydrazine displayed the most potent LSD1ꢀinhibitory
activity in enzyme assays. Kinetic study and mass spectrometric analysis indicated that peptide 3
is a mechanismꢀbased LSD1 inhibitor. Furthermore, peptides 37 and 38, which consist of cellꢀ
membraneꢀpermeable oligoarginine conjugated with peptide 3, induced a doseꢀdependent
increase of dimethylated Lys4 of histone H3 in HeLa cells, suggesting that they are likely to
exhibit LSD1ꢀinhibitory activity intracellularly. In addition, peptide 37 decreased the viability of
HeLa cells. We believe this new approach for targeting LSD1 provides a basis for development
of potent selective inhibitors and biological probes for LSD1.
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INTRODUCTION
Chromatin modification, including DNA methylation and histone modifications such as
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acetylation and methylation, plays a pivotal role in transcriptional regulation. These
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modifications epigenetically control biological functions such as cellular differentiation and
developmental changes of organisms. In addition, aberrations of chromatin modification are
closely associated with several diseases such as cancer and neurodegenerative disorders.
Therefore, modulators that can regulate chromatin modification have attracted a great deal of
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attention as therapeutic agents.
Among postꢀtranslation histone modifications, lysine methylation is one of the most widely
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studied modifications. Methylation of histone lysine residues is reversible and is regulated by
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histone lysine methyltransferases and histone lysine demethylases (KDMs). Lysineꢀspecific
demethylase 1 (LSD1, also known as KDM1A) is a demethylase that removes methyl groups
from mono and dimethylated Lys4 of histone H3 (H3K4me1/2) through flavin adenine
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dinucleotide (FAD)ꢀdependent enzymatic oxidation. LSD1 binds with several transcription
factors and regulates the expression of a number of genes such as synapsin and muscarinic
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acetylcholine receptor 4. LSD1 has also been reported to be associated with several disease
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states. For example, it is involved in proliferation of neuroblastoma cells and leukemia cells. It
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is also associated with latent infection of αꢀherpesvirus and globin disorder. Therefore, LSD1
inhibitors are of interest as chemical tools for studying the functions of LSD1 and as candidate
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therapeutic agents targeting LSD1.
LSD1 has been reported to interact with SNAIL1, a member of the SNAIL/SCRATCH family
of transcription factors, and to play a role in the expression of cancerꢀassociated SNAIL1 target
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genes. Further, inhibition of the interaction between LSD1 and SNAIL1 blocks cancer cell
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invasion. Therefore, inhibitors of the LSD1/SNAIL1 interaction appear to be novel anticancer
agent candidates. Indeed, Tortorici et al. recently reported SNAIL1ꢀderived peptides as reversible
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LSD1 inhibitors. On the other hand, we and other groups revealed that small molecules
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containing a propargyl amine, hydrazine, or phenylcyclopropylamine (PCPA) moiety (Chart 1)
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irreversibly inhibit LSD1.
Such mechanismꢀbased irreversible inhibitors are expected to
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maintain the pharmacological effect because of a long target residence time. In the light of
these findings, we initiated a search for SNAIL1 peptideꢀbased irreversible LSD1 inactivators.
Herein we report the design, synthesis, LSD1ꢀinhibitory activity, and cellular activity of a series
of SNAIL1ꢀderived peptides bearing various functional groups.
CHEMISTRY
The routes used for the synthesis of peptides 1–5 (Chart 2), which were prepared for this study,
are shown in Schemes 1–3. Scheme 1 shows the preparation of peptides 1 and 2 with a
propargylamine moiety. BocꢀAsp(OBn)ꢀOH 6 was treated with tertꢀBuOH in the presence of
t
EDCI and DMAP to give BocꢀAsp(OBn)ꢀO Bu 7. The benzyl group of compound 7 was removed
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by hydrogenolysis to give BocꢀAspꢀO Bu 8. Conversion of the carboxylic acid of 8 to mixed
anhydride by using ClCOOEt and subsequent reduction with NaBH yielded alcohol 9.
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Propargylamines 11 and 12 were synthesized from the alcohol 9 via the mesylate 10. Removal
of the Boc group and tertꢀbutyl group of 11 and 12 using hydrochloric acid afforded amino acids
13 and 14. Treatment of the amino acids 13 and 14 with FmocꢀOSu in the presence of NaHCO3
gave the Fmocꢀprotected amino acids 15 and 16, from which the propargylated peptides 1 and 2
were prepared using Fmocꢀbased solidꢀphase peptide synthesis.
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Scheme 2 shows the synthesis of peptides 3 and 4. Benzylation of aspartic acid 17 afforded
Bn ꢀAsp(OBn)ꢀOBn 18. Selective reduction of the lessꢀhindered benzyl ester of 18 using DIBAL
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yielded alcohol 19. The alcohol 19 was converted to aldehyde 20 by Swern oxidation. Reductive
amino alkylation of BocNHNH or BocNMeNH with the aldehyde 20 in the presence of sodium
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cyanoborohydride gave NꢀBoc hydrazine compounds 23 and 24. Fmocꢀprotected unnatural amino
acids 29 and 30 were prepared from compounds 23 and 24 in three steps: Boc protection using
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Boc) O, removal of the benzyl groups by hydrogenolysis, and Fmoc protection using FmocꢀOSu.
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The Fmocꢀprotected amino acids 29 and 30 were incorporated into peptides 3 and 4, respectively,
by means of Fmocꢀbased solidꢀphase peptide synthesis.
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Peptide 5 was prepared from alcohol 31 as outlined in Scheme 3. The alcohol 31 was reacted
with mesyl chloride to obtain mesylate 32. Treatment of the mesylate 32 with PCPA in the
presence of Cs CO gave compound 33. Deprotection of the Boc and tertꢀbutyl groups of 33
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afforded amino acid 34. The amino acid 34 was converted to Fmocꢀprotected PCPAꢀconjugated
amino acid 36 by Fmoc protection of 34 and Boc protection of 35. The PCPAꢀconjugated
SNAIL1ꢀbased peptide 5 was prepared from 36 via standard Fmocꢀbased solidꢀphase peptide
synthesis.
Oligoarginineꢀconjugated peptides 37–40 (Chart 3) were also synthesized by Fmocꢀbased
solidꢀphase peptide synthesis using a lysine derivative with ivDde protection (see Experimental
Section).
RESULTS AND DISCUSSION
We designed SNAIL1 peptideꢀbased LSD1 inactivators based on the crystal structure of LSD1
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complexed with a 20ꢀaminoꢀacid SNAIL1 Nꢀterminal peptide and based on the work of Cole
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and coꢀworkers on histone H3 peptideꢀbased LSD1 inactivators containing a propargyl amine, a
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hydrazine, or a cyclopropylamine moiety. The Xꢀray crystal structure of LSD1/SNAIL1 peptide
complex revealed that Phe 4 of the SNAIL1 peptide is positioned in front of the flavin in the
active site of LSD1 (Figure 1). On the basis of this structure, we designed SNAIL1ꢀbased
peptides 1–5 (Chart 2), in which the phenylalanine residue is replaced with nonꢀnatural amino
acid residues bearing propargyl amine (1 and 2), hydrazine (3 and 4), or PCPA (5). We expected
that the designed SNAIL1 peptides 1–5 would selectively bind LSD1 over LSD2, because it has
been reported that the affinity of LSD2 for a SNAIL1 peptide at least 10 times lower than that of
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LSD1. In addition, these SNAIL1ꢀbased peptides were expected to be recognized effectively by
LSD1 (Supporting Figure S1) and to inactivate LSD1 irreversibly through amine oxidation by
FAD and subsequent covalent bond formation with FAD in the active site of LSD1 (Figure 2).
Specifically, peptides 1 and 2 bearing propargyl amine were expected to be oxidized to an α,βꢀ
unsaturated iminium cation species that would be nucleophilically attacked by N5 of the reduced
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FAD, leading to LSD1 inactivation (Figure 2a). The expected mechanism of LSD1 inactivation
by peptides 3 and 4 bearing hydrazine is shown in Figure 2b. The first oxidation of the hydrazine
of peptide 3 by FAD would yield the diazene and the second oxidation of the diazene would
produce the primary diazonium species, which would inactivate LSD1 through S 2 reaction
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between the diazonium cation and FADH .
