Ieodomycins A-D, antimicrobial fatty acids from a marine Bacillus sp.

Article abstract of DOI:10.1021/np200223r

Bioassay-guided isolation of the EtOAc extract of a marine Bacillus sp., cultured in modified Bennett's broth medium, yielded four new antimicrobial fatty acids, named ieodomycins A-D (1-4). The planar structures of these new compounds were determined by extensive 1D and 2D NMR and HRESIMS spectroscopic data analysis. Their absolute configurations were elucidated by modified Mosher's method and literature data review. All four new compounds (1-4) demonstrated antimicrobial activities in vitro.

Full text of DOI:10.1021/np200223r

ARTICLE
pubs.acs.org/jnp
Ieodomycins AÀD, Antimicrobial Fatty Acids from a Marine Bacillus sp.
M. A. Mojid Mondol,†,‡ Ji Hye Kim,† Min ah Lee,† Fakir Shahidullah Tareq,†,‡ Hyi-Seung Lee,† Yeon-Ju Lee,†
and Hee Jae Shin*,†,‡
†Marine Natural Products Chemistry Laboratory, Korea Ocean Research & Development Institute, Ansan, P.O. Box 29, Seoul 425-600,
Republic of Korea
‡University of Science and Technology, 113 Gwahangno, Yuseong-gu, Daejeon, Republic of Korea
S Supporting Information
b
ABSTRACT: Bioassay-guided isolation of the EtOAc extract of a
marine Bacillus sp., cultured in modified Bennett’s broth medium,
yielded four new antimicrobial fatty acids, named ieodomycins
AÀD (1À4). The planar structures of these new compounds
were determined by extensive 1D and 2D NMR and HRESIMS
spectroscopic data analysis. Their absolute configurations were
elucidated by modified Mosher’s method and literature data
review. All four new compounds (1À4) demonstrated antimi-
crobial activities in vitro.
arine organisms represent a promising source for natural
products in the future due to the incredible diversity of
M
chemical compounds and bioactivities.1 Marine Bacillus species,
ubiquitous and diverse in marine ecosystems,2 are well known
for producing antimicrobial3 and anticancer compounds,4 bio-
surfactants,5 and so on. There is an urgent need for new
antimicrobial agents to combat the threats imposed by antibiotic-
resistant pathogenic bacteria, limited efficacy of antifungal drugs,
and the recent advent of bioterrorism.6 Marine bacteria isolated
from the sediments and the surface of marine algae and inverte-
brates have been shown to produce secondary metabolites that
display antibacterial properties.7
As a part of our continuing program on the discovery of
bioactive secondary metabolites from marine microorganisms,
we isolated a marine bacterium from a sediment sample collected
from Ieodo, Republic of Korea’s southern reef. This strain,
designated as 09ID194, was identified as Bacillus sp. based on
16S rDNA sequencing. The EtOAc extract of the culture broth of
strain 09ID194 showed potent antimicrobial activity. Bioassay-
guided isolation and repeated chromatographic steps led to the
isolation of four new antimicrobial compounds, ieodomycins
AÀD (1À4). This paper reports the isolation, structure elucida-
tion, and antimicrobial activities of these compounds (1À4).
’ RESULTS AND DISCUSSION
Ieodomycin A (1) was isolated as a yellowish, amorphous
solid. The molecular formula of 1 was determined to be C13-
H22O4 by extensive analysis of its combined HRESIMS and 1D
and 2D NMR data, which required three degrees of unsaturation.
The UV spectrum of 1 exhibited an absorption band at λmax
232 nm, which was assigned to a conjugated diene. The IR
absorptions at 3400 and 1715 cmÀ1 indicated the presence of
hydroxy (OH) and ester carbonyl (CdO) groups, respectively.
