Gageostatins A-C, antimicrobial linear lipopeptides from a marine Bacillus subtilis

Article abstract of DOI:10.3390/md12020871

Concerning the requirements of effective drug candidates to combat against high rising multidrug resistant pathogens, we isolated three new linear lipopeptides, gageostatins A-C (1-3), consisting of hepta-peptides and new 3-β-hydroxy fatty acids from the fermentation broth of a marine-derived bacterium Bacillus subtilis. Their structures were elucidated by analyzing a combination of extensive 1D, 2D NMR spectroscopic data and high resolution ESIMS data. Fatty acids, namely 3-β-hydroxy-11-methyltridecanoic and 3-β-hydroxy-9,11-dimethyltridecanoic acids were characterized in lipopeptides 1 and 2, respectively, whereas an unsaturated fatty acid (E)-7,9-dimethylundec-2-enoic acid was assigned in 3. The 3R configuration of the stereocenter of 3-β-hydroxy fatty acids in 1 and 2 was established by Mosher's MTPA method. The absolute stereochemistry of amino acid residues in 1-3 was ascertained by acid hydrolysis followed by Marfey's derivatization studies. Gageostatins 1-3 exhibited good antifungal activities with MICs values of 4-32 μg/mL when tested against pathogenic fungi (R. solani, B. cinerea and C. acutatum) and moderate antibacterial activity against bacteria (B. subtilis, S. aeureus, S. typhi and P. aeruginosa) with MICs values of 8-64 μg/mL. Futhermore, gageostatins 1-3 displayed cytotoxicity against six human cancer cell lines with GI₅₀ values of 4.6-19.6 μg/mL. It is also noteworthy that mixed compounds 1+2 displayed better antifungal and cytotoxic activities than individuals.

Full text of DOI:10.3390/md12020871

Mar. Drugs 2014, 12, 871-885; doi:10.3390/md12020871
OPEN ACCESS
marine drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Gageostatins A–C, Antimicrobial Linear Lipopeptides from a
Marine Bacillus subtilis
Fakir Shahidullah Tareq 1,2, Min Ah Lee 2, Hyi-Seung Lee 2, Jong-Seok Lee 2, Yeon-Ju Lee 2 and
Hee Jae Shin 1,2,
*
1
Department of Marine Biotechnology, University of Science and Technology, 176 Gajung-dong,
217 Gajungro Yuseong-gu, Daejeon, 305-350, Korea; E-Mail: stareqfakir@gmail.com
Marine Natural Products Chemistry Laboratory, Korea Institute of Ocean Science and Technology,
Ansan, 426-744, Korea; E-Mails: yimina82@kiost.ac (M.A.L.); hslee@kiost.ac (H.-S.L.);
jslee@kiost.ac (J.-S.L.); yjlee@kiost.ac (Y.-J.L.)
2
* Author to whom correspondence should be addressed; E-Mail: shinhj@kiost.ac;
Tel.: +82-31-400-6172; Fax: +82-31-400-6170.
Received: 29 November 2013; in revised form: 19 December 2013 / Accepted: 20 January 2014 /
Published: 31 January 2014
Abstract: Concerning the requirements of effective drug candidates to combat against high
rising multidrug resistant pathogens, we isolated three new linear lipopeptides, gageostatins
A–C (1–3), consisting of hepta-peptides and new 3-β-hydroxy fatty acids from the
fermentation broth of a marine-derived bacterium Bacillus subtilis. Their structures were
elucidated by analyzing a combination of extensive 1D, 2D NMR spectroscopic data and
high resolution ESIMS data. Fatty acids, namely 3-β-hydroxy-11-methyltridecanoic and
3-β-hydroxy-9,11-dimethyltridecanoic acids were characterized in lipopeptides 1 and 2,
respectively, whereas an unsaturated fatty acid (E)-7,9-dimethylundec-2-enoic acid was
assigned in 3. The 3R configuration of the stereocenter of 3-β-hydroxy fatty acids in 1 and
2 was established by Mosher’s MTPA method. The absolute stereochemistry of amino acid
residues in 1–3 was ascertained by acid hydrolysis followed by Marfey’s derivatization
studies. Gageostatins 1–3 exhibited good antifungal activities with MICs values of
4–32 µg/mL when tested against pathogenic fungi (R. solani, B. cinerea and C. acutatum)
and moderate antibacterial activity against bacteria (B. subtilis, S. aeureus, S. typhi and
P. aeruginosa) with MICs values of 8–64 µg/mL. Futhermore, gageostatins 1–3 displayed
cytotoxicity against six human cancer cell lines with GI50 values of 4.6–19.6 µg/mL. It is
also noteworthy that mixed compounds 1+2 displayed better antifungal and cytotoxic
activities than individuals.

