Bisebromoamide, a potent cytotoxic peptide from the marine cyanobacterium lyngbya sp.: Isolation, stereostructure, and biological activity

Article abstract of DOI:10.1021/ol9020546

A novel cytotoxic peptide, termed bisebromoamide (1), has been Isolated from the marine cyanobacterium Lyngbya sp. Its planar structure was determined by 1D and 2D NMR spectroscopy. The absolute stereostructure of 1 was determined by chemical degradation

Full text of DOI:10.1021/ol9020546

ORGANIC
LETTERS
2009
Vol. 11, No. 21
5062-5065
Bisebromoamide, a Potent Cytotoxic
Peptide from the Marine
Cyanobacterium Lyngbya sp.: Isolation,
Stereostructure, and Biological Activity
Toshiaki Teruya,† Hiroaki Sasaki,† Hidesuke Fukazawa,‡ and Kiyotake Suenaga†,*
Department of Chemistry, Faculty of Science and Technology, Keio UniVersity,
Hiyoshi 3-14-1, Kohoku-ku, Yokohama 223-8522, Japan, and National Institute of
Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan
suenaga@chem.keio.ac.jp
Received September 4, 2009
ABSTRACT
A novel cytotoxic peptide, termed bisebromoamide (1), has been isolated from the marine cyanobacterium Lyngbya sp. Its planar structure
was determined by 1D and 2D NMR spectroscopy. The absolute stereostructure of 1 was determined by chemical degradation followed by
chiral HPLC analysis. Bisebromoamide (1) exhibited potent protein kinase inhibition: the phosphorylation of ERK in NRK cells by PDGF-
stimulation was selectively inhibited by treatment with 10-0.1 µM of 1.
Natural products, especially those from terrestrial plants and
microbes, have long been a source of drug molecules, and
pharmacologically active compounds from plants and microbes
continue to play an important role in new investigational drugs.1
However, considerable attention has recently been given to
marine organisms due to their remarkable physiological activi-
ties.2 In particular, cyanobacteria are prolific producers of
bioactive secondary metabolites3 and have been recognized as
a source of potential pharmaceuticals.4 For example, TZT-1027,
a synthetic analogue of dolastatin 10, is currently being
evaluated in phase I clinical trials in Japan, Europe, and the
United States.5 Dolastatin 10 was originally isolated from the
sea hare Dolabella auricularia6 and was recently obtained from
a marine cyanobacterium.7 Cryptophycin-309 and cryptophycin-
249, which are synthetic analogues of the terrestrial cyanobac-
terial peptide cryptophycin-1, have undergone preclinical effi-
cacy studies, and there is sufficient interest to consider entering
them into a clinical trial.8
In our ongoing efforts toward finding novel marine
cyanobacterial metabolites with antitumor activity,9 we report
here the structure determination and preliminary biological
characterization of bisebromoamide (1), a marine cyanobac-
† Keio University.
‡ National Institute of Infectious Diseases.
(1) Newman, D. J.; Cragg, G. M. J. Nat Prod. 2007, 70, 461–477. (b)
Butler, M. S Nat. Prod. Rep. 2008, 25, 475–561.
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ReV. Drug DiscoVery 2009, 8, 69–85.
(5) Horti, J.; Juhasz, E.; Monostori, Z.; Maeda, K.; Eckhardt, S.; Bodrogi,
I. Cancer Chemother. Pharmacol. 2008, 62, 173–180.
(6) Pettit, G. R.; Kamano, Y.; Herald, C. L.; Tuiman, A. A.; Boettner,
E. F.; Kizu, H.; Schmidt, J. M.; Baczynskyj, L.; Tomer, K. B.; Bontems,
R. J. J. Am. Chem. Soc. 1987, 109, 6883–6885.
(3) Gerwick, W. H.; Tan, L. T.; Sitachitta, N. Alkaloids 2001, 57, 75–
184.
(7) Luesch, H.; Moore, R. E.; Paul, V. J.; Mooberry, P. S.; Corbett,
T. H. J. Nat. Prod. 2001, 64, 907–910.
(4) Tan, L. T. Phytochemistry 2007, 68, 954–979.
10.1021/ol9020546 CCC: $40.75
Published on Web 10/05/2009
 2009 American Chemical Society

