Gageotetrins A-C, noncytotoxic antimicrobial linear lipopeptides from a marine bacterium bacillus subtilis

Article abstract of DOI:10.1021/ol403657r

Gageotetrins A-C (1-3), a unique class of linear lipopeptides, consisting of di- and tetrapeptides and a new fatty acid were isolated from a Marine Bacillus subtilis. The structures of 1-3 were assigned by spectroscopic data and their absolute stereochemistries were ascertained by chemical derivatization. Compounds 1-3 displayed good antimicrobial activities with MIC values of 0.01-0.06 μM. However, these compounds failed to register any cytotoxicity (GI₅₀ > 30 μg/ml) against human cancer cell lines.

Full text of DOI:10.1021/ol403657r

Letter
pubs.acs.org/OrgLett
Gageotetrins A−C, Noncytotoxic Antimicrobial Linear Lipopeptides
from a Marine Bacterium Bacillus subtilis
Fakir Shahidullah Tareq,†,‡ Min Ah Lee,‡ Hyi-Seung Lee,‡ Yeon-Ju Lee,‡ Jong Seok Lee,‡
Choudhury M. Hasan,§ Md. Tofazzal Islam,∥ and Hee Jae Shin*,†,‡
†Department of Marine Biotechnology, University of Science and Technology, Republic of Korea
‡Marine Natural Products Laboratory, Korea Institute of Ocean Science and Technology, Republic of Korea
§Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Bangladesh
∥Department of Biotechnology, Bangabandhu Sheikh Muzibur Rahman Agricultural University, Dhaka, Bangladesh
S
* Supporting Information
ABSTRACT: Gageotetrins A−C (1−3), a unique class of linear lip-
opeptides, consisting of di- and tetrapeptides and a new fatty acid were
isolated from a Marine Bacillus subtilis. The structures of 1−3 were assigned
by spectroscopic data and their absolute stereochemistries were ascertained
by chemical derivatization. Compounds 1−3 displayed good antimicrobial
activities with MIC values of 0.01−0.06 μM. However, these compounds
failed to register any cytotoxicity (GI50 > 30 μg/ml) against human cancer
cell lines.
ipopeptides (LPs) have been reported to be produced by
Ldistinctively different groups of bacteria and fungi, and
these compounds were found to exert roles in antagonistic
interactions with other organisms, e.g., plant pathogenicity and
antifungal, antibacterial, antiviral, and antitumor properties.1 In
particular, LPs of the surfactin, iturin, and fengycin families are
important metabolites produced by the bacterial strain Bacillus
sp., and their involvement in disease control has been widely
reported.2,3 However, linear lipodipeptides, especially lip-
otetrapeptides possessing three Leu and Glu amino acids
along with a new fatty acid residue, have been unprecedented
from natural sources.
As part of our continuous exploration of bioactive secondary
metabolites from marine-derived bacteria, we isolated the strain
109GGC020 from marine sediment samples collected from
Gageocho, Republic of Korea, which revealed 100% 16S rRNA
sequencing similarity to Bacillus subtilis. The unicellular
bacterium was cultured, and the fermentation broth (100 L)
was extracted with EtOAC. Thereafter, the chromatographic
separation of the EtOAC extract using flash column
chromatography in combination with semipreparative and
analytical HPLC yielded a novel class of linear lipopeptides,
gageotetrins A (3.2 mg), B (5.6 mg), and C (2.8 mg) (Figure
1).
Figure 1. Structures of gageotetrins A−C (1−3).
of amides, a long aliphatic chain, and carbonyl groups,
respectively. Two NH signals at δH 7.57 and δH 8.38, α-
methines at δH 4.29 and δH 4.30 in the 1H NMR spectrum and
the carbonyl signals at δC 173.6−181.7 together with α-carbons
at δC 54.9 and δC 55.5 in the 13C NMR spectrum revealed the
peptidic nature of 1 (Table 1). In addition, a broad singlet at δH
1.29 and an oxygenated proton at δH 3.98 were observed in the
Gageotetrin A (1) was isolated as an amorphous solid with a
molecular formula of C25H46N2O7 as determined by HRESI-
MS at m/z 509.3203 [M + Na]+. The IR absorbencies of 1 at
3285, 2923, and 1583 cm−1 were consistent with the presence
Received: December 19, 2013
Published: January 23, 2014
© 2014 American Chemical Society
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Table 1. 1H and 13C NMR and ROESY data of 1 in CD3OH
individual amino acid residues were clearly observed in the
COSY and TOCSY spectra (partial structures a and b).