Mar. Drugs 2014, 12
Keywords: Bacillus subtilis; lipopeptides; antimicrobial activity; cytotoxicity
872
1. Introduction
Marine ecosystems comprise a productive and enormous resource of vast chemical entities with
immeasurable biological activities [1]. Given such a background, the chemistry of marine natural
products has been progressing at an unprecedented rate, resulting in discoveries of a wide range of
carbon skeletons and molecules that are not usually found from terrestrial sources [2]. In fact, marine
microorganisms are ubiquitous in the marine environment, and can tolerate adverse conditions such as
high temperature, pressure, salinity and pH, and possess unique metabolic pathways that are different
from their terrestrial counterparts [3]. Bacillus subtilis is an example of widely distributed bacterium,
capable of growth in many environments, and exhibits considerable genomic diversity [4]. A genomic
study on the widely distributed Bacillus strains revealed that about eight percent of its genome is
devoted to synthesizing antibiotics [5,6]; whereas, a genomic study on a marine-derived B. subtilis
subsp. spizisenii strain gtP20b, isolated from the Indian Ocean, revealed the presence of huge number
of genes for biosynthesis of secondary metabolites [7]. As a consequence, a large number of natural
product research on marine-derived Bacillus sp. resulted in the discovery of a diverse class of
secondary metabolites including lipopeptides, polypeptides, macrolactones, fatty acids, polyketides,
lipoamides, and isocoumarins [8]. Among these compounds, lipopeptides (LPs) of the surfactin, iturin,
and fengycin families have been found to exhibit a wide range of bioactivities including antimicrobial,
antiviral, anticancer, immunosuppressive, antituberculosis, antimycoplasmic and exceptional surfactant
properties [9–11].
LPs are nonribosomal oligopeptides produced by large multienzyme complexes in several
Bacillus species and share a common cyclic structure including a β-amino or β-hydroxyl fatty acid that
is integrated into the peptide moiety [12]. The mode of action of some of them is via perturbation of
the cell membrane of the microorganism, thus affecting the transmembrane electric potential [13]. The
major step involves the binding of cationic LPs to the negatively charged lipopolysaccharide of
Gram-negative bacteria or to the lipotechoic acid of Gram-positive bacteria [14,15]. In fungi, the LPs
bind to the negatively charged membrane phosphatidylinositol and to the negatively charged terminal
sialic acid moieties [16,17]. For example, echinocandins and pneumocandins display strong antifungal
activities by inhibiting the synthesis of 1,3-β-D-glucan, an essential cell wall homopolysaccharide in
pathogenic fungi including Aspergillus and Candida [18].
As the multidrug-resistant pathogens are emerging day by day with regularity, it has now become
an urgent issue to discover new antimicrobials to combat against them. Concerning the requirements
for the new antibiotics, we have focused our attention on the isolation of antimicrobial secondary
metabolites from the bacterium B. subtilis, whose crude extract showed potent antimicrobial activity.
Chemical investigations from the fermentation broth of the bacterium B. subtilis yielded three new
linear LPs, gageostatins A (2.8 mg), B (3.2 mg) and C (1.5 mg) (Figure 1). We report here the details
of isolation, structural characterization and antimicrobial activities of these new LPs.

Mar. Drugs 2014, 12
873
Figure 1. Structures of Gageosatins A–C (1–3).
COOH
Leu-3
Asp
HOOC
Glu
O
Leu-1
HO
14
O
O
O
OH
H
H
N
H
13
3
N
N
( )7
1
N
N
H
N
H
N
12
H
H
O
O
O
O
Val
Leu-4
Leu-2
1
COOH
HOOC
H
N
15
14
O
O
O
O
OH
3
H
N
H
13
HO
N
( )5
1
N
H
N
H
N
N
10
12
H
H
O
O
O
O
2
COOH
HOOC
H
N
13
12
O
O
O
O
H
H
11
3
HO
N
N
1
( )3
N
N
N
H
N
8
H
H
H
O
O
O
O
3
2. Results and Discussion
2.1. Isolation of Compounds
The bacterial strain 109GGC020 was isolated from a marine sediment sample collected from
Gageocho, in the Republic of Korea’s southern reef, and identified as B. subtilis by 16s rRNA
sequencing. As the physiological process of bacteria varies with the variation of surrounding living
environment and metabolic profiles can be changed with very small changes in cultivation
conditions [19,20] the growth conditions of the strain for maximum metabolites production were
optimized through culturing in combination of different salinity of water, pH, and temperature before
proceeding of large scale culture. The optimal salinity of water, pH and temperature were found to be
18.3 g/L, 7.02 and 24 °C, respectively, for the maximum growth of the strain. The strain was then cultured
following above conditions and the fermentation broth was extracted with EtOAc. Thereafter, three new
linear lipopeptdes gageostatins A–C (1–3), were isolated from the EtOAc extract by sequential
fractionations and purifications employing flash column chromatography followed by reversed phase
HPLC techniques.
2.2. Structure Determination
Gageostatin A (1) was isolated as an amorphous solid and showed a molecular ion peak at
m/z 1062.6691 [M + Na]+ in the HR-ESIMS spectrum corresponding to the molecular formula of
C52H93N7O14 (see Supplementary Information). The IR spectra of 1 gave prominent broad peaks at

