Kakeromamide A, a new cyclic pentapeptide inducing astrocyte differentiation isolated from the marine cyanobacterium Moorea bouillonii

Article abstract of DOI:10.1016/j.bmcl.2018.04.067

Kakeromamide A (1), a new cyclic pentapeptide encompassing a thiazole ring moiety and a β-amino acid, was isolated from the marine cyanobacterium Moorea bouillonii. Its structure was elucidated by the spectral analysis and the modified Marfey's method. Compound 1 induced differentiation of neural stem cells into astrocytes at the concentration of 10 μM.

Full text of DOI:10.1016/j.bmcl.2018.04.067

Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Kakeromamide A, a new cyclic pentapeptide inducing astrocyte
differentiation isolated from the marine cyanobacterium Moorea
bouillonii
a
a
a
b
c
d
Fumiaki Nakamura , Hiroshi Maejima , Midori Kawamura , Daisuke Arai , Tatsufumi Okino , Meng Zhao ,
d
e
e
b,f
a,b,
⇑
Tao Ye , Jungyeol Lee , Young-Tae Chang , Nobuhiro Fusetani , Yoichi Nakao
a
Department of Chemistry and Biochemistry, Graduate School of Advanced Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan
Research Institute for Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan
Graduate School of Environmental Science, Hokkaido University, Sapporo 060-0810, Japan
State Key Laboratory of Chemical Oncogenomics, Peking University Shenzhen Graduate School, Xili, Nanshan District, Shenzhen 518055, China
Department of Chemistry, POSTECH, Pohang 37673, Republic of Korea
b
c
d
e
f
Fisheries and Oceans Hakodate, 3-1-1 Minato-cho, Hakodate 041-8611, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Kakeromamide A (1), a new cyclic pentapeptide encompassing a thiazole ring moiety and a b-amino acid,
was isolated from the marine cyanobacterium Moorea bouillonii. Its structure was elucidated by the spec-
tral analysis and the modified Marfey’s method. Compound 1 induced differentiation of neural stem cells
into astrocytes at the concentration of 10 mM.
Received 5 April 2018
Revised 27 April 2018
Accepted 29 April 2018
Available online xxxx
Ó 2018 Published by Elsevier Ltd.
Keywords:
Pentapeptide
Cyanobacterium
Astrocyte differentiation
Neural stem cells
Marine natural products
Introduction
activity in compound 1 to induce differentiation of neural stem
cells into astrocytes at 10 mM in the in vitro differentiation
model using mouse ES cells. In this letter, we report the iso-
lation, structure elucidation, and biological activities of kakero-
mamide A (1).
5
,6
Marine cyanobacteria are known to be a rich source of pep-
tides such as lyngbyabellin B1 and dolastatin 10. In this study,
a new cyclic peptide named kakeromamide A (1) was isolated
from the marine cyanobacterium Moorea bouillonii collected at
Kakeroma Island in Kagoshima prefecture of the southern part
of Japan. This peptide shares the common amino acid sequence
with that of a cyclic depsipeptide ulongamide D4 in which
valine was replaced by hydroxy isovaleric acid. Ulongamide
D showed cytotoxicity against KB and LoVo cells with the
IC50 values of 1 and 5 mM, respectively, while compound 1
showed only a moderate cytotoxicity against HeLa cells with
the IC50 value of 10 mM. However, we found a unique biological
2,3
The frozen specimen of Moorea bouillonii (128 g wet weight),
collected by hand using SCUBA at Kakeroma Island in Kagoshima
0
0
prefecture (N 28° 04.67 , E 129° 18.42 ), was extracted with MeOH.
The combined methanolic extract was evaporated in vacuo and
2 3
partitioned between H O and CHCl . The organic layer was sub-
jected to ODS flash chromatography and followed by the
reversed-phase HPLC, yielding 1.2 mg of kakeromamide A (1) as
ꢁ6
the colorless amorphous solid (9.4 ꢀ 10 % yield based on the
wet weight).
⇑
Corresponding author at: Department of Chemistry and Biochemistry, Graduate
School of Advanced Science and Engineering, Waseda University, 3-4-1 Okubo,
Shinjuku-ku, Tokyo 169-8555, Japan.
E-mail address: ayocha@waseda.jp (Y. Nakao).
https://doi.org/10.1016/j.bmcl.2018.04.067
0
960-894X/Ó 2018 Published by Elsevier Ltd.
Please cite this article in press as: Nakamura F., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.04.067