The 13C NMR and HSQC spectra displayed four olefinic carbon
Received: March 10, 2011
Published: June 23, 2011
r
Copyright
2011 American Chemical Society and
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Journal of Natural Products
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Table 1. NMR Data for Compounds 1 and 2 in CD3OD
1
2
δH, mult. (J in Hz)b
HMBCc
δC
δH, mult. (J in Hz)b
HMBCc
a
a
position
δC
1
2
174.0
43.9
174.1
40.2
2.47, dd (10.0, 5.0)
1, 3, 4
2.36, dd (16.8, 7.3, H-2ax)
2.86, dd (16.8, 1.3, H-2eq)
4.19, dddd (10.4, 7.3, 3.5, 1.3, H-3ax)
2.24, ddd (13.5, 10.4, 7.3, H-4ax)
2.27, ddd (13.5, 4.2, 3.5, H-4eq)
4.24, dddd (10.4, 7.3, 4.2, 3.5, H-5ax)
1.80, m
3, 4
3
4
66.6
45.3
4.25, m
1.52, m
64.4
38.7
1
5
2, 3
5
6
7
68.8
37.5
37.0
3.76, m
1.56, m
2.10, m
2.19, m
3
78.5
34.9
36.1
4
4, 5, 7, 8
5, 6, 8, 9, 12
5, 7
2.18, m
5, 6, 8, 9, 12
2.25, m
8
140.1
127.0
134.7
115.1
139.0
127.6
134.5
115.6
9
5.86, d (10.5)
7, 10, 11, 12
8, 9
5.88, d (10.6)
7, 12
9, 10
7, 8, 9
10
11
6.57, ddd (16.7, 10.5, 10.5)
4.93, d (10.5, H-11b)
5.04, d (16.7, H-11a)
1.75, s
6.58, ddd (16.6, 10.6, 10.6)
4.96, dd (10.6, 2.2, H-11b)
5.07, dd (16.6, 2.2, H-11a)
1.77, s
9, 10
12
16.8
52.1
7, 8, 9
1
16.6
OCH3
3.67, s
a Measured at 125 MHz. b Measured at 500 MHz. c HMBC correlations are from proton(s) stated to the indicated carbon(s).
1
Figure 1. 1HÀ H COSY and HMBC correlations for compounds 1À4.
signals (δC 115.1À140.1) including an unusual terminal olefinic
methylene carbon (δC 115.1), four methylene carbons (δC 37.0À
45.3), two oxygenated methine carbons (δC 66.6 and 68.8),
two methyl carbons (δC 16.8 and 52.1), one of which was
oxygenated, and an ester carbonyl carbon (δC 174.0)
(Table 1). Analysis of the HÀ H COSY spectrum suggested
two isolated spin systems: one from H2-2 at δH 2.47 to H2-7 at
δH 2.10/2.19 and another from H-9 at δH 5.86 to H2-11 at δH
4.93/5.04. Their connectivity with C-8 (δC 140.1) was estab-
lished by long-range correlations of H2-7 with C-8 and C-9 (δC
127.0) (Figure 1). The connectivity of the methoxy (OCH3)
Figure 2. Key ROESY correlations for compounds 1À4.
group (δH 3.67, s) with carbonyl carbon C-1 (δC 174.0) was
readily determined, as its protons showed an HMBC correlation
with C-1 (Figure 1). Similarly, the remaining methyl group signal
(δH 1.75, s, H3-12) showed HMBC cross-peaks with C-7, C-8,
and C-9, and from these correlations, its connectivity with C-8
was confirmed (Figure 1). From these correlation data, the
planar structure of compound 1 was established. ROESY correla-
tions were observed between H-10 and H3-12 and between H-9
1
1
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Figure 3. Assignment of the relative configuration of the 3,5-diol of 1
based on Kishi’s Universal NMR Database (Database 2). ΔδC values
between the model system and 1 are shown. The relative configuration
of the 3,5-diol is assigned as anti on the basis of the best fit with the
model system.
and H-11b (δH 4.93), but no correlation was found between H-9
and H3-12 (Figure 2). From these correlations, the geometry
of the C-8/C-9 trisubstituted double bond was established as E.
The relative configuration of the 3,5-diol of 1 was assigned by
using Kishi’s Universal NMR Database(Database2).8À10 The 13C
NMRresonances ofC-3/C-5were in good agreement with an anti
arrangement of the 1,3-diol model system (Figure 3). Assign-
ment of the absolute configuration of the 3,5-diol of 1 was
carried out by the modified Mosher’s ester method.11À14 Com-
pound 1 was treated with (R)-(À)- and (S)-(+)-R-methoxy-
R-(trifluoromethyl)phenylacetyl chloride (MTPA-Cl) in dry
pyridine separately to yield bis-(S)- and (R)-MTPA ester deri-
vatives 1a and 1b, respectively. All proton signals of the two
Figure 4. ΔδH values (ΔδH = δS À δR) for MTPA esters of 1À4.