Mar. Drugs 2014, 12
874
3291 cm−1 (NH) and 1646–1737 cm–1 (CO), consistent with the presence of amide carbonyl groups
and a broad peak at 2930 cm−1 confirmed the presence of aliphatic chain. The 1H NMR data (Table 1),
recorded in both CD3OD and CD3OH of the chromatographically homogeneous material, revealed the
presence of a long aliphatic chain (CH2 at δH 1.29) and a peptide backbone by 7 NH signals at
δH 7.68–8.79 together with α-protons between δH 4.13 and 4.57. A resonance at δH 3.98 indicated the
presence of a further oxygenated proton, and CH3 signals were observed at δH 0.86–0.98. Furthermore,
13
the C NMR spectrum of 1 indicated the presence of 10 carbonyl carbons at δC 173.6–181.5, which
were attributable to amino acids. These detailed IR absorbencies together with 1H and 13C data analysis
indicated the lipopeptidic nature of 1.
Table 1. 1H and 13C NMR and HMBC data of 1–3 in CD3OD.
1
2
3
Units
No.
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
δC
Leu-1
1
2
180.3
53.5
41.6
26.1
21.7
23.5
180.2
53.3
41.6
26.1
21.7
23.4
180.2
54.8
41.6
26.1
21.8
23.4
4.37 m
1.65 m
4.37 m
1.65 m
4.30 m
1.63 m
3
4
1.65 m
1.65 m
1.63 m
5
0.91 m
0.91 m
0.89 m
6
0.91 m
0.91 m
0.89 m
NH
8.37 d (8.0) a
8.37 d (8.0) a
8.52 d (9.5) a
Leu-2
1
2
173.6
53.4
43.4
26.1
21.7
23.6
173.5
53.3
43.4
26.1
21.7
23.6
175.7
53.3
41.4
26.1
22.3
24.0
4.38 m
1.65 m
4.38 m
1.65 m
4.39 m
1.63 m
3
4
1.65 m
1.65 m
1.63 m
5
0.91 m
0.91 m
0.96 m
6
0.91 m
0.91 m
0.96 m
NH
7.96 d (8.0) a
7.97 d (8.0) a
7.67 d (9.0) a
Asp
1
2
3
174.3
53.1
40.1
174.2
53.0
40.0
174.3
53.2
40.1
4.57 t (6.0)
2.56 dd (16.5, 5.5)
2.76 dd (16.5, 6.0)
8.38 d (8.0) a
4.57 m
4.56 m
2.56 dd (16.5, 5.0)
2.75 dd (16.5, 5.5)
8.39 d (8.0) a
2.51 dd (16.5, 5.5)
2.75 dd (16.5, 6.0)
8.40 d (9.0) a
NH
COOH
178.2
178.0
178.2
Val
1
2
173.5
61.0
31.2
19.3
20.2
173.5
60.9
31.2
19.7
20.0
173.7
61.2
31.2
19.8
20.1
4.13 d (8.0)
2.18 m
4.13 m
2.17 m
4.06 m
2.13 m
3
4
0.93 m
0.93 m
0.96 m
5
0.93 m
0.93 m
0.96 m
NH
7.96 d (8.0) a
7.97 d (8.0) a
7.97 d (8.0) a

