Octaminomycins A and B, cyclic octadepsipeptides active against Plasmodium falciparum

Article abstract of DOI:10.1021/acs.jnatprod.6b00758

Two new cyclic octadepsipeptides, octaminomycins A (1) and B (2), were isolated from a microbial metabolite fraction library of Streptomyces sp. RK85-270 based on Natural Products Plot screening. Their structures were elucidated on the basis of HRESIMS, 1D and 2D NMR spectroscopic data, and MS/MS experiments for sequence analysis. The absolute configurations of the constituent amino acid residues were determined by a combination of single-crystal X-ray diffraction and Marfey's methodology. Notably, octaminomycins A (1) and B (2) showed good in vitro antiplasmodial activity against chloroquine-sensitive as well as chloroquine-resistant strains with no cytotoxicity up to 30 μM. (Chemical Equation Presented).

Full text of DOI:10.1021/acs.jnatprod.6b00758

Article
pubs.acs.org/jnp
Octaminomycins A and B, Cyclic Octadepsipeptides Active against
Plasmodium falciparum
†
,‡
§
§
§
⊥
Jun-Pil Jang, Toshihiko Nogawa, Yushi Futamura, Takeshi Shimizu, Daisuke Hashizume,
Shunji Takahashi,†,§ Jae-Hyuk Jang,‡ Jong Seog Ahn,* and Hiroyuki Osada*
,‡
,§
†
RIKEN Global Research Cluster, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
‡
Anticancer Agent Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju, 28116, South Korea
§
Chemical Biology Research Group, RIKEN Center for Sustainable Research Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
⊥Materials Characterization Support Unit, RIKEN Center for Emergent Matter Science (CEMS), 2-1 Hirosawa, Wako, Saitama
3
51-0198, Japan
*
S Supporting Information
ABSTRACT: Two new cyclic octadepsipeptides, octamino-
mycins A (1) and B (2), were isolated from a microbial
metabolite fraction library of Streptomyces sp. RK85-270 based
on Natural Products Plot screening. Their structures were
elucidated on the basis of HRESIMS, 1D and 2D NMR
spectroscopic data, and MS/MS experiments for sequence
analysis. The absolute configurations of the constituent amino
acid residues were determined by a combination of single-
crystal X-ray diffraction and Marfey’s methodology. Notably,
octaminomycins A (1) and B (2) showed good in vitro antiplasmodial activity against chloroquine-sensitive as well as
chloroquine-resistant strains with no cytotoxicity up to 30 μM.
econdary metabolites continue to provide a diverse and
unique source of biologically active substances for drug
discovery. Some synthesized drugs were inspired by the
efficiently and rapidly. In addition, the efforts for identifying
known compounds are reduced significantly. We recently
S
7
discovered novel compounds, namely, verticilactam, spirotoa-
8
9
10
scaffolds of natural products, and, even now, natural products
mides A and B, pyrrolizilactone, RK-270A, -B, and -C, RK-
11 12
1
,2
1
355A and -B, and unantimycin A, which were obtained
are undoubtedly useful for discovering pharmaceutical leads.
from NPPlot searches. Herein, we report the purification,
structure elucidation, and biological activities of new cyclic
octadepsipeptides, designated octaminomycins A (1) and B
Microorganisms, particularly those of the Streptomyces genus,
have played an important role as a major source of lead/seed
compounds. Approximately 40% of known bioactive com-
pounds of microbial origin are derived from the Streptomyces
(
2).
3
,4
genus. However, recently, the rate of reisolation of known
compounds far exceeds the isolation of novel structures because
the majority of metabolites have already been investigated. To
overcome this barrier, we have developed a microbial
metabolites fraction library and a spectral database, named
RESULTS AND DISCUSSION
■
The fraction library of microbial metabolites was constructed
from 36.0 g of EtOAc extract, which was prepared from 30 L of
culture broth. The EtOAc extract was subjected to MPLC on a
silica gel column using a stepwise gradient elution with
5
,6
the Natural Products Plot (NPPlot). The fraction library
consists of metabolites semipurified by basic chromatographic
techniques, such as medium-pressure liquid chromatography
CHCl −MeOH (50:1 to 0:1) to yield eight fractions. Each
3
MPLC fraction was further separated by C -HPLC with a
18
(
MPLC) and HPLC, and some fractions contain an almost
gradient from 5% to 100% MeOH with H O (acidified with
2
pure single metabolite. Each fraction is subjected to diode-array
detector (DAD)-LC/MS analysis to collect physicochemical
information including UV absorption and mass spectra of
metabolites within the fraction. NPPlot is a distribution map of
metabolites plotted on a two-dimensional (2D) area by HPLC
retention time on the x axis and m/z values on the y axis. Each
metabolite has UV information on maximum absorption values
for the z axis in NPPlot, which allows us to find specific or
distinctive metabolite groups easily. On the basis of the NPPlot
analysis, structurally unique metabolites can be identified
0.05% formic acid) into 48 fractions for the fraction library. All
isolated fractions were analyzed by LC/MS, and the
spectroscopic data of each peak within the fraction were stored
in the database. In the course of our screening for structurally
unique secondary metabolites using the NPPlot analysis, we
identified two unknown peaks with identical UV absorption
patterns, but slightly different mass spectra in fractions of
Received: August 24, 2016
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b00758
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
from Pro1 H-2 (δ 4.25) to Val C-1 (δ 171.5) extended this
H
C
sequence to Pro2-NMeTyr-Leu2-Pro1-Val. Similarly, the
HMBC correlations from Leu1 NH (δH 7.94) to Phe C-1
(
δ 170.6) and from Phe NH (δ 9.20) to Thr C-1 (δ 170.7)
C H C
established the partial sequence of Leu1-Phe-Thr. This partial
sequence was confirmed by NOESY correlations between Leu1
NH (δ 7.93) and Phe H-2 (δ 4.26), and Phe NH (δ 9.20)
H
H
H
and Thr H-2 (δ 3.90). A NOESY correlation between Val NH
H
(
δH 7.11) and Leu1 H-2 (δH 4.12) linked the two partial
sequences together into Pro2-NMeTyr-Leu2-Pro1-Val-Leu1-
Phe-Thr. The HMBC correlation between the β-proton of a
threonine residue and the carbonyl carbon of a Pro2 residue
suggested 1 to be a depsipeptide cyclized between the C-
terminal carboxyl and the side chain hydroxy group of the
threonine. The amino acid sequence of 1 was further confirmed
from a detailed analysis of the MS/MS data of hydrolysate 1a of
1
and its methyl ester 1b, following a reported method (Figure
13
S9). Consequently, the planar structure of 1 was elucidated as
an N-acyl cyclic octadepsipeptide with the sequence cyclo-(Pro-
N-MeTyr-Leu-Pro-Val-Leu-Phe-Thr).