On the other hand, peptide 4 with a
monomethylhydrazine group would be converted to peptide 3 through enzymatic demethylation
by LSD1. Then, it would inhibit LSD1 similarly to peptide 3. Introduction of the methyl group
into the hydrazine moiety of peptide 3 was expected to enhance the affinity for LSD1, because
methylated lysine is a substrate of LSD1. The expected mechanism of LSD1 inactivation by
peptide 5 involves formation of a PCPAꢀFAD adduct, following the oxidation by FAD and
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cyclopropyl ring opening (Figure 2c).
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The SNAIL1ꢀbased peptides 1–5 were prepared (Schemes 1–3) and evaluated for LSD1ꢀ
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inhibitory activity using a peroxidaseꢀcoupled assay as described before.
The results are
summarized in Table 1 as IC values. Among the tested peptides, peptide 3 with a hydrazine
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moiety was found to be the most potent LSD1 inactivator (3: IC = 0.44 ꢀM). The activity of
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peptide 4 with a monomethylhydrazine group was moderate (4: IC50 = 8.4 ꢀM). We also
examined the inhibitory activity of peptides 3 and 4 toward LSD2 which is a paralogue of LSD1,
monoamine oxidase (MAO) A/B which are other FADꢀdependent oxidases, and Jumonji ATꢀrich
interactive domain 1A (JARID1A) which is one of histone H3K4me3/me2ꢀspecific
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demethylases. As shown in Table 1, peptides 3 and 4 were inactive at concentrations up to 50
ꢁM in LSD2ꢀinhibitory assays. These results are consistent with the previous report that the
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affinity of LSD2 for SNAIL peptide is lower than that of LSD1. Furthermore, the two peptides
were also almost inactive against MAOs and JARID1A. These results demonstrated that peptides
3 and 4 are inhibitors with high selectivity for LSD1 over LSD2, MAO A/B and JARID1A.
Because peptide 3, which is the most potent LSD1 inhibitor, was designed to inactivate LSD1
irreversibly as shown in Figure 2b, we next investigated the mechanism of LSD1 inhibition by
peptide 3. We initially examined whether the inhibition is timeꢀdependent. The time course of
product formation was monitored in the absence or presence of peptide 3. As shown in Figure 3,
peptide 3 was found to be a timeꢀdependent inhibitor of LSD1.
To gain further insight into the mechanism of LSD1 inhibition by peptide 3, mass
spectrometric analysis of a mixture of LSD1 incubated with FAD and peptide 3 was performed.
If peptide 3 reacts with FAD as expected, peptide 3ꢀFAD adduct (Figure 2b) should be generated.
As depicted in Figure 4a, a significant peak at m/z 3123.4 was observed. This peak corresponds
to the predicted molecular weight of peptide 3ꢀFAD adduct. The peak was dependent on the
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presence of LSD1; that is, it was not detected in the absence of LSD1 (Figure 4b). In addition, an
excess of H3K4me2 peptide, a substrate of LSD1, inhibited the generation of the 3ꢀFAD adduct
(Supporting Figure S2). The data from the mass spectrometric analysis support the idea that
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ꢀ
peptide 3 oxidized by FAD is nucleophilically attacked by FAD in the active site of LSD1 to
afford peptide 3ꢀFAD conjugate, and this reaction results in inhibition of LSD1. Kinetic analysis
and mass analysis results indicate that peptide 3 is an irreversible LSD1 inactivator, as we had
hoped.
Mass analysis of peptide 4 bearing a methylated hydrazine moiety was also performed. As
shown in Supporting Figure S3, the peak corresponding to peptide 3, the demethylated form of
peptide 4, was observed dependently on the presence of LSD1. In addition, the peak of peptide 3ꢀ
FAD adduct was detected dependently on LSD1, though the signal intensity was weak (Figure 4c
and 4d). These results suggest that peptide 4 inhibits LSD1 through the generation of peptide 3
by demethylation of a part of peptide 4, followed by formation of peptide 3ꢀFAD adduct.
However, the LSD1ꢀinhibitory activity of peptide 4 was less than that of peptide 3 (Table 1).
These results indicate that the demethylation of methylated hydrazine in peptide 4 is unfavorable
as compared with the oxidation of hydrazine in peptide 3.
Although peptide compounds generally show poor cell permeability because of their high
polarity, previous studies have established that oligomers of arginine covalently attached to a
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variety of molecules can efficiently cross the cell membrane. For the application of SNAIL1ꢀ
based peptide 3, the most potent LSD1 inactivator in this study (Table 1), to cellꢀbased assays,
we therefore designed and synthesized oligoarginineꢀconjugated derivatives of peptide 3.
Oligoarginineꢀconjugated peptides 37–39 (Chart 3) were designed based on the Xꢀray crystal
structure of LSD1/SNAIL1 peptide complex (Figure 5). As shown in Figure 5, the side chain of
Lys 8 of the SNAIL1 peptide is located outside the active pocket of LSD1. Thus, peptides 37 and
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8, in which oligoarginine is conjugated with the side chain of Lys 8 of peptide 3, were expected
to show little or no decrease of LSD1ꢀinhibitory activity. In addition, we designed peptide 39 in
which oligoarginine is conjugated with the side chain of Lys 15 of peptide 3. It was reported that
residues 10–20 of the 20ꢀaminoꢀacid SNAIL1 Nꢀterminal peptide could not be identified in terms
of electron density in the Xꢀray crystal structure analysis of the LSD1/SNAIL1 peptide
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complex. This suggest that the residues 10–20 of the SNAIL1 peptide lack an ordered
conformation and Lys 15 of the SNAIL1 peptide is likely to be positioned out of the active
pocket of LSD1. Therefore, peptide 39 was also expected to show wellꢀmaintained LSD1ꢀ
inhibitory activity, as compared with peptide 3.
Oligoarginineꢀconjugated peptides 37–39 were prepared and initially evaluated for in vitro
LSD1ꢀinhibitory activity (Table 2). Interestingly, oligoarginineꢀconjugated peptides 37–39 were
found to be more potent LSD1 inhibitors than peptide 3. The increase in LSD1ꢀinhibitory activity
of 37 and 38 may be due to the interaction between the cationic oligoarginine and anionic amino
acid residues of LSD1, such as Asp 556, Asp 557, and Glu 559 (Figure 6). Furthermore, peptides
3
7–39 displayed only weak inhibitory activity toward LSD2, MAO A/B, and JARID1A (Table 2),
confirming their high selectivity for LSD1 over LSD2, MAO A/B, and JARID1A (LSD2
IC /LSD1 IC = 23–80; MAO A IC /LSD1 IC > 94; MAO B IC /LSD1 IC = 94–104;
5
0
50
50
50
50
50
JARID1A IC /LSD1 IC = 16–44).
5
0
50
We also carried out mass spectrometric analysis of a mixture of LSD1 incubated with peptides
3
7–39. The peak of peptide 37ꢀ, 38ꢀ or 39ꢀFAD adduct was detected dependently on LSD1 like
the case of peptide 3 (Supporting Figure S4).
Next, we examined the activity of peptides 37–39 and 40 in cellꢀbased assays. Peptide 40
(Chart 3), in which the hydrazine of peptide 37 is replaced with an amine, was prepared as a
reference compound to confirm that the covalent modification is essential for the inꢀcell activity
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of the peptides. We performed a cellular assay using western blot analysis. Because LSD1 is
4
a
known to catalyze demethylation of H3K4me2, H3K4me1 and H3K9me2, the effect of
oligoarginine peptides on the methylation levels of H3K4 and H3K9 in HeLa cells was examined.
As shown in Figure 7a, treatment with 20 ꢀM peptides 37 and 38 induced a large increase in
dimethylated H3K4 as compared with that induced by peptides 3, 39, and 40. Loss in H3K4
dimethylating activity of amine 40 suggests that the covalent bond formation of the peptide with
FAD is responsible for the activity in cells. Furthermore, the increase of H3K4me2 in the
presence of peptides 37 and 38 was doseꢀdependent without affecting the levels of H3K4me3,
H3K4me1, and H3K9me2 (Figure 7b). The similar results have been obtained with the reported
10
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1
2l
hydrazineꢀbased LSD1 inhibitor. The results in both enzyme assays and cellular assays suggest
that peptides 37 and 38 bearing oligoarginine moieties permeate through the cell membrane and
selectively inhibit LSD1 over JARID1 in the nucleus.