Mar. Drugs 2014, 12
875
Table 1. Cont.
Leu-3
1
175.0
54.8
41.4
26.1
22.2
23.9
174.9
54.7
41.4
26.1
22.1
23.8
175.5
2
4.40 m
1.65 m
4.40 m
1.65 m
4.35 m
1.65 m
53.5
40.4
26.1
22.4
23.8
3
4
1.65 m
1.65 m
1.63 m
5
0.93 m
0.93 m
0.92 m
6
0.93 m
0.93 m
0.92 m
NH
7.68 d (9.0) a
7.68 d (9.0) a
7.96 d (8.0) a
Leu-4
1
175.6
54.2
41.3
26.1
22.3
24.1
175.5
54.1
41.3
26.1
22.2
23.9
173.6
55.5
40.4
26.1
22.3
23.9
2
4.31 m
1.65 m
4.31 m
1.65 m
4.33 m
1.63 m
1.63 m
0.92 m
0.92 m
8.51 m
3
4
1.65 m
1.65 m
5
0.93 m
0.93 m
6
0.93 m
8.48 d (6.5) a
0.93 m
8.49 d (6.5) a
NH
Glu
1
175.0
55.5
29.6
174.9
55.4
29.5
175.1
55.5
29.8
2
4.34 m
1.95 m
4.34 m
1.95 m
2.05 m
2.29 m
4.35 m
1.95 m
2.03 m
2.26 m
3
2.05 m
4
2.29 t (7.0)
35.4
35.4
35.5
COOH
181.5
181.4
181.5
NH
8.79 d (6.5) a
8.81 d (6.5) a
8.81 brd.
3-OH acid
1
2
175.4
45.0
175.3
44.7
169.1
124.7
2.33 dd (9.0, 14.5)
2.46 dd (4.0, 14.5)
3.98 m
2.33 dd (8.5, 14.0)
2.47 dd (4.0, 14.5)
3.98 m
6.01 d (15.5)
3
4
70.2
26.9
70.0
26.9
6.79 dt(15.5, 8.5)
2.18 m
146.5
33.2
1.34 m
1.34 m
1.48 m
1.47 m
5
6
1.49 m
38.6
1.49 m
38.5
1.45 m
1.16 m
29.7
40.4
29.3
30.8
35.8
37.9
1.29 brs.
28.1–31.3
1.29 brs.
28.1–31.3
7
1.52 m
8
1.17 m
1.54 m
40.3
29.2
30.8
1.28 brds.
1.28 brds.
1.12 m
9
10
1.17 m
40.4
1.29 brs.
1.28 m
11
12
1.51 m
29.3
38.5
1.29 brs.
1.12 m
1.29 m
0.87 m
0.86 m
0.87 m
35.7
37.8
0.87 m
11.9
14.6
1.29 brs.
0.86 m
13
14
15
0.86 m
0.87 m
14.6
23.1
11.9
14.5
23.1
0.87 m
23.2
a 1H NMR data recorded in CD3OH.

Mar. Drugs 2014, 12
876
The detailed analysis of COSY, TOCSY and HMBC correlations enabled us to identify partial
substructures a–h corresponded to amino acid residues and a fatty acid moiety that constituted 1
(Figure 2). The spin systems of the α-H and NH resonances of Glu (δH 4.34/ 8.79), Asp (δH 4.57/8.38),
Val (δH 4.13/7.96) and Leu (δH 4.37/8.37, δH 4.38/7.96, δH 4.40/7.68, δH 4.37/8.48), and the
correlations from the α-methine resonances to the side chains of the individual amino acid residues
were observed in the COSY spectrum. Excitation of the amide proton signal at δH 8.79 showing
TOCSY correlations with α-methine at δH 4.34 and methylene signals at δH 1.95 and 2.05, in addition,
α-methine signals showing correlations with methylene signals at δH 1.95/2.05 and 2.29, revealed the
presence of a glutamic acid unit. Correlations observed from the methylene protons at δH 1.95/2.05 and
2.29 to a carboxylic carbon resonated at δC 181.5 and the protons at δH 1.95/2.05 to carbonyl carbon at
δC 175.0 in a HMBC spectrum confirmed the glutamic acid as a substructure “a”. Likewise, an amide
proton signal at δH 8.38 showed connectivities with methine and methylene protons resonated at
δH 4.57 and 2.56/2.76 in TOCSY experiment, further connectivity of these protons with carbonyl and
carboxylic carbons at δC 174.3 and 178.2 observed in HMBC experiment revealed the presence of Asp
unit (substructure b). In an entirely analogous way, substructures c–g (only COSY and TOCSY
correlations are shown in Figure 2) were assigned by using 1D and 2D NMR data. Moreover, the
TOCSY and HMBC spectra allowed identifying a 3-β-hydroxyl fatty acid residue in the molecule 1 as
a substructure h. In the 13C NMR spectra of 1, there were 12 methyl carbons and among these carbons
10 were attributed to amino acid residues and therefore, the remaining 2 methyl carbons should be
present in the aliphatic chain. One methyl carbon resonated at δC 14.6 was assigned as a terminal
methyl and another methyl carbon signal resonated at δC 23.1 formed side chain with the fatty acid.
The position of the later methyl group was located at C-11 of the fatty acid unit by the analysis of
TOCSY and HMBC correlations. Based on the composition of the peptide moiety and the molecular
weight of the molecule, the fatty acid chain was primarily determined to be a C14 fatty acid amidated to
the N-terminal amine of the peptide. The chain length of the fatty acid was further confirmed from the
acid hydrolysis of 1 followed by APCI-MS analysis (m/z at 243.02 [M − H]−) (SI). Thus, the
3-β-hydroxyl fatty acid was found as 3-β-hydroxy-11-methyltridecanoic acid in 1 with an optical
rotation value [α]D27 −29 (0.1, MeOH) (SI). The lipopeptide structure of 1 was then corroborated
constructing the sequence of Leu-Leu-Asp-Val-Leu-Leu-Glu-fatty acid by two dimensional ROESY
and HMBC experiments which showed long range proton-proton and proton-carbon correlations and
correlations between amide protons and α-protons.
Gageostatin B (2) was isolated as an amorphous solid and the molecular formula was determined to
be C53H95N7O14 based on the high-resolution ESIMS peak at m/z 1076.6831 [M + Na]+. Preliminary,
NMR analysis showed a close similarity between the spectra of compounds 1 and 2, indicating a
lipopeptide nature of 2. However, a methyl and a methine carbons resonated at δC 11.9 and 37.8 were
observed in the 13C NMR spectra of 2, which were not observed in compound 1. These carbons could
be located either on the alkyl chain or on the peptide chain. Therefore, a detailed investigation on the
structure of compound 2 was undertaken by running a series of 1D and 2D NMR experiments. The
detailed NMR data analysis pointed to the same amino acids sequence between the two compounds.
Therefore, these additional methyl and methine groups must be located in the aliphatic chain. The
position of the methyl group was then corroborated at C-9 of the fatty acid unit by the analysis of
TOCSY and HMBC correlations. Furthermore, the LC-MS analysis (m/z at 257.08 [M − H]−) of the

