Jahanyne, an apoptosis-inducing lipopeptide from the marine cyanobacterium lyngbya sp.

Article abstract of DOI:10.1021/ol5036722

An acetylene-containing lipopeptide, jahanyne, was isolated from the marine cyanobacterium Lyngbya sp. Its gross structure was established by spectroscopic analyses, and the absolute configuration was clarified based on a combination of chiral HPLC analyses, spectroscopic analyses, and derivatization reactions. Jahanyne significantly inhibited the growth of human cancer cells and induced apoptosis in HeLa cells.

Full text of DOI:10.1021/ol5036722

Letter
pubs.acs.org/OrgLett
Jahanyne, an Apoptosis-Inducing Lipopeptide from the Marine
Cyanobacterium Lyngbya sp.
Arihiro Iwasaki, Osamu Ohno, Shinpei Sumimoto, Hidetoshi Ogawa, Kim Anh Nguyen,
and Kiyotake Suenaga*
Department of Chemistry, Keio University, 3-14-1, Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan
S
* Supporting Information
ABSTRACT: An acetylene-containing lipopeptide, jahanyne,
was isolated from the marine cyanobacterium Lyngbya sp. Its
gross structure was established by spectroscopic analyses, and
the absolute configuration was clarified based on a
combination of chiral HPLC analyses, spectroscopic analyses,
and derivatization reactions. Jahanyne significantly inhibited
the growth of human cancer cells and induced apoptosis in
HeLa cells.
arious secondary metabolites have been isolated from
Vmarine sources over the past several decades. In a series of
investigations, marine microorganisms such as cyanobacteria,
fungi, and bacteria have been proven to be a rich source of new
biologically active substances.1,2 In particular, marine cyanobac-
teria have been recognized as prolific producers of novel
bioactive peptides,3 and we have isolated several compounds,
including bisebromoamide,4 kurahamide,5 kurahyne,6 and
maedamide,7 from marine cyanobacteria. In our continuing
The molecular formula of jahanyne (1) was found to be
search for novel bioactive substances, we investigated the
constituents of the marine cyanobacterium Lyngbya sp. and
isolated a novel acetylene-containing lipopeptide, jahanyne (1).
1 Possessed two rare partial structures, a 2-(1-oxo-ethyl)-
pyrrolidine moiety (Oep) and a 2,4-dimethyldec-9-ynoic acid
moiety (fatty acid). Although 2-(1-oxo-ethyl)-pyrrolidine itself
has been reported to be a constituent of tobacco,8 1 is the first
discovered compound that contains an Oep moiety as a partial
structure. To the best of our knowledge, a 2,4-dimethyl fatty
acid moiety containing a terminal acetylene group has been
reported in only one other natural product, and the
stereochemistry of the fatty acid moiety in the precedent
compound, carmabin A,9 has not been determined to date.
Here, we describe the isolation, structure elucidation, and
preliminary biological characterization of jahanyne (1).
The marine cyanobacterium Lyngbya sp. (900 g, wet weight)
was collected at the coast near Jahana, Okinawa, and extracted
with methanol. The extract was filtered, concentrated, and
partitioned between EtOAc and H2O. The organic extract was
further partitioned between 90% aqueous MeOH and hexane.
The material obtained from the aqueous MeOH portion was
subjected to fractionation with reversed-phase column
chromatography (ODS silica gel, MeOH−H2O) and repeated
reversed-phase HPLC (Cosmosil 5C18-MS-II, MeOH−H2O;
Cosmosil Cholester, MeCN−H2O) to afford 18.5 mg of
jahanyne (1).