Finally, we tested the effects of peptides 37–40 on the viability of cancer cells (Figure 8). In
this study, we used HeLa cells because it has been reported that LSD1 is overexpressed in HeLa
12d–f
cells, and LSD1 inhibition suppresses HeLa cell growth.
Peptide 37 reduced the cell viability
at concentrations of 25–100 ꢀM. Although peptides 38 and 39 were moderately effective, their
activity was weaker than that of peptide 37. On the other hand, peptide 40 did not inhibit the cell
viability strongly. Thus, peptide 37 showed the strongest antiproliferative activity against HeLa
cells among the three peptides.
CONCLUSIONS
We have designed SNAIL1ꢀbased peptides bearing a propargyl amine (1 and 2), hydrazine (3
and 4), or phenylcyclopropane (5) moiety based on the Xꢀray crystal structure of LSD1
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complexed with a 20ꢀaminoꢀacid SNAIL1 Nꢀterminal peptide. Among these five peptides,
hydrazineꢀcontaining SNAIL1ꢀbased peptide 3 showed the most potent LSD1ꢀinhibitory activity
with an IC of 0.44 ꢀM. In addition, kinetic study and mass spectrometric analysis demonstrated
5
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that peptide 3 inhibits LSD1 through covalent bond formation with FAD after oxidation of the
hydrazine moiety in the active site of LSD1. Furthermore, cellꢀpenetrating peptides 37 and 38,
which were designed based on peptide 3, increased H3K4ꢀmethylation, suggesting that they are
likely to inhibit intracellular LSD1 activity. In addition, peptide 37 decreased the viability of
HeLa cells. To our knowledge, this is the first report of cellꢀactive peptideꢀbased LSD1
inactivators. These SNAIL1ꢀbased peptide derivatives represent an entry into a new class of
LSD1ꢀselective inhibitors, and should be useful tools for probing the biology of LSD1.
EXPERIMENTAL SECTION
Chemistry. Melting points were determined using a Yanagimoto micro melting point
1
apparatus or a Büchi 545 melting point apparatus. Proton nuclear magnetic resonance spectra ( H
1
3
NMR) and carbon nuclear magnetic resonance spectra ( C NMR) were recorded on a JEOL
JNMꢀLA500, JEOL JNMꢀA500, BURKER AVANCE 300, or BRUKER AVANCE600
spectrometer in the indicated solvent. Chemical shifts (δ) are reported in parts per million relative
to the internal standard tetramethylsilane. Electron ionization (EI) mass spectra and fast atom
bombardment (FAB) mass spectra were recorded on a JEOL JMSꢀSX102A mass spectrometer.
Electrospray ionization (ESI) mass spectra were recorded on a BRUKER HCTplus mass
®
TM
spectrometer (ESIꢀIT) or a Waters MICROMASS LCT PREMIER (ESIꢀTOF). The specific
rotation was measured on a HORIBA SEPAꢀ300 polarimeter. HPLC analysis for unnatural
amino acids was performed on a Cosmosil 5C ꢀARꢀII analytical column (Nacalai Tesque, 4.6 ×
1
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2
50 mm, flow rate 1.0 mL/min) and eluted products were detected by UV absorbance
measurement at 254 nm. A solvent system consisting of 0.1% (v/v) TFA aqueous solution
solvent A) and 0.1% (v/v) TFA in MeCN (solvent B) was used for HPLC elution. For HPLC
separation of synthetic peptides, a Cosmosil 5C ꢀARꢀII analytical column (Nacalai Tesque, 4.6 ×
(
10
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37
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49
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60
1
8
2
50 mm, flow rate 1.0 mL/min) or a Cosmosil 5C ꢀARꢀII preparative column (Nacalai Tesque,
18
2
0 × 250 mm, flow rate 10 mL/min) was employed and eluted products were detected by UV
absorbance measurement at 220 nm. A solvent system consisting of 0.1% (v/v) TFA aqueous
solution (solvent A) and 0.1% (v/v) TFA in MeCN (solvent B) was used for HPLC elution.
Chiral HPLC was performed on a CHIRALPAK IA column (4.6 × 250 mm, flow rate 1 mL/min)
and eluted products were detected by UV absorbance measurement at 254 nm. A solvent system
consisting of hexane/iPrOH = 90/10 (v/v) was used for chiral HPLC elution. The purity of the
unnatural amino acid compounds for peptide synthesis and the tested peptides was determined by
HPLC and the purity level was ≥95%. Reagents and solvents were purchased from Aldrich,
Tokyo Kasei Kogyo, Wako Pure Chemical Industries, and Kanto Kagaku and used without
purification. Flash column chromatography was performed using silica gel supplied by Merck or
Toyota Silica Gel.
Fmocꢀbased solidꢀphase peptide synthesis. An Fmocꢀprotected amino acid (5.0 eq.) was
®
coupled to NovaSyn TGR resin (0.25 mmol amine/g), which affords peptide amide, with the aid
of N,N'ꢀdiisopropylcarbodiimide (5.0 eq.) and 1ꢀhydroxyꢀ1Hꢀbenzotriazole monohydrate (5.0
eq.) in DMF at room temperature for 2 h. Then the Fmoc group was removed with 20% (v/v)
piperidine in DMF at room temperature for 10 min. After peptide chain elongation, BocꢀProꢀOH
was introduced as an Nꢀterminal residue. The resulting resin was treated with TFA/mꢀ
cresol/thioanisole/1,2ꢀethanedithiol/H O (80/5/5/5/5 (v/v), 50 ꢁL/1 mg resin) at room
2
temperature for 1.5 h. After removal of the resin by filtration, cooled Et O was added to the
2
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filtrate, and the precipitate was collected by centrifugation. The precipitate was washed with
Et O, and the obtained crude peptide was purified by preparative HPLC and then lyophilized. All
2
purified peptides were obtained as white lyophilized powders. For introduction of an
oligoarginine unit, an Nꢀεꢀ[1ꢀ(4,4ꢀdimethylꢀ2,6ꢀdioxocyclohexꢀ1ꢀylidene)ꢀ3ꢀmethylbutyl] (ivDde)
group was used as a protecting group for a side chain of lysine. FmocꢀLys(ivDde)ꢀOH was
incorporated at the site of Lys8 or Lys15. After sequence elongation, the ivDde group was
removed by treatment with 4% (v/v) hydrazine monohydrate in DMF at room temperature for 5 h.
0
1
2
3
4
5
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8
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0
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0
TM
Then, oligoarginine (R8) or an oligoarginine (R8)ꢀminiPEG was introduced on the generated
ε–amine using standard Fmocꢀbased solidꢀphase peptide synthesis as described above. Results of
characterization of peptides are presented in the supporting information.
SNAIL1ꢀ20ꢀpeptideꢀbased propargylbenzylamine (1). Step 1: Preparation of (S)ꢀ5ꢀbenzyl
1ꢀtertꢀbutyl 2ꢀtertꢀbutoxycarbonylaminopentanedioate (7). A solution of (S)ꢀ5ꢀbenzyloxyꢀ2ꢀ
tertꢀbutoxycarbonylaminoꢀ5ꢀoxopentanoic acid (6) (6.00 g, 17.8 mmol), tertꢀbutanol (1.70 mL,
18.0 mmol), EDCI (3.45 g, 18.0 mmol), and DMAP (2.20 g, 18.0 mmol) in CH Cl (60 mL) was
2
2
stirred at room temperature for 12 hours. The reaction mixture was washed with saturated
aqueous NaHCO and brine, and dried over Na SO . Filtration, concentration in vacuo, and
3
2
4
purification of the residue by flash column chromatography (AcOEt/n–hexane = 1/9) gave 6.50 g
1
(
93%) of 7 as a colorless oil: H NMR (CD OD, 500 MHz, δ; ppm) 7.35–7.30 (5H, m), 5.12 (2H,
3
s), 4.03–4.00 (1H, m), 2.47–2.44 (2H, m), 2.14–2.07 (1H, m), 1.89–1.84 (1H, m), 1.45 (9H, s),
+
24
1
.43 (9H, s); MS (EI) m/z: 393 (M ); [α] ꢀ6.74 (c 0.089, MeOH).
D
Step 2: Preparation of (S)ꢀ5ꢀtertꢀbutoxyꢀ4ꢀtertꢀbutoxycarbonylaminoꢀ5ꢀoxopentanoic acid
(8). To a solution of 7 (6.00 g, 15.3 mmol) in MeOH (3 mL) was added 850 mg of 5% Pd/C. The
®
mixture was stirred under H at room temperature for 8 hours, and then filtered through Celite .