terial metabolite with antiproliferative activity at nanomolar
levels from a cyanobacterium of the genus Lyngbya sp.
connectivities were obtained from the NMR analysis, the Opp
and C-terminus of the N-Me-Phe residue were unambiguously
connected via an amide linkage, based on its molecular formula
and degree of unsaturation. Thus, the planar structure of
bisebromoamide (1) was determined (Figure 1).
The marine cyanobacterium Lyngbya sp. was directly
harvested in Okinawa Prefecture. A crude organic extract
of this material was subjected to bioassay-guided fraction-
ation by solvent partition, ODS-HPLC, to yield bisebromoa-
mide (1) as a colorless oil.
Figure 1. Gross structure of 1 determined by 2D-NMR spectroscopy
1
(bold lines, H-1H COSY; arrows, HMBC correlations).
The HRESIMS spectrum of bisebromoamide (1) gave
an [M + Na] pseudomolecular ion at m/z 1044.4212 that
was consistent with the pseudomolecular formula C51-
H7279BrN7O8SNa (calcd for C51H7279BrN7O8SNa, 1044.4244).
To assign the absolute configuration of the eight chiral
centers, we sought to generate optically active fragments,
for which some enantiomeric standards are commercially
available (Ala, Leu), while others required laboratory
synthesis by standard methods [N-Me-Tyr, Me-Pro, 2-me-
thylcystine, N-Me-Phe, and Opp]. Acid hydrolysis of 1
generated Ala, N-Me-Tyr, Me-Pro, 2-methylcystine, Leu,
N-Me-Phe, and Opp. The bromine atom of the N-Me-Br-
Tyr moiety was lost during acid hydrolysis of 1. The
hydrolysate could be separated into single compounds except
for a mixture of Ala and 2-methylcystine. Chiral HPLC
established the stereochemistry of N-Me-Tyr, N-Me-Phe, and
Leu to be D, L, and D, respectively. Treatment of Me-Pro,
Opp, and the mixture of Ala and 2-methylcystine with
Marfey’s reagent,10 followed by C18 HPLC, determined that
the stereochemistries of Ala and 2-methylcystine were L and
D, respectively. However, the Marfey derivatives of both Me-
Pro and Opp from 1 completely epimerized during acid
hydrolysis. On the other hand, the ozonolysis-acid hydroly-
sis sequence, which was developed to determine the stere-
ochemistry of thiazole amino acids,11 provided diastereo-
merically enriched 4(S)-Me-Pro [2S:2R ) 7:3] (Scheme 1).
1
The H and 13C NMR analysis showed that it was peptidic
in nature; however, there were several resonances that were
not attributable to the common ribosomally encoded amino
acids, implying that 1 possessed a highly functionalized
structure. The 1H NMR data in CDCl3 showed the presence
of two amide NH groups (δ 7.47, 6.37) and two N-
methylamide groups (δ 3.14, 3.06). The 1H NMR spectrum
showed a prominent intense singlet at δ 1.17 that was
attributed to a tert-butyl group. A COSY analysis in CDCl3
revealed that these exchangeable protons connected to alanine
(Ala) and leucine (Leu) residues, respectively. Further two-
dimensional NMR analysis in CD3OD using COSY, HMQC,
and HMBC data suggested the presence of N-methyl-3-
bromotyrosine (N-Me-Br-Tyr), modified 4-methylproline
(Me-Pro), N-methylphenylalanine (N-Me-Phe), and 2-(1-oxo-
propyl)pyrrolidine (Opp) residues (Table 1). Furthermore,
HMBCs from a methyl singlet (H-4; Me-Tzn, δ 1.52) to
carbonyl C-1 (Me-Tzn, δ 175.7), quaternary carbon C-2 (Me-
Tzn, δ 84.9), and methylene carbon C-3 (Me-Tzn, δ 43.5),
combined with H-3 (Me-Tzn, δ 3.23 and 3.37) to C-1 (Me-
Pro, δ 180.3), suggested the presence of a 2-substituted
thiazoline-4-methyl-4-carboxylic acid unit (Tzn ) thiazoline).
Finally, detailed HMBC experiments were used to determine
the connectivity between six amino acids residues (Table 1).
HMBC correlations H3 (pivalic acid)/C1 (pivalic acid) and H2
(Ala)/C1 (pivalic acid) suggested the connectivity of C1 (pivalic
acid)-C2 (Ala) via an amide linkage and revealed that 1
possesses an N-pivalamide moiety. Although no additional
Scheme 1. Degradation Strategy To Liberate Chiral Subunits
(8) Liang, J.; Moore, R. E.; Moher, E. D.; Munroe, J. E.; Al-awar, R. S.;
Hay, D. A.; Varie, D. L.; Zhang, T. Y.; Akins, J. A.; Martinelli, M. J.;
Shih, C.; Ray, J. E.; Gibson, L. L.; Vasudevan, V.; Polin, L.; White, K.;
Kushner, J.; Simpson, C.; Pugh, S.; Corbett, T. H. InVestig. New Drugs
2005, 23, 213–224.
(9) Teruya, T.; Sasaki, H.; Kitamura, K.; Nakayama, T.; Suenaga, K.
Org. Lett. 2009, 11, 2421–2424.
Org. Lett., Vol. 11, No. 21, 2009
5063