The absolute configurations of the amino acids were
determined by Marfey’s method after acid hydrolysis.
Compound 1 was hydrolyzed and derivatized with L-FDLA
and analyzed by an LC-MS comparison between the two
derivatives. Seven of the eight amino acids (Pro × 2, NMeTyr,
Leu, Val, Phe, and Thr) were assigned an L configuration, while
the second Leu residue was the D configuration (Table S1,
Supporting Information). The positions of the different
enantiomers of Leu in the amino acid sequence in 1 were
further supported by a single-crystal X-ray crystallography
experiment. Compound 1 was crystallized by slow evaporation
of a solution in an EtOAc−n-hexane mixture over 2 weeks.
Although the resulting crystals were not suitable in size and
quality for single-crystal X-ray data allowing determination of
the absolute configuration, the relative 3D structure of 1 was
obtained (Figure 2). The X-ray structure is consistent with the
LC-MS analyses of the acid hydrolysates of 1, generated using
Marfey’s method. Therefore, compound 1 was confirmed as a
new acyl cyclic octadepsipeptide and was named octaminomy-
cin A.
Streptomyces sp. RK85-270 (Figure S20, Supporting Informa-
tion). The related fractions were purified by C -HPLC to
afford compounds 1 (50 mg) and 2 (14 mg).
1
8
Octaminomycin A (1) was isolated as a white, amorphous
powder. The molecular formula of 1 was established as
C H N O on the basis of HRESIMS data. The IR spectrum
5
3
76
8
11
−1
suggested the presence of amine or hydroxy (3295 cm ), ester
−1
−1
carbonyl (1750 cm ), and amide carbonyl (1650 cm )
functionalities, and the UV spectrum indicated that 1 was a
peptide with aromatic residues, having absorption bands at 216
and 277 nm. The peptidic nature of 1 was suggested by the
1
signal distribution pattern observed in the H NMR spectrum
(
DMSO-d ), which included exchangeable amide NH signals
6
(δ 6−10), amino acid α-proton signals (δ 4−6), and aliphatic
Octaminomycin B (2) was obtained as a yellowish powder.
H
H
13
methylene and methyl signals (δ 0−3) (Table 1). The
C
The molecular formula was established as C52
basis of HRESIMS data, which was less than that of
octaminomycin A (1) by a CH unit. The molecular formula
H N O11 on the
74 8
H
NMR spectrum for 1 displayed nine distinguishable signals for
ester/amide-type carbonyls (δC 167.9−175.4). Combined
analysis of the DQF-COSY, HSQC-TOCSY, and HMBC
NMR spectra identified the structures of eight standard amino
acids, including two leucines, two prolines, one valine, one
phenylalanine, one threonine, and one tyrosine. An HMBC
correlation observed from N-Me (δH 2.97) to Tyr C-2 (δC
2
showed the absence of a methylene group, compared to
compound 1, which indicates that octaminomycin B is an
analogue of 1 with slight modifications. A comparison of the
1D NMR data (Table 1) between compounds 1 and 2
indicated that their planar structures were very similar, except
5
3.0) indicated that the Tyr residue was N-methylated.
that the signal of the methylene group H
2
-2′ (δ 27.9/δ 2.21;
C H
1
Moreover, regarding the Thr residue, the DQF-COSY
2.37) in 1 was absent in the spectra of 2. Furthermore, the H
and 13C NMR signals of the N-propionyl methyl in 1 were
deshielded from δ 9.4/δ 0.97 to δ 22.9/δ 1.90 in 2. These
correlation between H -3′ (δ 0.97) and H -2′ (δ 2.21;
3
H
2
H
2
.37) and further HMBC correlations from H -3′ (δ 0.97),
3
H
C
H
C
H
H -2′ (δ 2.21; 2.37), and Thr NH (δ 8.43) to the amide
data suggested that the threonine residue in 2 was N-acetylated.