2
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1
The filtrate was concentrated in vacuo to give 4.50 g (97%) of 8 as a colorless oil: H NMR
(CD OD, 500 MHz, δ; ppm) 4.02–3.99 (1H, m), 2.38 (2H, t, J = 7.5 Hz), 2.08–2.04 (1H, m),
3
+
23
1
.88–1.81 (1H, m), 1.47 (9H, s), 1.44 (9H, s); MS (EI) m/z: 303 (M ); [α]
ꢀ12.22 (c 0.090,
D
10
11
12
13
14
15
16
17
18
19
20
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23
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59
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MeOH).
Step 3: Preparation of (S)ꢀtertꢀbutyl 2ꢀtertꢀbutoxycarbonylaminoꢀ5ꢀhydroxypentanoate
9). To a solution of 8 (1.53 g, 5.06 mmol) and triethylamine (3.5 mL, 25.1 mmol) in THF (21
(
mL) was added ethyl chloroformate (2.5 mL, 26.1 mmol) at –10 °C. The mixture was stirred at –
0 °C for 1 hour and insoluble materials were filtered off. To a solution of NaBH (956 mg, 25.3
1
4
mmol) in THF (20 mL) and water (5 mL) was added the filtrate at 0 °C, and the reaction mixture
was stirred at room temperature for 5 hours. After acidification with 1 N aqueous HCl (to pH 3),
the mixture was extracted with AcOEt. The organic layer was separated, washed with 10%
aqueous solution of NaOH, water, and brine, and dried over Na SO . Filtration, concentration in
2
4
vacuo, and purification of the residue by flash column chromatography (AcOEt/n–hexane = 1/3)
1
gave 1.08 g (74%) of 9 as a colorless oil: H NMR (CD OD, 500 MHz, δ; ppm) 3.96–3.94 (1H,
3
m), 3.56–3.53 (2H, m), 1.86–1.80 (1H, m), 1.68–1.64 (1H, m), 1.60–1.55 (2H, m), 1.46 (9H, s),
+
23
1.44 (9H, s); MS (EI) m/z: 289 (M ); [α] ꢀ5.62 (c 0.169, MeOH).
D
Step
4:
Preparation
of
(S)ꢀtertꢀbutyl
2ꢀtertꢀbutoxycarbonylaminoꢀ5ꢀ
methylsulfonyloxypentanoate (10). To a solution of 9 (500 mg, 1.73 mmol), triethylamine (3.5
mL, 25.1 mmol), and DMAP (21.0 mg, 0.173 mmol) in CH Cl (3 mL) was added dropwise
2
2
methanesulfonyl chloride (270 ꢀL, 3.46 mmol) at –20 °C. The reaction mixture was stirred at –
20 °C for 1 hour, then poured into water and extracted with AcOEt. The organic layer was
separated, washed with brine, and dried over Na SO . Filtration, concentration in vacuo, and
2
4
purification of the residue by flash column chromatography (AcOEt/n–hexane = 1/1) gave 370
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4
4
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5
5
5
5
5
5
5
5
6
1
mg (58%) of 10 as a colorless oil: H NMR (CD OD, 500 MHz, δ; ppm) 4.25 (2H, t, J = 6.5 Hz),
3
4.00–3.98 (1H, m), 3.06 (1H, s), 1.92–1.85 (1H, m), 1.83–1.77 (2H, m), 1.74–1.66 (1H, m), 1.46
+
(
9H, s), 1.44 (9H, s); MS (EI) m/z: 367 (M ).
0
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Step
5:
Preparation
of
(S)ꢀtertꢀbutyl
5ꢀbenzyl(propꢀ2ꢀynꢀ1ꢀyl)aminoꢀ2ꢀtertꢀ
butoxycarbonylaminopentanoate (11). To a solution of 10 (368 mg, 1.00 mmol) in DMF (1
mL) was added benzylpropargylamine (217 mg, 1.50 mmol) and Cs CO (500 mg, 1.50 mmol)
2
3
and the reaction mixture was stirred at 65 °C for 2 hours. The reaction mixture was poured into
water and extracted with CHCl . The organic layer was separated and washed with saturated
3
aqueous NaHCO and brine, and dried over Na SO . Filtration, concentration in vacuo, and
3
2
4
purification of the residue by flash column chromatography (AcOEt/n–hexane = 1/1) gave 70 mg
1
(
19%) of 11 as a colorless oil: H NMR (CD OD, 500 MHz, δ; ppm) 7.34 (2H, d, J = 7.5 Hz),
3
7
.28–7.25 (3H, m), 4.58 (2H, s), 4.16 (2H, s), 4.04–4.03 (2H, s), 4.00–3.99 (2H, m), 2.65 (1H, d,
+
J = 2.5 Hz), 1.70–1.68 (4H, m), 1.46 (9H, s), 1.44 (9H, s); MS (EI) m/z: 416 (M ).
Step 6: Preparation of (S)ꢀ2ꢀaminoꢀ5ꢀbenzyl(propꢀ2ꢀynꢀ1ꢀyl)aminopentanoic acid
hydrochloric acid salt (13·HCl). To a solution of 11 (500 mg, 1.20 mmol) in CH Cl (2 mL)
2
2
was added dropwise 4 N HCl in dioxane (5 mL, 20.0 mmol) at 0 °C. The reaction mixture was
stirred at 0 °C for 1 hour, and then concentrated in vacuo to give 330 mg (80%) of 13·HCl as a
1
colorless amorphous solid: H NMR (CD OD, 500 MHz, δ; ppm) 7.58 (2H, broad s), 7.33 (2H, d,
3
J = 7.5 Hz), 7.29–7.28 (3H, m), 4.58 (2H, s), 4.21 (2H, s), 4.04–3.98 (4H, m), 2.66 (1H, d, J =
+
2
.5 Hz), 2.11–1.98 (4H, m); MS (EI) m/z: 260 (M ).
Step 7: Preparation of (S)ꢀ2ꢀ[(9Hꢀfluorenꢀ9ꢀyl)methoxy]carbonylaminoꢀ5ꢀbenzyl(propꢀ2ꢀ
ynꢀ1ꢀyl)aminopentanoic acid (15). To a solution of 13·HCl (330 mg, 1.27 mmol) and NaHCO3
640 mg, 7.61 mmol) in water (2 mL) and dioxane (1 mL) was added Nꢀ(9ꢀ
(
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fluorenylmethoxycarbonyloxy)succinimide (427 mg, 1.27 mmol) in dioxane (1 mL) at 0 °C. The
reaction mixture was stirred at room temperature for 15 hour. After acidification with 2 N
aqueous HCl (to pH 6), the mixture was extracted with AcOEt. The organic layer was separated,
washed with brine and dried over Na SO . Filtration, concentration in vacuo, and purification of
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
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53
54
55
56
57
58
59
60
2
4
the residue by flash column chromatography (CHCl /MeOH = 1/9) gave 120 mg (20%) of 15 as a
3
1
colorless solid: H NMR (CD OD, 500 MHz, δ; ppm) 7.79 (2H, d, J = 7.5 Hz), 7.68 (2H, d, J =
3
7.5 Hz), 7.39 (2H, d, J = 7.5 Hz), 7.31–7.28 (5H, m), 7.27–7.25 (2H, m), 4.57 (2H, s), 4.37–4.35
(2H, m), 4.22–4.17 (4H, m), 4.05–3.98 (2H, m), 2.65 (1H, d, J = 2.5 Hz), 2.12–1.98 (2H, m),
+
1
.81–1.77 (2H, m); MS (EI) m/z: 482 (M ); HPLC, retention time = 43.4 min, purity 95.6%
(analytical HPLC conditions: a linear gradient, 0 min (5% B)ꢀ45 min (50% B)ꢀ55 min (50% B)ꢀ
20
60 min (90% B)); [α]
+6.94 (c 0.072, MeOH).
D
Step 8: Preparation of SNAIL1ꢀ20ꢀpeptideꢀbased propargylbenzylamine (1). By
employing 15, peptide 1 was obtained in 17% isolated yield based on the starting resin: HPLC,
retention time = 19.4 min, purity 96.0% (analytical HPLC conditions: linear gradient of solvent B
in solvent A, 5 to 30% over 30 min); preparative HPLC conditions: linear gradient of solvent B
in solvent A 13% to 23% over 30 min); LRMS (ESIꢀTOF) m/z calcd for C H N O ([M +
1
11 177 35 30
3+
3
H] ): 827.8, found: 827.6.