1
diester derivatives were assigned by 1HÀ H COSY experiments,
and the 1H chemical shift values of the bis-(R)-MTPA ester (1b)
were subtracted from the corresponding values of the bis-(S)-
MTPA ester (1a). The ΔδH values [ΔδH = δS À δR] are shown
in Figure 4. Negative ΔδH values for H-3 (À0.11), H-4 (À0.12/
À0.13), and H-5 (À0.17), corresponding to the ΔδH pattern
for diesters of anti-1,3-diols reported by Riguera,13 indicated that
the absolute configurations of C-3 and C-5 of 1 were S and R,
respectively. The structure of 1 was therefore conclusively
determined to be (3S,5R,8E)-methyl 3,5-dihydroxy-8-methylun-
deca-8,10-dienoate, and 1 was named ieodomycin A.
was observed between H-3ax and H-4ax, which also supported
the equatorial position of the 3-OH group. The axial position of
H-5 was indicated by its coupling constants with H-4ax (J = 7.3)
and H-6ax (J = 10.4) and also supported by a ROESY correlation
between H-4eq and H-5ax (Figure 2). ROESY correlations
between H-2ax and H-4ax and between H-3ax and H-5ax
confirmed the assignments. These correlation data suggested a
chair conformation for the δ-lactone ring.16 The absolute con-
figuration of 2 at C-3 was determined by application of the
modified Mosher’s method as for 1. Acylation of 2 with (R)-(À)-
and (S)-(+)-MTPA-Cl furnished the mono-(S)- and (R)-MTPA
Ieodomycin B (2) was isolated as an optically active, white,
amorphous solid. The molecular formula of 2 was determined to
be C12H18O3 (one carbon, four hydrogens, and one oxygen atom
less than 1) from the HRESIMS spectrum, which gave a
molecular ion peak ([M + Na]+) at m/z 233.1145. Its IR
spectrum indicated the presence of hydroxy (3365 cmÀ1) and
δ-lactone (1715 cmÀ1) groups. Comparison of its UV, IR, and
1H and 13C NMR (Table 1) data with those of 1 indicated that 2
had a similar skeleton to 1, except that the methoxy (OCH3)
group attached to C-1 in 1 was absent and instead C-1 was
attached to the oxygen at C-5 in 2, forming a δ-lactone ring. By
detailed analysis of UV, IR, and 1D and 2D NMR (Figure 1) data,
the planar structure of 2 was unequivocally established. The
1
esters (2a and 2b), respectively. Analysis of the H NMR data
(Figure 4) for these MTPA esters indicated that the absolute
configuration at C-3 was S. The configuration of C-5 in com-
pound 2 was determined as R based on the anti relationship
between C-3 and C-5, which was suggested by the coupling
constants and ROESY correlations.16 Comprehensive 1D and
2D spectroscopic data analysis led to the determination of
structure of 2 as (3S,5R,8E)-3-hydroxy-5-(8-methylhexa-8,10-
dienyl)tetrahydro-1H-pyran-1-one (ieodomycin B).
Ieodomycin C (3), a pale, amorphous solid, analyzed for
C12H20O4 on the basis of its parent ion in the HRESIMS (m/z
227.1288 [M À H]À) and the 1H and 13C NMR data (Table 2),
indicating three degrees of unsaturation. Its IR spectrum dis-
closed absorption bands at 3349 and 1711 cmÀ1 assignable to
hydroxy (OH) and carbonyl (CdO) groups, respectively. The
UV spectrum exhibited an absorption maximum at λmax 230 nm,
which was attributed to a conjugated diene. The proton and
carbon signals of 3 (Table 2) were unambiguously assigned by
3
relative configuration of 2 was deduced on the basis of JHÀH
coupling constants and ROESY spectroscopic data. The ROE
correlations of H-10 with H3-12, and H-9 with H-11b, as well as
no correlation between H-9 and H-10, suggested the E geometry
of the C-8/C-9 double bond (Figure 2). The splitting pattern and
chemical shifts of H2-2 (δH 2.36, dd, J = 16.8, 7.3, H-2ax, and
δH 2.86, dd, J = 16.8, 1.3, H-2eq) of the δ-lactone ring clearly
established the anti relative configuration of C-3 and C-5.15 The
large vicinal coupling constant (J = 7.3) between H-2ax and
H-3ax indicated that the 3-OH group was in an equatorial
position, and this was confirmed by a ROESY correlation
between H-2eq and H-3ax. A trans-diaxial coupling (J = 10.4)
1
2D NMR experiments (1HÀ H COSY, HSQC, and HMBC).