Further interpretation of the HSQC and HMBC spectroscopic
data of 2 confirmed this hypothesis. The HMBC correlations
from H -2′ (δ 1.90) and Thr NH (δH 8.52) to the amide
2
H
H
carbonyl carbon C-1′ (δ 175.4) indicated the threonine
C
nitrogen in 1 was acylated with a propionyl group. The
assignment of the amino acid sequence was carried out by a
combination of HMBC, NOESY (Figure 1), and MS/MS
analysis (Figure S11). HMBC correlations from Pro2 H-2 (δH
3
H
carbonyl carbon at C-1′ (δ 172.0) indicated that the threonine
C
residue in 2 was N-acetylated. The amino acid sequence was
also established by combined analysis of the HMBC and
NOESY spectra (Figure 1). The absolute configurations of the
amino acid residues composing cyclopeptide 2 were
determined by Marfey’s method and found to be identical to
4
2
.82) to NMeTyr C-1 (δ 167.9), from NMeTyrN-Me (δH
C
.97) to Leu2 C-1 (δ 172.7), and from Leu2 NH (δ 8.16)
C
H
and H-2 (δ 4.60) to Pro1 C-1 (δ 168.8) suggested a partial
H
C
sequence of Pro2-NMeTyr-Leu2-Pro1. An HMBC correlation
B
DOI: 10.1021/acs.jnatprod.6b00758
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 1. H (500 MHz) and 13C (125 MHz) NMR Data for Octaminomycins A (1) and B (2) in DMSO-d6
1
octaminomycin A (1)
octaminomycin B (2)
position
N-Pr Thr
δC
δH mult. (J in Hz)
position
N-Ac Thr
δC
δH, mult. (J in Hz)
1
170.7, C
58.7, CH
69.5, CH
15.8, CH3
1
170.6, C
58.8, CH
69.3, CH
15.8, CH3
2
3.90, dd (10.9, 4.6)
4.46, m
2
3.92, dd (10.9, 4.6)
4.46, m
3
3
4
0.43, d (5.7)
8.43, d (4.6)
4
0.43, d (5.7)
8.52, d (4.3)
NH
NH
1′
2′
1
′
′
175.4, C
172.0, C
2
27.9, CH2
2.21, m
22.9, CH3
1.90, s
2
.37, m
3
1
2
3
′
9.4, CH3
170.6, C
0.97, t (7.4)
Phe
Phe
1
2
3
170.5, C
55.2, CH
34.6, CH2
55.4, CH
34.5, CH2
4.26, m
4.26, m
3.27, dd (14.9, 11.4)
3.27, dd (14.9, 11.4)
3.40, dd (14.9, 5.1)
3
.41, dd (14.9, 5.1)
4
5
6
7
138.9, C
4
138.9, C
/5′
/6′
128.8, CH
128.1, CH
126.1, CH
7.12, m
5/5′
6/6′
7
128.8, CH
128.1, CH
126.1, CH
7.12, m
7.17, m
7.17, m
7.11, m
7.10, m
NH
1
9.20, d (6.3)
NH
1
9.20, d (6.3)
Leu-1
171.5, C
52.6, CH
39.0, CH2
Leu-1
171.6, C
52.7, CH
39.1, CH2
2
4.12, m
1.44, m
2
4.10, m
3
3
1.43, m
1
.82, m
1.74, m
4
24.3, CH
20.6, CH3
23.2, CH3
1.74, m
4
24.3, CH
20.6, CH3
23.2, CH3
1.72, m
5
0.83, d (6.9)
0.91, d (7.0)
7.94, d (7.4)
5
0.83, d (6.3)
0.91, d (6.3)
7.93, d (7.4)
6
6
NH
1
NH
1
Val
171.5, C
Val
171.5, C
2
54.6, CH
29.8, CH
16.2, CH3
19.5, CH3
4.63, m
2
54.7, CH
29.8, CH
16.2, CH3
19.5, CH3
4.64, m
3
2.13, m
3
2.14, m
4
0.86, d (6.3)
0.83, d (6.3)
7.11, m
4
0.86, d (6.3)
0.83, d (6.3)
7.10, m
5
5
NH
1
NH
1
Pro-1
168.8, C
59.1, CH
24.4, CH2
Pro-1
168.8, C
59.1, CH
24.4, CH2
2
4.25, m
1.83, m
2
4.28, m
1.84, m
2.34, m
1.84, m
3.36, m
3.59, m
3
3
2
.34, m
4
5
25.1, CH2
47.6, CH2
1.80, m
3.36, m
4
5
25.1, CH2
47.6, CH2
3
.58, m
Leu-2
1
2
3
172.7, C
46.0, CH
42.0, CH2
Leu-2
1
2
3
172.7, C
46.0, CH
42.0, CH2
4.60, dd (10.3, 2.8)
1.16, m
4.61, dd (10.3, 2.8)
1.16, m
1
.29, m
1.30, m
4
23.8, CH
21.3, CH3
23.0, CH3
1.36, m
4
23.8, CH
21.3, CH3
23.0, CH3
1.36, m
5
0.87, d (5.2)
0.79, d (6.8)
8.16, d (9.1)
5
0.87, d (5.2)
0.79, d (6.8)
8.14, d (9.8)
6
6
NH
1
NH
N-Me-Tyr
167.9, C
N-Me-Tyr
1
167.9, C
2
52.9, CH
33.7, CH2
126.6, C
5.50, dd (9.2, 5.8)
2.74, m
2
53.0, CH
33.7, CH2
126.6, C
5.49, dd (9.8, 6.3)
2.74, m
3
3
4
4
5
6
7
/5′
/6′
129.9, CH
114.4, CH
155.8, C
6.92, d (8.0)
6.47, d (8.6)
5/5′
6/6′
7
129.9, CH
114.5, CH
155.7, C
6.92, d (8.0)
6.45, d (8.6)
NMe
29.5, CH3
170.7, C
2.97, s
NMe
1
29.5, CH3
170.7, C
2.97, s
̀
Pro-2
1
2
3
Pro-2
58.6, CH
30.9, CH2
4.82, dd (9.2, 1.2)
1.84, m
2
58.6, CH
30.8, CH2
4.84, br d (8.1)
1.84, m
3
2
.14, m
2.14, m
C
DOI: 10.1021/acs.jnatprod.6b00758
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 1. continued
octaminomycin A (1)
octaminomycin B (2)
position
δC
δH mult. (J in Hz)
position
δC
δH, mult. (J in Hz)
4
5
21.8, CH2
1.60, m
4
5
21.8, CH2
1.61, m
1.80, m
3.37, m
1
.78, m
46.4, CH2
3.37, m
46.4, CH2
Figure 1. Key 2D NMR correlations of octaminomycins A (1) and B (2).
