Synthesis of a bis-cationic α,α-disubstituted amino acid (9-amino-bispidine-9-carboxylic acid) and its effects on the conformational properties of peptides

Article abstract of DOI:10.1016/j.tet.2015.02.076

A new bis-cationic cyclic amino acid, 9-amino-3,7-diazabicyclo[3.3.1]nonane-9-carboxylic acid (9-amino-bispidine-9-carboxylic acid; Abp), which is available for both solution phase and solid phase peptide synthesis, was designed and synthesized. Furthermore, a heterotripeptide Cbz-Leu-Abp-Ala-OMe (9) containing Abp was prepared, and its dominant conformation was analyzed by examining its nuclear magnetic resonance and infrared spectra and performing molecular modeling. The tripeptide 9 formed a β-turn structure as its preferred conformation in solution.

Full text of DOI:10.1016/j.tet.2015.02.076

Tetrahedron 71 (2015) 2241e2245
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of a bis-cationic a,a-disubstituted amino acid (9-amino-
bispidine-9-carboxylic acid) and its effects on the conformational
properties of peptides
a
,
b
a
,
a
a
*
Hiroko Yamashita , Yosuke Demizu , Takashi Misawa , Takuji Shoda ,
Masaaki Kurihara
a
a,b,
*
Division of Organic Chemistry, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya, Tokyo 158-8501, Japan
Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 February 2015
Received in revised form 19 February 2015
Accepted 21 February 2015
Available online 26 February 2015
A new bis-cationic cyclic amino acid, 9-amino-3,7-diazabicyclo[3.3.1]nonane-9-carboxylic acid (9-
amino-bispidine-9-carboxylic acid; Abp), which is available for both solution phase and solid phase
peptide synthesis, was designed and synthesized. Furthermore, a heterotripeptide Cbz-Leu-Abp-Ala-
OMe (9) containing Abp was prepared, and its dominant conformation was analyzed by examining its
nuclear magnetic resonance and infrared spectra and performing molecular modeling. The tripeptide 9
formed a
b
-turn structure as its preferred conformation in solution.
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
Cationic amino acid
a,a-disubstituted amino acid
Peptide
Conformational analysis
1
. Introduction
acids, such as lysine and arginine, and is also used as a helical
promoter in antimicrobial peptides. On the other hand, the bis-
cationic acyclic amino acid bis-ornithine [bis(Orn)] causes pep-
Naturally basic (cationic) amino acids, e.g., arginine, histidine,
8
and lysine, in proteins and peptides play important roles in bi-
ological and chemical activities. Cationic peptides have unique
properties, e.g., they can exhibit cell-penetrating and antimicrobial
activity.1 The stabilization of peptides’ secondary structures, par-
ticularly their helical structures, has been demonstrated to be ef-
tides to adopt fully extended conformations, and [bis(Orn)] oli-
9
gopeptides are also used for cargo delivery. Thus, these non-
proteinogenic cationic amino acids are widely used as building
blocks for functional peptides. We speculated that a new bis-
cationic cyclic amino acid with cationic and peptide helix-
promoting properties could become a useful building block for
functional peptides. In this study, we designed a bis-cationic cyclic
dAA containing a bispidine moiety, 9-amino-3,7-diazabicyclo[3.3.1]
nonane-9-carboxylic acid (9-amino-bispidine-9-carboxylic acid;
Abp), which is available for both solution phase and solid phase
peptide synthesis (Fig. 1). That is to say, N-terminal Cbz- and Fmoc-
2
fective at enhancing their biological activities. Non-proteinogenic
3
amino acids, such as
cyclic
stabilization. dAA, in which the
acids are replaced with alkyl substituents, are often incorporated
into biologically active peptides to stabilize their helical structures
a
,a
-disubstituted
a
-amino acids (dAA) and
b
-amino acids,4 are often utilized as tools for peptide-helix
-hydrogen atoms of -amino
a
a
5
or fully extended conformations, and peptides containing dAA are
also more resistant to proteolytic degradation. To date, several
6
cationic acyclic and cyclic dAA have been synthesized, and the
conformations and properties of peptides containing these residues
H
H
N
N
H
N
have been studied. Among cationic cyclic dAA, 4-aminopiperidine-
H N
2
NH2
7
4
-carboxylic acid (Api), which contains a piperidine ring, has been
commonly used as a mimic of naturally occurring cationic amino
H N
2
CO H
H N
2
CO H
H N
2
CO H
2
2
2
Api
bis(Orn)
Abp
*
Corresponding authors. Tel.: þ81 3 3700 1141; fax: þ81 3 3707 6950; e-mail
addresses: demizu@nihs.go.jp (Y. Demizu), masaaki@nihs.go.jp (M. Kurihara).