The resonances in the 1H and 13C NMR spectra (Table 2) were
well separated, and a single spin system from H2-2 at δH 2.45 to
H3-12 at δH 1.13 of 3 was determined in a stepwise fashion by
1
analysis of the 1HÀ H COSY spectrum (Figure 1). Both of the
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Table 2. NMR Data for Compounds 3 and 4 in CD3OD
3
4
δH, mult. (J in Hz)b
HMBCc
δC
δH, mult. (J in Hz)b
HMBCc
a
a
position
δC
1
2
3
4
5
6
7
175.6
43.7
171.5
121.3
146.5
130.0
145.3
34.0
2.45, d (6.0)
1, 3, 4
1, 2, 4, 5
2, 3, 6
3, 6, 7
5, 8
5.78, d (15.3)
7.21, dd (15.3, 10.5)
6.24, dd (15.3, 10.5)
6.16, m
1, 4
70.2
4.50, m
1, 2, 4, 5
2, 3, 5, 6
3, 4, 6, 7
4, 5, 7, 8
5, 6, 8, 9
133.6
132.1
131.0
136.1
5.60, dd (15.3, 6.5)
6.22, dd (15.3, 10.5)
6.03, dd (15.3, 10.5)
5.70, dt (15.3, 7.0)
2.20, m
5, 8, 9
26.2
1.45, m
1.56, m
8
9
33.7
26.7
2.10, m
6, 7, 9, 10
8, 11
39.7
68.4
1.43, m
7, 9, 10
7
1.38, m
3.72, m
1.50, m
10
11
12
39.8
68.5
23.6
1.42, m
23.6
1.14, d (6.0)
8, 9
3.70, m
9
1.13, d (6.0)
10, 11
a Measured at 125 MHz. b Measured at 500 MHz. c HMBC correlations are from proton(s) stated to the indicated carbon(s).
double bonds at C-4/C-5 and C-6/C-7 had an E geometry, which
was supported by large scalar coupling constants between H-4
and H-5 (J = 15.3 Hz) and between H-6 and H-7 (J = 15.3 Hz).
This was also confirmed by ROESY correlations between H-4 and
H-6 and between H-5 and H-7 (Figure 2). Like compounds 1
and 2, conversion of compound 3a into the Mosher’s diesters
(S)-MTPA (3b) and (R)-MTPA (3c) followed by analysis of the
1H NMR data (Figure 4) indicated the S and R absolute config-
urations at the C-3 and C-11 stereocenters in 3, respectively. The
structureof 3 was establishedconclusivelyas(3S,4E,6E,11R)-3,11-
dihydroxydodeca-4,6-dienoic acid (ieodomycin C).
Saccharomyces cerevisiae (KCTC 7913). Compounds 1À4 ex-
hibited activity against Bacillus subtilis and Escherichia coli with
minimum inhibitory concentrations (MICs) of 32À64 μg/mL,
but showed only weak growth inhibition against the yeast
Saccharomyces cerevisiae, with an MIC of 256 μg/mL. In compar-
ison, long-chain and branched polyunsaturated fatty acids
isolated from freshwater sponges showed no antimicrobial
activity against E. coli and S. cerevisiae and weak activity against
B. subtilis.17 An unsaturated dihydroxy acid isolated from the
fungus Mycosphaerella rubella, 6S,13R-dihydroxy-2E,4E,8E-tetra-
decatrienoic acid, which has a similar structure to 2, showed weak
antibacterial but no antiyeast activities.12
Polyunsaturated branched and unbranched fatty acids are
widely found in marine organisms including bacteria, cyanobacteria,
algae,18 and dinoflagellates,19 and most of them are biologically
inactive. By comparing the antibacterial activity among different
fatty acids, it has been shown that unsaturated and hydroxy fatty
acids showed better antibacterial activities.20À22 Even though there
have been several reports regarding the mode of action of long-
chain unsaturated fatty acids, the precise mechanism for the
antimicrobial activity remains unclear. Recently, it was suggested
that the antimicrobial activity of unsaturated fatty acids is related
to their inhibition of bacterial fatty acid synthesis.23
Ieodomycin D (4) was obtained as an optically active, light
yellowish, amorphous solid. The molecular formula of 4 was
established as C10H16O3 by HRESIMS and 13C NMR data,
having two carbons, four hydrogens, and one oxygen atom less
than 3 and the same degrees of unsaturation. Its UV and IR
spectra were quite similar to those of 3, indicating the presence of
a carbonyl group (1684 cmÀ1), a hydroxy group (3365 cmÀ1),
and a conjugated diene moiety (λmax 254 nm). Assignments of all
the protonated carbons were done by the analysis of the HSQC
1
1
spectrum as shown in Table 2. The HÀ H COSY data of 4
revealed only one spin system starting from H-2 (δH 5.78) and
ending at H3-10 (δH 1.14). An R,β,γ,δ-unsaturated carbonyl
moiety was established as H-2 at δH 5.78 and H-4 at δH 6.24
showed long-range correlations with C-1 (δC 171.5) and C-3
(δC 146.5), respectively. The E geometries of the C-2/C-3 and
C-4/C-5 double bonds were assigned on the basis of the large
coupling constants between H-2 and H-3 (J = 15.3 Hz) and
between H-4 and H-5 (J = 15.3 Hz) (Table 2) and were
confirmed by ROESY correlations (Figure 2). The absolute
configuration of the stereogenic center at C-9 in 4 was addressed
by the modified Mosher’s method; each mono-(S)- and (R)-MTPA
In conclusion, we discovered four new rare hydroxy unsatu-
rated fatty acids, ieodomycins AÀD (1À4), which showed
antibacterial activity against both Gram-positive and Gram-negative
pathogens. Interestingly compounds 1À4 were produced by this
Bacillus sp. only in low salinity (12 g/L) but not in high salinity
(32 g/L) culture medium.