C60H94N8O9 by HRESIMS (m/z 1093.7046, calcd for
C60H94N8O9Na [M + Na]+ 1093.7041). The NMR data for 1
1
are summarized in Table 1. The H NMR spectrum revealed
the presence of five singlets corresponding to N-methyl amide
substituents (δ 3.10, 3.03 2.96, 2.92, 2.14), seven α-methines of
amino acids (δ 5.88, 5.30, 5.06, 5.03. 4.93, 4.84, 4.72), and five
aromatic protons consistent with a phenyl group of a
phenylalanine (δ 7.22−7.29). Additionally, 1 possessed two
13C NMR absorptions consistent with a terminal acetylene
group (δ 85.0, 69.6) and nine carbonyl signals (δ 208.3, 179.3,
174.7, 172.5, 172.1, 171.8, 171.1, 170.9, 170.5). These data
suggested an acetylene-containing peptide structure. Detailed
1
analyses of the H NMR, 13C NMR, COSY, HMQC, and
HMBC spectra revealed the presence of two prolines, one N-
methylphenylalanine, three N-methylvalines, and one N-
methylalanine. Furthermore, the presence of two residues
derived from 2-(1-oxo-ethyl)-pyrrolidine and 2,4-dimethyldec-
9-ynoic acid (fatty acid) was clarified. The existence of the
terminal alkyne moiety was confirmed based on the IR band
(2116 cm−1).
The sequence of these partial structures was determined
based on the HMBC and NOESY data (Figure 1, Table S1).
Five HMBC correlations, N-Me of N-Me-Phe/C-1 of N-Me-
Received: December 20, 2014
© XXXX American Chemical Society
A
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1
Table 1. H and 13C NMR Data for Jahanyne (1) in CD3OD
a
b
a
b
position
Oep
δC
δH (J in Hz)
position
δC
δH (J in Hz)
4
5
20.4, CH3
18.3, CH3
30.9, CH3
0.87, d (6.8)
0.76, d (6.8)
2.92, s
1
26.8, CH3
208.3, C
2.18, s
2
N-Me
3
66.8, CH
28.7, CH2
4.61, dd (9.0, 5.0)
2.23, m
N-Me-Val3
4a
1
172.1, C
4b
1.90, m
2
60.0, CH
28.2, CH
18.8, CH3
19.9, CH3
30.8, CH3
5.06, d (11.2)
2.32, m
c
5a
25.81, CH2
48.3, CH2
2.06, m
3
5b
1.96, m
4
0.91, d (7.2)
0.83, d (6.8)
3.10, s
6a
3.84, m
5
6b
3.65, m
N-Me
Pro1
Pro2
1
172.5, C
1
174.7, C
58.8, CH
29.8, CH2
c
2
59.78, CH
29.4, CH2
4.72, dd (9.1, 3.8)
2.29, m
2
4.84, dd (8.9, 4.3)
2.26, m
3a
3a
3b
2.02, m
3b
1.72, m
c
c
4a
25.78, CH2
48.9, CH2
2.09, m
4a
25.78, CH2
2.04, m
4b
1.96, m
4b
1.97, m
5a
3.73, m
5a
48.4, CH2
3.66, m
5b
3.58, m
5b
3.55, m
N-Me-Phe
N-Me-Ala
1
170.5, C
56.6, CH
35.1, CH2
1
171.8, C
2
5.88, dd (11.9, 4.5)
3.10, m
2
52.6, CH
14.2, CH3
31.4, CH3
5.30, q (7.4)
1.27, d (7.4)
3.03, s
3a
3
3b
3.02, m
N-Me
4
138.3, C
fatty acid
5/9
130.5, CH
129.6, CH
128.1, CH
31.3, CH3
7.22, m
7.29, m
7.25, m
2.96, s
1
179.3, C
34.8, CH
42.5, CH2
6/8
2
2.95, m
1.52, m
1.30, m
1.43, m
1.33, m
1.12, m
1.48, m
1.33, m
1.48, m
2.17, m
7
3a
3b
4
N-Me
N-Me-Val1
31.8, CH
37.5, CH2
1
171.1, C
5a
5b
6a
6b
7
2
60.0, CH
28.1, CH
20.2, CH3
18.0, CH3
30.2, CH3
5.03, d (10.8)
2.15, m
3
27.1, CH2
4
0.83, d (6.8)
0.64, d (7.2)
2.14, s
5
29.8, CH2
18.9, CH2
85.0, C
N-Me
8
N-Me-Val2
9
1
2
3
170.9, C
10
11
12
69.6, CH
17.8, CH3
20.1, CH3
2.19, m
c
59.83, CH
4.93, d (11.1)
1.07, d (6.8)
0.89, d (6.4)
28.2, CH
2.31, m
a
b
c
Measured at 100 MHz. Measured at 400 MHz. These carbon signals are interchangeable, respectively.
