Macrolactin W, a new antibacterial macrolide from a marine Bacillus sp.

Article abstract of DOI:10.1016/j.bmcl.2010.12.050

Bioactivity-guided isolation for the new/novel metabolites from the EtOAc extract obtained from the culture broth of a marine Bacillus sp. 09ID194 followed by chromatographic fractionations and subsequently HPLC purifications led to the isolation of two known macrolides, macrolactins A (1) and Q (2), together with a new glycosylated marcrolide, macrolactin W (3). The chemical structures of compounds 1-3 were assigned based on extensive MS and NMR spectral data analysis and literature review. Compound 3 showed potent antibacterial activity against both Gram-positive and Gram-negative pathogenic bacteria.

Full text of DOI:10.1016/j.bmcl.2010.12.050

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3832–3835
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Macrolactin W, a new antibacterial macrolide from a marine Bacillus sp.
a,b
a
a
a
a,b,
⇑
M. A. Mojid Mondol , Ji Hye Kim , Hyi-Seung Lee , Yeon-Ju Lee , Hee Jae Shin
a
Marine Natural Products Chemistry Laboratory, Korea Ocean Research & Development Institute, Ansan, PO Box 29, Seoul 425-600, Republic of Korea
University of Science and Technology, 113 Gwahangno, Yuseong-gu, Daejeon, Republic of Korea
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Bioactivity-guided isolation for the new/novel metabolites from the EtOAc extract obtained from the
culture broth of a marine Bacillus sp. 09ID194 followed by chromatographic fractionations and subse-
quently HPLC purifications led to the isolation of two known macrolides, macrolactins A (1) and Q (2),
together with a new glycosylated marcrolide, macrolactin W (3). The chemical structures of compounds
Received 13 August 2010
Revised 2 December 2010
Accepted 13 December 2010
Available online 16 December 2010
1
–3 were assigned based on extensive MS and NMR spectral data analysis and literature review.
Compound 3 showed potent antibacterial activity against both Gram-positive and Gram-negative path-
ogenic bacteria.
Keywords:
Macrolactins
MIC
Ó 2011 Published by Elsevier Ltd.
NMR
Antibacterial activity
Macrolides
1
13
Terrestrial microorganisms have been extensively studied for
the discovery of novel bioactive metabolites than marine microor-
ganisms. Marine microbial metabolites exhibit a broad spectrum of
biological activities because of living in quite different environ-
ment compared to terrestrial counterparts and may have unique
metabolic pathway to produce metabolites which possess novel
[M+Na]+;
D 0.0 mmu), H and C spectral data (Table 1), implying
1
13
11 degrees of unsaturation. The H and C NMR signals of 3 were
characteristics of both macrolide and sugar moiety. The infrared
spectrum of 3 (in MeOH) showed absorbance bands at 3372, 1742,
1700 and 1070 cm , indicative of hydroxyl (OH), olefinic (C@C)
and ester (C@O) functionalities. Ultraviolet absorbances at kmax
(log e) 226 (4.74) and 258 (4.35) nm were assigned to a chromo-
phore with extended conjugation. Analysis of C NMR spectrum
(Table 1) showed three ester carbonyl carbon resonances between
ꢀ1
1
structures and activities. 24-Membered macrolactins were gener-
1
3
ally produced by Bacillus sp. and exhibited antibacterial, anticancer
2
and antiviral activities. As a part of our ongoing research on
isolation of bioactive secondary metabolites from marine
microorganisms, we isolated a marine bacterium 09ID194 from a
sediment sample collected from Ieodo, South Korea’s southern reef,
d
C
168.0 and 173.4, twelve methine carbons between d
144.5 assigned to six double bonds, nine oxygenated methine car-
bons between d 69.5 and 96.0, one oxygenated methylene carbon
at d 62.3, and nine aliphatic carbons between d 20.3 and 44.3. In
C
118.7 and
C
3
and later identified as Bacillus sp. based on 16S rDNA sequencing.
C
C
Macrolactins (1–3) (Fig. 1) were purified from the EtOAc extract of
the culture broth of this strain by C-18 reversed-phase HPLC.
