Ohmyungsamycins A and B: Cytotoxic and antimicrobial cyclic peptides produced by Streptomyces sp. from a volcanic island

Article abstract of DOI:10.1021/jo401974g

Ohmyungsamycins A and B (1 and 2), which are new cyclic peptides, were isolated from a marine bacterial strain belonging to the Streptomyces genus collected from a sand beach on Jeju, a volcanic island in the Republic of Korea. Based on the interpretation of the NMR, UV, and IR spectroscopic and MS data, the planar structures of 1 and 2 were elucidated as cyclic depsipeptides bearing unusual amino acid units, including N-methyl-4-methoxytrytophan, β-hydroxyphenylalanine, and N,N-dimethylvaline. The absolute configurations of the α-carbons of the amino acid residues were determined using the advanced Marfey's method. The configurations of the additional stereogenic centers at the β-carbons of the threonine, N-methylthreonine, and β-hydroxyphenylalanine units were assigned by GITC (2,3,4,6-tetra-O-acetyl- β-d-glucopyranosyl isothiocyanate) derivatization and the modified Mosher's method. We have developed a new method utilizing PGME (phenylglycine methyl ester) derivatization coupled with chromatographic analysis to determine the absolute configuration of N,N-dimethylvaline. Our first successful establishment of the absolute configuration of N,N-dimethylvaline using PGME will provide a general and convenient analytical method for determining the absolute configurations of amino acids with fully substituted amine groups. Ohmyungsamycins A and B showed significant inhibitory activities against diverse cancer cells as well as antibacterial effects.

Full text of DOI:10.1021/jo401974g

Featured Article
pubs.acs.org/joc
Ohmyungsamycins A and B: Cytotoxic and Antimicrobial Cyclic
Peptides Produced by Streptomyces sp. from a Volcanic Island
Soohyun Um,† Tae Joon Choi,† Heegyu Kim,‡ Byung Yong Kim,§ Seong-Hwan Kim,† Sang Kook Lee,†
Ki-Bong Oh,‡ Jongheon Shin,† and Dong-Chan Oh*,†
†Natural Products Research Institute, College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742,
Republic of Korea
‡Department of Agricultural Biotechnology, College of Agriculture and Life Science, Seoul National University, 1 Gwanak-ro,
Gwanak-gu, Seoul 151-921, Republic of Korea
§Department of Agricultural Microbiology, National Academy of Agricultural Science, Suwon 441-707, Republic of Korea
S
* Supporting Information
ABSTRACT: Ohmyungsamycins A and B (1 and 2), which
are new cyclic peptides, were isolated from a marine bacterial
strain belonging to the Streptomyces genus collected from a
sand beach on Jeju, a volcanic island in the Republic of Korea.
Based on the interpretation of the NMR, UV, and IR
spectroscopic and MS data, the planar structures of 1 and 2
were elucidated as cyclic depsipeptides bearing unusual amino
acid units, including N-methyl-4-methoxytrytophan, β-hydrox-
yphenylalanine, and N,N-dimethylvaline. The absolute config-
urations of the α-carbons of the amino acid residues were
determined using the advanced Marfey’s method. The configurations of the additional stereogenic centers at the β-carbons of the
threonine, N-methylthreonine, and β-hydroxyphenylalanine units were assigned by GITC (2,3,4,6-tetra-O-acetyl-β-D-
glucopyranosyl isothiocyanate) derivatization and the modified Mosher’s method. We have developed a new method utilizing
PGME (phenylglycine methyl ester) derivatization coupled with chromatographic analysis to determine the absolute
configuration of N,N-dimethylvaline. Our first successful establishment of the absolute configuration of N,N-dimethylvaline using
PGME will provide a general and convenient analytical method for determining the absolute configurations of amino acids with
fully substituted amine groups. Ohmyungsamycins A and B showed significant inhibitory activities against diverse cancer cells as
well as antibacterial effects.
ioactive secondary metabolites from marine organisms are
now generally considered to be promising and developable
and form distinctive seashores that differ from common quartz-
rich sand beaches7 and that have the potential to harbor unique
microorganisms that genetically encode new bioactive com-
pounds. During our recent studies of microorganisms collected
around Jeju Island, we discovered a new lasso peptide,
sungsanpin, from a deep-sea Streptomyces sp.8 and new polyene
polyols, separacenes A−D, from a seashore Streptomyces sp.9 Our
continued isolation and chemical screening of marine actino-
mycetes led to the discovery of a Streptomyces strain (SNJ042,
most closely related to Streptomyces cheonanensis) that was
isolated from Shinyang Beach on Jeju Island; this strain produces
new cyclic peptides, ohmyungsamycins A and B (1 and 2). The
ohmyungsamycins were found to be structurally unique due to
the incorporation of unusual amino acids such as N-methyl-4-
methoxytryptophan, β-hydroxyphenylalanine, and N,N-dime-
thylvaline. Here, we report the spectroscopic structural
elucidation, the determination of absolute configuration,
including the application of a newly developed chromatographic
B
drug sources.1 Since the approval of zinconotide from a tropical
cone snail as an analgesic agent in the United States in 2004,
several secondary metabolites from marine invertebrates and
their synthetic analogues, such as ecteinascidin-743 and
halichondrin, have been marketed for clinical use.1 In addition
to marine macroorganisms, marine microorganisms have been
more recently spotlighted as chemical treasures.2,3 A statistical
overview of marine natural products between 1985 and 2008
clearly demonstrates that the pace of discovery of bioactive
secondary metabolites from marine microbes has accelerated
over the last 15 years.4 Leading the drug candidates discovered