SNAIL1ꢀ20ꢀpeptideꢀbased propargylmethylamine (2). Preparation of (S)ꢀ2ꢀ({[(9Hꢀ
fluorenꢀ9ꢀyl)methoxy]carbonyl}amino)ꢀ5ꢀ[methyl(propꢀ2ꢀynꢀ1ꢀyl)amino]pentanoic acid (16).
Compound 16 was prepared from 10 using the procedure described for 1 (steps 5–7, yield 4.0%
1
from 10): H NMR (500 MHz, CD OD, δ; ppm): 7.88 (2H, d, J = 7.5 Hz), 7.71–7.64 (2H, m),
3
7.37 (2H, t, J = 7.5 Hz), 7.29 (2H, t, J = 7.5 Hz), 4.33 (2H, m), 4.19 (1H, t, J = 6.5 Hz), 4.07 (1H,
q, J = 4.5 Hz), 3.78 (2H, d, J = 2.5 Hz), 3.05 (1H, t, J = 2.5 Hz), 2.90–2.87 (2H, m), 2.65 (3H, s),
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5
5
5
5
5
5
6
13
1
.86–1.83 (1H, m), 1.72–1.61 (2H, m), 1.41–1.38 (2H, m); C NMR (125 MHz, CDCl , δ;
3
ppm): 177.88, 158.40, 145.26, 128.22, 128.20, 126.30, 120.96, 78.64, 75.44, 68.01, 57.01, 56.51,
+
5
6.37, 45.96, 41.06, 33.11, 26.10, 24.06; MS (FAB) m/z: 423 (MH ); HPLC, retention time =
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
5.2 min, purity 99.2% (analytical HPLC conditions: a linear gradient, 0 min (5% B)ꢀ25 min
2
2
(50% B)ꢀ35 min (50% B)ꢀ40 min (90% B)ꢀ50 min (90% B)); [α]
+2.19 (c 0.228, MeOH).
D
Preparation of SNAIL1ꢀ20ꢀpeptideꢀbased propargylmethylamine (2). By employing 16,
peptide 2 was obtained in 16% isolated yield based on the starting resin: HPLC, retention time =
1
5.9 min, purity 96.8% (analytical HPLC conditions: linear gradient of solvent B in solvent A, 5
to 30% over 30 min); preparative HPLC conditions: linear gradient of solvent B in solvent A 6%
3
+
to 16% over 30 min); LRMS (ESIꢀTOF) m/z calcd for C H N O ([M + 3H] ): 802.4,
1
05 173 35 30
found: 802.2.
SNAIL1ꢀ20ꢀpeptideꢀbased hydrazine (3). Step 1: Preparation of (S)ꢀdibenzyl 2ꢀ
(dibenzylamino)pentanedioate (18). To a solution of Lꢀglutamic acid (17) (4.26 g, 29.0 mmol),
K CO (16.2 g, 118 mmol), and NaOH (2.33 g, 58.0 mmol) in water (100 mL) was slowly added
2
3
benzyl bromide (20.2 g, 118 mmol). The reaction mixture was stirred at reflux temperature for 3
hours, and then extracted with AcOEt. The organic layer was separated and dried over Na SO .
2
4
Filtration, concentration in vacuo, and purification of the residue by flash column
1
chromatography (AcOEt/n–hexane = 1/9) gave 4.31 g (29%) of 18 as a colorless oil: H NMR
(
300 MHz, CDCl , δ; ppm): 7.45–7.18 (20H, m), 5.23 (1H, d, J = 12.3 Hz), 5.22 (1H, d, J = 12.3
3
Hz), 5.00 (1H, d, J = 12.6 Hz), 5.00 (1H, d, J = 12.6 Hz), 4.95 (1H, d, J = 12.6 Hz), 3.88 (2H, d,
J = 13.8 Hz), 3.50 (2H, d, J = 13.8 Hz), 3.41 (1H, t, J = 7.7 Hz), 2.50 (1H, m), 2.34 (1H, m), 2.05
13
(
2H, m); C NMR (75 MHz, CDCl , δ; ppm): 173.53, 172.91, 139.95, 136.70, 129.65, 129.37,
3
1
29.28, 129.24, 129.11, 129.05, 128.92, 127.82, 66.95, 66.90, 60.60, 55.23, 31.42, 25.05; MS
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+
23
(
ESIꢀIT) m/z: 508 [(M+H) ]; [α] ꢀ83.52 (c 0.182, MeOH).
D
Step 2: Preparation of (S)ꢀbenzyl 2ꢀ(dibenzylamino)ꢀ5ꢀhydroxypentanoate (19). To a
solution of 18 (1.92 g, 3.78 mmol) in THF (12.5 mL) was slowly added DIBAL (8.32 mL of a 1
M solution in toluene, 8.32 mmol) at –15 °C. The reaction mixture was stirred at 0 °C for 30
minutes, and then water (7 mL) was added. Stirring was continued at 0 °C for 30 minutes.
Filtration, concentration in vacuo, and purification of the residue by flash column
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
chromatography (AcOEt/n–hexane = 1/4 to 1/2) gave 1.40 g (92%) of 19 as a colorless oil: H
NMR (300 MHz, CDCl , δ; ppm): 7.45–7.34 (5H, m), 7.33–7.18 (10H, m), 5.26 (1H, d, J = 12.3
3
Hz), 5.14 (1H, d, J = 12.3 Hz), 3.93 (2H, d, J = 14.1 Hz), 3.50 (1H, d, J = 13.5 Hz), 3.54–3.43
1
3
(4H, m), 3.38 (1H, t, J = 7.5 Hz), 1.87–1.61 (3H, m), 1.58–1.39 (1H, m), 1.30 (1H, br); C NMR
(
75 MHz, CDCl , δ; ppm): 173.48, 140.18, 136.83, 129.68, 129.37, 129.28, 129.02, 127.81,
3
+
24
66.81, 63.18, 61.36, 55.29, 30.10, 26.58; MS (ESIꢀIT) m/z: 404 [(M+H) ]; [α]
ꢀ111.54 (c
D
0
.117, MeOH).
Step 3: Preparation of (S)ꢀbenzyl 2ꢀ(dibenzylamino)ꢀ5ꢀoxopentanoate (20). To a solution
of oxalyl chloride (690 ꢀL, 7.92 mmol) in CH Cl (3mL) was slowly added a solution of DMSO
2
2
(
675 ꢀL, 9.50 mmol) in CH Cl (2 mL) at –78 °C. The mixture was stirred for 25 minutes at –
2
2
7
8 °C, then a solution of 19 (1.28 g, 3.17 mmol) in CH Cl (2 mL) at –78 °C was slowly added,
2 2
and stirring was continued for 2 hours at –78 °C. To the mixture was added Et N (1.3 mL, 9.38
3
mmol), and stirring was continued for 1 hour at –78 °C. The mixture was further stirred for 15
minutes 0 °C, then poured into water, and extracted with CH Cl . The organic layer was
2
2
separated, washed with brine, and dried over Na SO . Filtration, concentration in vacuo and
2
4
purification of the residue by flash column chromatography (AcOEt/n–hexane = 1/9 to 1/2) gave
1
1
.08 g (85%) of 20 as a colorless oil: H NMR (300 MHz, CDCl , δ; ppm): 9.59 (1H, t, J = 1.2
3
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Hz), 7.46–7.35 (5H, m), 7.34–7.20 (10H, m), 5.27 (1H, d, J = 12.3 Hz), 5.17 (1H, d, J = 12.3 Hz),
3
2
1
.87 (2H, d, J = 13.8 Hz), 3.50 (2H, d, J = 13.8 Hz), 3.35 (1H, t, J = 7.8 Hz), 2.60–2.32 (2H, m),
1
3
.08–1.98 (2H, m); C NMR (75 MHz, CDCl , δ; ppm): 201.38, 172.12, 139.12. 135.95, 128.93,
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
28.67, 128.58, 128.45, 128.36, 127.19, 66.27, 59.87, 54.54, 40.47, 21.66; MS (ESIꢀIT) m/z: 402
+
22
[(M+H) ]; [α] ꢀ72.22 (c 0.126, MeOH).