Fig. 1. Cationic amino acids.
http://dx.doi.org/10.1016/j.tet.2015.02.076
0
040-4020/Ó 2015 Elsevier Ltd. All rights reserved.

2242
H. Yamashita et al. / Tetrahedron 71 (2015) 2241e2245
protected amino acids, Cbz-Abp-OH and Fmoc-Abp-OH, were
synthesized. Furthermore, a tripeptide containing Abp (Boc-Leu-
Abp-Ala-OMe) was prepared using the solution phase method, and
its preferred conformation was analyzed by examining its Fourier
transform infrared (FTIR) and nuclear magnetic resonance (NMR)
spectra and performing molecular modeling.
Table 1
Di-Boc protection of the hydantoin 3
Entry
Conditions
Yield of 4 (%)
Yield of 5 (%)
1
2
3
4
5
6
DIPEA, DMAP, THF
Et N, DMAP, THF
3
DBU, DMAP, THF
DABCO, DMAP, THF
9
16
35
37
0
28
46
26
24
75
0
K
2
CO , acetone
3
2
2
. Results and discussion
TMP, THF
93
.1. Synthesis of bis-cationic amino acids (Cbz-Abp-OH and
(HATU)/1-hydroxy-7-azabenzotriazole (HOAt) as a coupling re-
Fmoc-Abp-OH) and heteropeptide
agent (Scheme 2).
Our strategy for synthesizing N-terminal protected Cbz-Abp-OH
(6) and Fmoc-Abp-OH (7) is shown in Scheme 1. In the first step, 1-
2.2. Conformational analysis of the tripeptide 9
Boc-4-piperidone was subjected to cyclization in the presence of
benzylamine and paraformaldehyde to afford the 3,7-diazabicyclo
The FTIR spectra of peptides 8 and 9 in the NH-stretching region
10
ꢀ1
[3.3.1]nonan-9-one derivative 1. The N-Benzyl group of 1 was
(amide A 3250e3500 cm ) were measured in CDCl
3
solution, as
ꢀ1
replaced with a Boc group to give 2, and then the hydantoin 3 was
obtained from 2 via a B u€ cherereBergs procedure that resulted in
a high yield. The conditions employed for the di-Boc protection of
shown in Fig. 2. The band of the tripeptide 9 seen in the 3427 cm
region was assigned to free peptide NH groups, and the band ob-
ꢀ
1
served in the 3350 cm region was assigned to a peptide NH group
with an NeH/O]C intramolecular hydrogen bond (Fig. 2a). This
IR spectrum was very similar to those exhibited by 310-helical
the hydantoin 3 are summarized in Table 1. As shown in entries
1
e4, the use of several organic bases gave a mixture of the di- and
12
mono-Boc protected hydantoins 4 and 5, but the use of potassium
carbonate, an inorganic base, gave only the mono-Boc product 5
peptides containing b-turn structures in solution. Whereas, in the
ꢀ1
spectrum of the dipeptide 8, the single band at the 3427 cm re-
gion was observed, indicating that the dipeptide 8 was not folded
into the turn structure.
(
entry 5). Conversely, the di-Boc product 4 was exclusively obtained
when trimethylpyridine (TMP) was used as a base (entry 6). The
hydrolysis of 4 with aqueous LiOH gave an N- and C-terminal-free
amino acid,11 and subsequent Cbz protection afforded the side-
chain Boc-protected amino acid Cbz-Abp-OH (6). The Fmoc-
protected amino acid Fmoc-Abp-OH (7) was obtained by using
Fmoc-OSu instead of Cbz-OSu.