Figure 2. ORTEP drawing of octaminomycin A (1) at the 50% probability level. Black, blue, and red ellipsoids and small circles denote carbon,
nitrogen, oxygen, and hydrogen atoms, respectively.
those identified in 1 (Table S2). The positions of the different
enantiomers of Leu in the amino acid sequence in 2 were
proposed by comparison of the specific rotation value and H
and 13C NMR data with those of 1. The similar physical data
and similar overall chemical shifts of 2 to those of 1 revealed
that they share the same macrocyclic scaffold. Therefore, the
1
D
DOI: 10.1021/acs.jnatprod.6b00758
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
structure of 2 was established as N-acetyl cyclo-(L-Pro-N-Me-L-
Tyr-D-Leu-L-Pro-L-Val-L-Leu-L-Phe-L-Thr) and named octami-
nomycin B.
of our methodology (constructing a fraction library and using
NPPlot) to discover novel natural product.
Octaminomycins A (1) and B (2) showed no significant
cytotoxicity at a concentration of 30 μM against human cervical
cancer cells (HeLa), human promyelocytic leukemia cells (HL-
EXPERIMENTAL SECTION
■
General Experimental Procedures. The melting point was
recorded on a Yanaco melting point apparatus. UV spectra and specific
rotations were recorded on a Beckman DU 530 Life Science UV/vis
spectrophotometer and a Horiba SEPA-300 high-sensitive polarimeter,
respectively. IR spectra were recorded on a Horiba FT-720 IR
spectrometer with a DuraSampl IR II ATR instrument. NMR spectra
were recorded on a JEOL ECA-500 FT-NMR spectrometer at 500
6
0), mouse temperature-sensitive cdc2 mutant cells (tsFT210),
ts
and rat kidney cells that were infected with ts25 (src -NRK).
They were also evaluated for antimicrobial activity against
Staphylococcus aureus 209, Escherichia coli HO141, Aspergillus
fumigatus Af293, Pyricularia oryzae kita-1, and Candida albicans
JCM1542 and for antimalarial activity against Plasmodium
falciparum 3D7, Dd2, and K1 strains. Chloroquine was less
effective to resistant strains Dd2 and K1. On the contrary
octaminomycins A (1) and B (2) showed the same in vitro
antimalarial activities against chloroquine-sensitive 3D7 and
chloroquine-resistant Dd2 and K1 strains with no antimicrobial
activity up to 30 μM (Table 2).
1
13
MHz for H NMR and 125 MHz for C NMR. Chemical shifts were
referenced to a residual solvent signal (DMSO-d6 1H δ 2.50, C δC
13
H
3
9.51). Mass spectra were obtained on an AB Sciex Qtrap (ESIMS)
and ABI3200, and HRESIMS was accomplished on a Waters Synapt
GII spectrometer. DAD-LC/MS analysis was performed using a
Waters Alliance 2965 HPLC system, attached to a Waters 2996 PDA
detector, with a Waters Xterra C -column (5 μm, 2.1 mm i.d. × 150
18
mm) that was connected to an AB Sciex Qtrap MS/MS system
equipped with an ESI probe. MPLC was accomplished using a
Teledyne ISCO CombiFlash Companion. Preparative HPLC was
performed using a Waters 600E pump system with a Senshu Pak
Pegasil ODS column (5 μm, 10 mm i.d. × 250 mm). All solvents and
reagents were of analytical grade and were purchased from commercial
sources.
Table 2. Antimalarial Activities for Octaminomycins A (1)
and B (2) Compared to Chloroquine against Chloroquine-
Sensitive (3D7) and -Resistant (Dd2 and K1) Strains of
Plasmidium falciparum (IC50 in μM)
Strain Identification. Strain RK85-270 was isolated from a soil
sample collected in Java, Indonesia. The strain exhibited the highest
octaminomycin A
1)
octaminomycin B
(
(2)
chloroquine
1
6S rRNA gene sequence (GenBank accession number: KY039979)
P. falciparum 3D7
P. falciparum Dd2
P. falciparum Kl
1.5
1.6
1.3
1.5
0.041
0.52
0.46
similarities to Streptomyces endus NRRL 2339 (100%), Streptomyces
sporocinereus NBRC 100766 (100%), and Streptomyces demainii NRRL
B-1478 (99.86%). Therefore, the strain RK85-270 was identified and
named Streptomyces sp. RK85-270. Strain RK85-270 was deposited in
the Chemical Biology Research Group, RIKEN Center for Sustainable
Research Science.