Mar. Drugs 2014, 12
877
hexane phase of the acid hydrolysate of 2, confirmed the chain length of the fatty acid in compound 2.
These extensive NMR data along with LC-MS results confirmed the presence of 3-β-hydroxy-9,
11-dimethyltridecanoic acid in 2 with an optical rotation value [α]2D7 −6 (0.25, MeOH). The search
result from the natural products database system suggested this fatty acid is a new fatty acid derivative.
Finally, the complete chemical structure of compound 2 was constructed by the detailed analysis of
COSY, TOCSY, HMBC and ROESY correlations (Supplementary Information).
Figure 2. Assignment of partial structures (a–h) by COSY and TOCSY correlations and
complete structure by HMBC correlations of gageostatins A.
COOH
HOOC
HN
HN
HN
HN
O
O
O
O
d
c
b
a
COSY
TOCSY
HMBC
HN
HN
HN
O
e
O
O
g
f
O
OH
( )n
h
COOH
HOOC
O
O
O
O
OH
H
H
H
N
HO
N
N
( )n
N
N
N
N
H
H
H
H
O
O
O
O
1
Gageostatin 3 was isolated as an amorphous solid and the molecular formula of C51H89N7O13 was
1
deduced based on high-resolution ESIMS measurement at m/z 1030.6423 [M + Na]+. The detailed H
and 13C data analyses were highly indicative of the lipopeptide nature of 3 and found to possess same
amino acids as 2. However, the existence of signals at δH 6.01 (d, J = 15.5) and 6.79 (dt, J = 15.5, 8.5)
in the 1H NMR data, which showed HSQC cross peaks with carbons resonated at δC 124.7 and 146.5,
respectively, suggested the presence of a conjugated double bond in 3. As the amino acids sequence
was found to be same as 2, corroborated by the TOCSY, HMBC and ROESY correlations, the double
bond must be located at the fatty acid moiety. The structure of the fatty acid was then established with
the help of TOCSY and HMBC correlations. The olefinic protons at δH 6.01 and 6.79 showed HMBC
correlations to the carbonyl carbon resonated at δC 169.1. Furthermore, the olefinic proton at δH 6.01
showed TOCSY and HMBC correlations with the later part of fatty acid chain. These correlations were
sufficient to locate the position of the conjugated diene C-2/C-3 position of the fatty acid moiety in 3.
The large coupling constant value of this conjugated diene suggested E configuration. Thus, the
unsaturated fatty acid was assigned as (E)-7,9-dimethylundec-2-enoic acid in compound 3. This fatty