Fig. 3 shows a solvent perturbation experiment in which the
tripeptide 9 was treated with the strong H-bond acceptor solvent
dimethyl sulfoxide (DMSO) (0e10% (v/v)). Two NH (N
eH) chemical shifts were found to be sensitive (solvent-exposed
NH groups) to the addition of DMSO. The latter results demonstrate
1
eH and
N
2
Scheme 1. Synthesis of bis-cationic cyclic a,a-disubstituted amino acids.
Then, the dipeptide Cbz-Abp-Ala-OMe (8) and the tripeptide
Cbz-Leu-Abp-Ala-OMe (9), both of which contained Abp residues,
that these two NH protons were exposed by the solvent used for the
NMR, suggesting that are not intramolecularly hydrogen bonded.
were synthesized by conventional solution phase methods using 1-
3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), hydrochlo-
ride/1-hydroxybenzotriazole (HOBt) hydrate, or O-(7-azabenz
otriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
These findings are indicative of a
which the two NH groups (N eH and N
the peptide are freely solvated (not intramolecularly hydrogen
b
-turn or a 310-helical structure, in
(
1
2
eH) at the N-terminus of
1
3
bonded) peptide NH groups (Fig. 4).

H. Yamashita et al. / Tetrahedron 71 (2015) 2241e2245
2243
O
H
O
N
H
H-Ala-OMe
EDC, HOBt, DIPEA
N
Cbz
N
Cbz
N
H
CO
2
Me
N
N
H
2
CO Me
1) H
2) Cbz-Leu-OH, HATU, HOAt,
DIPEA, CH Cl , rt, 15h
2 2
, Pd(OH) , MeOH, 15h
H
6
O
2 2
CH Cl , rt, 36h
Boc
Boc
2
2
Boc
N
Boc
N
N
81%
8
41%
9
Scheme 2. Synthesis of the dipeptide 8 and the tripeptide 9.
Fig. 2. IR spectra of (a) the tripeptide 9 and (b) the dipeptide 8 in CDCl
3
solution. Peptide concentration: 2.0 mM.
8
N -H
7
6
5
.5
N -H
7
H bond
.5
6
O
H Leu O H AbpO H Ala O
BnO
N
1
N
2
N
3
OMe
free N-H
N -H
.5
5
0
2
4
6
8
10
%
DMSO in CDCl
Fig. 3. 1H NMR experiment involving the addition of DMSO to a CDCl
of DMSO (v/v) added to the CDCl solution. Peptide concentration: 1.0 mM.
solution of tripeptide 9. Plots of NH chemical shifts in the 1H NMR spectra of 9 as a function of the percentage
3
3
Conformational calculations for the tripeptide 9 were per-
formed using the software package MacroModel ver. 10.0 (Schro-
dinger, Inc.). The Monte Carlo multiple minimum method and
AMBER* force field were used to find the peptide’s global minimum
energy conformation. The peptide’s extended conformation was
used as the initial structure, and 20,000 conformers were opti-
mized. The global minimum energy conformation of 9 was found to
be a type III b-turn structure; i.e., a single 310-helix turn. The N-
terminal carbonyl group (Boc group) served as thy hydrogen bond
acceptor. Thus, the N-terminal protecting groups of helical peptides
(
including b-turn peptides) can generally work as the hydrogen
3
d
bond acceptor in solution and in the crystalline state. This cal-
culated structure was supported by our findings regarding the
conformation of 9 in solution.
H bond
O
O
H
Bn
N
O
N
N
H
CO Me
2
H
3. Conclusion
O
Boc
Boc
In summary, we have designed and synthesized a new bis-
cationic cyclic amino acid, 9-amino-3,7-diazabicyclo[3.3.1]nonane-
N
N
9-carboxylic acid (9-amino-bispidine-9-carboxylic acid; Abp), which
is available for both solution phase and solid phase peptide synthesis
and has peptide helix-promoting abilities. Furthermore, a tripeptide
Fig. 4. The calculated conformation and hydrogen bonding pattern of the tripeptide 9.