Moreover, the NH signal at δH 8.23 showed TOCSY
connectivities with α-methine and methylene protons at δH
4.30 and δH 1.91/2.12, and further HMBC connectivities of
methylene signals at δH 1.91/2.12 and δH 2.27 with a carbonyl
carbon at δC 181.7 confirmed the presence of the Glu residue in
1. The oxygenated proton at δH 3.98 showed HMBC
connectivities with the carbonyl carbon at δC 174.7 and also
with the aliphatic chain. These HMBC correlations together
with TOCSY correlations allowed the assignment of a 3-
hydroxy fatty acid (partial structure c) in 1.
position
δH, mult (J, Hz)
δC
179.7
ROESY
Leu
1
2
3
4.29, m (overlapped)
1.56, m
54.9
43.6
NH, 3, 4
2
1.63, m
4
1.67, m
26.1
22.6
23.9
2, 5, 6
3, 4
3, 4
2
5
0.93, d (2.0)
0.93, d (2.0)
7.57, d (8.0)
6
NH
1
Glu
173.6
55.5
29.6
2
4.30, m (overlapped)
1.91, m
3, 4
2, 4
Based on the composition of the peptide moiety and the
molecular weight of the molecule, the fatty acid was primarily
determined to be a C14 fatty acid, amidated to the N-terminal
amine of the peptide. The molecular formula of the fatty acid
was determined as C14H28O3 by LC−MS analysis (m/z 243.02
[M − H]−) of the acid hydrolysate of 1 (Supporting
Information). In the 13C spectrum, four methyl carbons were
observed, among them two were attributed to a Leu residue
and the remaining two were assigned to be present in the fatty
acid chain. The methyl carbon at δC 14.5 was assigned as a
terminal group and the second methyl (δC 23.8) was attached
at C-11 of the fatty acid chain, corroborated by the analysis of
TOCSY and HMBC correlations. Finally, the fatty acid was
found as a 3-hydroxy-11-methyltridecanoic acid (HTDA) with
3
2.12, m
4
2.27, t (7.5)
8.38, d (7.0)
35.4
2, 3
NH
COOH
181.7
3-OH acid
(HTDA)
1
2
174.7
44.9
2.32, dd (14.0, 8.5)
2.42, dd (13.5, 4.0)
3.98, m
3, 4
3
4
5
69.9
38.5
26.8
2, 4, 5
2, 3
3
1.47, m
1.47, m
1.30, m (overlapped)
1.28, brds.
an optical rotation value of [α]27 −22.0 (0.1, MeOH).
D
6−9
30.7−30.8
Collectively, the lipopeptide structure of 1 was then determined
constructing the sequence of Leu-Glu-HTDA by ROESY and
HMBC experiments (Figure 2).
10
1.29, m (overlapped)
1.17, m
33.1
14
13
11
12
13
14
1.29, m (overlapped)
1.29, m (overlapped)
0.89, t (7.5)
38.5
30.8
14.5
23.8
Gageotetrin B (2) was isolated as an amorphous solid, and its
molecular formula was determined to be C38H70N4O9 by
HRESIMS at m/z 749.5020 [M + Na]+. A series of 13C
resonances at δC 174.1−175.1 were assigned to amide or ester
0.91, d (2.0)
1
carbonyls. In addition, the H NMR spectrum of 2 contained
1H NMR spectrum, revealing the presence of aliphatic chain in
the molecule. These detailed IR and NMR results clearly
indicated the lipopeptidic nature of 1.