The absolute configurations of all of the amino acid moieties
in 1 were assigned to be L-form by a comparison of the results
of chiral HPLC of the acid hydrolysate of 1 with D and L amino
acid standards (see Supporting Information, pp S10−S13).
With regard to the Oep moiety, a direct acid hydrolysis of 1 can
result in the epimerization of C-3 of the Oep moiety. To
prevent this epimerization, reduction of ketone with NaBH4
followed by acid hydrolysis afforded 2-(1-hydroxyethyl)-
pyrrolidine derived from the Oep moiety as a mixture of two
diastereomers. The absolute stereochemistry of Oep was
determined by reversed-phase HPLC analysis based on a
comparison of the retention times of Marfey derivatives10 of the
obtained diastereomeric alcohols to those of authentic samples.
As a result, the absolute configuration of the Oep moiety was
elucidated to be 3S (see SI, pp S14−S15).
Figure 1. Gross structure of jahanyne (1), based on 2D NMR
correlations.
Val1, N-Me of N-Me-Val1/C-1 of N-Me-Val2, N-Me of N-Me-
Val2/C-1 of N-Me-Val3, N-Me of N-Me-Val3/C-1 of Pro2, and
N-Me of N-Me-Ala/C-1 of the fatty acid, revealed two partial
structures, N-Me-Phe-N-Me-Val1-N-Me-Val2-N-Me-Val3-Pro2
and N-Me-Ala-Fatty acid. Furthermore, three NOESY
correlations, H-6a of Oep/H-2 of Pro1, H-5 of Pro1/H-2 of
N-Me-Phe, and H-5 of Pro2/H-2 of N-Me-Ala, allowed us to
determine the gross structure of 1 as shown in Figure 1.
The absolute configuration of C-2 of the fatty acid moiety
was determined by applying the PGME method.11 Catalytic
reduction of 1 followed by acid hydrolysis afforded 2,4-
B
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dimethyldecanoic acid derived from 1. The obtained fatty acid
was condensed with (R)- or (S)-phenylglycine methyl ester
1
(PGME) to afford 2a and 2b, respectively. The H NMR
spectra of the obtained diastereomers were compared, and
calculated Δδ values revealed that the stereochemistry of C-2
was 2R (Scheme 1; see SI, pp S16−S19).
Scheme 1. Preparation of the PGME Amides of the Fatty
Acid from Jahanyne (1)
Figure 2. Induction of apoptosis by jahanyne (1) in HeLa cells. (A)
HeLa cells were preincubated (solid column) or not (open column)
with 50 μM of Z-VAD-FMK. They were then treated with the
indicated concentrations of 1. After further incubation for 48 h, cell
viability was determined based on trypan blue dye exclusion. Values
are the mean SD of quadruplicate determinations. (B) HeLa cells
were incubated with the indicated concentrations of 1 for 24 h.
Cellular DNA was then extracted and electrophoresed on agarose gels.