2244
H. Yamashita et al. / Tetrahedron 71 (2015) 2241e2245
containing an Abp residue, Cbz-Leu-Abp-Ala-OMe (9), was also
prepared, and its dominant conformation was analyzed by examin-
ing its NMR and IR spectra and performing molecular modeling. The
85.4, 85.0, 79.5, 67.3, 66.7, 46.7, 45.8, 45.1, 43.4, 35.5, 28.6, 28.3, 27.2,
24.2, 20.1; [HR-ESI(þ)]: m/z calcd for C24
H
46
N
4
O
8
Na [MþNa]þ
633.3106, found 633.3041.
tripeptide 9 formed a
in solution. Various acyclic and cyclic
having the helix promoting property generally act as
b
-turn structure as its preferred conformation
-disubstituted amino acids
-turn inducers
in the tripeptide sequence. In this work, the tripeptide Cbz-Leu-
Abp-Ala-OMe (9) containing Abp also formed a type III -turn
a,a
4.1.4. Spectroscopic data for the mono-Boc protected hydantoin
ꢁ
ꢀ1
b
5. White solid; Mp 260e262 C; IR (CDCl
3
, cm ) 3310, 2976, 2930,
7.79 (br s, 1H), 3.98e4.19
14
1
1770, 1690; H NMR (600 MHz, CDCl
3
)
d
b
(m, 4H), 3.72 (dd, J¼18, 48 Hz, 2H), 3.24 (dd, J¼18, 54 Hz, 2H), 1.88
13
structure as its preferred conformation in solution and in the global
minimum energy conformation. This alternating Leu-Abp-Ala seg-
ment forming the turn structure would give a helical structure in the
longer sequences. The helix-promoting amino acid Abp is expected
to be a valuable tool for producing biologically active molecules, such
as drug delivery system carriers and antimicrobial peptides.
(br s, 2H), 1.51 (s, 9H), 1.48 (s, 18H); C NMR (100 MHz, CDCl
3
)
d
169.9, 154.9, 154.6, 152.1, 145.7, 86.3, 80.3, 80.0, 69.6, 60.5, 53.8,
45.7, 45.3, 44.6, 44.1, 35.2, 29.3, 28.4, 27.8, 21.1; [HR-ESI(þ)]: m/z
Na [MþNa]þ 533.2582, found 533.2537.
38
calcd for C24H N
4
O
8
4.1.5. Synthesis of compound 6. A solution of 4 (100 mg, 0.16 mmol)
ꢁ
in 1 M aqueous LiOH (1.0 mL) and THF (1.0 mL) was stirred at 70 C
4
4
. Experimental
for 18 h. After the solution had been neutralized with 3% aqueous
HCl, the THF was evaporated. Then, the aqueous solution was
.1. General methods
washed with Et
2
O, before being concentrated in vacuo. Cbz-OSu
(1.2 mL), and
(122.4 mg, 0.49 mmol), saturated aqueous NaHCO
3
1H and 13C NMR spectra were recorded on a Varian AS 400 or
1,4-dioxane (2.4 mL) were added to the resultant residue, and the
solution was stirred at room temperature for 15 h. After removing
the 1,4-dioxane, the solution was acidified with 3% aqueous HCl,
a JNM-ECA 600 spectrometer in CDCl
spectra were recorded on a JASCO FT/IR-4100 spectrometer at
3 3 6
, CD OD, or DMSO-d . FTIR
ꢀ1
a resolution of 1 cm . A mean of 64 scans were obtained for the
solution (CDCl ) method, and a NaCl cell path length of 0.1 mM was
employed. Electrospray ionization mass spectrometry spectra were
acquired on a SHIMADZU liquid chromatography mass spectrome-
try ion-trap time-of-flight spectrometer.