Analysis of 1D and 2D NMR spectra allowed construction of
three partial structures that constituted 1 (Figure 2). Two
amino acid residues were deduced as glutamic acid (Glu) and
leucine (Leu), while a third, nonamino acid moiety, was
identified as a 3-hydroxy fatty acid. The spin systems of the α-
methines and NH resonances of Leu (δH 4.29/7.57) and Glu
(δH 4.30/8.38) and the α-methines to the side chain of the
resonances of four amide signals at δH 7.79−8.24, α-methines at
δH 4.26−4.44 and side-chain functionalities, suggesting 2
contained a peptide component (Table 2, Supporting
Information). Consistent with this observation, detailed
analysis of the COSY, TOCSY, and HMBC data led to the
identification of three Leu and a methoxy-Glu (OMeGlu)
residues in 2 (Figure 1). The methoxy signal at δH 3.67
(singlet) did not show COSY and TOCSY correlations but
displayed an HMBC correlation to the carboxylic carbon at δC
175.1, corresponding to the presence of an OMeGlu residue in
2. A 3-hydroxy fatty acid residue was also assigned by TOCSY
and HMBC correlations from the observation of a distinct
proton signal at δH 3.98 which showed HSQC cross peak with
the carbon at δC 70.0. Subtraction of the atoms attributed to the
four amino acid residues from the molecular formula of 1
(C38H70N4O9) showed that the remaining fragment, i.e., the
fatty acid of the molecule had a molecular formula of C14H28O3.
The molecular formula of the fatty acid was further confirmed
by the acid hydrolysis followed by LC−MS analysis (m/z
243.423 [M − H]−) with an optical rotation value of [α]27
D
−27.8 (0.1, MeOH). This result confirmed the presence of the
same fatty acid residues in compounds 1 and 2. The sequence
of residues in 2 was then confirmed as Leu-Leu-Leu-OMeGlu-
HTDA on the basis of the TOCSY, ROESY and HMBC
experiments.
Gageotetrin C (3) was isolated as an amorphous solid, and
its molecular formula was assigned to be C37H68N4O9 with the
aid of HRESI-MS [M + Na]+ at m/z 735.4883. Preliminary, the
Figure 2. COSY, TOCSY, and HMBC correlations of 1 in CD3OH.
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NMR analysis showed a close similarity between the spectra of
2 and 3 (Table 2, Supporting Information), indicating a
lipopeptide nature of 3. The difference between these
compounds was identified from the lack of methoxy peak
that was appeared as singlet at δH 3.67 in 2. Detailed analysis of
NMR data of 3 revealed that it contained same amino acid
residues as 2 except for the presence of Glu instead of OMeGlu.
Similar to compounds 1 and 2, the presence of a 3-hydroxy
fatty acid was identified and the LC−MS analysis of the hexane
phase of the hydrolysate of 3 revealed that it had the same
molecular formula of C14H28O3 like in 1 and 2. But,
interestingly the 13C NMR spectrum of 3 displayed nine
methyl carbons at δC 11.9−23.2; six of them were attributed to
three Leu residues, and as the amino acids sequence of both 2
and 3 was found to be same by 2D data analysis the remaining
three methyls must be located in the fatty acid chain. The
methyl carbon at δC 11.9 was located as a terminal methyl and
the carbons at δC 19.8 and 23.2 were located at C-8 and C-11 in
the fatty acid moiety, respectively, by the TOCSY and HMBC
correlations. Finally, the fatty acid was determined as a 3-
hydroxy-8,10-dimethyldodecanoic acid (HDDA) with an
Figure 4. Absolute stereochemistry determination of 3-hydroxy fatty
acid in 1. (A) 1H NMR data of S-MTPA and (B) R-MTPA esters; (C)
ΔδH (δS − δR) values of derivatized products; (D) R-configuration of
C-3 of fatty acid in 1.
catalytic unit, referred to as a module (Figure 5).8 Each module
is composed of specific domains that are responsible for
optical rotation value of [α]27 −31 (0.1, MeOH). The search
D
results from natural products database confirmed this fatty acid
as a new fatty acid derivative from natural sources. The
complete structure of 3 was then elucidated on the basis of the
1D and 2D NMR data, constructing the sequence of Leu-Leu-
Leu-Glu-HDDA.