D
Step
oxopentylidene]hydrazinecarboxylate (21). To a solution of 20 (382 mg, 0.951 mmol) in THF
10 mL) was added tertꢀbutyl carbazate (257 mg, 1.94 mmol). The mixture was stirred for 5
hours at room temperature, then diluted with AcOEt, washed with brine, and dried over Na SO .
4:
Preparation
of
(S)ꢀtertꢀbutyl
2ꢀ[5ꢀ(benzyloxy)ꢀ4ꢀ(dibenzylamino)ꢀ5ꢀ
(
2
4
Filtration, concentration in vacuo, and purification of the residue by flash column
1
chromatography (AcOEt/n–hexane = 1/5 to 1/2) gave 459 mg (94%) of 21 as a colorless oil: H
NMR (300 MHz, CDCl , δ; ppm), small rotamers observed but not reported: 7.43–7.32 (5H, m),
3
7.30–7.18 (10H, m), 6.78 (1H, m), 5.27 (1H, d, J = 12.3 Hz), 5.17 (1H, d, J = 12.3 Hz), 3.88 (2H,
d, J = 13.8 Hz), 3.50 (2H, d, J = 13.8 Hz), 3.38 (1H, m), 2.50–2.36 (1H, m), 2.28–2.16 (1H, m),
1
3
2.05–1.84 (2H, m), 1.49 (9H, s); C NMR (75 MHz, CDCl , δ; ppm), small rotamers observed
3
but not reported: 171.37, 138.40, 135.09, 128.08, 128.01, 127.80, 127.73, 127.64, 127.49, 127.42,
+
1
26.18, 80.12, 65.26, 58.99, 53.54, 27.84, 27.39, 25.44; MS (ESIꢀIT) m/z: 516 [(M+H) ].
Step
5:
Preparation
of
(S)ꢀtertꢀbutyl
2ꢀ[5ꢀ(benzyloxy)ꢀ4ꢀ(dibenzylamino)ꢀ5ꢀ
oxopentyl]hydrazinecarboxylate (23). To a solution of 21 (230 mg, 0.446 mmol) in EtOH (5
mL) were added acetic acid (500 ꢀL, 8.74 mmol) and sodium cyanoborohydride (138 mg, 1.98
mmol). The reaction mixture was stirred for 12 hours at room temperature, then diluted with
AcOEt, washed with aqueous saturated sodium bicarbonate and brine, and dried over Na SO .
2
4
Filtration, concentration in vacuo, and purification of the residue by flash column
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chromatography (AcOEt/n–hexane = 1/4 to 1/1) gave 160 mg (69%) of 23 as a colorless oil: H
NMR (300 MHz, CDCl , δ; ppm): 7.45–7.35 (5H, m), 7.32–7.18 (10H, m), 5.85 (1H, br), 5.26
3
(
1H, d, J = 12.3 Hz), 5.14 (1H, d, J = 12.3 Hz), 3.88 (2H, d, J = 13.8 Hz), 3.50 (2H, d, J = 13.8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
3
Hz), 3.35 (1H, t, J = 7.5 Hz), 2.68 (2H, t, J = 7.0 Hz), 1.86–1.73 (2H, m), 1.60 (13H, m); C
NMR (75 MHz, CDCl , δ; ppm): 168.82, 135.65, 132.21, 124.69, 124.58, 124.41, 124.34, 123.07,
3
+
7
6.47, 62.06, 56.78, 50.58, 47.61, 24.46, 23.04, 20.48; MS (ESIꢀIT) m/z: 518 [(M+H) ].
Step
6:
Preparation
of
(S)ꢀtertꢀbutyl
2ꢀ[5ꢀ(benzyloxy)ꢀ4ꢀ(dibenzylamino)ꢀ5ꢀ
oxopentyl]hydrazinecarboxylate (25). To a solution of 23 (160 mg, 0.309 mmol) in CH Cl (5
2
2
mL) were added diꢀtertꢀbutyl dicarbonate (82.1 mg, 0.376 mmol) and 4ꢀdimethylaminopyridine
(10.2 mg). The mixture was stirred at room temperature for 7 hours, then diluted with CH Cl ,
2
2
washed with water and brine, and dried over Na SO . Filtration, concentration in vacuo, and
2
4
purification of the residue by flash column chromatography (AcOEt/n–hexane = 1/4) gave 148
1
mg (78%) of 25 as a colorless oil: H NMR (300 MHz, CDCl , δ; ppm) 7.52–7.35 (5H, m), 7.32–
3
7
.18 (10H, m), 5.26 (1H, d, J = 12.3 Hz), 5.12 (1H, d, J = 12.3 Hz), 4.38 (1H, br), 3.89 (2H, d, J
= 13.8 Hz), 3.49 (2H, d, J = 13.8 Hz), 3.35 (1H, t, J = 7.3 Hz), 2.70 (2H, t, J = 7.3 Hz), 1.86–
1
3
1
.35 (22H, m); C NMR (75 MHz, CDCl , δ; ppm): 172.64, 152.37, 139.50, 136.12, 128.84,
3
1
28.60, 128.49, 128.31, 128.26, 126.98, 83.06, 65.97, 60.79, 54.46, 50.88, 28.20, 28.09, 27.11,
+
24.51; MS (ESI) m/z: 618 [(M+H) ].
Step 7: Preparation of (S)ꢀ2ꢀaminoꢀ5ꢀ[1,2ꢀbis(tertꢀbutoxycarbonyl)hydrazinyl]pentanoic
acid (27). To a solution of 25 (185 mg, 0.299 mmol) in MeOH (20 mL) was added a solution of
formic acid (260 ꢀL) in MeOH (8.7 mL) and palladium black (100 mg, high surface area, 99.8%)
at –40 °C. The reaction mixture was stirred at room temperature for 2 hours. The palladium was
filtered off and the filtrate was concentrated in vacuo to give 125 mg of crude 27 which was used
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
25
for the next step without further purification: MS (ESIꢀIT) m/z: 348 [(M+H) ]; [α] ꢀ50.00 (c
D
0.030, MeOH).
Step 8: Preparation of (S)ꢀ2ꢀ[(9Hꢀfluorenꢀ9ꢀylmethoxycarbonyl)amino]ꢀ5ꢀ[1,2ꢀbis(tertꢀ
butoxycarbonyl)hydrazinyl]pentanoic acid (29). To a solution of crude 27 (125 mg) and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NaHCO (151 mg, 1.80 mmol) in water (1.5 mL) and dioxane (3 mL) was added Nꢀ(9ꢀ
3
fluorenylmethoxycarbonyloxy)succinimide (242 mg, 0.720 mmol) in dioxane (1 mL) at 0 °C.
The reaction was stirred at room temperature for 3 hours. After acidification with 10% citric acid,
the mixture was extracted with AcOEt. The organic layer was separated, washed with brine and
dried over Na SO . Filtration, concentration in vacuo, and purification of the residue by flash
2
4
column chromatography (MeOH/CH Cl = 1/99 to 1/6) followed by preparative TLC
2
2
1
(
MeOH/CH Cl = 1/12) gave 151 mg (89%, 2 steps) of 29 as a colorless solid: H NMR (CDCl ,
2
2
3
3
00 MHz, δ; ppm), small rotamers observed but not reported: 7.74 (2H, d, J = 7.2 Hz), 7.59 (2H,
m), 7.38 (2H, t, J = 7.2 Hz), 7.29 (2H, t, J = 7.2 Hz), 5.89 (1H, brd, J = 7.2 Hz), 4.60–4.32 (3H,
13
m), 4.20 (1H, m), 2.87 (2H, m), 2.02–1.85 (2H, m), 1.81–1.69 (2H, m), 1.50 (18H, s); C NMR
(
75 MHz, CDCl , δ; ppm), small rotamers observed but not reported: 156.21, 152.32, 143.97,
3
1
43.84, 141.84, 127.66, 127.08, 125.20, 119.92, 83.94, 67.09, 53.86, 50.36, 47.16, 29.96, 28.05,
+
2
3.06; MS (ESIꢀIT) m/z: 570 [(M+H) ]; HPLC, retention time = 20.2 min, purity 96.7% (rotamer
mixture) (analytical HPLC conditions: a linear gradient, 0 min (50% B)ꢀ30 min (50% B)ꢀ35 min
20
(90% B)ꢀ40 min (90% B)); [α]
+1.90 (c 0.079, MeOH); Chiral HPLC, >99% ee, retention
D
time = 59.0 min (major, S form), 28.0 min (minor, R form) (This compound was protected as
2
0
methyl ester by using trimethylsilyldiazomethane and the ester form was analyzed by chiral
HPLC).