’ EXPERIMENTAL SECTION
1
ester was separately prepared and subjected to H NMR data
General Experimental Procedures. Optical rotations were
measured on a JASCO (DIP-1000) digital polarimeter. UV spectra were
obtained on a Shimadzu UV-1650PC spectrophotometer. IR spectra
were recorded on a JASCO FT/IR-4100 spectrophotometer. 1D and 2D
NMR spectra were acquired on a Varian Unity 500 spectrometer in
CD3OD. High-resolution mass spectra were recorded on a hybrid ion-
trap time-of-flight mass spectrometer (Shimadzu LCMS-IT-TOF).
analysis. The R configuration of C-9 in 4 was determined on the
basis of the guidelines.11 Compound 4 was named ieodomycin D,
and the structure was unambiguously established as (2E,4E,9R)-
9-hydroxydeca-2,4-dienoic acid.
The antimicrobial activities of 1À4 were tested against Bacillus
subtilis (KCTC 1021), Escherichia coli (KCTC 1923), and
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Analytical HPLC was conducted on a PrimeLine binary pump with RI-
101 (Shodex) and variable UV detector (M 525). Continuous centri-
fugation was done on a centrifugal separator (Kansai Centrifugal
Separator Manufacturing Co. Ltd.). Semipreparative HPLC was per-
formed using ODS (YMC-Pack-ODS-A, 250 Â 10 mm, i.d. 5 μm) and
silica (YMC-Pack-SIL, 250 Â 10 mm, i.d. 5 μm) columns. Analytical
HPLC was conducted on an ODS column (YMC-Pack-ODS-A, 250 Â
4.6 mm, i.d. 5 μm). All solvents used were either spectral grade or distilled
prior to use.
Isolation and Taxonomy of Strain 09ID194. The strain
09ID194 was isolated from a sediment sample collected from Ieodo,
Republic of Korea’s southern reef, during an expedition in 2009. In brief,
one gram of the sediment sample was diluted in sterilized seawater
(10À1, 10À2, 10À3, and 10À4) under aseptic conditions, and 100 μL from
each dilution was spread onto modified Bennett’s agar (0.1% yeast
extract, 0.1% beef extract, 0.2% tryptone, 1% dextrose, 100% natural
seawater, and 1.8% agar, pH adjusted to 7.2 before sterilization). The
plates were incubated for 14 days at 30 °C, and the resulting colony of
strain 09ID194 was isolated and maintained on modified Bennett’s agar.
The strain 09ID194 formed well-developed, yellowish colonies on
modified Bennett’s agar. The strain was identified as Bacillus sp. on
the basis of 16S rDNA sequence analysis. The sequence was deposited in
GenBank under accession number JN048684. This strain is currently
preserved in the Microbial Culture Collection, KORDI, with the name
Bacillus sp. 09ID194 under the curatorship of H.J.S.
Seed and Large-Scale Cultures of Strain 09ID194. The seed
and large-scale cultures were carried out in modified Bennett’s medium
(0.1% yeast extract, 0.1% beef extract, 0.2% tryptone, and 1% dextrose,
salinity 12 g/L, pH 7.6). The medium (200 mL) was dispensed in a
500 mL conical flask and sterilized. A single colony of 09ID194 strain
from the agar plate was inoculated aseptically into the flask and
incubated at 24 °C for 2 days on a rotary shaker at 120 rpm. An aliquot
(0.2% v/v) from the seed culture was inoculated aseptically into 2 L
flasks (total 100 flasks) containing 1 L of sterilized culture medium. The
production culture was incubated under the same conditions as the seed
culture for 7 days and then harvested.
data (CD3OD), see Table 2; HRESIMS m/z 227.1288 [M À H]À (calcd
for C12H19O4, 227.1283).
Ieodomycin D (4): light yellowish, amorphous solid; [R]23 +15
D
(c 0.8, CHCl3); UV (MeOH) λmax (log ε) 254 (2.98) nm; IR (MeOH)
νmax 3365 (br), 2932, 1684, 1255, 1004 cmÀ1; 1H and 13C NMR data
(CD3OD), see Table 2; HRESIMS m/z 183.1029 [M À H]À (calcd for
C10H15O3, 183.1021).