Here we report the isolation, structure determination and
biological activities of compounds 1–3.
the macrolactin ring, six double bonds, one ester carbonyl carbon,
and its ring accounted for a total of eight degrees of unsaturation
and the remaining three degrees of unsaturation, of which two were
attributedto two succinate ester carbonylcarbons (onefor each) and
leaving one degree of unsaturation for cyclic structure of glucose
moiety, accounted for 11 degrees of unsaturation required by the
molecular formula of 3. The extensive COSY and HMBC (Fig. 2) spec-
troscopic data analysis revealedthat 3 has three spin systems: H-2 to
4
,5
The molecular formulas of compounds 1 and 2 were assigned to
1
be C24
H
34
O
5
and C30
H
44
O
10 respectively, based on their MS, H and
1
3
C NMR spectral data analysis. Compounds 1 and 2 gave molecu-
lar ion adduct peaks in ESIMS at m/z 425.34 ([M+Na] ) and 587.38
[M+Na] ), respectively. The MS, H and C NMR spectral data and
+
+
1
13
0
0
00
00
(
H-24, H-2 to H-3 and H-1 to H-6 and in these three spin systems,
the coupling to each adjacent protonated center were determined
sequentially in a stepwise fashion. The geometries (Z and E form)
specific rotation values ([ ) of compounds 1 and 2 were identical
a]
D
6
7
to those of known macrolactins A and Q respectively, hence the
identities of 1 and 2 were established.
of the carbon-carbon double bonds in the a-, b-, c-, d-unsaturated
Compound3 was isolated as an amorphous solidand analyzed for
ester (C-1 through C-5) and the two pairs of conjugated dienes
48
the molecular formula C34H O13 by its HRESIMS (m/z 687.2987
(C-8 to C-11 and C-16 to C-19) were assigned on the basis of their
H coupling constants (Table 1) and ROESY correlations (Fig. 3).
1
⇑
Corresponding author. Tel.: +82 31 400 6172; fax: +82 31 400 6170.
E-mail address: shinhj@kordi.re.kr (H.J. Shin).
The coupling constant (J = 11.3 Hz) of H-2 with H-3 and ROESY
correlation (Fig. 3) between H-2 and H-3 clearly established the
0
960-894X/$ - see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.12.050

M. A. Mojid Mondol et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3832–3835
3833
OH
6
'
O
5
'
OH
OH
O
1'
OH
3
'
HO
7
7
1
1
O
O
O
O
1
3
13
2
3
23
HO
24 HO
24
19
19
15
15
HO
HO
1
2
OH
O
6
''
O
5''
O
OH
1
''
OH
1'
4'
3''
HO
O
O
O
7
1
O
1
3
23
HO
24
1
5
19
HO
3
Figure 1. Chemical structures of macrolactins A (1), Q (2) and W (3).
OH
OH
O
O
6''
6
''
O
5''
O
5''
O
O
OH
OH
OH
OH
1'
1'
4'
1''
3''
4'
1''
3''
HO
HO
O
O
O
O
O
O
7
7
1
1
O
O
13
2
3
13
23
HO
24
HO
24
15
19
15
1
9
HO
HO
Figure 3. Key ROESY correlations of macrolactin W (3).
1
1
H- H COSY
geometry of C-2/C-3 double bond to be Z. The E geometry of the C-4/
C-5 olefin was supported by large vicinal proton–proton coupling
constant (J = 14.8 Hz) of H-4 with H-5. The large scalar coupling
HMBC
Figure 2. Key 1H– H COSY and HMBC correlations of macrolactin W (3).
1

3
834
M. A. Mojid Mondol et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3832–3835
0
0
00
Table 1
which was also supported by ROESY correlations (H-1 /H-5 ,
1
H and 13C NMR data of compound 3 in CD
3
OD-d
4
00
00
00
00
H-1 /H-3 , and H-2 /H-4 ) (Fig. 3). Compound 3 was assigned to
be a new derivative of 7-O-succinyl macrolactin A and so named
8
Position
d
C
d
H
, m (J in Hz)
HMBC
macrolactin W. In 24-membered macrolactins, compound 3 is first
1
2
3
4
5
6
7
8
9
168.0
118.7
144.5
131.1
140.5
40.2
5.55, d (1H, 11.3)
6.62, t (1H, 11.3)
7.21, dd (1H, 11.3, 14.8)
6.06, m (1H)
2.56, m (2H)
5.46, m (1H)
5.71, dd (1H, 5.0, 15.3)
6.58, dd (1H, 11.3, 15.3)
6.09, t (1H, 11.3)
5.61, m (1H)
C-1
C-1, C-5
macrolactin where succinic acid moiety was fully esterified.