1.1
0.83
14
Previously reported depsipeptides, such as dolastatin 10,
Culture Condition. Streptomyces sp. RK85-270 was cultured in a
1
5
16
17
venturamides, chaiyaphumines, and dragonamides, were
evaluated for their antiplasmodial activity. While some of these
compounds are potently toxic to mammalian cells as well as to
the malarial parasite, others such as venturamide and
chaiyaphumines A and C show relatively weak toxicity to
mammalian cells. In addition, chaiyaphumines A and C were
selected for bioactivity testing to compare the effect of the
benzyl and aliphatic derivatives at the N-terminal of the
threonine residue, and both showed activity against P. falcipa-
rum. Although the positive controls were several times more
potent, the activity of 1 and 2 is still interesting because they
show no cytotoxicity (IC50 > 30 μM) against mammalian cell
lines. The selective antimalarial activity of both 1 and 2 makes
these compounds exciting lead structures for further possible
development.
5
00 mL cylindrical flask (K1 flask) containing 70 mL of culture
medium (glucose 1%, soluble starch 2%, soybean meal 1.5%, malt
extract 0.5%, vegetable extract 10%, potato dextrose 2.5%, KH PO
0.05%, and MgSO ·7H O 0.05%) for 96 h at 28 °C on a rotary shaker
2
4
4 2
with agitation of 150 rpm. A 140 mL amount of each preculture was
used to inoculate two 30 L jar fermenters that contained 15 L of the
same culture medium, which were cultured at a stirring speed of 100
rpm and an aeration rate of 10 L/min for 4 days.
Construction of Fraction Library. The fraction library of
microbial metabolites was constructed from 36.08 g of EtOAc extract,
which was prepared from 30 L of culture broth. The methodology of
fraction library construction was followed as in the previously
1
6
5
−7
described method.
Extraction and Isolation. The 35th fraction of the fifth MPLC
fraction was purified by semipreparative reversed-phase HPLC
(
Senshu Pak Pegasil C18 column, 5 μm, 10 × 250 mm, 4 mL/min)
using isocratic 50% aqueous CH CN. Octaminomycin A (1, 14 mg)
3
In summary, we have obtained two new cyclic octadepsipep-
tides, octaminomycins A (1) and B (2), from the fraction
library by NPPlot screening, each containing an N-acetyl or N-
propionyl threonine. Their planar structures were determined
by analysis of the HRESIMS, MS/MS, and 1D and 2D NMR
data. The stereoconfigurations of the amino acid residues were
assigned by single-crystal X-ray diffraction analysis and a
combination of Marfey’s methodology and LC/MS analysis.
Compounds 1 and 2 showed good in vitro antimalarial activities
against chloroquine-sensitive 3D7 as well as chloroquine-
resistant Dd2 and K1 strains with no cytotoxicity and
antimicrobial activities up to 30 μM. Cyclic octapeptides are
was eluted at 34 min, and octaminomycin B (2, 50 mg) was eluted at
4
5 min.
Octaminomycin A (1): colorless crystals (EtOAc−hexane); mp
25
1
2
95−197 °C; [α]
−160 (c 0.1, MeOH); UV (MeOH) λ (log ε)
589
max
16 (4.31) 277 (3.19) nm; IR (ATR) ν 3295, 2960, 2870, 1750,
max
−1
1
13
1685, 1630, 1515, 1165 cm ; H and C NMR data, Table 1;
HRESIMS m/z 1001.5672 [M + H] (calcd for C H N O ,
+
5
3
77
8
11
1001.5712).
Octaminomycin B (2): yellowish powder; [α]25 −160 (c 0.1,
589
MeOH); UV (MeOH) λmax (log ε) 212 (4.29) 277 (3.23) nm; IR
−
1 1
(
ATR) ν 3280, 2955, 2875, 1750, 1685, 1620, 1515, 1170 cm ; H
max
13
+
and C NMR data, Table 1; HRESIMS m/z 987.5546 [M + H]
calcd for C H N O , 987.5555).
(
18
52 75
8
11
rare among microbial secondary metabolites. The discovery
of 1 and 2 represents a significant addition to the current
knowledge of bioactive cyclic peptides. Notably, the isolation of
the new natural products, octaminomycins, validates the ability
Hydrolysis of 1. A suspension of 1 (3.3 mg) in 0.1 N NaOH
aqueous solution (500 μL) was stirred for 4 h at room temperature
(rt). The mixture was neutralized with 1 N HCl and extracted with
CHCl (5 mL × 3). The combined organic layer was washed with
3
E
DOI: 10.1021/acs.jnatprod.6b00758
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
H O and brine, dried over Na SO , and evaporated to afford a
microliters of this sample was then analyzed by LC-MS selected ion
chromatography on a reversed-phase column (Waters Coretecs, C18
column; 2.1 × 100 mm, 1.6 μm, 0.5 mL/min) with a linear gradient
2
2
4
colorless solid. The crude material was purified by preparative TLC
CHCl −MeOH−AcOH, 100:10:1) to provide the acid 1a (0.8 mg)
(
3
1
as a colorless, amorphous powder: H NMR (500 MHz, CD OD) δ
from 5% to 100% aqueous CH CN containing formic acid over 8 min.