from marine microbes is salinosporamide A, a potent proteasome
inhibitor from the obligate marine actinomycete Salinispora
tropica, which has advanced to phase II clinical trials for the
treatment of myeloma patients.5
Investigations of unique environments that have rarely been
studied are necessary to efficiently discover new bioactive
compounds.6 Jeju Island is a volcanic island in the subtropical
region of the Republic of Korea. Its volcanic bedrocks, which are
rich in basalt and trachyte, provide feldspar and mafic minerals
Received: September 5, 2013
Published: November 22, 2013
© 2013 American Chemical Society
12321
dx.doi.org/10.1021/jo401974g | J. Org. Chem. 2013, 78, 12321−12329

The Journal of Organic Chemistry
Featured Article
a
Table 1. NMR Data for Ohmyungsamycin A (1) in Pyridine-d5
C/H
δC, type
δH, mult (J, Hz)
C/H
39
δC, type
δH, mult (J, Hz)
Val-1
1
174.3, C
58.3, CH
Val-6
173.2, C
54.3, CH
b
2
4.70, dd (8.5, 8.5)
9.28, d (8.5)
2.23, m
40
4.96, m
2-NH
3
40-NH
41
9.39, d (9.0)
2.40, m
32.8, CH
19.3, CH3
18.6, CH3
172.7, C
31.9, CH
19.0, CH3
19.7, CH3
171.7, C
b
b
4
1.12, m
42
1.14, m
b
5
0.97, m
43
1.03, d (6.0)
β-hydroxy-Phe-2
6
N-Me-Leu-7
44
7
60.0, CH
5.49, dd (8.0, 2.5)
9.66, d (8.0)
45
54.8, CH
38.9, CH2
5.65, dd (8.0)
1.74, m
7-NH
8
46a
46b
47
73.2, CH
143.0, C
5.94, d (2.5)
1.58, m
9
25.1, CH
23.1, CH3
21.9, CH3
31.3, CH3
173.5, C
1.43, m
10
11
12
13
14
15
16
16-NH
17
18
19
20
21
22a
22b
23
24
24-NH
25
26
27
28
29
30
31
32
33
34
35
36
37
38
127.0, CH
128.6, CH
127.5, CH
128.6, CH
127.0, CH
174.5, C
7.68, d (7.5)
48
0.67, d (6.0)
0.67, d (6.0)
3.52, s
7.41, dd (7.5, 7.5)
7.27, d (7.5)
49
50
7.41, dd (7.5, 7.5)
7.68, d (7.5)
Val-8
51
52
55.6, CH
5.29, dd (8.5, 8.5)
8.06, d (8.5)
2.57, m
Val-3
52-NH
53
58.3, CH
5.41, dd (8.5, 8.5)
8.02, d (8.5)
2.66, m
31.0, CH
19.0, CH3
19.7, CH3
170.6, C
54
1.20, d (7.0)
1.16, d (7.0)
33.1, CH
19.8, CH3
20.0, CH3
169.9, C
55
1.36, d (6.5)
1.20, d (7.5)
N-Me-Thr-9
Thr-10
56
57
62.4, CH
66.5, CH
20.5, CH3
34.5, CH3
171.4, C
5.61, d (3.5)
b
N-Me-4-methoxy-Trp-4
58
5.05, m
70.6, CH
27.1, CH2
4.59, dd (11.0, 4.5)
4.45, dd (13.5, 4.5)
4.32, dd (13.5, 11.0)
59
1.34, d (6.5)
3.68, s
60
61
112.9, C
62
52.5, CH
5.86, dd (8.5, 2.0)
10.02, d (8.5)
b
124.3, CH
7.19, m
62-NH
63
11.80, d (1.5)
69.4, CH
16.8, CH3
173.4, C
6.04, qd (6.5, 2.0)
1.52, d (6.5)
139.5, C
64
105.8, CH
122.7, CH
99.5, CH
154.9, C
7.30, m
Val-11
65
7.27, m
66
57.9, CH
5.24, dd (9.5, 7.5)
8.65, d (9.5)
2.26, m
6.67, d (7.0)
66-NH
67
31.8, CH
19.8, CH3
18.8, CH3
174.2, C
b
55.4, CH3
118.4, C
3.83, s
2.51, s
68
0.96, m
b
69
0.96, m
40.8, CH3
169.4, C
N-Me-Val-12
70
N-Me-Val-5
71
71.4, CH
32.2, CH
19.1, CH3
19.1, CH3
35.9, CH3
3.11, d (6.0)
2.18, m
70.9, CH
29.0, CH
21.9, CH3
19.7, CH3
39.9, CH3
3.19, m
72
3.03, m
73
1.09, m
1.22, d (7.0)
74
1.09, m
b
1.02, m
75
2.54, s
3.18, s
a
b
1H and 13C data were recorded at 900 and 225 MHz, respectively. Overlapped signals.
analysis using phenylglycine methyl ester (PGME) derivatives
for N,N-dimethylvaline, and the biological activities of the
ohmyungsamycins.
culture, isolate the compound, and elucidate the structure.
During our scale-up process, we discovered two new cyclic
peptides, ohmyungsamycins A and B.
Ohmyungsamycin A (1) was isolated as a white powder, and
the molecular formula was established as C75H119N13O16 by
HRFABMS data coupled with 1H and 13C NMR spectra (Table
RESULTS AND DISCUSSION
■
The Streptomyces strain SNJ042 was isolated from a sediment
sample collected from Shinyang Beach, Jeju Island. Strain
SNJ042 was cultivated in a seawater-based liquid medium, and its
production of secondary metabolites was chemically analyzed by
LC/MS. In our chemical analysis, we detected that SNJ042
produced a major compound with the UV spectra indicating an
indole moiety (λmax at 203 and 281 nm). Our dereplication
process based on its indole signature and molecular ion (m/z at
1458.8) indicated that this secondary metabolite is possibly
unknown. This initial evaluation prompted us to scale up the
1
1). The H NMR spectrum showed seven downfield amide
proton signals at δH 10.1−8.0 and five N-methyl groups at δH
3.7−2.5, indicating an amino acid-based structure bearing 12
residues. The 13C NMR spectrum consistently displayed 12
amide or ester carbonyl signals at δC 175−169. In addition to
these peptide features, ohmyungsamycin A showed unusual
1
signals in the H, 13C, and HSQC NMR spectra that were not
associated with standard amino acids. The methyl group at δC
55.4 - δH 3.83 is clearly a signal resulting from a methoxy group.
12322
dx.doi.org/10.1021/jo401974g | J. Org. Chem. 2013, 78, 12321−12329

The Journal of Organic Chemistry
Featured Article
The oxygenated methine correlation at δC 73.2 - δH 5.94 also
suggested the presence of additional oxidation modifying a
typical amino acid unit.
at C-29 on the basis of the HMBC correlation from H3-30 to C-
29. Lastly, further HMBC couplings from the N-methyl group at
C-32 to C-21 and from H-21 to the C-20 carbonyl carbon (δC
169.9) established this residue as an N-methyl-4-methoxytrypto-
phan unit (Figure 1b).
The connectivity of the 12 identified amino acid units was
established on the basis of the HMBC correlations (Figure 2).
First, the 2-NH (δH 9.28) amide proton of Val-1 correlated with
the carbonyl carbon (C-6; δC 172.7) of β-hydroxy-Phe-2 in the
HMBC NMR spectrum, establishing the linkage between Val-1
to β-hydroxy-Phe-2. The NH (7-NH; δH 9.66) of β-hydroxy-
Phe-2 clearly showed a heteronuclear correlation to C-15 (the
amide carbon of Val-3; δC 174.5), thus establishing the
connectivity between β-hydroxy-Phe-2 and Val-3. The next
amino acid was deduced as N-methyl-4-methoxy-Trp-4 on the
basis of the HMBC correlation from 16-NH (δH 8.02) to C-20
(the carbonyl carbon of N-methyl-4-methoxy-Trp-4; δC 169.9).