Preparation of Bis-(S)-MTPA Ester (1a). Compound 1 (0.6 mg)
was dissolved in 200 μL of pyridine and stirred at room temperature (rt)
for 10 min. For preparation of the bis-(S)-MTPA ester (1a) of 1, 20 μL
of (R)-(À)-MTPA-Cl was added to the reaction vial, and the mixture
was stirred at rt for 16 h. Completion of the reaction was monitored by
LC/MS. The reaction mixture was dried in vacuo, redissolved in EtOAc,
washed with H2O, and purified by a silica column using 10% MeOH in
CHCl3 as eluent to obtain 1a (0.45 mg).
Compound 1a: amorphous solid; 1H NMR data (CD3OD) δH 2.64
(H2-2, dd, J = 10.0, 6.5 Hz), 5.34 (H-3, m), 1.92 (H-4b, m), 1.94 (H-4a,
m), 4.88 (H-5, m), 1.74 (H2-6, m), 1.91 (H-7b, m), 1.96 (H-7a, m), 5.73
(H-9, d, J = 10.5 Hz), 6.51 (H-10, ddd, J = 16.5, 10.5, 10.5 Hz), 4.96
(H-11b, d, J = 10.5 Hz), 5.04 (H-11a, d, J = 16.5 Hz), 1.67 (H3-12, s),
3.58 (OCH3, s), 3.54 (2 Â OCH3, s) 7.38À7.64 (10H, m); ESIMS m/z
697.34 [M + Na]+.
Compound 1b: In an entirely analogous way, bis-(R)-MTPA ester
(1b) was obtained using (S)-(+)-MTPA-Cl. Amorphous solid (0.5 mg);
1H NMR data (CD3OD) δH 2.66 (H2-2, dd, J = 9.0, 6.5 Hz), 5.45 (H-3,
m), 2.04 (H-4b, m), 2.07 (H-4a, m), 5.05 (H-5, m), 1.71 (H2-6, m), 1.90
(H-7b, m), 1.92 (H-7a, m), 5.72 (H-9, d, J = 10.0 Hz), 6.03 (H-10, ddd,
J = 16.5, 10.0, 10.0 Hz), 4.96 (H-11b, d, J = 10.0 Hz), 5.04 (H-11a, d,
J = 16.5 Hz), 1.67 (H3-12, s), 3.59 (OCH3, s), 3.60 (2 Â OCH3, s) 7.44À
7.57 (10H, m); ESIMS m/z 697.45 [M + Na]+.
Preparation of Mono-(S)- and (R)-MTPA Esters (2a and 2b).
Compounds 2a (0.5 mg) and 2b (0.62 mg) were prepared in an
analogous way from (R)-(À)- and (S)-(+)-MTPA-Cl, respectively, as
described above for 1a and 1b.
Compound 2a: amorphous solid; 1H NMR data (CD3OD) δH 2.53
(H-2b, dd, J = 16.8, 6.0), 2.06 (H-2a, dd, J = 16.8, 6.0), 5.40 (H-3, m),
2.50 (H-4b, m), 2.52 (H-4a, m), 4.41 (H-5, m), 1.80 (H2-6, m), 2.17
(H-7b, m), 2.26 (H-7a, m), 5.90 (H-9, d, J = 11.0), 6.59 (H-10, ddd,
J = 16.5, 11.0, 11.0), 4.97 (H-11b, d, J = 11.0), 5.07 (H-11a, d, J = 16.5),
1.77 (H3-12, s), 3.53 (OCH3, s), 7.37À7.50 (5H, m); ESIMS m/z
449.24 [M + Na]+.
Extraction and Isolation. The production culture broth (100 L)
was centrifuged, and the supernatant was extracted with EtOAc (2 Â
100 L). The EtOAc layer was concentrated to dryness using rotary
evaporators at 40 °C. The residual suspension (20 g) was subjected to
ODS open column chromatography followed by stepwise gradient
elution with MeOHÀH2O (v/v) (1:4, 2:3, 3:2, 4:1, and 100:0) as
eluent. The fraction eluted with MeOHÀH2O (3:2, v/v) was subjected
to further fractionations by semipreparative ODS HPLC (50% MeOH
in H2O; flow rate, 1.5 mL/min; detector, RI) to obtain nine fractions
(F-1À9). Compounds 1À4 were purified by normal-phase semiprepara-
tive HPLC (flow rate, 1.3 mL/min; detector, UV) from F-8, F-6, F-3, and
F-4 using the following isocratic programs: 13% MeOHÀEtOAc; 5%
MeOHÀCHCl3;10%MeOHÀCHCl3;6%MeOHÀCHCl3, to yieldpure
compounds 1 (3.4 mg), 2 (3.4 mg), 3 (3.3 mg), and 4 (5.5 mg),
respectively.