2
F. Nakamura et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
kakeromamide A (1)
2
3.8
Kakeromamide A (1) with the optical rotation of [
a
58
]
D
+ 6.2°
6 7
O S as
(
c 0.065, MeOH), had a molecular formula of C42
H
N
+
determined by HRFABMS analysis (m/z 791.4171 [M+H] , calcd
for C42 S m/z 791.4166, +0.5 mDa). The IR absorption at
647, 1612, and 1541 cm indicated the presence of amide bonds.
H
59
N
6
O
7
D
ꢁ1
1
Fig. 1. Substructure of kakeromamide A (1).
Table 1
NMR spectral data for kakeromamide A (1) in CD
3
CN (400 MHz and 600 MHz).
mult. (J in Hz)
Unit
Val
Position
d
C
d
H
COSY
HMBC
1
2
3
4
5
176.8
57.2
32.0
18.9
20.2
4.30 dd (9.8, 7.3)
1.80 dqq (9.8, 6.8, 6.7)
0.79 d (6.8)
NH-2, H-3
H-2, H-4, H-5
H-3
H-3
H-2
C-2, C-5
C-2, C-4
C-32
0.89 d (6.7)
a
2
-NH
6.53 d (7.3)
N,O-diimethyl-Tyr-1
6
7
8
174.0
52.3
33.4
a
a
5.53 dd (11.3, 4.6)
1.38 dd (16.4, 4.6)
H-8
H-7
H-7
C-6 , C-8
a
C-7 , C-9
2
.70 dd (16.4, 11.3)
9
1
1
1
130.1
130.0
114.9
159.2
31.7
0/14
1/13
2
6.88 d (8.7)
6.77 d (8.7)
H-11/13
H-10/14
C-8, C-12
C-9
N-CH
O-CH
3
3.05 s
3.73 s
C-1, C-7
C-12
3
55.9
N,O-dimethyl-Tyr-2
15
169.9
63.7
34.6
a
a
16
7
5.25 dd (9.7, 5.2)
2.61 dd (14.4, 9.7)
H-17
H-16
H-16
C-15 , C-17
a
1
C-16 , C-18
2
.97 dd (14.4, 5.2)
1
1
2
2
8
131.0
131.3
115.2
159.5
29.7
9/23
0/22
6
6.99 d (8.6)
6.56 d (8.6)
H-20/22
H-19/23
C-17, C-21
C-18
N-CH
O-CH
3
2.86 s
3.48 s
C-6, C-16
C-21
3
55.7
Val-thz-ca
24
161.4
150.4
123.6
170.0
57.2
36.8
17.8
20.8
2
2
2
2
2
3
3
2
5
6
7
8
9
0
1
8.01 s
C25 , C-27a
a
a
5.33 dd (9.3, 5.5)
2.01 dqq (5.5, 6.8, 6.8)
0.78 d (6.8)
NH-28, H-29
H-28, H-30, H-31
H-29
C-27 , C-29
C-28, C-31
C-28, C-30
0.94 d (6.8)
H-29
8-NH
8.60 d (9.3)
H-28
Amha
32
173.3
44.6
52.8
31.9
20.2
14.6
14.4
3
3
3
3
3
3
3
3
4
5
6
7
8
2.60 dq (3.4, 7.0)
H-34, H-38
H-33, H-35, NH-34
H-34, H-36
H-35, H-37
H-36
4.09 dddd(12.0, 10.4, 3.4, 2.5)
1.07 m, 1.70 dddd (14.1, 9.2, 7.0, 2.5)
1.27 m, 1.45 m
C-33
0.97 t (7.5)
C-35, C-36
1.09 d (7.0)
H-33
C-32, C-33, C-34
4-NH
8.51 d (10.4)
H-34
a
observed only in the spectrum recorded on the spectrometer (600 MHz).
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F. Nakamura et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
3
The 1H NMR spectrum of compound 1 (Table 1) measured in
CD CN indicated its peptidal nature, i.e. three amide protons (d
.53, 8.51, 8.60), two singlets for N-methyl protons (d 2.86,
.05) and signals for four -protons (d 4.30, 5.25, 5.33, 5.53), as
-proton of the b-amino acid residue at d
COSY analysis revealed the presence of seven spin systems
3
H
6
3
H
a
H
2.60. 1H- H
1
well as an
a
H
7
8
(
Fig. 1). Analysis of HMQC and HMBC spectra identified three
a-amino acid residues; valine (Val) and two N,O-dimethyl-tyrosi-
nes (N,O-diMeTyr-1, 2). The fourth amino acid, 2-(1-amino-2-
methylpropyl)thiazole-4-carboxylic acid (Val-thz-ca), biogeneti-
cally derived from valine and cysteine, was deduced as follows; a
downfield shifted singlet proton signal at d
carbon resonances at d 123.6, 150.8, 161.4, and 170.0 were
characteristic of a 2-alkylthiazole-4-carboxylic acid unit (C-24 to
8.01 and the sp2
H
C
9
1
C-27). A characteristic
JCH value of 194 Hz between H-26
10
and C-26 supported the existence of a thiazole ring. HMBC
correlations between H-26/C-27 and H-28/C-27 connected the
thiazole unit to Val-like unit, to from a Val-thz-ca residue. The
Fig. 2. Key COSY and HMBC correlations for kakeromamide A (1).
Fig. 3. Astrocyte differentiation assay. (a): The schematic drawings of the astrocyte differentiation assay. (b): Fluorescent microscopy photograph of differentiated cells from
neural stem cells (NSCs). Cells were treated with DMSO (left) or 10 mM compound 1 (right, magnification ꢀ20, blue: fluorescent labeled nucleus by Hoechst 33342, green:
fluorescent labeled Glial Fibrillary Acidic Protein by chromeo 488, scale bar: 50 mm). (c): Rates for NSCs differentiating into astrocytes (calculated by counting of GFAP-