Table 1. NMR Spectral Data of 1 in CD3OD
position
1Ha (ppm)
13Cb (ppm)
HMBC (selected, HfC)
Opp
1
2
3
4
5
6
7
3.54 m
1.88 m
1.82 m, 2.20 m
4.56 dd (8.8, 4.4)
48.7
25.7
29.1
62.6
211.6
33.6
7.8
4 (Opp)
5 (Opp)
2.54 q (7.3)
1.03 t (7.3)
5 (Opp)
N-Me-Phe
1
2
3
170.4
56.5
35.7
5.82 dd (10.3, 5.4)
3.06 m
1 (N-Me-Phe)
4, 5 (N-Me-Phe)
4
138.3
5,9
6,8
7
8
7.27 dd (7.3, 2.4)
7.20 dd (7.8, 7.3)
7.09 dd (7.8, 2.4)
3.07 s
130.7
129.4
127.7
31.1
2 (N-Me-Phe), 1 (Leu)
Leu
1
2
3
4
5
6
174.3
50.2
40.0
25.7
21.7
23.7
4.52 dd (10.8, 3.4)
0.67 m, 1.57 m
1.52 m
0.81 d (6.4)
0.79 d (6.4)
1 (Leu), 1 (Me-Tzn)
Me-Tzn
1
2
3
175.7
84.9
43.5
3.37 d (11.2)
24.9
3.23 d (11.2),
1.52 s
1 (Me-Pro), 2 (Me-Tzn)
1, 2 (Me-Tzn)
4
Me-Pro
1
2
3
4
5
6
180.3
66.5
39.6
35.1
55.7
16.7
4.83 dd (10.7, 7.8)
1.67 m, 2.37 m
2.25 m
2.94 m, 3.56 m
1.04 d (6.8)
1 (N-Me-Br-Tyr)
1 (Me-Pro)
2 (Me-Pro)
N-Me-Br-Tyr
1
2
3
171.4
57.8
34.5
5.58 dd (9.3, 6.4)
2.96 m
1 (N-Me-Br-Tyr)
4, 5, 9 (N-Me-Br-Tyr)
4
5
6
7
8
9
10
131.2
135.1
110.3
154.1
117.1
131.0
32.3
7.36 d (2.0)
6.81 d (8.3)
7.07 d (8.3, 2.0)
3.12 s
2 (N-Me-Br-Tyr), 1 (Ala)
Ala
1
2
3
175.2
46.8
16.7
4.65 m
0.95 d (7.3)
1 (Ala), 1 (pivalic acid)
pivalic acid
1
2
3
180.7
39.3
27.7
1.14 s
1, 2 (pivalic acid)
a Recorded at 400 MHz. Coupling constants (Hz) are in parentheses. b Recorded at 100 MHz.
To prevent the racemization of Opp, reduction of the ketone
with NaBH4 followed by acid hydrolysis afforded 2(S)-(1-
hydroxypropyl)piperidine [6S:6R ) 1:1]. These analyses
identified Me-Pro and Opp as (2S,4S) and (2S), respectively.
5064
Org. Lett., Vol. 11, No. 21, 2009