2
D
3
Compound 3 showed optical rotation ½
a
ꢁ
ꢀ56.7° (c 0.10, MeOH)
C-2, C-3, C-6
C-6, C-7
C-5, C-7, C-8
C-5, C-6, C-8, C-9, C-1
C-6, C-7
C-7, C-11
C-8, C-9, C-12
C-9, C-12
7
which is similar to macrolactin Q and it can, therefore, be as-
sumed that compound 3 has the same absolute stereochemistry
as macrolactin Q.
0
74.6
132.2
127.7
130.8
129.9
36.3
The absolute configurations of the sugar and the aglycon part in
9
3
were determined by acid hydrolysis, where the glycon portion
1
1
1
0
1
2
was converted to glucose and the aglycon to macrolactin A, fol-
lowed by TLC comparison with authentic sample and measure-
ment of optical rotation after purification. The glucose isolated
2.22, m (1H)
C-13, C-14
2.59, m (1H)
1
1
1
1
1
1
1
2
3
4
5
6
7
8
9
0
69.5
44.3
69.9
135.1
131.5
131.6
135.5
33.26
3.77, m (1H)
1.63, m (2H)
4.33, m (1H)
5.54, dd (1H, 7.0, 15.3)
6.19, dd (1H, 11.5, 15.3)
6.05, t (1H, 11.5)
5.69, m (1H)
C-11, C-14, C-15
C-12, C-13
C-13, C-14, C-16, C-17
C-14, C-17
C-19
C-16
from the acid hydrolysis of 3 gave a positive specific rotation
23
½
a
ꢁ
+23° (c 0.15, H
2
O), indicating that it was
value with authentic
sample. Both the glucose isolated and authentic sample exhibited
same R value (0.55) in same condition. The aglycon portion ob-
tained from 3 showed optical rotation ½ ꢀ13.2° (c 1.5, MeOH)
D-glucose. This fact
D
was further confirmed by comparing its R
f
f
2
D
3
C-20, C-21
C-18, C-19, C-21, C-22
a
ꢁ
2.12, m (1H)
6
which is similar to macrolactin A. Together with the agreement
2.19, m (1H)
1
13
of the H and C NMR data with the literature values, this result
suggested that the aglycon portion in 3 has the same absolute ste-
reochemistry as macrolactin A.
2
2
1
2
26.0
36.25
1.50, m (2H)
1.58, m (1H)
C-22
C-20, C-21
1.64, m (1H
2
2
1
2
3
3
4
72.5
20.3
173.4
29.97
29.98
4.99, m (1H)
1.25, d (3H, 6.0)
C-1, C-22, C-24
C-23
The minimum inhibitory concentration (MIC) of compound 3
10
was determined by serial dilution technique. The MIC of com-
0
0
0
0
0
0
0
pound 3 against Bacillus subtilis (KCTC 1021), Staphylococcus aureus
(KCTC 1916), Escherichia coli (KCTC 1923) and Pseudomonas
2.73, m (2H)
2.64, m (1H)
C-1 , C-3
C-2 , C-4
1
1
2.68, m (1H)
aeruginosa (KCTC 2592) was 64 lg/mL. The cytoxicity of com-
0
4
1
2
3
4
5
6
173.1
96.0
74.1
77.9
78.9
71.0
62.3
pound 3 was evaluated against a panel of cancer cell lines: ACHN
human renal cancer, HCT 15 human colon cancer, MDA-MB-231
human breast cancer, NCI-H23 human lung cancer, NUGC-3 human
stomach cancer, and PC-3 human prostate cancer cell lines. The
new compound 3 failed to register any cytotoxicity at a concentra-
0
0
0
0
0
0
0
0
0
0
0
0
0
5.49, d (1H, 8.0)
C-4
00
00
00
3.33, dd (1H, 8.0, 8.4)
3.42, dd (1H, 8.4, 8.8)
3.35, m (1H)
3.36, m (1H)
3.66, dd (1H, 4.0, 12.2)
C-1 , C-3
00
C-2 , C-5
00
00
C-4
C-4
tion of 10 lg/mL against cancer cell lines tested.