1
The long-range H−13C coupling from H3-32 (N-methyl of N-
methyl-4-methoxy-Trp-4; δH 2.51) to C-21 (α-carbon of N-
methyl-4-methoxy-Trp-4; δC 70.6) and C-33 (the carbonyl
carbon of N-methyl-Val-5; δC 169.4) connected this residue to
N-methyl-Val-5. The N-methyl protons (H3-38; δH 3.18) of N-
methyl-Val-5 displayed an HMBC correlation with C-39 (the
carbonyl carbon of Val-6; δC 173.2), thereby establishing the
sequence from N-methyl-Val-5 to Val-6. Val-6 was then
connected to N-methyl-Leu-7 on the basis of the HMBC
coupling of 40-NH (δH 9.39) with C-44 (δC 171.7). The N-
methyl group of N-methyl-Leu-7 displayed strong correlations to
C-45 (δC 54.8) and C-51 (δC 173.5), thus confirming the
extension of the peptide chain to Val-8. The NH of Val-8 at δH
8.06 coupled with the carbonyl carbon of N-methyl-Thr-9 (C-56;
δC 170.6), which connected the N-methyl-Thr-9 unit to Val-8.
Further analysis of the HMBC spectrum enabled us to establish
the connectivity between N-methyl-Thr-9 and Thr-10 on the
basis of the heteronuclear coupling of H3-60 (δH 3.68) and C-61
(δC 171.4). The next residue was assigned as Val-11 on the basis
of the HMBC correlation from the NH proton (δH 10.02) of
Thr-10 to the amide carbon (C-65) of Val-11 at δC 173.4. The
last unit of the peptide, N-methyl-Val-12, was then connected by
the long-range coupling of 66-NH (δH 8.65) and C-70 (δC
174.2).
Further analysis of the COSY, TOCSY, HSQC, and HMBC
NMR spectra enabled the straightforward identification of 10
standard amino acids, including five valines, two N-methylva-
lines, one threonine, one N-methylthreonine, and one N-
methylleucine. One of the remaining two amino acid units was
β-hydroxyphenylalanine. The COSY and TOCSY correlations of
7-NH (δH 9.66), H-7 (δH 5.49), and H-8 (δH 5.94) and a HMBC
correlation from H-7 to C-6 (δC 172.7) established that the
backbone of the amino acid bears an oxygenated β-carbon (C-8;
δC 73.2). The HMBC correlation from H-8 to the aromatic
carbons C-9 (δC 143.0), C-10 (δC 127.0), and C-14 (δC1127.0)
revealed that this unit is an aromatic residue. The H−1H
couplings between the aromatic protons H-10 to H-14, along
with their coupling constants (J = 7.5 Hz), established this as a
six-membered aromatic ring and thus identified this unusual
residue as β-hydroxyphenylalanine (Figure 1a). The last amino
The elucidated structure thus far bore a phenyl ring, an indole
ring, and 12 carbonyl groups, which accounted for 22 of the 23
degrees of unsaturation deduced from the molecular formula.
Therefore, we deduced that the last double bond equivalent must
be a result of the presence of an additional ring. Careful analysis
of the HMBC spectrum revealed a correlation from H-63 (β-
proton of Thr-10; δH 6.04) to C-1 (the carbonyl carbon of Val-1;
δC 174.3), thus indicating a macrolide tethered through Thr-10
and Val-1. The planar structure of ohmyungsamycin A (1) was
thus defined as a cyclic depsipeptide composed of 12 amino acid
residues.
Ohmyungsamycin B (2) was purified as an amorphous white
powder, and the molecular formula was determined to be
C76H121N13O16 through analysis of the HRFABMS data and the
1H and 13C NMR spectra (Table 2). The molecular formula of 2
possesses one more carbon and two more proton atoms than that
of 1, which indicates that ohmyungsamycin B is an analogue of 1
with slight modifications. The 1H and 13C NMR spectra of 2 had
very similar features compared to those of 1, as expected. The
only difference was an additional N-methyl group at δH 2.54 and
δC 35.9. Further analysis of the COSY, TOCSY, HSQC, and
Figure 1. Structure elucidation of the unusual amino acids in 1 on the
basis of COSY and HMBC NMR spectra: (a) β-hydroxyphenylalanine;
(b) N-methyl-4-methyoxytryptophan.
acid was assigned as N-methyl-4-methoxytryptophan. The
COSY correlation between H-21 (δH 4.59) and H2-22 (δH
4.45, 4.32) connected the α-carbon (C-21; δC 70.6) and β-
carbon (C-22; δC 27.1) of the amino acid unit. The HMBC
correlation from H2-22 to C-23 (δC 112.9), C-24 (δC 124.3), and
C-31 (δC 118.4) indicated that this unit had an aromatic group in
the side chain. The proton−proton couplings in the COSY and
TOCSY NMR spectra between H-26 (δH 7.30) and H-27 (δH
7.27) and between H-27 and H-28 (δH 6.67, J = 7.0 Hz) allowed
us to construct a partial structure of a six-membered aromatic
ring. The strong long-range heteronuclear couplings from H-27
and H-24 to C-25 (δC 139.5), from H-28 and H-24 to C-31, and
from H-27 to C-29 (δC 154.9), along with the COSY correlation
between 24-NH (δH 11.80) and H-24, indicated the presence of
an indole ring in the side chain. The methoxy group was assigned
12323
dx.doi.org/10.1021/jo401974g | J. Org. Chem. 2013, 78, 12321−12329

The Journal of Organic Chemistry
Featured Article
Figure 2. Key HMBC correlations for the determination of the planar structure of ohmyungsamycin A (1).
HMBC spectra revealed that the last unit of ohmyungsamycin A,
N-methyl-Val-12, was replaced with N,N-dimethyl-Val-12.
Because ohmyungsamycins A and B are composed of α-amino
acids, the advanced Marfey’s method using FDAA (1-fluoro-2,4-
dinitrophenyl-5-alanine amide) was applied to determine the
absolute configurations of the amino acid α-carbons.10 Acid
hydrolysis of 1 was performed for 1 h to minimize degradation of
the fragile amino acids such as β-hydroxyphenylalanine and N-
methyl-4-methoxytryptophan.11 LC/MS analyses of the L- and D-
FDAA derivatives allowed us to determine that the absolute
configurations of all α-carbons found in 1 are S (L).
Because threonine has an additional stereogenic center at the
β-carbon, its configuration in 1 was established through
comparison of the retention time of its GITC (2,3,4,6-tetra-O-
acetyl-β-D-glucopyranosyl isothiocyanate) derivative with the
retention times of the GITC derivatives of authentic standards of
L-allo-Thr and L-Thr as well as standards of N-methyl-L-allo-Thr
and N-methyl-L-Thr.12 The LC/MS analysis of the GITC
derivatives established that ohmyungsamycin A possesses L-Thr
and N-methyl-L-Thr and not the allo-forms.