2 4
extracted with AcOEt, and dried over Na SO . The solvent was then
3
removed, and the residue was purified by column chromatography
on silica gel (n-hexane: AcOEt¼1: 3 in the presence of 1% AcOH) to
give the Cbz-protected amino acid Cbz-Abp-OH 6 (29 mg, 34%) as
ꢁ
ꢀ1
a white solid. White solid; Mp 156e158 C; IR (CDCl
2
3
, cm ) 3309,
7.21e7.29
1
986, 2880, 1740, 1680, 1529; H NMR (600 MHz, CDCl
3
)
d
4
2
.1.1. Synthesis of compound 2. A suspension of 1 (3.3 g,10.0 mmol),
(m, 5H), 6.06 (br s,1H), 5.02 (s, 2H), 3.78e4.12 (m, 4H), 3.23e3.36 (m,
13
0% Pd(OH)
2
(200 mg), and Boc
2
O (2.6 g, 12.0 mmol) in AcOEt
4H), 2.67 (br s, 1H), 2.47 (br s, 1H) 1.35 (s, 18H); C NMR (100 MHz,
DMSO-d 173.3, 155.6, 154.9, 154.6, 137.5, 129.1, 128.6, 128.4, 79.1,
ꢁ
(
50 mL) was vigorously stirred under an H
2
atmosphere at 60 C for
6
) d
2
4 h. After filtration of the Pd-catalyst, the filtrate was concentrated
66.1, 58.6, 46.9, 45.9, 45.5, 44.5, 32.6, 32.3, 28.8; [HR-ESI(þ)]: m/z
in vacuo, and the resultant residue was purified by column chro-
matography on silica gel (n-hexane:AcOEt¼3:1) to give 2 (3.2 g,
calcd for C26H N O
37 3 8
Na [MþNa]þ 542.2473, found 542.2424.
ꢁ
ꢀ1
)
9
2
4
2
7
3
5%) as a white solid. White solid; Mp 163e165 C; IR (CDCl
3
, cm
4.1.6. Spectroscopic data for the Fmoc-protected amino acid
1
ꢁ
ꢀ1
976, 2938, 1736, 1690; H NMR (600 MHz, CD
3
OD)
d
3.94e4.03 (m,
7. White solid; Mp 257e259 C; IR (CDCl
3
, cm ) 3291, 2978, 2931,
7.73 (d, J¼7.2 Hz, 2H),
1
H), 3.32 (dd, J¼9, 27 Hz, 2H), 3.23 (dd, J¼12, 42 Hz, 2H), 1.90 (br s,
1802, 1767, 1686; H NMR (600 MHz, CDCl
3
)
d
H), 1.42 (s, 18H); 13C NMR (100 MHz, CD
OD)
d
155.7, 94.2, 91.1,
7.52 (d, J¼7.2 Hz, 2H), 7.38 (t, J¼7.6 Hz, 2H), 7.28 (t, J¼7.6 Hz, 2H),
5.39 (br s, 1H), 4.46 (br s, 1H), 4.20 (d, J¼7.2 Hz, 2H), 3.94e4.06 (m,
4H), 3.01e3.40 (m, 4H), 2.74 (br s, 1H), 2.51 (br s, 1H), 1.43 (s, 9H),
3
9.6, 36.4, 27.4; [HR-ESI(þ)]: m/z calcd for C17
H
28
N
2
O
5
Na [MþNa]þ
63.1890, found 363.1874.
1
3
1
.39 (s, 9H); C NMR (100 MHz, CDCl
3
) d 175.6, 155.1, 154.8, 141.5,
4.1.2. Synthesis of compound 3. A mixture of 2 (2.5 g, 7.3 mmol),
127.9, 127.7, 124.8, 120.0, 80.1, 71.7, 66.9, 58.9, 47.2, 46.3, 45.2, 44.9,
(
NH
4
)
2
CO
3
(2.3 g, 24.2 mmol), and KCN (774 mg, 11.9 mmol) in 50%
28.6, 28.4, 22.7, 20.8; [HR-ESI(þ)]: m/z calcd for C33
41 3 8
H N O Na
ꢁ
[MþNa]þ 630.2786, found 630.2707.
aqueous EtOH (10 mL) was stirred at 120 C in a sealed tube for 15 h.