protein at 10-0.1 µM. Some tubulin modulators have an
effect on the phosphorylation of ERK. The pattern of
differential cytotoxicity of 1 was evaluated by the Compare
Program and was revealed not to be correlated with that
shown by tubulin modulators. Immunoblotting analysis using
an antiacetylated lysine antibody supported this result.
Tubulin acetylation is one marker of microtubule stability
and is affected after treatment with some tubulin modulators.
The total levels of acetylated tubulin remained unchanged
by treatment with 1. Therefore, the ERK signaling pathways
may be one of the intracellular targets of 1. Aberrant
activation of the Ras/Raf/MEK/ERK pathway is commonly
observed in various cancers.12 Thus, the therapeutic targeting
of individual components of the Ras/Raf/MEK/ERK pathway
has attracted much attention in the development of anticancer
drugs. Recently, potent small-molecule inhibitors that target
the components of the Ras/Raf/MEK/ERK pathway have
been developed. Among them, RAF265, BAY 43-9006, and
AZD6244 have reached the clinical-trial stage.13 Bisebro-
moamide (1) is a promising lead agent in cancer drug
discovery.
Figure 2. Effect of bisebromoamide (1) on PDGF-signaling. NRK
cells were preincubated with the indicated concentrations of 1 for
3 h and then stimulated with PDGF for 10 min. The cells were
lysed, and proteins were subjected to SDS-PAGE and analyzed by
immunoblotting using a cocktail that contained antibodies against
phosphorylated forms of the components. A detailed procedure of
immunoblotting analysis is described in the Supporting Information.
In summary, bisebromoamide (1), a marine cyanobacterial
metabolite with antiproliferative activity at nanomolar levels,
was isolated from a cyanobacterium of the genus Lyngbya
sp. Further studies on the mode of action, cancer chemo-
therapeutic potential, and synthesis of bisebromoamide (1)
are in progress.
Therefore, the absolute stereostructure of bisebromoamide
(1) was determined to be as shown in formula 1.
Acknowledgment. We thank Professor Isao Inoue and Dr.
Takeshi Nakayama (University of Tsukuba) for identifying
the cyanobacterium. We thank Dr. T. Yamori (Screening
Committee of Anticancer Drugs supported by a Grant-in-
Aid for Scientific Research on Priority Area “Cancer” from
The Ministry of Education, Culture, Sports, Science and
Technology, Japan) for screening in the human cancer cell
line panel. This work was supported by a Grant-in-Aid for
Young Scientists (B) and Scientific Research (C) and a Grant
for Private Education Institute Aid from the Ministry of
Education, Culture, Sports, Science and Technology, Japan,
Suntory Institute for Bioorganic Research, and the Naito
Foundation.
Bisebromoamide (1) contains a high degree of D-amino
acids and N-methylated amino acids along with several other
modified amino acid residues of nonribosomal origin.
Furthermore, bisebromoamide (1) possesses a dense com-
bination of unusual structural features, including a substituted
Me-Tzn fused to a Me-Pro. Another unusual structural
element is the Opp and N-Me-Br-Tyr. The Opp unit in 1 is
unprecedented in natural products. In addition, 1 possesses
an N-pivalamide moiety.
Bisebromoamide (1) exhibited cytotoxicity against HeLa
S3 cells with an IC50 value of 0.04 µg/mL. Bisebromoamide
(1) was evaluated against a panel of 39 human cancer cell
lines (termed JFCR39) at the Japanese Foundation for Cancer
Research (see the Supporting Information). The average 50%
growth inhibition (GI50) value across all of the cell lines
tested was 40 nM. In addition, bisebromoamide (1) exhibited
potent protein kinase inhibition: the phosphorylation of ERK
(extracellular signal regulated protein kinase) in NRK cells
by PDGF (platelet-derived growth factor)-stimulation was
selectively inhibited by treatment with 10 to 0.1 µM of 1
(Figure 2). Bisebromoamide (1) had no effect on the
phosphorylation of AKT, PKD, PLCγ1, or S6 ribosomal
Supporting Information Available: Protein abbrevia-
tions, detailed experimental procedures, spectroscopic data,
and HCC panel data for 1. This material is available free of
charge via the Internet at http://pubs.acs.org.
OL9020546
(10) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591–596.
(11) McDonald, L. A.; Ireland, C. M. J. Nat. Prod. 1992, 55, 376–379.
(12) Roberts, P. J.; Der, C. J. Oncogene 2007, 26, 3291–3310.
(13) Montagut, C.; Settleman, J. Cancer Lett. 2009, 283, 125–134.
Org. Lett., Vol. 11, No. 21, 2009
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