The absolute configuration of the amino acids in 1−3 was
determined by Marfey’s method4 (Figure 3), and the
Figure 5. Hypothetical biosynthetic pathways of gageotetrins.
catalyzing different enzymatic activities.9 The adenylation (A)
domain is responsible for amino acid recognition and
adenylation at the expense of ATP to form an acyl adenylate
intermediate. The intermediate covalently binds to a
phosphopantetheine carrier of the adjacent thiolation (T)
domain. Peptide bond formation of two consecutively bound
amino acids is catalyzed by the condensation (C) domain. An
additional C-domain, the N-acyl domain, is located at the N-
terminal domain of the first module, suggesting that the first
amino acid is initially N-acylated with a 3-hydroxy fatty acid in
this domain.10,11 This proposed biosynthetic pathways are quite
consistent with the chemical synthetic procedure of surfactin
B.12
Antimicrobial activities of 1−3 were investigated against both
bacteria and pathogenic fungi by broth dilution assay.13
Compounds 1−3 were found more active against fungi
compared to bacteria with MIC values of 0.02−0.04 μM
(Figure 6). Moreover, compounds 2 and 3 displayed good time
course motility inhibition and lytic activity against the late
blight pathogen Phytophthora capsici, which causes enormous
economic damage in cucumber, pepper, tomato and beans, at
0.02 μM (Figure 7). P. capsici caused huge losses to numerous
vegetable producers in Michigan.14 The pathogen is also
important on a global scale and is potentially the most
destructive organism of peppers in Spain.15 In addition, the
Figure 3. Acid hydrolysis and derivatization studies of 1.
configurations of all Leu and Glu residues were found to be
the L-form (Supporting Information). In addition, the absolute
configuration at C-3 of the fatty acid in 1 was determined by
Mosher’s method5 (Figure 3). A consistent distribution of
positive and negative ΔδH values (ΔδH = δS − δR) obtained
from S-MTPA and R-MTPA esters around C-3 allowed the
assignment of the R-configuration at the C-3 position (Figure
4). The absolute stereochemistry at C-3 of the fatty acids in 2
and 3 was also determined to be R-form by comparing optical
rotation values of the fatty acids obtained from 1−3 and
literature reviews.6,7
Gageotetrins A−C might be biosynthetically produced in a
ribosome-independent manner with megaenzymes called
nonribosomal peptide synthetases via the function of the
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Letter
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: shinhj@kiost.ac. Tel.: +82-31-400-6172. Fax: +82-31-
400-6170.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported in part by the Korea Institute of
Ocean Science and Technology (Grant No. PE99121) and the
Ministry of Oceans and Fisheries (Grant No. PM57420),
Korea.
Figure 6. Antimicrobial activities of 1−3 and positive control P
(azithromycin for bacteria and amphotericin B for fungi).
REFERENCES
■
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Figure 7. Time−course motility inhibitory and lytic activities of 2 and
3 against P. capsici at 0.02 μM. Compound 3 failed to lyse zoospores at
a concentration of 0.02 μM but showed activity when the
concentration was increased (Supporting Information, Table 1).
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cancer, and PC-3 prostate cancer cell lines by sulforhodamine B
(SBR) assay16 but failed to register any cytotoxicity at a
concentration of 30 μg/mL. Noncytotoxic activity of 1−3 was
further confirmed by WST-1 assay17 against the human
leukemia cell line (K-562).
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To the best of our knowledge, gageotetrins A−C represent
the first example of rare bioactive linear lipopeptides consisting
of a Leu-rich peptide backbone and a new 3-hydroxy fatty acid
(HDDA) from a marine-derived bacterium. Several lip-
opeptides containing a 3-hydroxy fatty acid have been reported
including surfactins and biosurfactants and showed that the
amino acid residue composition and the fatty acid play an
important role for their activity.18 In fact, 3-hydroxy fatty acid
itself displays good antifungal properties.19 Moreover, caspo-
fungin, which possesses a fatty acid slightly resembling the fatty
acid present in 3, has been approved as an antifungal agent
exhibiting a unique mechanism of action relative to the other
currently approved antifungal agents.20 Consequently, anti-
microbial activity of 1−3 resulting from this study could be
promising for finding new frontiers to develop nontoxic
antibiotics.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental and spectroscopic data for 2 and 3. This material
is available free of charge via the Internet at http://pubs.acs.org.
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