After removing the EtOH, the suspension was filtered off, and the
precipitate was washed with a small portion of water to give the
hydantoin 3 (2.5 g, 88%) as a white solid. White solid; Mp
4.1.7. Synthesis of the dipeptide 8. A mixture of EDC (83 mg,
0.47 mmol), HOBt (66 mg, 0.47 mmol), DIPEA (183 L,1.08 mmol),
alanine methyl ester hydrochloride (50 mg, 0.36 mmol), and 7
(62.2 mg, 0.12 mmol) in CH Cl (2 mL) was stirred at room tem-
perature for 36 h. The solution was then washed with 3% aqueous
HCl, saturated aqueous NaHCO , and brine, before being dried over
Na SO . After the solvent had been removed, the residue was pu-
m
L-
ꢁ
ꢀ1
1
2
(
(
34e236 C; IR (CDCl
600 MHz, DMSO-d
d, 1H), 3.82 (d, 1H), 3.66 (d, 1H), 3.54 (d, 1H), 3.34 (br s, 1H), 3.27 (d,
3
, cm ) 3248, 2979,1765,1729, 1683; H NMR
6
)
d
10.87 (s, 1H), 8.66 (s, 1H), 3.90 ( , 1H), 3.83
d
2
2
13
1
H), 3.15 (d, 1H), 1.92 (d, 2H), 1.38 (s, 6H), 1.35 (s, 12H); C NMR
3
(
100 MHz, DMSO-d
6
)
d
176.6, 156.5, 154.7, 79.1, 61.4, 46.4, 45.7, 45.3,
2
4
4
4
4.8, 34.8, 28.7; [HR-ESI(þ)]: m/z calcd for C19
H
30
N
4
O
6
Na [MþNa]þ
rified by column chromatography on silica gel (n-
hexane:AcOEt¼2:1) to give the dipeptide 8 (58 mg, 81%). White
33.2058, found 433.2023.
ꢁ
ꢀ1
solid; Mp 166e168 C; IR (CDCl
3
, cm ) 3427, 2979, 2929, 2872,
1
4
.1.3. Synthesis of compound 4. A solution of 3 (1.0 g, 2.4 mmol),
O (10.6 g, 48.7 mmol) in THF
20 mL) was stirred at room temperature for 16 h. After removing
1741, 1688; H NMR (400 MHz, CDCl
3
)
d
7.28e7.37 (m, 6H), 5.38 (br
TMP (1.6 mL, 12.2 mmol), and (Boc)
(
2
s, 0.5H), 5.33 (br s, 0.5H), 5.09 (dd, J¼12, 18.8 Hz, 2H), 4.48e4.51 (m,
1H), 3.97e4.17 (m, 4H), 3.71 (s, 3H), 3.24e3.42 (m, 4H), 2.63 (br s,
13
the THF and TMP, the resultant residue was purified by column
chromatography on silica gel (n-hexane:AcOEt¼3:1) to give the di-
Boc protected hydantoin 4 (1.4 g, 93%) as colorless crystals. White
solid; Mp 248e250 C; IR (CDCl
NMR (600 MHz, CDCl
2H), 1.42 (s, 9H), 1.41 (s, 9H), 1.28 (s, J¼4.4 Hz, 3H); C NMR
(100 MHz, CDCl 173.0, 170.3, 156.0, 155.2, 154.7, 135.9, 128.6,
3
) d
128.4, 128.1, 79.8, 67.2, 60.4, 58.6, 52.4, 48.2, 46.4, 45.2, 44.8, 43.9,
ꢁ
ꢀ1
1
3
, cm ) 2980, 2920, 1770, 1660; H
4.03 (dd, J¼18, 36 Hz, 2H), 3.92 (t, J¼18 Hz,
H), 3.66 (dd, J¼12, 48 Hz, 2H), 3.48 (dd, J¼18, 36 Hz, 2H), 2.18 (br s,
H), 2.01 (br s, 1H), 1.53 (s, 9H), 1.51 (s, 9H), 1.43 (s, 9H), 1.41 (s, 9H);
32.6, 32.4, 31.6, 28.5, 28.4, 28.3, 21.1; [HR-ESI(þ)]: m/z calcd for
)
d
C
30
H
44
N
4
O
9
Na [MþNa]þ 627.3001, found 627.2963.