Although the absolute configurations of common amino acid
residues were readily assigned via the advanced Marfey’s method,
β-hydroxyphenylalanine bears one additional stereogenic center
at the β-carbon (C-8), whose absolute configuration could not be
determined by the advanced Marfey’s method or GITC
derivatization. Thus, we applied the modified Mosher’s method
to determine the absolute configuration of this chiral center.13 S-
and R-MTPA esters (3 and 4) at C-8 were obtained through
derivatization of 1 with R- and S-α-methoxy-α-(trifluoromethyl)-
phenylacetyl chloride (MTPA-Cl). The 1H chemical shifts
around the β-carbon of β-hydroxyphenylalanine were deter-
mined from the 1H and COSY NMR spectra, and ΔδS−R values
were calculated. On the basis of the consistent distribution of
ΔδS‑R values, the absolute configuration of the β-carbon of β-
hydroxyphenylalanine (C-8) was assigned as R (Figure 3).
Ohmyungsamycin B (2) incorporates N,N-dimethylvaline,
which does not contain an NH to react with FDAA or GITC for
the determination of its absolute configuration. We therefore
developed a new method utilizing phenylglycine methyl ester
(PGME) derivatization of the carboxylic acid side of N,N-
dimethylvaline and subsequent chromatographic analysis by LC/
MS. PGME was originally developed as an anisotropic reagent
with which to generate diastereomers for determining the
absolute configuration of chiral carboxylic acids on the basis of
1H NMR spectroscopic analysis in a manner analogous to the
method employing MTPA for secondary alcohols.14 Instead of
NMR analysis, which requires the preparative separation of
esterified products, a chromatographic application using MTPA
esters has been reported for determining the absolute
configuration of 2-hydroxyisoleucic acid.15 Thus, we developed
a new method for chromatographic assignment of the absolute
configuration of the S- and R-PGME adducts of N,N-dimethylva-
line in 2 by LC/MS analysis (Figure 4a).
After acid hydrolysis, the free amino acids were coupled with S-
and R-PGME. S- and R-PGME derivatives (5 and 6) of N,N-
dimethylvaline were clearly separated during our LC/MS
analysis (Figure 4b). The absolute configuration of N,N-
dimethylvaline in 2 was determined to be L (S) by comparison
of the retention times of its PGME derivatives with those of
authentic N,N-dimethyl-L- and D-valine-PGME adducts (Figure
4b). The remaining stereogenic centers of 2 were deduced to be
identical to those in 1 on the basis of the high similarity of the 1H
1
and 13C chemical shifts, the H−1H coupling constants, the
optical rotation values, and the CD spectra (see the Supporting
Information).
After establishing the absolute configuration of 1 completely,
1
we analyzed trans-annular ROESY correlations of 1. The H
NMR spectrum of 1 actually displayed an array of minor signals
(5:1 major to minor ratio), indicating that this compound has
another conformer in the solution caused by the amide bond
heterogeneity. The assignment of 1H NMR chemical shifts of the
conformer and the careful analysis of trans-annular ROESY
correlations of both conformers revealed that the main
differences between the two conformers reside mainly in the
orientations of the N-methyl groups at C-38 and C-60 in N-Me-
Val-5 and N-Me-Thr-9. In the major conformer, H3-38 are
positioned on the outside of the macrolactone ring and H3-60 are
oriented toward the inside of the ring. However, in the minor
conformer, the orientations of these methyl groups are opposite,
12324
dx.doi.org/10.1021/jo401974g | J. Org. Chem. 2013, 78, 12321−12329

The Journal of Organic Chemistry
Featured Article
a
Table 2. NMR data for Ohmyungsamycin B (2) in Pyridine-d5
C/H
δC, type
δH, mult (J, Hz)
C/H
39
δC, type
δH, mult (J, Hz)
Val-1
1
174.3, C
58.3, CH
Val-6
173.2, C
54.3, CH
b
2
4.70, dd (8.5, 8.5)
9.26, d (8.5)
2.23, m
40
4.96, m
2-NH
3
40-NH
41
9.39, d (9.0)
2.40, m
32.8, CH
19.3, CH3
18.6, CH3
172.7, C
31.9, CH
19.0, CH3
19.7, CH3
171.7, C
b
b
4
1.12, m
42
1.14, m
b
5
0.97, m
43
1.03, d (6.0)
β-hydroxy-Phe-2
6
N-Me-Leu-7
44
7
60.0, CH
5.49, dd (8.0, 2.5)
9.66, d (8.0)
45
54.8, CH
38.9, CH2
5.65, dd (8.0)
1.74, m
7-NH
8
46a
46b
47
73.2, CH
143.0, C
5.94, d (2.5)
1.58, m
9
25.1, CH
23.1, CH3
21.9, CH3
31.3, CH3
173.5, C
1.43, m
10
11
12
13
14
15
16
16-NH
17
18
19
20
21
22a
22b
23
24
24-NH
25
26
27
28
29
30
31
32
33
34
35
36
37
38
127.0, CH
128.6, CH
127.5, CH
128.6, CH
127.0, CH
174.5, C
7.68, d (7.5)
48
0.67, d (6.0)
0.67, d (6.0)
3.52, s
7.41, dd (7.5, 7.5)
7.27, d (7.5)
49
50
7.41, dd (7.5, 7.5)
7.68, d (7.5)
Val-8
51
52
55.6, CH
5.29, dd (8.5, 8.5)
8.06, d (8.5)
2.57, m
Val-3
52-NH
53
58.3,CH
5.41, dd (8.5, 8.5)
8.02, d (8.5)
2.66, m
31.0, CH
19.0, CH3
19.7, CH3
170.6, C
54
1.20, d (7.0)
1.16, d (7.0)
33.1, CH
19.8, CH3
20.0, CH3
169.9, C
55
1.36, d (6.5)
1.20, d (7.5)
N-Me-Thr-9
Thr-10
56
57
62.4, CH
66.5, CH
20.5, CH3
34.5, CH3
171.4, C
5.61, d (3.5)
b
N-Me-4-methoxy-Trp-4
58
5.07, m
70.6, CH
27.1, CH2
4.59, dd (11.0, 4.5)
4.45, dd (13.5, 4.5)
4.32, dd (13.5, 11.0)
59
1.34, d (6.5)
3.68, s
60
61
112.9, C
62
52.5, CH
5.86, dd (8.5, 2.0)
10.17, d (8.5)
b
124.3, CH
7.19, m
62-NH
63
11.80, d (1.5)
69.4, CH
16.8, CH3
173.4, C
6.04, qd (6.5, 2.0)
1.52, d (6.5)
139.5, C
64
105.8, CH
122.7, CH
99.5, CH
154.9, C
7.30, m
Val-11
65
7.27, m
66
57.9, CH
5.24, dd (9.5, 7.5)
8.65, d (9.5)
2.05, m
6.67, d (7.0)
66-NH
67
27.6, CH
19.8, CH3
18.8, CH3
174.2, C
b
55.4, CH3
118.4, C
3.83, s
2.51, s
68
0.95, m
b
69
0.95, m
40.8, CH3
169.4, C
N,N-diMe-Val-12
70
N-Me-Val-5
71
71.5, CH
32.2, CH
19.1, CH3
19.1, CH3
35.9, CH3
35.9, CH3
3.08, d (6.0)
2.16, m
1.11, m
1.11, m
2.54, s
70.9, CH
29.0, CH
21.9, CH3
19.7, CH3
39.9, CH3
3.19, m
72
3.03, m
73
1.22, d (7.0)
74
b
1.02, m
75
3.18, s
76
2.54, s
a
b
1H and 13C data were recorded at 900 and 225 MHz, respectively. Overlapped signals.