Step 9: Preparation of SNAIL1ꢀ20ꢀpeptideꢀbased hydrazine (3). By employing 29, peptide
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7
8
9
3
was obtained in 16% isolated yield based on the starting resin: HPLC, retention time = 18.0
min, purity 97.7% (analytical HPLC conditions: linear gradient of solvent B in solvent A, 1 to
3
1
7
0% over 30 min); preparative HPLC conditions: linear gradient of solvent B in solvent A 6% to
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3+
6% over 30 min); LRMS (ESIꢀTOF) m/z calcd for C H N O ([M + 3H] ): 790.1, found:
1
01 170 36 30
89.9.
SNAIL1ꢀ20ꢀpeptideꢀbased hydrazine (4): Preparation of (S)ꢀ2ꢀ({[(9Hꢀfluorenꢀ9ꢀ
yl)methoxy]carbonyl}amino)ꢀ5ꢀ[1,2ꢀbis(tertꢀbutoxycarbonyl)ꢀ2ꢀmethylhydrazinyl]pentanoic
acid (30). Compound 30 was prepared from 20 using the procedure described for 3 (steps 5–8,
1
yield 28% from 20): H NMR (DMSO, 300 MHz, δ; ppm), small rotamers observed but not
reported: 12.61 (1H, br), 7.89 (2H, d, J = 7.6 Hz), 7.65–7.63 (3H, m), 7.41 (2H, t, J = 7.6 Hz),
7.32 (2H, t, J = 7.6 Hz), 4.26–4.21 (3H, m), 4.07–3.96 (1H, m), 2.95 (3H, m), 1.83–1.68 (1H, m),
13
1.66–1.49 (3H, m), 1.41–1.34 (18H, m); C NMR (75 MHz, CDCl , δ; ppm), small rotamers
3
observed but not reported: 175.95, 156.66, 155.91, 155.24, 143.91, 141.34, 127.76, 127.13,
125.19, 120.01, 81.61, 67.27, 53.64, 48.19, 47.19, 37.45, 29.86, 28.33, 24.34; MS (ESIꢀIT) m/z:
+
584 (MH ); HPLC, retention time = 18.6 min, purity 98.3% (analytical HPLC conditions: a linear
2
0
gradient, 0 min (50% B)ꢀ30 min (90% B)ꢀ40 min (90% B)); [α] +4.11 (c 0.073, MeOH).
D
Preparation of SNAIL1ꢀ20ꢀpeptideꢀbased hydrazine (4): By employing 30, peptide 4 was
obtained in 29% isolated yield based on the starting resin: HPLC, retention time = 18.2 min,
purity 96.0% (analytical HPLC conditions: linear gradient of solvent B in solvent A, 1 to 30%
over 30 min); preparative HPLC conditions: linear gradient of solvent B in solvent A 6% to 16%
3
+
over 30 min); LRMS (ESIꢀTOF) m/z calcd for C H N O ([M + 3H] ): 794.8, found: 794.6.
1
02 172 36 30
SNAIL1ꢀ20ꢀpeptideꢀbased phenylcyclopropylamine (5). Preparation of (2S)ꢀ2ꢀ({[(9Hꢀ
fluorenꢀ9ꢀyl)methoxy]carbonyl}amino)ꢀ6ꢀ[(tertꢀbutoxycarbonyl)(2ꢀphenylcyclopropyl)
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
16
amino]hexanoic acid (36). Compound 36 was prepared from 31 using the procedure described
1
for 1 (steps 4–7) and 3 (step 6): yield 2%; H NMR (500 MHz, CD OD, δ; ppm) 7.90 (2H, t, J =
3
7
.5 Hz), 7.78 (2H, d, J = 7.5 Hz), 7.39 (2H, d, J = 7.0 Hz), 7.29–7.27 (2H, m), 7.22–7.19 (2H,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
m), 7.12 (2H, d, J = 8.0 Hz), 7.09–7.06 (1H, m), 4.59–4.58 (1H, m), 4.20–4.18 (1H, m), 4.04–
4.01 (1H, m), 2.57–2.56 (1H, m), 2.07–2.06 (1H, m), 1.51–1.42 (6H, m), 1.40 (9H, s), 1.28–1.12
＋
13
(
4H, m); MS (FAB): m/z 585 [(M＋H) ]; C NMR (500 MHz, CD OD, δ; ppm) HPLC,
3
retention time = 25.8 min, purity 97.0% (analytical HPLC conditions: a linear gradient, 0 min
20
(
10% B)ꢀ15 min (75% B)ꢀ35 min (75% B); [α] +0.197 (c 0.254, MeOH).
D
Preparation of SNAIL1ꢀ20ꢀpeptideꢀbased phenylcyclopropylamine (5). By employing 36,
peptide 5 was obtained in 14% isolated yield from the starting resin: HPLC, retention time = 15.8
min, purity 95.2% (analytical HPLC conditions: linear gradient of solvent B in solvent A, 5 to
45% over 30 min); preparative HPLC conditions: linear gradient of solvent B in solvent A 15%
3+
to 25% over 30 min); LRMS (ESIꢀTOF) m/z calcd for C H N O ([M + 3H] ): 828.8,
1
11 179 35 30
found: 828.5.
Oligoarginineꢀconjugated peptide 37. Peptide 37 was prepared according to the general
procedure and was obtained in 10% isolated yield from the starting resin: retention time = 19.2
min, purity 95.0% (analytical HPLC conditions: linear gradient of solvent B in solvent A, 1 to
30% over 30 min); preparative HPLC conditions: linear gradient of solvent B in solvent A 8% to
4+
1
8% over 30 min); LRMS (ESIꢀTOF) m/z calcd for C H N O ([M + 4H] ): 905.0, found:
149 266 68 38
9
04.8.
Oligoarginineꢀconjugated peptide 38. Peptide 38 was prepared according to the general
procedure and was obtained in 10% isolated yield from the starting resin: HPLC, retention time =
9.6 min, purity 95.5% (analytical HPLC conditions: linear gradient of solvent B in solvent A, 1
1
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to 30% over 30 min); preparative HPLC conditions: linear gradient of solvent B in solvent A 8%
4
+
to 18% over 30 min); LRMS (ESIꢀTOF) m/z calcd for C H N O ([M + 4H] ): 941.3,
155 277 69 41
found: 941.1.
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Oligoarginineꢀconjugated peptide 39. Peptide 39 was prepared according to the general
procedure and was obtained in 21% isolated yield from the starting resin: HPLC, retention time =
18.8 min, purity 97.5% (analytical HPLC conditions: linear gradient of solvent B in solvent A, 1
to 30% over 30 min); preparative HPLC conditions: linear gradient of solvent B in solvent A 7%
4
+
to 17% over 30 min); LRMS (ESIꢀTOF) m/z calcd for C H N O ([M + 4H] ): 905.0,
1
49 266 68 38
found: 904.9.
Oligoarginineꢀconjugated peptide 40. Peptide 40 was prepared according to the general
procedure and was obtained in 33% isolated yield from the starting resin: HPLC, retention time =
20.0 min, purity 97.3% (analytical HPLC conditions: linear gradient of solvent B in solvent A, 1
to 30% over 30 min); preparative HPLC conditions: linear gradient of solvent B in solvent A 8%
4
+
to 30% over 30 min); LRMS (ESIꢀTOF) m/z calcd for C H N O ([M + 4H] ): 904.8,
1
50 267 67 38
found: 904.3.