3
H
3
7
(
4
.19 (2H, m), 7.14 (2H, m), 7.13 (1H, m), 6.95 (2H, d, J = 8.5), 6.57
2H, d, J = 8.0), 5.30 (1H, dd, J = 9.5, 5.5), 4.70 (1H, dd, J = 11.0,
.0), 4.35 (1H, t, J = 8.0), 4.34 (1H, d, J = 8.5), 4.28, (2H, m), 4.22
Amino acid standards were prepared by dissolving amino acids in 20
μL of H
O (w/v) followed by the addition of 50 μL of FDLA and 50
2
μL of 1 M NaHCO
, reacted at 40 °C for 1.5 h, and neutralized with
3
(
(
1H, d, J = 6.5), 4.17 (1H, dd, J = 10.5, 4.0), 3.97 (1H, t, J = 5.5), 3.70
1H, m), 3.58 (1H, m), 3.45 (1H, t, J = 6.5), 3.00, (1H, m), 2.98 (2H,
50 μL of 1 M HCl. Mixtures were then processed for LC-MS in a
fashion similar to that used for cyclopeptide analyses; the retention
times for FDLA derivatives of L-Thr, D-Thr, L-allo-Thr, D-allo-Thr, L-
Phe, D-Phe, L-Val, D-Val, L-Pro, D-Pro, L-Leu, D-Leu, and N-Me-L-Tyr
were 3.96, 4.46, 4.04, 4.24, 5.05, 5.64, 4.73, 5.44, 4.42, 4.70, 5.04, 5.81,
and 3.92 min, respectively. Accordingly, the amino acids in 1 and 2
were L-Thr (3.96 min), L-Phe (5.04 min), L-Val (4.73 min), L-Pro
(4.40 min), L-Leu (5.03 min), D-Leu (5.81 min), and N-Me-L-Phe
(3.93 min).
m), 2.96 (3H, s), 2.84 (1H, dd, J = 14.5, 7.5), 2.23 (2H, m), 2.09 (2H,
m), 1.97 (1H, m), 1.91 (2H, m), 1.83 (2H, m), 1.73 (1H, m), 1.64
(
1H, m), 1.44 (1H, m), 1.39 (1H, m), 1.29 (2H, m), 1.05 (3H, t, J =
7
0
.5), 1.04 (3H, d, J = 6.5), 0.93 (3H, d, J = 7.0), 0.85 (3H, d, J = 6.5),
.84 (3H, d, J = 6.5), 0.83 (3H, d, J = 6.5); HRESIMS m/z 1041.5659
+
[
M + Na] (calcd for C H N O Na, 1041.5659).
53
78
8
12
Methanolysis of 1. K CO3 (0.3 mg) was added to a stirred
2
solution of 1 (3.3 mg) in MeOH. After stirring for 1 h at rt, the
mixture was heated for 2 h under reflux. The mixture was cooled to rt,
and the solvent was concentrated. The residue was purified by
preparative TLC (CHCl −MeOH, 10:1) to provide the methyl ester
1
CDCl ) δ 7.27 (2H, m), 7.20 (2H, m), 7.19 (1H, m), 7.00 (2H, d, J =
8
4
(
Antimalarial Activity Assay. Plasmodium falciparum 3D7, Dd2,
and K1 strains were cultured at 37 °C under 5.0% CO in human
2
erythrocytes (3% hematocrit) in RPMI1640 (Thermo Fisher
Scientific), supplemented with 10% human plasma. The assay
3
10
1
b (1.0 mg) as a colorless, amorphous powder: H NMR (500 MHz,
methodology was followed as in the previously described method.
In Vitro Cytotoxicity Assay. The human promyelocytic leukemia
3
H
2
1
cell line HL-60 was cultured at 37 °C in RPMI-1640, supplemented
with 10% fetal bovine serum (FBS; Sigma-Aldrich). The human cervix
epidermoid carcinoma cell line HeLa was cultured at 37 °C in DMEM
.0), 6.79 (2H, d, J = 8.5), 5.68 (1H, m), 4.80 (1H, m), 4.65 (1H, m),
.44 (2H, m), 4.39, (1H, d, J = 6.0), 4.20 (1H, m), 3.74 (3H, s), 3.65
1H, d, J = 5.5), 3.60 (1H, m), 3.57 (1H, m), 3.19, (1H, m), 3.09 (1H,
(
Thermo Fisher Scientific), supplemented with 10% FBS. tsFT210
m), 3.04 (2H, m), 3.00 (3H, s), 2.34 (1H, m), 2.27 (2H, m), 2.18 (2H,
m), 2.04 (2H, m), 1.97 (2H, m), 1.91 (2H, m), 1.66 (2H, m), 1.61
22
cells, the mouse temperature-sensitive cdc2 mutant cell line of
mammary carcinoma FM3A, was cultured at 32 °C in RPMI-1640,
supplemented with 5% calf serum (CS; PAA Laboratories GmbH).
src -NRK cells, the rat kidney cells infected with ts25, a T-class
mutant of Rous sarcoma virus Prague strain, were cultured at a
permissive temperature (32 °C) in MEM (Sigma-Aldrich) and
supplemented with 10% CS. Each cell line was seeded into a 96-well
(
2H, m), 1.45 (1H, m), 1.35 (2H, m), 1.14 (3H, t, J = 7.5), 1.07 (3H,
d, J = 6.0), 0.96 (3H, d, J = 7.0), 0.91 (3H, d, J = 6.0), 0.90 (3H, d, J =
.0), 0.87 (3H, d, J = 7.0), 0.85 (3H, d, J = 6.0), 0.83 (3H, d, J = 6.5);
ts
23
7
+
HRESIMS m/z 1055.5760 [M + Na] (calcd for C H N O Na,
1
54
80
8
12
055.5760).