3
2
1
4.1.8. Synthesis of the tripeptide 9. A mixture of 8 (104 mg,
0.17 mmol) and 20% Pd(OH) (10 mg) in MeOH (10 mL) was
1
3
C NMR (100 MHz, CDCl
3
)
d
176.6, 155.1, 155.0, 154.9, 150.8, 146.2,
2

H. Yamashita et al. / Tetrahedron 71 (2015) 2241e2245
2245
rigorously stirred under an H
2
atmosphere at room temperature for
Shoda, T.; Okuhira, K.; Sekino, Y.; Naito, M.; Kurihara, M. Bioconjugate Chem.
014, 25, 1921e1924.
. (a) Brown, R. A.; Diemer, V.; Webb, S. J.; Clayden, J. Nat. Chem. 2013, 5, 853e860;
b) Tanaka, M. Chem. Pharm. Bull. 2007, 55, 349e358; (c) Royo, S.; Borggraeve,
2
15 h and then filtrated. The filtrate was concentrated in vacuo to
3
give a crude amine, which was used for the next reaction without
(
further purification. A mixture of HATU (233 mg, 0.61 mmol), HOAt
W. M. D.; Peggion, C.; Formaggio, F.; Crisma, M.; Jim eꢀ nez, A. I.; Cativiela, C.;
Toniolo, C. J. Am. Chem. Soc. 2005, 127, 2036e2037; (d) Demizu, Y.; Yamashita,
H.; Yamazaki, N.; Sato, Y.; Doi, M.; Tanaka, M.; Kurihara, M. J. Org. Chem. 2013,
(
83 mg, 0.61 mmol), DIPEA (260
mentioned amine, and Cbz-protected
.52 mmol) in CH Cl (10 mL) was stirred at room temperature for
5 h. The solution was washed with 3% aqueous HCl, saturated
aqueous NaHCO , and brine, before being dried over Na SO . After
m
L, 1.53 mmol), the above-
L-leucine (137 mg,
78, 12106e12113.
0
1
2
2
4. (a) Wang, S.; Otani, Y.; Liu, X.; Kawahata, M.; Yamaguchi, K.; Ohwada, T. J. Org.
Chem. 2014, 79, 5287e5300; (b) Imamura, Y.; Umezawa, N.; Osawa, S.; Shi-
mada, N.; Higo, T.; Yokoshima, S.; Fukuyama, T.; Iwatsubo, T.; Kato, N.; Tomita,
T.; Higuchi, T. J. Med. Chem. 2013, 56, 1443e1454; (c) Horne, W. S.; Gellman, S.
H. Acc. Chem. Res. 2008, 41, 1399e1408; (d) Seebach, D.; Gardiner, J. Acc. Chem.
Res. 2008, 41, 1366e1375.
. (a) Tanaka, M.; Nishimura, S.; Oba, M.; Demizu, Y.; Kurihara, M.; Suemune, H.
Chem.dEur. J. 2003, 9, 3082e3090; (b) Imawaka, N.; Tanaka, M.; Suemune, H.
Helv. Chim. Acta 2000, 83, 2823e2835; (c) Benedetti, E. Biopolymers (Pept. Sci.)
3
2
4
the solvent had been removed, the residue was purified by column
chromatography on silica gel (n-hexane:AcOEt¼1:1) to give the
5
ꢁ
tripeptide 9 (52 mg, 41%). White solid; Mp 180e182 C; IR (CDCl
3
,
ꢀ
1
1
cm ) 3427, 3350, 2958, 2932, 1734, 1691; H NMR (400 MHz,
CDCl 7.72 (br s, 1H), 7.27e7.33 (m, 5H), 6.83 (br s, 1H), 5.30 (br s,
1996, 40, 3e44.