generating a different set of trans-annular ROESY correlations
(see Table S4 and Figure S23 in the Supporting Information).
Ohmyungsamycins A and B (1 and 2) are structurally most
similar to the cyclic peptides isolated from Nonomuraea sp., a
terrestrial actinomycete, reported in a patent.16 However, the
ohmyungsamycins have different numbers of amino acid
residues, and the absolute configurations of the cyclic peptides
from Nonomuraea sp., particularly the β-carbons of β-
hydroxyphenylalanine, threonine, and N-methylthreonine, and
the α-carbon of N,N-dimethylvaline were not rigorously
determined.
ohmyungsamycin A (1) exhibited potent antiproliferative effects
against HCT-116, A549, SNU-638, MDA-MB-231, and SK-
HEP-1 cells, with IC50 values ranging from 359 to 816 nM. In
particular, ohmyungsamycin A was 78 times more potent against
the human colon cancer cell line HCT-116 than the positive
control compound, etoposide. However, ohmyungsamycin B (2)
displayed weak cytotoxicity against the tested cancer cells, with
IC50 values of 12.4 to 16.8 μM. In the case of normal lung
epithelial (MRC-5) cells, ohmyungsamycins A and B (1 and 2)
both showed virtually no cytotoxicity (IC50 > 40 μM), indicating
that these compounds exhibit relatively selective antiproliferative
activity against cancer cells compared to normal cells (see Table
3 and Tables S2 and S3, Supporting Information). This
difference in the antiproliferation activity against cancer cells
compared to normal cells by the compounds may be associated
To evaluate the effects of ohmyungsamycin A and B on the
growth of various human cancer cells, the growth inhibitory
potential was determined using a colorimetric sulforhodamine B
(SRB) protein dye staining method.17 As summarized in Table 3,
12325
dx.doi.org/10.1021/jo401974g | J. Org. Chem. 2013, 78, 12321−12329

The Journal of Organic Chemistry
Featured Article
spread onto agar plates containing various media. Strain SNJ042 was
isolated from A6 isolation medium (18 g/L agar containing 3.4%
seawater and 5 μg/mL of polymyxin B sulfate). The strain was deposited
in the Korea Collection for Type Cultures (accession no.
KCTC18240P). The 16S rDNA (1469 bp) of SNJ042 was sequenced
for phylogenetic analysis. On the basis of the 16S rDNA sequences of
SNJ042 and type strains of related Streptomyces spp., strain SNJ042 is
most closely related to Streptomyces cheonanensis (97% similarity, see the
Supporting Information, Figure S20).
Cultivation and Extraction. Strain SNJ042 was cultivated on solid
YEME medium (4 g of yeast extract, 10 g of malt extract, 4 g of glucose,
and 18 g of agar per 1 L of sterilized 3.4% seawater) at 25 °C. The
bacterium was transferred to liquid A1+C medium (10 g of starch, 4 g of
yeast, 2 g of peptone, and 1 g of CaCO3 per 1 L of sterilized 3.4%
seawater) in a 100-mL Erlenmeyer flask containing 50 mL of the
medium. After incubation at 30 °C with shaking at 200 rpm for 3 days, 5
mL of the liquid culture was inoculated to each 1 L of A1+C in 2.8-L
Fernbach flasks. The culture was cultivated at 30 °C with shaking at 200
rpm for 6 days. The entire culture (84 L) was extracted twice with 126 L
of distilled EtOAc using a separation funnel. After anhydrous sodium
sulfate was added to remove residual water, the organic phase was
concentrated in vacuo to yield 9.8 g of dry extract.
Figure 3. ΔδS‑R values in ppm around the β-carbon of β-
hydroxyphenylalanine (C-8) for S- and R-MTPA esters (3 and 4) of 1
in pyridine-d5.
with the consequence of genetic background or of the
biologically various characteristics between cancer and normal
cells. However, the exact molecular mechanism behind the
selectivity should be further investigated.
Isolation of Ohmyungsamycins A and B (1 and 2). A portion of
the dry crude extract (2.3 g) was resuspended with Celite in MeOH and
dried in vacuo to generate Celite-adsorbed extract. The Celite-adsorbed
extract was loaded onto 2 g of prepacked C18 Sepak resin. The extract
was fractionated by elution with a step gradient composed of water and
MeOH. Ohmyungsamycins A and B (1 and 2) eluted in the 80% MeOH
fraction. To obtain pure ohmyungsamycins A and B, the material from
the 80% MeOH fraction was purified by semipreparative reversed-phase
HPLC (Kromasil, C18, 10 × 250 mm, flow rate: 2 mL/min, detection:
UV 280 nm, gradient solvent system: 70−85% aqueous MeOH over 50
min). Ohmyungsamycins A and B eluted at 35.1 and 35.3 min,
respectively. The entire procedure was repeated six times to furnish 14.5
mg of 1 and 4.6 mg of 2.
Ohmyungsamycin A (1) also displayed significant inhibitory
activity against both Gram-positive and Gram-negative bacteria,
including Bacillus subtilis, Kocuria rhizophila, and Proteus hauseri
(MIC = 1.07−4.28 μM) (Table 4). However, ohmyungsamycin
A did not inhibit Staphylococcus aureus, Salmonella enterica, or
Escherichia coli. Ohmyungsamycin B (2) was less active than 1,
exhibiting MIC values of 8.50 to 34.0 μM against B. subtilis, K.
rhizophila, and P. hauseri. Ohmyungsamycin A (1) is much more
potent than ohmyungsamycin B (2) with regard to its
cytotoxicity and antibacterial activity. Thus, the presence of the
additional N-methyl group at the terminus of 2 could lead to a
significant decrease the biological activity of the ohmyungsamy-
cins.