Mar. Drugs 2014, 12
878
acid was also found as a new derivative, which has never been described before from the natural
sources. The fatty acid moiety was then connected with the peptide sequence at the glutamic acid side
with the help of HMBC correlations.
The absolute configuration of the amino acid residues in 1–3 was determined by acid hydrolysis and
Marfey’s method (SI) [21]. This method unambiguously established the configuration of each of Leu,
Asp, and Glu to be L-form and Val to be D-form. In addition, the absolute configuration of 1 at selected
stereocenter at C-3 of fatty acid moiety was determined by acid hydrolysis, followed by Mosher’s
MTPA method [22] and assigned based on proton resonances of the S–MTPA and R–MTPA ester
derivatives. A consistent distribution of positive and negative ΔδH values (ΔδH = δS − δR)
around C-3 allowed the assignment of R-configuration for C-3 position (Figure 3) [23]. This result was
also supported by the literature reviews, as (R)-3-hydroxy fatty acid displays negative molecular
rotation in MeOH [24,25]. The absolute stereochemistry at 3-hydroxy position of the fatty acid in 2
was determined to possess R configuration by the comparison of optical rotation value with that of
fatty acid in 1 (Supplementary Information).
Figure 3. Absolute stereochemistry determination of 3-hydroxy fatty acid in 1.
1
(A) H 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.
1.290
1.292
(CH2)n
(CH2)n
1.597
1.557
1.352
1.499
1.320,
1.458
2.567
2.663
2.542
2.638
OCH3
CF3
OCH3
CF3
O
O
HOOC
HOOC
5.484
5.483
O
O
H
H
(B)
(A)
(CH2)n
+0.04
+0.03
+0.04
O
OH
-0.03
-0.03
OCH3
CF3
O
(R)
HO
(CH2)n
-0.001
HOOC
(R)
O
H
(D)
(C)
2.3. Antimicrobial Activities
Minimum growth inhibitory activity of compounds 1–3 and 1+2 was evaluated against both Gram
positive and Gram negative bacteria and fungi following broth dilution assay [26]. Different growth
conditions of bacteria and fungi were maintained while culturing these microorganisms [27,28]. It was
found that mixed compounds 1 + 2 displayed better activity than individuals (Table 2). Moreover,
compound 3 possessing an unsaturated fatty acid displayed less activity than 1, 2 and 1 + 2. It is
noteworthy that these compounds 1–3 and 1 + 2 showed better activity against fungi compared to bacteria.

Mar. Drugs 2014, 12
879
Table 2. Minimum Inhibitory Concentrations (MICs) of 1–3.
MICs (µg/mL)
Microorganisms
1
2
1 + 2
3
P.C.
Fungi
R. solani
C. acutatum
B. cinera
4
8
4
8
8
8
4
4
4
32
16
32
1
1
1
Gram Positive Bacteria
S. aureus
B. subtilis
16
16
16
32
8
64
32
2
2
16
Gram Negative Bacteria
S. Typhi
16
16
32
16
32
32
64
2
2
P. aeruginosa
8
P.C.: Positive control (Azithromycin for Bacteria and Amphotericin B for Fungi).
2.4. Cytotoxic Properties
The cytotoxicity of compounds 1–3 and mixed compounds 1 + 2 was also evaluated against a panel
of six human cancer cell lines (breast cancer: MDA-MB-231, colon cancer: HCT15, prostate cancer:
PC-3, lung cancer: NCI-H23, stomach cancer: NUGC-3, and renal cancer:ACHN) according to a
sulforhodamine B (SBR) assay [29] and found to display moderate cytotoxicity (Table 3). Compounds
1–3 showed similar activity against all cell lines but mixed compounds 1 + 2 showed better
inhibitory activity. In particular, mixed compounds 1 + 2 displayed significant activity against lung
cancer (NCI-H23) with a GI50 value of 4.6 µg/mL.
Table 3. Human Cancer Cell Line Inhibition Values (GI50) of 1–3.
(GI50, µg/mL)
Cancer Cell Lines
1
2
1 + 2
10.5
10.9
12.0
4.6
3
ADR a
0.56
0.33
0.91
0.71
0.53
0.51
Breast cancer: MDA-MB-231
Colon cancer: HCT-15
Prostate cancer: PC-3
14.9
11.4
10.8
11.2
11.8
11.5
16.1
18.3
19.4
11.7
13.9
18.4
11.2
23.2
11.7
10.9
10.5
12.3
Lung cancer: NCI-H23
Stomach cancer: NUGC-3
Renal cancer: ACHN
10.1
10.7
a ADR means adriamycin as standard.
3. Experimental Section
3.1. General Experimental Procedures
UV spectra were obtained on a Shimadzu UV-1650PC spectrophotometer. IR spectra were recorded
on a JASCO FT/IR-4100 spectrophotometer. Optical rotations were measured on a JASCO (DIP-1000)
digital polarimeter. Nuclear magnetic resonance spectroscopic data were acquired on a Varian Unity
500 spectrometer. High-resolution ESIMS were recorded on a hybrid ion-trap time-of-flight mass
spectrometer (Shimadzu LC/MS-IT-TOF). HPLC was conducted with a PrimeLine Binary pump with