3
) d
6
. (a) Yeo, D. J.; Warriner, S. L.; Wilson, A. J. Chem. Commun. 2013, 9131e9133; (b)
Yamaguchi, H.; Kodama, H.; Osada, S.; Kato, F.; Jelokhani-Niaraki, M.; Kondo, M.
Biosci. Biotechnol. Biochem. 2003, 67, 2269e2272.
1
5
1
H), 5.09 (dd, J¼8.4, 23.2 Hz, 2H), 4.41e4.46 (m, 1H), 3.93e4.16 (m,
H), 3.64 (s, 3H), 3.13e3.48 (m, 4H), 2.94 (br s, 1H), 2.59 (br s, 1H),
.66e1.71 (m, 3H), 1.40e1.47 (m, 21H), 0.92 (br s, 6H); 13C NMR
7. (a) Cho, J.; Tanaka, M.; Sato, S.; Kinbara, K.; Aida, T. J. Am. Chem. Soc. 2010, 132,
3176e13178; (b) Ousaka, N.; Inai, Y.; Kuroda, R. J. Am. Chem. Soc. 2008, 130,
2266e12267; (c) Oba, M.; Tanaka, M.; Takano, Y.; Suemune, H. Tetrahedron
005, 61, 593e598; (d) Yokum, T. S.; Elzer, P. H.; McLaughlin, M. L. J. Med. Chem.
1
1
2
(
100 MHz, CDCl
3
)
d
173.9; 173.0, 170.2, 156.6, 156.2, 154.7; 136.3,
1
4
28.7, 128.3, 128.2, 79.9, 67.6, 67.1, 59.8, 54.2, 52.4, 48.2, 46.4, 45.2,
4.9, 41.5, 40.2, 28.6, 28.4, 24.8, 22.9, 22.8, 21.9; [HR-ESI(þ)]: m/z
1996, 39, 3603e3605.
8. Aussedat, B.; Chassaing, G.; Lavielle, S.; Burlina, F. Tetrahedron Lett. 2006, 47,
10Na [MþNa]þ 740.3841, found 740.3804.
calcd for C36
55 5
H N O
3723e3726.
9
. Aussedat, B.; Dupont, E.; Sagan, S.; Joliot, A.; Lavielle, S.; Chassaing, G.; Burlina,
F. Chem. Commun. 2008, 1398e1400.
Acknowledgements
1
1
0. Huttenloch, O.; Laxman, E.; Waldmann, H. Chem. Commun. 2002, 673e675.
1. Wysong, C. L.; Yokum, T. S.; Morales, G. A.; Gundry, R. L.; McLaughlin, M. L.;
Hammer, R. P. J. Org. Chem. 1996, 61, 7650e7651.
2. (a) Crisma, M.; Bonora, G. M.; Toniolo, C.; Benedetti, E.; Bavoso, A.; Di Blasio, B.;
Pavone, V.; Pedone, C. Int. J. Biol. Macromol. 1988, 10, 300e304; (b) Benedetti, E.;
Barone, V.; Bavoso, A.; Di Blasio, B.; Lelj, F.; Pavone, V.; Pedone, C.; Bonora, G.
M.; Toniolo, C.; Leplawy, M. T.; Kaczmarek, K.; Redlinski, A. Biopolymers 1988,
This study was supported, in part, by JSPS KAKENHI Grant
Number 26460169 (Y.D.), by a Grant-in-Aid from the Tokyo Bio-
chemical Research Foundation (Y.D.), and scholarship support from
the Shoshisha Foundation (H.Y.).
1
27, 357e371.
13. (a) Polese, A.; Formaggio, F.; Crisma, M.; Valle, G.; Toniolo, C.; Bonora, G. M.;
Broxterman, Q. B.; Kamphuis, J. Chem.dEur. J. 1996, 2, 1104e1111; (b) Demizu,
Y.; Doi, M.; Kurihara, M.; Maruyama, T.; Suemune, H.; Tanaka, M. Chem.dEur. J.
References and notes
2
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2
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