In summary, a chemical study of marine actinomycete strains
from a volcanic island sand beach led to the discovery of
ohmyungsamycins A and B, which bear unusual amino acids,
including β-hydroxyphenylalanine, N-methyl-4-methoxytrypto-
phan, and N,N-dimethylvaline. We developed a new chromato-
graphic method using PGME derivatization to determine the
absolute configuration of N,N-dimethylvaline. Our new method
will serve as a general and convenient analytical procedure for
determining the absolute configurations of amino acids that are
fully substituted at the amine position. The structural uniqueness
and significant biological activities of the ohmyungsamycins
indicate that the chemical and biological investigation of unique
environments, including volcanic islands, can be an effective
strategy for discovering new biologically active compounds.
Ohmyungsamycin A (1): white powder; [α]2D5 −42 (c 0.2, MeOH);
UV (MeOH) λmax (log ε) 203 (3.42), 281 (2.83) nm; CD (MeOH)
(Δε) 211 (−36.6); IR (neat) νmax 3297, 2963, 1640, 1535, 1509, 1201
1
cm−1; H and 13C NMR (pyridine-d5) see Table 1; HRFABMS m/z
1458.8983 [M + H]+ (calcd for C75H120N13O16, 1458.8976).
Ohmyungsamycin B (2): white powder; [α]2D5 −39 (c 0.2, MeOH);
UV (MeOH) λmax (log ε) 203 (3.43), 281 (2.81) nm; CD (MeOH)
(Δε) 212 (−45.6); IR (neat) νmax 3297, 2963, 1640, 1531, 1509, 1199
1
cm−1; H and 13C NMR (pyridine-d5) see Table 2; HRFABMS m/z
1472.9106 [M + H]+ (calcd for C76H122N13O16, 1472.9133).
Determination of the Absolute Configurations of α-Carbons
of the Amino Acid Residues in Ohmyungsamycin A (1). One
milligram of ohmyungsamycin (1) was hydrolyzed in 0.5 mL of 6 N HCl
at 115 °C for 1 h, and the reaction mixture was subsequently cooled in
ice−water for 3 min. The reaction solvent was evaporated in vacuo, and
residual HCl was completely removed by the addition of 0.5 mL of water
and removal of the solvent three times. The hydrolysate was lyophilized
to complete dryness for 24 h. The hydrolysate from the reaction
containing the free amino acids was divided into two portions, and each
portion was transferred into an 8 mL vial. The hydrolysate was then
dissolved in 100 μL of 1 N NaHCO3. Either 50 μL of 10 mg/mL L-
FDAA (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide) or 50 μL of D-
FDAA in acetone was added to each of the two vials containing the
dissolved free amino acids. The reaction mixtures were incubated at 80
°C for 3 min. A 50-μL aliquot of 2 N HCl was added to neutralize the
reaction, and 300 μL of aqueous 50% CH3CN was added to the vials. A
20-μL aliquot of each reaction mixture was analyzed by LC-MS using a
gradient solvent system (20 to 60% CH3CN containing 0.1% formic acid
over 40 min, C18 reversed-phase column: 100 × 4.6 mm, detection: UV
340 nm). L-FDAA derivatives eluted before D-FDAA derivatives for all
amino acid residues in the hydrolysate. Thus, the absolute
configurations of all of the amino acid residues in 1 were determined
to be L (see Table S1, Supporting Information).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
recorded in MeOH at 589 with a 1-cm cell. UV spectra were measured
with UV−Vis spectrometer from 200 to 600 nm in a 1-cm cuvette. IR
spectra were recorded with 4 cm−1 resolution. 1H NMR (900 MHz) and
13C NMR (225 MHz) spectral data were obtained on a 900-MHz NMR
spectrometer. Low-resolution LC/MS data were obtained on an HPLC
coupled to a quadrupole mass spectrometer. High-resolution fast-atom
bombardment (HR-FAB) mass spectrometry data were recorded with
high-resolution mass spectrometer.
Isolation of the Strain SNJ042 and Phylogenetic Analysis.
Marine sediments were collected from Shinyang Beach, Jeju Island,
Republic of Korea, in 2010. The sediment (1 g) was diluted in 30 mL of
sterilized artificial seawater, and the suspension was heated in a water
bath for 5 min (55 °C). Two hundred microliters of the solution was
Determination of the Absolute Configuration of the β-
Carbon of β-Hydroxyphenylalanine. The absolute configuration of
12326
dx.doi.org/10.1021/jo401974g | J. Org. Chem. 2013, 78, 12321−12329

The Journal of Organic Chemistry
Featured Article
Figure 4. Chromatographic determination of the absolute configuration of N,N-dimethylvaline in 2 by PGME derivatization. (a) Procedure for the
chemical reaction with PGME. (b) LC/MS analysis (ion extraction for M.W. 292) of S- and R-PGME derivatives of the hydrolysate of 2 (top), N,N-
dimethyl-L-valine (middle), and N,N-dimethyl-D-valine (bottom).
the stereogenic center at the β-carbon of β-hydroxyphenylalanine was
determined using the modified Mosher’s method with R- and S-α-
methoxy-(trifluoromethyl)phenylacetyl chloride (MTPA-Cl). Oh-
myungsamycin A (1, 2 mg, dried under high vacuum for 24 h) was
dissolved in 1 mL of distilled pyridine after a catalytic amount of 4-
dimethylaminopyridine (DMAP) was added. The reaction mixture was
stirred for 10 min. Fifty microliters of R-MTPA chloride was carefully
added to the reaction vial. After 2 h, 50 μL of MeOH was added to the
vial to quench the reaction. S-MTPA ester (3) was purified by
semipreparative HPLC (Kromasil, C18, 10 × 250 mm, gradient solvent
system: 30−100% aqueous MeOH over 50 min, tR 32 min). The R-
MTPA ester (4) was obtained using the same procedure (tR: 32 min).
S-MTPA ester 3: 1H NMR (500 MHz, pyridine-d5) δH 11.80 (1H, br
s), 10.29 (1H, d, J = 8.5), 9.75 (1H, d, J = 8.5), 9.38 (1H, d, J = 9.0), 9.20
(1H, d, J = 8.5), 8.57 (1H, d, J = 9.5), 8.09 (1H, d, J = 8.5), 8.02 (1H, d, J
= 9.0), 7.92 (2H, d, J = 7.0), 7.62 (2H, d, J = 8.0), 7.44−7.24 (9H, m),
6.67 (1H, d, J = 7.0), 6.63 (1H, br s), 6.40 (1H, m), 6.00 (1H, m), 5.86
(2H, m), 5.72 (1H, m), 5.65 (1H, m), 5.45 (1H, m), 5.39 (1H, dd, J =
9.5, 9.5), 5.27 (1H, dd, J = 7.5, 7.5), 5.21 (1H, m), 4.65 (1H, dd, J = 8.0,
8.0), 4.58 (1H, m), 4.45 (1H, dd, J = 13.5, 4.5), 4.32 (1H, dd, J = 13.5,
11.5), 3.81 (3H, s), 3.69 (3H, s), 3.67 (1H, d, J = 8.0), 3.55 (3H, s), 3.38
(3H, s), 3.20 (1H, d, J = 8.0), 3.17 (3H, s), 3.03 (1H, m), 2.66 (1H, m),
2.53 (1H, m), 2.49 (3H, s), 2.48 (3H, s), 2.37 (1H, m), 2.25−2.17 (3H,
m), 1.73 (1H, m), 1.63 (1H, m), 1.47 (3H, d, J = 6.5), 1.46 (1H, m), 1.35
(3H, d, J = 6.5), 1.29 (3H, d, J = 6.5), 1.22−1.15 (12H, m), 1.12 (3H, d, J
= 6.5), 1.09−1.05 (9H, m), 1.01 (6H, m), 0.93 (9H, m), 0.69 (6H, J =
7.0). The molecular formula of 3 was confirmed as C85H126F3N13O18 by
ESIMS analysis ([M + H]+ at m/z 1674).