Mar. Drugs 2014, 12
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RI-101(Shodex) and Variable UV Detector (M 525). Semi-preparative HPLC was performed using
ODS (YMC-Pack-ODS-A, 250 × 10 mm i.d, 5 µm) and silica (YMC-Pack-SIL, 250 × 10 mm i.d,
5 µm) columns. Analytical HPLC was conducted on an ODS column (YMC-Pack-ODS-A, 250 × 4.6 mm
i.d, 5 µm). All solvents used were either spectral grade or distilled prior to use. Natural sea water was
collected from East Sea of South Korea at depths of 20 m.
3.2. Isolation and Identification of the Strain 109GGC020
The strain 109GGC020 was isolated from a sediment sample collected from Gageocho, in the
Republic of Korea, in 2010, by serial dilution techniques and grown on Bennett’s media agar plate
(For 1 L 100% natural sea water media composition was 1% dextrose, 0.1% yeast extract, 0.1% beef
extract, 0.2% tryptone, 1.8% agar and pH adjusted to 7.1). The strain was then identified as
Bacillus subtilis on the basis of 16S rRNA sequence analysis. The sequence was deposited in GenBank
under accession number JQ927413. This strain is currently preserved in the Microbial Culture
Collection, KIOST, with the name Bacillus subtilis 109GGC020 under the curatorship of Hee Jae Shin.
3.3. Seed and Mass Cultures of the Strain
The seed culture of the strain 109GGC020 was performed in triplicate into 100 mL flasks,
containing 50 mL medium. An aliquot (0.2% v/v) from the seed culture was inoculated aseptically into
2 L flasks (total 50 flasks) containing 1.2 L sterilized culture medium (same composition as above).
The production culture was incubated at 28 °C for 7 days.
3.4. Extraction and Isolation of Compounds
The cells were separated from the culture broth by centrifugation and the broth was extracted with
EtOAc (2 times). The EtOAc layer was evaporated to dryness under reduced pressure at 40 °C. The
obtained crude extract (11.8 g) was subjected to an ODS open column chromatography followed by
stepwise gradient elution with MeOH–H2O (v/v) (1:4, 2:3, 3:2, 4:1 and 100:0) as eluent. The 100%
MeOH fraction was again subjected to further fractionations and purification by C18 semi-preparative
and analytical HPLC using solvent system 95% MeOH in H2O to yield pure compounds 1–3.
Gageostatin A (1): HPLC retention time (42 min); amorphous solid; [α]2D7 +52 (c 0.1, MeOH);
1
13
IR (MeOH) νmax 3291 cm−1 (NH) and 1646–1737 cm−1 (CO), 2930 cm−1; H and C NMR data
(CD3OD), Table 1; HRESIMS m/z 1062.6691 [M + Na]+.
Gageostatin B (2): HPLC retention time (44 min); amorphous solid; [α]2D7 +53 (c 0.1, MeOH); IR
(MeOH) νmax 3293 (br), 2927, 1742 cm−1; 1H and 13C NMR data (CD3OD), Table 1;
HRESIMS m/z 1076.6831 [M + Na]+.
Gageostatin C (3): HPLC retention time (50 min); amorphous solid; [α]2D7 +16 (c 0.1, MeOH); IR
1
13
(MeOH) νmax 3373 (br), 2970, 1660 cm−1; H and C NMR data (CD3OD), Table 1; HRESIMS m/z
1030.6423 [M + Na]+.