*
positive cells) compared to that of control (n = 4, mean ± S.D., p < 0.05). d: Percentages of cell numbers after sample treatment (n = 4, mean ± S.D.). (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)
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4
F. Nakamura et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
remaining b-amino acid was deduced as 3-amino-2-methylhex-
anoic acid (Amha) by an HMBC cross peak from H-38 of the spin
system (CH -38 to CH -37) to C-32.
3 3
Connectivities among these amino acids were established on
the basis of inter-residual HMBC cross peaks (Figs. S6 and S7).
In conclusion, a new cyclic peptide kakeromamide A (1) con-
taining unique amino acid residues of Val-thz-ca and b-amino acid
(Amha) was isolated from the marine cyanobacterium Moorea
bouillonii. Compound 1 induced differentiation of neural stem cells
into astrocytes without marked cytotoxicity. The detailed mecha-
nism for this biological activity is under the investigation.
H C
Correlation between 2-NH (d 6.53) and C-32 (d 173.3) clearly
indicated the amide bond formation between Val (NH) and Amha
(
CO). An HMBC cross-peak between N-methyl protons at d
H
2.86
Acknowledgments
(
N,O-diMeTyr-2) and d 174.0 (C-26) connected N,O-diMeTyr-2 to
C
N,O-diMeTyr-1, while an HMBC correlation between the N-methyl
signal at d 3.05 (N,O-diMeTyr-1) and C-1 (d 176.2) established
the linkage between N,O-diMeTyr-1 and Val. Although no inter-
We appreciate Prof. K. Hori, Dr. M. Hirayama, and the crews of
R/V Toyoshiomaru of Hiroshima University for their support in the
sample collection. We are also thankful for Emeritus Prof. J. Kimura
of Aoyama Gakuin University for providing synthetic samples of
Amha. This paper is a part of the outcome of research performed
under a Waseda University Grant-in-Aid for Scientific Research,
the Strategic Research Platforms for Private University, a Matching
Fund Subsidy from the Ministry of Education, Science, Sports, and
Technology (MEXT), Japan, and JSPS KAKENHI Grant Numbers
26221204, 25303008, and 19310138; Shenzhen Peacock Plan
(KQTD2015071714043444). This work was supported by JSPS
Core-to-Core Program, A. Advanced Research Networks. This work
was inspired by the international and interdisciplinary environ-
ments of the JSPS Asian CORE Program, Asian Chemical Biology
Initiative (ACBI) and JSPS A3 Foresight Program.
H
C
residual HMBC cross-peak was observed among Val-thz-ca and
other residues, the lower field shifted d values for H-28 (d
and H-34 (d 4.09) as well as typical values for amide protons of
8-NH (d 8.60) and 34-NH (d 8.51) strongly suggested that the
H
5.33)
H
2
H
H
nitrogen atoms at 28 and 34 form amide bonds. Therefore, position
of Val-thz-ca was between Amha and N,O-diMeTyr-2, completing
the planar structure of kakeromamide A (1) (Fig. 2). The detailed
NMR spectral data are listed in Table 1.
Absolute configuration of 5 amino acid residues in kakero-
mamide A (1) could be established by the modified Marfey’s
1
1,12
method.
thz-ca, and Amha) obtained by hydrolysis of compound 1 were
derivatized with -FDLA. LC-MS analysis of the -FDLA derivatives
Amino acid residues (Val, N,O-diMeTyr-1 and 2, Val-
L
L
using reversed phase column (C18) revealed the absolute configura-
tion of each residue.
A. Supplementary data
Kakeromamide A (1) exhibited a moderate cytotoxicity with an
IC50 value of 10 mM against HeLa cells, while it showed no cytotox-
icity against P388 cells at 10 mM. Cyclic depsipeptides ulongamide
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmcl.2018.04.067.
4
D encompassing the similar amino acid sequence in which Val is
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Please cite this article in press as: Nakamura F., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.04.067