Crystallization and X-ray Crystallographic Analysis of
4
3
Octaminomycin A (1). Compound 1 was crystallized by slow
evaporation of the solution in EtOAc−hexane. A colorless, block-
shaped, single crystal with dimensions of 0.10 × 0.09 × 0.05 mm was
mounted on a glass capillary and set on a Rigaku AFC-8 with a
Saturn70 CCD diffractometer. The measurements were carried out by
an oscillation method at 90 K using Mo Kα radiation. The unit cell
dimensions were determined using 62 343 reflections with 1.16° ≤ θ ≤
plate (1.5 × 10 cells/well for HL-60, 4 × 10 cells/well for HeLa, 1.6
4 4 ts
× 10 cells/well for tsFT210, and 1.0 × 10 cells/well for src -NRK)
and then exposed to test compounds for 48 h. Following 48 h
exposures to test compounds, cell proliferation was determined using a
Cell Count Reagent SF (Nacalai Tesque) according to the
manufacturer’s instructions. Briefly, following a 48 h exposure, a 1/
1
0 volume of WST-8 solution was added to each well, and the plates
0.46°. The diffraction data of 61 176 up to (sin θ/λ)max = 0.595 Å−1
were incubated for 1 h. Then, cell growth was measured as the
absorbance at 450 nm on a microplate reader (PerkinElmer).
Antimicrobial Activities Assay. The assay was performed as
3
were collected. Lorentz, polarization, and numerical absorption
corrections were applied during the scaling processes, affording 9800
unique reflections with an Rint of 0.1055. The initial structure of 1 was
solved by a direct method using the SIR2004 program and refined by
a full matrix least-squares method using the SHELXL-2014 program
1
0
previously described.
19
2
0
ASSOCIATED CONTENT
Supporting Information
■
(Figure 2). The crystallographic information file (CIF) of 2 has been
*
S
deposited with the Cambridge Crystallographic Data Centre (deposit
no. CCDC 1495124). Copies of the data can be obtained, free of
charge, on application to the Director, CCDC, 12 Union Road,
Cambridge CB21EZ, U.K. (fax: + 44-(0)223-336033 or e-mail:
deposit@ccdc.cam.ac.uk).
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00758.
X-ray crystallograpic data of compound 1 (CIF)
Crystal data of 1: C H O O ·1.5CH COOC H ·H O, fw =
53
76
8
11
3
2
5
2
1
D and 2D NMR, HRESIMS, and LC/MS results from
1
1
151.38, orthorhombic, space group P2 2 2 , a = 11.1520(8) Å, b =
1 1 1
3
6.7802(12) Å, c = 35.226(3) Å, V = 6591.9(8) Å ; D = 1.160 Mg
Marfey’s analysis of compounds 1 and 2 (PDF)
X
−3
−1
2
m ; Z = 4; μ(Mo Kα) = 0.084 mm , R(F) = 0.1145, wR(F ) =
0
with I > 2σ(I). Δρ
LC-MS Analysis of the Acid Hydrolysates of 1 and 2 Using
Marfey’s Method. Compounds 1 (0.6 mg) and 2 (0.8 mg) were each
dissolved in 6 N HCl (1 mL) and heated at 110 °C for 12 h. After
cooling to rt, the solvent was removed under reduced pressure. The
−1
.2633, and S = 1.522 for 5297 reflections of (sin θ/λ) = 0.595 Å
max
AUTHOR INFORMATION
−3
,
= −0.427, 0.475 e Å .
■
min max
Corresponding Authors
Tel (J. S. Ahn): +82-43-240-6160. Fax: +82-43-240-6169. E-
mail: jsahn@kribb.re.kr.
Tel (H. Osada): +81-48-467-9541. Fax: +81-48-462-4669. E-
*
*
remaining hydrolysate was resuspended in 50 μL of H O and treated
with 50 μL of 1% (w/v) 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide
2
mail: hisyo@riken.jp.
(
FDLA) in acetone and 50 μL of 1 M NaHCO . The mixture was
ORCID
3
heated at 40 °C for 1.5 h. After cooling to rt, the contents were
neutralized with 50 μL of 1 M HCl, and the resulting mixture was
added to 300 μL of MeOH to afford a final hydrolysate volume of 500
μL. From this sample, a 50 μL aliquot was withdrawn, and its solvent
removed followed by redissolution in 50 μL of MeOH. Two
Jae-Hyuk Jang: 0000-0002-4363-4252
Hiroyuki Osada: 0000-0001-7400-8388
Notes
The authors declare no competing financial interest.
F
DOI: 10.1021/acs.jnatprod.6b00758
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(
23) Osada, H.; Koshino, H.; Isono, K.; Takahashi, H.; Kawanishi, G.