3.81, dd (1H, 2.0, 12.2)
6
Gustafson and co-workers reported that macrolactin A, which
was isolated from unclassified marine bacterium, inhibited S. aureus
and B. subtilis in standard ‘disc diffusion assay’ at a concentration of 5
constant (J = 15.3 Hz) between H-8 and H-9, and small coupling con-
stant (J = 11.3 Hz) of H-10 with H-11, and ROESY correlations (Fig. 3)
between H-9 and H-10 and between H-10 and H-11, definitely
certified the geometries of C-8/C-9 and C-10/C-11 double bonds as
E and Z, respectively. The relative configurations of the disubstituted
C-16/C-17 and C-18/C-19 conjugated olefins were deduced to be
E and Z, respectively, in similarfashion simply by theanalysis of their
coupling constants (JH-16/H-17 = 15.3 Hz, JH-18/H-19 = 11.5 Hz) (Table
1
2
and 20
lg/disc, respectively. Macrolactins F and K in which C-15
was a ketone carbonyl group showed weak antibacterial activity.
1
2
Macrolactins G–M inhibited S. aureus, regardless of the positions
of hydroxyl group at C-7 or C-9 or of the number of ring members.
So the position of hydroxyl group at C-15 may play an important role
in the antibacterial activity of macrolactins. Macrolactins O–R7
inhibited S. aureus PDF in dose-dependent manners with IC50 (lM)
13
1
) and ROESY correlations (H-16/H-18 and H-18/H-19) (Fig. 3).
The point of cyclization of the ester in macrolactone ring of 3
values of 53.5, 57.7, 12.1 and 61.5, respectively. Macrolactin S iso-
lated from a marine B. subtilis, showed significant antimicrobial
activity against E. coli but weak activity against B. subtilis and S.
was indicated by the low-field shift of H-23 at d
was clearly coupled to the H-24 methyl group at d
J = 6.0 Hz) and firmly supported by showing long range HMBC
correlation (Fig. 2) of H-23 with ester carbonyl carbon at d 168.0
C-1). The COSY correlation between H-2 and H-3 and the HBMC
correlations of H-2 with C-1 (d
73.1) indicated the presence of succinyl moiety. The down-field
shift of H-7 (d
the HMBC correlation of the anomeric proton at d
H
4.99, which
1
4
15
H
1.25 (d,
aureus. Macrolactin T, and V exhibited antifungal and significant
antibacterial activity, respectively. Compound 3 and 7-O-succinyl
8
C
macrolactin A showed almost similar antibacterial activities. This
0
0
(
result suggested that full esterification of succinic acid moiety in
macrolactins does not affect antibacterial activity. The strain
09ID194 produced more amount of compounds 1–3 only in low
salinity (12 psu) compare to high salinity (32 psu) and it may be
due to maintaining osmoregulation in their physiological process.
In conclusion, macrolactin W (3) is a new 24-membered glycos-
ylated lactone compound isolated from the culture broth of a marine
Bacillus sp. 09ID194. Macrolactin W showed potent antibacterial
activity against both Gram-positive and Gram-negative bacteria
which may serve for the development of new antibacterial agent.
0
0
0
0
C
173.4) and H-3 with C-4 (d
C
1
0
H
5.46, m) and its HMBC connectivity with C-1 and
5.49 (d,
H
00
0
J = 8.0 Hz, H-1 ) with C-4 corroborated that the succinic acid
moiety was esterified to macrolactone ring at C-7 and sugar moiety
0
0
at C-1 . A six carbons pyranose glycoside constituent was estab-
lished by an acetal carbon resonance at d 96.0, four methine
carbon signals between d 71.0 and 78.9 and a methylene carbon
62.3. Appropriate H NMR resonances for a b-pyranose sugar
including the anomeric (axial) proton at d 5.49, were also
C
C
1
at d
C
H
Acknowledgements
observed. Coupling constants analysis revealed diaxial couplings
ranging from 8.0 to 8.8 Hz between all of the glycoside ring
protons, thus defining the presence of b-glucopyranosyl moiety
The authors express gratitude to Dr. C. Kun, Korea Basic Science
Institute, Ochang, Korea, for providing mass data. This research

M. A. Mojid Mondol et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3832–3835
3835
was supported in part by the Ministry of Land, Transport and Mar-
itime Affairs, Korea and Korea Ocean Research and Development
Institute (Grants PE98472 to D.H.K. and PE98494 to H.S.P.).
concentrated to dryness using rotary evaporator in vacuo at 40 °C. The residual
suspension (3.0 g) was subjected to ODS open column chromatography
followed by stepwise gradient elution with MeOH/H
:1 and 100:0) as eluant. The fraction eluted with MeOH/H
purified by reversed-phase HPLC (YMC ODS-A column, 250 ꢂ 10 mm i.d.: 70%
MeOH in H O; flow rate: 1.5 mL/min; detector: RI) to yield pure compounds 1
28 mg), 2 (5 mg) and 3 (17.5 mg).