Compound 2b: amorphous solid; 1H NMR data (CD3OD) δH 2.67
(H-2b, dd, J = 17.0, 5.5), 3.10 (H-2a, dd, J = 17.0, 5.5), 5.56 (H-3, m),
2.44 (H-4b, m), 2.46 (H-4a, m), 4.35 (H-5, m), 1.61 (H2-6, m), 2.16 (H-
7b, m), 2.22 (H-7a, m), 5.88 (H-9, d, J = 10.5), 6.58 (H-10, ddd, J = 16.5,
10.5, 10.5), 4.97 (H-11b, d, J = 10.5), 5.07 (H-11a, d, J = 16.5), 1.77 (H3-
12, s), 3.52 (OCH3, s), 7.40À7.57 (5H, m); ESIMS m/z449.26 [M + Na]+.
Preparation of Compound 3a. Compound 3 (2.0 mg) was
dissolved in 1 mL of anhydrous MeOH, and 200 μL of 2 M
(trimethylsilyl)diazomethane was added to the solution. The reaction
mixture was stirred at rt, and completion of the reaction was confirmed
by LC/MS analysis in 1 h. The reaction mixture was dried in vacuo, and
purification by analytical ODS HPLC using 65% MeOH in H2O
provided the methyl ester of compound 3 (3a, 1.8 mg).
Ieodomycin A (1): yellowish, amorphous solid; [R]23 +19 (c 0.9,
D
CHCl3); UV (MeOH) λmax (log ε) 232 (4.08) and 201 (2.92) nm; IR
1
(MeOH) νmax 3400 (br), 2928, 1715, 1251, 1065, 839 cmÀ1; H and
Compound 3a: pale, amorphous solid; 1H NMR data (CD3OD) δH
2.50 (H2-2, d, J = 6.5 Hz), 4.51 (H-3, m), 5.59 (H-4, dd, J = 15.3, 7.0 Hz),
6.22 (H-5, dd, J = 15.3, 10.3 Hz), 6.04 (H-6, dd, J = 15.3, 10.3 Hz), 5.71
(H-7, dt, J = 15.3, 7.0 Hz), 2.10 (H2-8, m), 1.38 (H-9b, m), 1.50 (H-9a,
m), 1.42 (H2-10, m), 3.71 (H-11, m), 1.14 (H3-12, d, J = 6.5 Hz), 3.67
(OCH3, s); ESIMS m/z 241.33 [M À H]À.
13C NMR data (CD3OD), see Table 1; HRESIMS m/z 243.1587 [M + H]+
(calcd for C13H23O4, 243.1596).
Ieodomycin B (2): white, amorphous solid; [R]23 +21 (c 0.9,
D
CHCl3); UV (MeOH) λmax (log ε) 233 (3.17) and 201 (2.80) nm;
IR (MeOH) νmax 3365 (br), 2924, 1715, 1251, 1065 cmÀ1; 1H and 13
C
NMR data (CD3OD), see Table 1; HRESIMS m/z 233.1145 [M + Na]+
(calcd for C12H18O3Na, 233.1154).
Bis-(S)-MTPA Ester Preparation (3b). Compound 3a (0.9 mg)
was dissolved in 1 mL of pyridine and stirred at rt for 30 min in a vial. A
few crystals of 4-dimethylamiopyridine were added, and the solution was
stirred for another 30 min. For preparation of the bis-(S)-MTPA
Ieodomycin C (3): pale, amorphous solid; [R]23D +18 (c 0.1, MeOH);
UV (MeOH) λmax (log ε) 230 (4.00) and 201 (2.67) nm; IR (MeOH)
νmax 3349 (br), 2924, 1711, 1407, 1278, 993 cmÀ1; 1H and 13C NMR
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Journal of Natural Products
ARTICLE
ester (3b) of 3a, 10 μL of (R)-(À)-MTPA-Cl was added, and the
reaction was stirred at rt for 3 h. Completion of the reaction was
monitored by LC/MS. The reaction was quenched by adding 200 μL of
MeOH. The reaction mixture was dried in vacuo, redissolved in H2O,
and extracted with EtOAc (3 Â 3 mL). The extract was purified on an
ODS analytical column using 67% MeOH in H2O as eluent to obtain 3b
(0.7 mg).
tests were performed in nutrient broth and yeast maltose broth, respec-
tively. A serial double dilution of each compound was prepared in 96-well
microtiter plates over the range 0.5À512.0 μg/mL. An overnight broth
culture of each strain was prepared, and the final concentration of
organisms in each culture was adjusted to 1.5 Â 108 cfu/mL by
comparing the culture turbidity with the 0.5 McFarland standard.