Biology. LSD1 Inhibition Assay. LSD1 activity was assayed at 25°C by using the peroxidaseꢀ
1
1d,e,f
coupled method, as described previously.
the first 21 amino acid residues of histone H3, incorporating dimethylated lysine at position 4
H3K4me2 peptide) (SigmaꢀAldrich), was used as the substrate of LSD1. The reaction mixture
The chemically synthesized peptide consisting of
(
contained 50 mM HEPESꢀNaOH, pH 7.5, 0.1 mM 4ꢀaminoantipyrine, 1 mM 3,5ꢀdichloroꢀ2ꢀ
hydroxybenzenesulfonic acid, 5.5 units/ml horseradish peroxidase, 20 ꢀM H3K4me2 peptide,
and an appropriate amount of LSD1. To assess the inhibitory effect of test compounds on the
LSD1 activity, we used partially purified LSD1 (obtained at the purification step of HisTrap HP
chromatography) dissolved in buffer C. Reaction in the absence of inhibitors was also done as a
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control. Reaction mixtures (18 ꢀL), containing all the materials except H3K4me2 peptide were
first incubated for 5 minutes. Then, the reactions were initiated by adding 2 ꢀl of 0.2 mM peptide
solution to the assay mixtures. Absorbance at 515 nm was monitored for 30 minutes in a 384ꢀ
0
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0
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0
e
well plate (Nunc) by using a microplate reader (SpectraMax M2 ; Molecular Devices). Enzyme
activity was determined from the linear part of the reaction curve. The ratio of the enzyme
activity measured in the presence of inhibitor to the activity of the control was plotted against
log[Inh].
LSD2 Inhibition Assay. Production and purification of recombinant Hisꢀtagged human fullꢀ
21
length LSD2 was performed as described previously. The histone demethylation reaction for
assaying LSD2 carried out for 1 hour at 37 C in 20 ꢀL of 50 mM HEPESꢀNaOH (pH 8.5)
containing 0.1% BSA, 2 ꢀg of Hisꢀtagged fullꢀlength LSD2, and 10 ꢀM H3K4me2, the
chemically synthesized peptide consisted of the first 21 amino acid residues of histone H3,
incorporating dimethylated lysine at position 4. The reaction was stopped by adding 20 ꢀL of
100% acetonitrile including 0.1% trifluoroacetic acid. 0.5 ꢀL of 10mg/ml αꢀCyanoꢀ4ꢀ
hydroxycinnamic acid was added to the 0.5 ꢀL of reaction mixture, and 1 ꢀL of this mixture was
spotted on the sample plate, dried and analyzed by matrixꢀassisted laser desorption ionization
timeꢀofꢀflight mass spectrometry (MALDIꢀTOFMS) using JMSꢀS3000 (JEOL). The enzymatic
removal of H3K4me0/1 from H3K4me2 substrate was quantified by this protocol. The LSD2
inhibition activity of the test compounds was calculated from the remaining amount of
H3K4me2. The 50% inhibitory concentration (IC ) of the test compounds was calculated as the
5
0
concentration at which the half amount of H3K4me2 was removed compared to that removed
when the enzyme was added.
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TM
MAO Inhibition Assays. The MAO activity assays were performed using a MAOꢀGlo assay
kit (Promega), according to the supplier’s protocol. MAO A (18 unit/mg) or MAO B (6 unit/mg)
(
SigmaꢀAldrich) (25 ꢀL/well), 160 ꢀM (for MAOꢀA) or 16 ꢀM (for MAOꢀB) (4S)ꢀ4,5ꢀdihydroꢀ
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2
ꢀ(6ꢀhydroxybenzothiazolyl)ꢀ4ꢀthiazolecarboxylic acid (12.5 ꢀL/well), a MAO substrate, and
various concentrations of inhibitors (12.5 ꢀL/well) were incubated at room temperature.
Reactions were stopped after 60 min by adding reconstituted Luciferin Detection Reagent (50
ꢀ
L/well). Then, 20 min after addition of this reagent, the fluorescence at 562 nm of the wells was
measured with a fluorescence reader. The value of % inhibition was calculated from the
fluorescence readings of inhibited wells relative to those of control wells.
JARID1A Inhibition Assay. The JARID1A activity was measured as described in ref 22.
Kinetic Analysis. The kinetic analysis was performed using a LSD1 fluorometric drug
discovery kit (Enzo #NMLꢀAK544ꢀ0001). Peptide 3, H3K4me2 peptide (final concentration, 20
ꢀ
M) and HRP/CELLestial Red were mixed in assay buffer (total volume, 80 ꢀL) on each well of
a 96ꢀplate. The reactions (in 100 ꢀL) were initiated by adding LSD1 (10 ꢀg) in 20 ꢀL of assay
buffer to each well. After incubation at room temperature for 5 minutes, the fluorescence in each
TM
well was measured with an ARVO X3 microplate reader (excitation at 540 nm, emission, at
590 nm) every 30 seconds for 15 minutes.
MALDI-TOF/MS Analysis. LSD1 (12 ꢀM) was incubated with 100 ꢀM peptides 3 and 4 for
22 hours at room temperature in 50 mM sodium phosphate buffer (pH 7.5), 4% glycerol and 1%
DMSO. The control was run with FAD instead of the enzyme. The reaction mixtures (10 ꢀL)
were then denatured with 6 M guanidine hydrochloride (10 ꢀL) and acidified with 1% TFA (5
ꢀ
L). An aliquot of the reaction mixture (10 ꢀL) was desalted and concentrated using ZipTip ꢀꢀ
C18 (Millipore) (eluted with 5 ꢀL of 50% MeCN containing 0.1% TFA). The eluate was
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analyzed on a 5800 MALDIꢀTOF/TOF mass spectrometer (AB SCIEX). MALDI mass spectra
were acquired between m/z 800 and 4000 in the positive ion mode. αꢀCyanoꢀ4ꢀhydroxycinnamic
acid (CHCA, LaserBio) was used as the MALDI matrix without further purification. CHCA was
dissolved in 50% MeCN containing 0.1% TFA to a concentration of 5 mg/mL. Samples for
MALDIꢀTOF MS analysis were prepared by mixing the desalted reaction mixture (2 ꢀL) with the
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matrix solution (2 ꢀL). The applied samples (1 ꢀL) were allowed to dry at room temperature.
Western Blot Analysis. Human cervical carcinoma HeLa cells were cultured in RPMI 1640
containing 10% heatꢀinactivated fetal bovine serum (FBS), penicillin and streptomycin mixture
5
at 37 °C in a humidified atmosphere of 5% CO in air. The HeLa cells (5 x 10 ) were plated in 6ꢀ
2
well plates (Iwaki) and cultured for 24 hours. After removal of the medium, the cells were
washed with PBS. The cells were incubated with synthetic peptides at the indicated
concentrations in RPMI1640 medium without FBS for 24 hours, then collected and extracted
with SDS buffer. Protein concentrations of the lysates were determined using a BCA protein
assay. Equivalent amounts of protein from each lysate were resolved in 5–20% SDSꢀ
polyacrylamide gels and transferred onto PVDF membranes. After blocking with TBSꢀT
containing 5% skim milk, the transblotted membranes were probed with rabbit monoclonal
H3K4me3 antibody (Abcam, #ab8580) (1:500 dilution), rabbit polyclonal H3K4me2 antibody
(Abcam, #ab32356) (1:1000 dilution), rabbit polyclonal H3K4me1 antibody (Abcam,
#ab194691) (1:500 dilution), rabbit polyclonal H3K9me2 antibody (Abcam, #ab194680) (1:500
dilution) or rabbit polyclonal H3 antibody (Abcam, #ab1791) (1:200000 dilution) in TBSꢀT.
Each probed membrane was washed with TBSꢀT, incubated with ECL rabbit IgG, HRPꢀlinked
whole antibody (GE Healthcare Life Sciences, #NA934) (1:2500 dilution), and again washed
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with TBSꢀT. The immunoblots were visualized by enhanced chemiluminescence with
TM
Immobilon Western Chemiluminescent HRP Substrate (Millipore, #P90718).
3
Cell viability assay. The cells were plated in 96ꢀwell plates at the initial density of 1 x 10
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cells/well (50 ꢀL/well, HeLa) and incubated at 37°C. After 24 hours, the medium was removed
and the cells were washed with PBS. The cells were incubated with synthetic peptides at the
indicated concentrations in 50 ꢀL of RPMI1640 medium without FBS for 24 hours. Then, 50 ꢀL
of RPMI1640 culture medium was added to each well and the plates were incubated at 37 °C
under 5% CO in air for a further 48 hours. The mixtures were then treated with 10 ꢀL of
2
AlamarBlue® (AbD Serotec, #BUF012A), and incubation was continued at 37 °C for 3 hours.
TM
The fluorescence in each well was measured with an ARVO X3 microplate reader (excitation
at 540 nm, emission, at 590 nm). The cell viability (%) was calculated from the fluorescence
readings.
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