The remaining stereochemistry at C-4 of the fatty acid was
established based on the differences in 1H NMR chemical shifts
between two geminal protons at C-3. According to Schmidt et
al.,12 it is possible to determine the relative configuration of 1,3-
dimethyl-substituted alkyl chains by interpreting 1H NMR shift
differences between two geminal protons sandwiched between
two methyl groups. The Δδ value of H-3a and H-3b in 1, 0.22
ppm, apparently indicated that the relative stereochemistry of
the fatty acid moiety was anti, (2R,4S). To confirm this
stereochemistry, we synthesized the (R)-PGME amide of
(2S,4R)-2,4-dimethyldecanoic acid (3), the enantiomer of 2b,
for comparison purposes. Hydrolysis of the known amide 413
followed by condensation with (R)-PGME afforded 3 (Scheme
2). The 1H NMR spectrum of 3 was identical to that of 2b (see
SI, pp S20−S21). Therefore, the absolute stereochemistry of
jahanyne was established as shown in 1.
amino acids were clarified based on the chiral HPLC analyses of
the acid hydrolysate. The absolute stereochemistry of the
characteristic 2,4-dimethyl fatty acid moiety was established
based on a combination of the PGME method, a spectroscopic
analysis developed by Schmidt et al., and a synthetic method.
Through these experiments, we demonstrated that the method
by Schmidt et al.12 was effective for determining the structures
of natural products. Additionally, 1 is the first discovered
compound that contains a 2-(1-oxo-ethyl)-pyrrolidine as a
partial structure, and it is indicated that the marine
cyanobacterium Lyngbya sp. is the prolific source of natural
products possessing characteristic structures. With respect to
biological activity, 1 significantly inhibited the growth of both
HeLa cells and HL60 cells, with IC50 values of 1.8 μM and 0.63
μM, respectively. Furthermore, 1 was revealed to induce
apoptosis in HeLa cells. According to the previous paper,14 the
structure-related compound, carmabin A, exhibited good
antimalarial activity. The acyclic nature and the presences of
a 2,4-dimethyldec-9-ynoic acid moiety and an aromatic amino
acid moiety are consistent with each other. Therefore, jahanyne
(1) may exhibit antimalarial activity. A detailed investigation of
the bioactivity of 1 is in progress.
Scheme 2. Synthesis of the (R)-PGME Amide of (2S,4R)-
2,4-Dimethyldecanoic Acid (3)
To evaluate the growth-inhibitory activities of jahanyne (1),
an MTT assay with HeLa cells and HL60 cells was used. The
cells were treated in 96-well plates with various concentrations
of the compounds (0.01−10 μM for HeLa cells, 0.001−10 μM
for HL60 cells) for 72 h. The data from these assays revealed
that 1 inhibited the growth of both HeLa cells and HL60 cells,
with IC50 values of 1.8 μM and 0.63 μM, respectively.
Additionally, 1 showed cytotoxicity against HeLa cells, as
determined using the trypan blue dye exclusion assay (Figure
2A). Furthermore, the cell death of these cells induced by 1 was
suppressed in the presence of Z-VAD-FMK, an irreversible and
cell-permeable inhibitor of caspases (Figure 2A). Significant
DNA laddering in these cells was observed in the presence of 1
(Figure 2B). These results indicated that 1 induced apoptosis in
HeLa cells.
ASSOCIATED CONTENT
* Supporting Information
■
S
1H, 13C, COSY, NOESY, HMQC, and HMBC NMR spectra in
CD3OD for jahanyne (1). HPLC chromatograms for
determination of the absolute configurations. Detailed exper-
imental procedures. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
In conclusion, jahanyne (1), a novel acetylene-containing
lipopeptide, was isolated from the marine cyanobacterium
Lyngbya sp. The gross structure of 1 was established by
spectroscopic analyses, and the absolute configurations of
*E-mail: suenaga@chem.keio.ac.jp.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was supported in part by Grants-in-Aid from the
Ministry of Education, Culture, Sports, Science and Technol-
ogy of Japan (24310160 and 20436992), Shimadzu Science
Foundation, Novartis Foundation (Japan) for the Promotion of
Science, the Uehara Memorial Foundation, and Keio Gijuku
Fukuzawa Memorial Fund for the Advancement of Education
and Research.
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