2
O (v/v) (1:4, 2:3, 3:2,
4
2
O (4:1, v/v) was
2
References and notes
(
6
7
8
.
.
.
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3
.
The strain 09ID194 was isolated from a sediment sample collected from Ieodo,
South Korea’s southern reef, during expedition in 2009 by serial dilution
technique. In brief, one gram of the sediment sample was diluted in sterilized
9.
Compound 3 (3 mg) was refluxed in 1 N HCl (1 mL) at 90 °C for 2 h. The
completion of hydrolysis was confirmed by LC/MS analysis (in ESIMS, sugar m/z
+
+
2
03.05 [M+Na] ; macrolactin A m/z 425.16 [M+Na] ). After cooling, the reaction
ꢀ
1
ꢀ2
ꢀ3
ꢀ4
sea water (10 , 10 , 10 and 10 ) in aseptic conditions and 100 lL from
mixture was extracted with EtOAc (2 mL ꢂ 2). The EtOAc extract was
each dilution was spread on modified Bennett’s agar medium (0.1% yeast
extract, 0.1% beef extract, 0.2% tryptone, 1% dextrose, 100% natural sea water,
evaporated and the residue was purified through a silica column (YMC-Pack-
SIL, 250 ꢂ 10 mm i.d.: 10% CHCl
3
in EtOAc; flow rate: 1.5 mL/min; detector:
1
.8% agar and pH adjusted to 7.2 before sterilization). The plates were
UV) to yield pure aglycon (macrolactin A, 0.8 mg). The aqueous phase was
neutralized with 1 N NaOH and evaporated to dryness and purified through
ODS analytical column (YMC-Pack-ODS-A, 250 ꢂ 4.6 mm i.d.: 55% MeOH in
incubated for 14 days at 30 °C, and the resulting colony of the strain 09ID194
was isolated and maintained on the modified Bennett’s agar. The strain
0
9ID194 formed well-developed reddish substrate and aerial mycelium on
2
H O; flow rate: 0.6 mL/min; detector: RI) to obtain pure glycon (D-glucose,
modified Bennett’s agar medium. The strain was identified as Bacillus sp. on the
basis of 16S rDNA sequence analysis. This strain is currently deposited in the
Microbial Culture Collection, KORDI, with the name of Bacillus sp. 09ID194
under the curatorship of H.J.S.
The seed and mass culture were carried out in modified Bennett’s broth
medium. The composition and pH of the seed culture medium (0.1% yeast
extract, 0.1% beef extract, 0.2% tryptone, 1% dextrose, 1.2% psu, pH 7.6) were
same as mass culture medium. 50 mL of the medium was dispensed in 250 mL
conical flask. A single colony of 09ID194 strain from the agar plate was
inoculated into the flask and incubated at 30 °C for 2 days on a rotary shaker at
0
.7 mg). The glycon was analyzed by TLC (Kieselgel, eluting solvent CHCl
MeOH-1:1, sprayed with 1% H SO /vanillin and heated) to reveal the presence
of -glucose as its R value (0.55) was coincident with the authentic sample.
3
/
2
4
D
f
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Rahman, M. M. J. Nat. Prod. 2008, 71, 1427.
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.
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1
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Prod. Res. 2008, 22, 342.
1
20 rpm. An aliquot (0.2% v/v) from the seed culture was inoculated into a 20 L
1
1
4. Xue, C.; Tian, L.; Xu, M.; Deng, Z.; Lin, W. J. Antibiot. 2008, 61, 668.
5. Gao, C. H.; Tian, X. P.; Qi, S. H.; Luo, X. M.; Wang, P.; Zhang, S. J. Antibiot. 2010,
fermenter containing 15 L culture medium (15 L ꢂ 2). The culture was carried
out at 30 °C, 120 rpm for 7 days and then harvested.
6
3, 191.
5
.
The fermentation broth (15 L ꢂ 2) was centrifuged by continuous centrifuge,
and the supernatant was extracted with EtOAc (30 L ꢂ 2). The EtOAc layer was