ACKNOWLEDGMENTS
■
J. Antibiot. 1991, 44, 259−261.
We thank Dr. T. Nakamura and Dr. Y. Hongo at RIKEN for
the HRESIMS measurements and Ms. H. Aono, Ms. N. Morita,
Mr. K. Yamamoto, and Dr. H. Hayase from RIKEN for the
bioactivity tests. We also thank Dr. H. Kusakabe from RIKEN
for the collection of the strain Streptomyces sp. RK85-270. This
work was supported in part by a Grant-in-Aid for Scientific
Research (A) from the Ministry of Education, Culture, Sports
and Technology of Japan, the Program for Promotion of Basic
and Applied Researches for Innovations in Bio-oriented
Industry, and the Research Program on Hepatitis from the
Japan Agency for Medical Research and Development, AMED.
This work was also supported in part by grants from the KRIBB
Research Initiative Program and the International Corporation
Research Program of NST funded by the Ministry of Science,
ICT and Future Planning of Korea.
REFERENCES
■
(
(
1) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2016, 79, 629−661.
2) Cragg, G. M.; Newman, D. J. Biochim. Biophys. Acta, Gen. Subj.
2
(
2
(
013, 1830, 3670−3695.
3) Clardy, J.; Fischbach, M. A.; Walsh, C. T. Nat. Biotechnol. 2006,
4, 1541−1550.
4) Geniloud, O.; Gonzal
́
ez, I.; Salazar, O.; Martín, J.; Tormo, J. R.;
Vicente, F. J. Ind. Microbiol. Biotechnol. 2011, 38, 375−389.
(
(
5) Osada, H.; Nogawa, T. Pure Appl. Chem. 2011, 84, 1407−1420.
6) Kato, N.; Takahashi, S.; Nogawa, T.; Saito, T.; Osada, H. Curr.
Opin. Chem. Biol. 2012, 16, 101−108.
7) Nogawa, T.; Okano, A.; Takahashi, S.; Uramoto, M.; Konno, H.;
Saito, T.; Osada, H. Org. Lett. 2010, 12, 4564−4567.
8) Nogawa, T.; Takahashi, S.; Okano, A.; Kawatani, M.; Uramoto,
M.; Saito, T.; Osada, H. J. Antibiot. 2012, 65, 123−128.
9) Nogawa, T.; Kawatani, M.; Uramoto, M.; Okano, A.; Aono, H.;
Futamura, Y.; Koshino, H.; Takahashi, S.; Osada, H. J. Antibiot. 2013,
6, 621−623.
10) Jang, J.-P.; Nogawa, T.; Uramoto, M.; Okano, A.; Futamura, Y.;
Shimizu, T.; Takahashi, S.; Jang, J.-H.; Ahn, J. S.; Osada, H. J. Antibiot.
015, 68, 293−295.
11) Lim, C.-L.; Nogawa, T.; Uramoto, M.; Okano, A.; Hongo, Y.;
(
(
(
6
(
2
(
Nakamura, T.; Koshino, H.; Takahashi, S.; Ibrahim, D.; Osada, H. J.
Antibiot. 2014, 67, 323−329.
(
12) Lim, C.-L.; Nogawa, T.; Okano, A.; Futamura, Y.; Kwatani, M.;
Takahashi, S.; Ibrahim, D.; Osada, H. J. Antibiot. 2016, 69, 456−458.
13) Honma, M.; Tanaka, K.; Konno, K.; Tsuge, K.; Okuno, T.;
Hashimoto, M. Bioorg. Med. Chem. 2012, 20, 3793−3798.
14) Fennell, B. J.; Carolan, S.; Pettit, G. R.; Bell, A. J. Antimicrob.
Chemother. 2003, 51, 833−841.
15) Linington, R. G.; Gonzal
Ortega-Barría, E.; Gerwick, W. H. J. Nat. Prod. 2007, 70, 397−401.
16) Grundmann, F.; Kaiser, M.; Schiell, M.; Batzer, A.; Kurz, M.;
Thanwisai, A.; Chantratita, N.; Bode, H. B. J. Nat. Prod. 2014, 77,
79−783.
17) McPhail, K. L.; Correa, J.; Linington, R. G.; Gonzalez, J.;
Ortega-Barria, E.; Capson, T. L.; Gerwick, W. H. J. Nat. Prod. 2007, 70,
84−988.
18) Caboche, S.; Pupin, M.; Lecler
Kucherov, G. Nucleic Acids Res. 2008, 36, D326−331.
19) Burla, M. C.; Caliandro, R.; Camalli, M.; Carrozzini, B.;
(
(
(
́
̃
es, J.; Urena, L. D.; Romero, L. I.;
(
7
(
9
(
̀
e, V.; Fontaine, A.; Jacques, P.;
(
Cascarano, G. L.; De Caro, L.; Giacovazzo, C.; Polidori, G.; Spagna, R.
J. Appl. Crystallogr. 2005, 38, 381−388.
(
20) Sheldrick, G. M. Acta Crystallogr., Sect. A: Found. Adv. 2015, 71,
3
(
4
(
−8.
21) Osada, H.; Magae, J.; Watanabe, C.; Isono, K. J. Antibiot. 1988,
1, 925−931.
22) Osada, H.; Cui, C. B.; Onose, R.; Hanaoka, F. Bioorg. Med.
Chem. 1997, 5, 193−203.
G
DOI: 10.1021/acs.jnatprod.6b00758
J. Nat. Prod. XXXX, XXX, XXX−XXX