Culture broth (20 μL) was added to each dilution of compounds
1À4, the final volume of each well was adjusted to 200 μL using the
respective culture medium, and the plates were incubated for 24 h at
37 °C for bacteria and 48 h at 30 °C for the yeast.25,26 The minimum
inhibitory concentration is the lowest concentration of a sample at which
the microorganism did not demonstrate visible growth, as indicated by
the presence of turbidity.
Compound 3b: pale, amorphous solid; 1H NMR data (CD3OD) δH
3.01 (H2-2, d, J = 6.5 Hz), 4.51 (H-3, m), 5.55 (H-4, dd, J = 15.0, 7.5 Hz),
6.20 (H-5, dd, J = 15.0, 10.0 Hz), 5.92 (H-6, dd, J = 15.0, 10.0 Hz), 5.56
(H-7, dt, J = 15.0, 6.0 Hz), 1.98 (H2-8, m), 1.35 (H-9b, m), 1.50 (H-9a,
m), 1.48 (H2-10, m), 3.71 (H-11, m), 1.14 (H3-12, d, J = 6.5 Hz), 3.67
(OCH3, s), 3.54 (2 Â OCH3, s), 7.35À7.60 (10H, m); ESIMS m/z
697.33 [M + Na]+.
Bis-(R)-MTPA Ester Preparation (3c). Bis-(R)-MTPA ester of
compound 3a (0.9 mg) was prepared from (S)-(+)-MTPA-Cl similarly
to 3b. Bis-(R)-MTPA ester of compound 3a was purified on an ODS
analytical column using 65% MeOH in H2O as eluent to obtain 3c (0.6 mg).
Compound 3c: pale, amorphous solid; 1H NMR data (CD3OD) δH
2.50 (H2-2, d, J = 6.5 Hz), 4.51 (H-3, m), 5.85 (H-4, dd, J = 15.0, 8.5 Hz),
6.22 (H-5, dd, J = 15.0, 10.0 Hz), 6.05 (H-6, dd, J = 15.0, 10.0 Hz), 5.70
(H-7, dt, J = 15.0, 6.0 Hz), 2.05 (H2-8, m), 1.39 (H-9b, m), 1.53 (H-9a,
m), 1.51 (H2-10, m), 3.71 (H-11, m), 1.13 (H3-12, d, J = 6.5 Hz), 3.67
(OCH3, s), 3.54 (2 Â OCH3, s), 7.36À7.60 (10H, m); ESIMS m/z
673.48 [M À H]À.
’ ASSOCIATED CONTENT
S
Supporting Information. 1D, 2D NMR and HRESIMS
b
data of compounds 1À4. This material is available free of charge
via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: +82-31-400-6172. Fax: +82-31-400-6170. E-mail: shinhj@
kordi.re.kr.
Preparation of Compound 4a. Compound 4 (3.0 mg) was
dissolved in 2 mL of anhydrous MeOH. The methyl ester of compound
4 was prepared according to the above-described method. The reaction
mixture was dried in vacuo and purified on an analytical ODS HPLC
using 60% MeOH in H2O to obtain the methyl ester of compound 4 (4a,
2.3 mg).
’ ACKNOWLEDGMENT
The authors thank Dr. C. Kun, Korea Basic Science Institute,
Ochang, Republic of Korea, for providing mass data. This research
was supported in part by the Ministry of Land, Transport and
Maritime Affairs, Korea, and Korea Ocean Research and Develop-
ment Institute (Grants PE98472 to D.H.K. and PE98494 to H.S.P.).
Compound 4a: yellowish, amorphous solid; 1H NMR data
(CD3OD) δH 5.83 (H-2, d, J = 15.2 Hz), 7.26 (H-3, dd, J = 15.2, 10.3
Hz), 6.25 (H-4, dd, J = 15.2, 10.3 Hz), 6.21 (H-5, m), 2.20 (H2-6, m),
1.45 (H-7b, m), 1.56 (H-7a, m), 1.42 (H2-8, m), 3.71 (H-9, m), 1.14
(H3-10, d, J = 6.5 Hz), 3.70 (OCH3, s); ESIMS m/z 197.22 [M À H]À.
(S)-MTPA Ester Preparation (4b). The (S)-MTPA ester of
compound 4a (1 mg) was prepared from (R)-(À)-MTPA-Cl according
to the above-described method. The reaction mixture was dried in vacuo,
redissolved in H2O, and extracted with EtOAc (3 Â 3 mL). The extract
was purified on an ODS analytical column using 65% MeOH in H2O as
eluent to obtain 4b (0.8 mg).
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3.71 (OCH3, s), 3.54 (OCH3, s), 7.38À7.60 (5H, m); ESIMS m/z
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