R-MTPA ester 4: 1H NMR (500 MHz, pyridine-d5) δH 11.85 (1H, br
s), 10.36 (1H, d, J = 8.5), 9.75 (1H, d, J = 8.5), 9.37 (1H, d, J = 9.0), 9.19
(1H, d, J = 8.5), 8.60 (1H, m), 8.02 (1H, d, J = 8.5), 7.93 (1H, d, J = 9.0),
7.87 (2H, d, J = 7.0), 7.62 (2H, d, J = 8.0), 7.44−7.24 (9H, m), 6.67 (1H,
d, J = 7.0), 6.56 (1H, br s), 6.44 (1H, m), 6.01 (1H, m), 5.83 (2H, d, 2.5),
5.72 (1H, m), 5.67 (1H, m), 5.46 (1H, m), 5.39 (1H, dd, J = 9.5, 9.5),
5.27 (1H, m), 5.22 (1H, m), 4.65 (1H, dd, J = 8.0, 8.0), 4.58 (1H, dd, J =
11.0, 4.5), 4.44 (1H, dd, J = 13.5, 4.5), 4.34 (1H, m), 3.81 (3H, s), 3.69
12327
dx.doi.org/10.1021/jo401974g | J. Org. Chem. 2013, 78, 12321−12329

The Journal of Organic Chemistry
Featured Article
Determination of the Absolute Configuration of N,N-
Dimethylvaline. Two milligrams of ohmyungsamycin B (2) was
hydrolyzed and dried under high vacuum for 24 h. The hydrolysate was
dissolved in 2 mL of tetrahydrofuran (THF) and was split equally into
two vials. The vials were treated with 6 mg of S-PGME or R-PGME, and
7 mg of ethyl-(N,N-dimethylamino)propylcarbodiimide hydrochloride
(EDC) was added to each vial. After the reaction mixtures were stirred at
25 °C for 6 h, S- and R-PGME amide products ([M + H]+ m/z at 293)
were analyzed with LC/MS (Phenomenex, C18, 250 × 4.6 mm, gradient
system: 10−100% aqueous CH3CN over 40 min). S- and R-PGME
amide products eluted at 15.5 and 17.7 min, respectively. The absolute
Table 3. Inhibitory Effects of Ohmyungsamycins A and B (1
and 2) on the Proliferation of Human Cancer Cells and
Normal Cells
IC50 (μM)
ohmyungsamycin ohmyungsamycin
a
cell line classification
A
B
etoposide
28.1
HCT-
116
colon cancer
cells
0.359
12.4
A549
lung cancer
cells
0.551
0.532
15.6
13.5
1.36
1.37
configuration of N,N-dimethylvaline in 2 was determined to be
L
SNU-
638
stomach
cancer
cells
through comparison of the retention times of the S- and R-PGME amide
products with those of the authentic standards of N,N-dimethyl- L- and
D-valine.
MDA-
MB-
231
breast
cancer
cells
0.688
0.816
12.7
16.8
19.0
5.04
36.2
Cell Culture. Human lung cancer (A549), colon cancer (HCT116),
stomach cancer (SNU638), breast cancer (MDA-MB-231), liver cancer
(SK-HEP-1), and lung fibroblast (MRC-5) cells were provided by the
Korean Cell Line Bank (Seoul, Korea). Cells were cultured in medium
(DMEM for MDA-MB-231 and SK-HEP-1 cells; RPMI 1640 medium
for A549, HCT116, and SNU638 cells; MEM for MRC-5 cells)
supplemented with 10% heat-inactivated FBS and antibiotics−
antimycotic solution (100 units/mL penicillin G sodium, 100 μg/mL
streptomycin, and 250 ng/mL amphotericin B). Cells were maintained
at 37 °C under a humidified atmosphere containing 5% CO2.
Cell Proliferation Assay. Cells (3.5 × 103 cells/mL for HCT-116
and SK-HEP-1; 4 × 103 cells/mL for A549; 5 × 103 cells/mL for MDA-
MB-231, and SNU-638; 7 × 103 cells/mL for MRC-5 cells) were treated
with various concentrations of ohmyungsamycin A and B for 3 days.
After treatment, cells were fixed with 10% TCA solution, and cell
viability was determined using the sulforhodamine B (SRB) assay. The
results were expressed as percentages relative to solvent-treated control
incubations, and IC50 values were calculated using nonlinear regression
analysis (percent survival versus concentration).
Antibacterial Assay. Six bacterial strains (Staphylococcus aureus,
Bacillus subtilis, Kocouria rhizophila, Proteus hauseri, Salmonella enterica,
and Escherichia coli) were used for antibacterial activity assays. Bacteria
were grown overnight in Luria−Bertani (LB) broth at 37 °C. Cells were
harvested by centrifugation and washed twice with sterile distilled water.
Ohmyungsamycins A and B (1 and 2) were dissolved in 100% dimethyl
sulfoxide (10 mg/mL) and diluted in 96-well plates with m Plate Count
Broth to prepare serial 2-fold dilutions in the range of 100 to 0.39 μg/
mL. Test bacteria were added to each well (1 × 105 cells/mL) and
incubated for 16 h at 37 °C. The minimum inhibitory concentration
(MIC) values were determined as the lowest concentration of test
compounds that inhibited bacterial growth. Ampicillin was used as a
positive control.
SK-
HEP-
1
liver cancer
cells
MRC-5 lung normal
cells
>40
>40
a
Etoposide was used as a positive control.
Table 4. Inhibitory Activities of Ohmyungsamycins A and B (1
and 2) against Bacterial Strains
MIC (μM)
ohmyungsamycin ohmyungsamycin
strain
A
B
ampicillin
1.14
Staphylococcus aureus
ATCC6538p
not active
not active
Bacillus subtilis
4.28
34.0
1.14
1.14
2.28
1.14
8.87
ATCC6633
Kocuria rhizophila
NBRC12708
1.07
8.50
Proteus hauseri
NBRC3851
2.14
17.0
Salmonella enterica
ATCC14028
not active
not active
not active
not active
Escherichia coli
ATCC35270
(3H, s), 3.61 (1H, m), 3.54 (3H, s), 3.29 (3H, s), 3.19 (1H, d, J = 8.5),
3.17 (3H, s), 3.02 (1H, m), 2.66 (1H, m), 2.50 (6H, s), 2.37 (1H, m),
2.18−2.11 (4H, m), 1.73 (1H, m), 1.63 (1H, m), 1.50 (3H, m), 1.44
(1H, m), 1.34−1.17 (21H, m), 1.14−1.05 (9H, m), 1.01−0.84 (15H,
m), 0.67 (3H, J = 7.0), 0.66 (3H, J = 7.0). The molecular formula of 4
was confirmed as C85H126F3N13O18 by ESIMS analysis ([M + H]+ at m/z
1674).
ASSOCIATED CONTENT
* Supporting Information
■
S
NMR spectra of 1 and 2, detailed Marfey’s analysis results, and
identification of strain SNJ042. This material is available free of
charge via the Internet at http://pubs.acs.org.
Determination of the Absolute Configurations of the β-
Carbons of Threonine and N-Methylthreonine (GITC Derivati-
zation). Two milligrams of authentic standards of N-methyl-L-allo-Thr
and N-methyl-L-Thr were each dissolved in 2 mL of water. Then, 200 μL
of 6% triethylamine and 200 μL of 1% GITC in acetone were added, and
the reaction mixture was stirred at room temperature for 15 min. Next,
100 μL of 5% acetic acid was added to neutralize the reaction mixture.
The product was analyzed by LC/MS with a gradient solvent system
(10% to 100% CH3CN containing 0.1% formic acid over 20 min, C18
reversed-phase HPLC column: 250 × 4.6 mm). The GITC derivatives of
N-methyl-L-allo-Thr and N-methyl-L-Thr eluted at 15.9 and 15.1 min,
respectively. The hydrolysate of 1 was also derivatized with GITC using
an identical method. The GITC derivative of the hydrolysate of 1 eluted
at 15.1 min in the LC/MS analysis, which indicated that the unit in 1 is
N-methyl-L-Thr, not N-methyl-L-allo-Thr. The same procedure was
performed for L-Thr and L-allo-Thr. The GITC derivatives of allo-L-Thr
and L-Thr eluted at 15.5 and 15.8 min, respectively, and the retention
time of the GITC derivative of L-Thr from the hydrolysate of 1 was 15.8,
indicating that the threonine residue in 1 is L-Thr.
AUTHOR INFORMATION
Corresponding Author
*Tel: 82 2 880 2491. Fax: 82 2 762 8322. E-mail: dongchanoh@
snu.ac.kr.
■
Notes
The authors declare the following competing financial
interest(s): The patent application for the ohmyungsamycins
has been submitted.
ACKNOWLEDGMENTS
■
This work was supported by a National Research Foundation of
Korea Grant funded by the Korean Government (MEST)
(M1A5A1-2010-0020429 and 2009-0083533).
12328
dx.doi.org/10.1021/jo401974g | J. Org. Chem. 2013, 78, 12321−12329

The Journal of Organic Chemistry
Featured Article
REFERENCES
■
(1) Molinski, T. F.; Dalisay, D. S.; Lievens, S. L.; Saludes, J. P. Nat. Rev.
Drug Discov. 2009, 8, 69−85.
(2) Gulder, T. A. M.; Moore, B. S. Curr. Opin. Microbiol. 2009, 12,
252−260.
(3) Fenical, W.; Jensen, P. R. Nat. Chem. Biol. 2006, 2, 666−673.
(4) Hu, G.-P.; Yuan, J.; Sun, L.; She, Z.-G.; Wu, J.-H.; Lan, X.-J.; Zhu,
X.; Lin, Y.-C.; Chen, S.-P. Mar. Drugs 2011, 9, 514−525.
(5) Fenical, W.; Jensen, P. R.; Palladino, M. A.; Lam, K. S.; Lloyd, G. K.;
Potts, B. C. Bioorg. Med. Chem. 2009, 17, 2175−2180.
(6) Clardy, J.; Fischbach, M. A. Wiley Encyc. Chem. Biol. 2009, 3, 240−
249.
(7) Won, C.-K. Genozoic Igneous Rocks: Geology of Korea; Lee, D.-S.,
Ed.; Kyohak-sa: Seoul, 1988; pp 329−330.
(8) Um, S.; Kim, Y.-J.; Kwon, H.; Wen, H.; Kim, S.-H.; Kwon, H. C.;
Park, S.; Shin, J.; Oh, D.-C. J. Nat. Prod. 2013, 76, 873−879.
(9) Bae, M.; Kim, H.; Shin, Y.; Kim, B. Y.; Lee, S. K.; Oh, K.-B.; Shin, J.;
Oh, D.-C. Mar. Drugs 2013, 11, 2882−2893.
(10) Fujii, K.; Ikai, Y.; Oka, H.; Suzuki, M.; Harada, K. Anal. Chem.
1997, 69, 5146−5151.
(11) Oh, D.-C.; Poulsen, M.; Currie, C. R.; Clardy, J. Nat. Chem. Biol.
2009, 5, 391−393.
(12) Hess, S.; Gustafson, K. R.; Milanowski, D. J.; Alvira, E.; Lipton, M.
A.; Pannell, L. K. J. Chromatogr. A 2004, 1035, 211−219.
́
(13) Seco, J. M.; Quinoa, E.; Riguera, R. Tetrahedron: Asymmetry 2001,
̃
12, 2915−2925.
(14) Yabuuchi, T.; Kusumi, T. J. Org. Chem. 2000, 65, 397−404.
(15) Tripathi, A.; Puddick, J.; Prinsep, M. R.; Rottmann, M.; Tan, L. K.
J. Nat. Prod. 2010, 73, 1810−1814.
(16) Kim, J. W.; Lee, S. W.; Park, S. J.; Suh, J. W.; Lee, I. A.; Yoon, T.
M.; Choi, J. K.; Lee, J. E.; Kim, J. Y.; Jin, Y. Y.; Franzblau, S.; Cho, S.;
Gao, W.; Guido, P.; McAlpine, J.; Napolitano, J.; Jaki, B.; Friesen, B.;
Rodriguez Brasco, M. F.; Lankin, D. PCT Int. WO 2012/144790 A1,
2012.
(17) Cho, S. K.; Hong, J. Y.; Park, H. J.; Lee, S. K. Biomol. Therap. 2012,
20, 513−519.
12329
dx.doi.org/10.1021/jo401974g | J. Org. Chem. 2013, 78, 12321−12329