Mar. Drugs 2014, 12
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3.5. Acid Hydrolysis of Compounds 1–3
Compound 1 (2.1 mg) was dissolved in 6N HCl (0.50 mL) and stirred at 120 °C for 23 h. After
cooling, the solution was diluted with water, and the products were extracted with chloroform. The
chloroform extract was concentrated under a stream of N2 and aqueous part was evaporated to dryness
under reduced pressure. The chloroform extract containing fatty acid was derivatized with Mosher’s
reagent to determine absolute configuration at the stereocenter C-3 of 3-hydroxy fatty acid and the
aqueous part was subjected for amino acids analysis by Marfey’s method. In the similar manner,
compounds 2 (0.8 mg) and 3 (0.2 mg) were hydrolyzed and analyzed.
1
Compound 1a (1.5 mg): colorless oil; H NMR data (CD3OD) δH 2.35 (H-2a, dd, J = 15.0, 8.5),
2.44 (H-2b, dd, J = 15.5, 5.0), 3.97 (H-3, m), 1.34 (H-4a, m, overlapped), 1.45 (H-4b, m, overlapped),
1.47 (H2-5, m, overlapped), 1.29 (H2-6-H2-9, brds.), 1.17 (H-10, m), 1.51 (H-11, m, overlapped),
1.29 (H-12, m, overlapped), 0.86 (H3-13, t, J = 5.5), 0.87 (H3-14, d, J = 6.5); APCI-MS m/z 243.04
[M − H]−; molecular rotation [α]2D7 −29 (0.1, MeOH).
3.6. Preparation of the (S)- and (R)-MTPA Esters of 3-Hydroxy Fatty Acids (1b and 1c)
Compound 1a (0.7 mg) was dissolved in 200 µL of pyridine into a 4 mL reaction vial and stirred at
room temperature (rt) for 10 min. To the vial, 20 µL of (R)-(−)-MTPA-Cl was added to prepare the
(S)-MTPA ester (1b) of 1a, 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, re-dissolved in EtOAc, washed with
H2O, and purified by an analytical HPLC using 90% MeOH in H2O as eluent to obtain 1b (0.4 mg). In
an entirely analogous way, (R)-MTPA ester (1c) of 1a was prepared by using (S)-(+)-MTPA-Cl.
Analysis of the ΔδH (δS − δR) values obtained by 1H NMR spectrum for each couple of diastereomeric
MTPA esters, according to the Mosher model pointed to an R-configuration at C-3 of the fatty acid.
3.7. Advanced Marfey’s Analysis of 1–3
The hydrolysates of 1–3 were re-suspended in H2O (100 µL). A 0.1% 1-fluoro-2,4-dinitrophenyl-5-
L-alaninamide solution in acetone (Marfey’s reagent, 20 µL) and 1N NaHCO3 (10 µL) were added to a
portion of the hydrolysate, and the mixture was heated at 40 °C for 2 h. The solution was cooled to
room temperature, neutralized with 2N HCl (5 µL), and evaporated to dryness. The residue was
re-suspended in H2O (50 µL) and analyzed by reversed-phase HPLC (YMC-Pack ODS, 250 × 4.6 mm,
5 µm, flow rate of 0.5 mL/min) using a linear gradient of 40% CH3CN in H2O containing 0.5%
TFA in 60 min at 28 °C. Similarly, standard amino acids (both L and D) were derivatized with
Marfey’s reagent and analyzed. The derivatized Leu, Asp, Glu residues in the hydrolysate of 1 were
eluted at the same retention time as the derivatized standard respective L-amino acids but not that of
D-Val (30.0 min).
3.8. Antimicrobial Assays
The antimicrobial activity of compounds 1–3 and 1 + 2 was determined by using a standard
broth dilution assay against Gram positive and Gram negative bacteria and fungi. Antibacterial and

Mar. Drugs 2014, 12
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antifungal tests were performed in nutrient broth and yeast maltose broth, respectively. A serial double
dilution of each compound was prepared in 96-microtiter plates over the range of 0.5–256 µg/mL. An
overnight broth culture of each strain was prepared and final concentration of organisms in each
culture was adjusted to 1.5 × 108 cfu/mL by comparing the culture turbidity with the 0.5 McFarland
Standard. Culture broth (30 µL) was added to each dilution of compounds 1–3 and 1 + 2 and the final
volume of each well was adjusted to 200 µL using the respective culture medium, and the plates were
incubated 24 h at 37 °C for bacteria and 48 h at 30 °C for yeast. The minimum inhibitory concentration
(MIC) is the lowest concentration of a sample at which the microorganism did not demonstrate visible
growth, as indicated by the presence of turbidity.
3.9. Cytotoxicity Test
Cancer cell growth inhibitory activity of compounds 1–3 and mixed compounds 1 + 2 was
performed according to a sulforhodamine B (SBR) assay. In brief, 96-well plate was loaded with
selected cell lines (MDA-MB-231, HCT-15, PC-3, NCI-H23, NUGC-3, and ACHN) and tested
samples (30, 10, and 3 µg/mL) were added. After incubation for 48 h, anchorage-dependant cells are
fixed with 50% (wt/vol) trichloroacetic acid and stained for 60 min. Access dye was washed with SRB
solution (0.4% sulforhodamine B in 1% acetic acid). The protein-bound dye is dissolved in 10 mM
Tris base solution and absorbance was measured at 510 nm using a microplate reader. GI50 values of
compounds 1–3 and 1 + 2 were then calculated using graphed prism software.
4. Conclusions
In summary, we discovered three new linear LPs from the culture broth of B. subtilis. The detailed
structural analysis of these compounds indicated the same amino acid residues and sequence to the
reported surfactins A–D [30]. However, the major differences between compounds 1–3 and surfactins
were found in their molecular mass and structures. Compounds 1–3 were obtained as a linear form
with all L-Leu, whereas surfactins A–D are cyclic peptides with L- and D-Leu. Moreover, the search
result in a natural products database confirmed the characterization of new fatty acid moieties in
compounds 2 and 3. Surfactins have been reported to display wide range of bioactivities including
antibacterial, antifungal, and anticancer activities [31–34]. Recently, surfactin like cyclic LPs have also
been discovered and reported to display strong and dose-dependent antifungal activity against the plant
pathogenic fungus Fusarium oxysporum [35]. Together with the pharmaceutical applications, LPs are
also used as biosurfactants such as emulsifier in the food and cosmetic industries and bioremediation
and dispersion of oil spills due to their biodegradability, low toxicity and environmental compatibility
properties [36,37]. In our study, the antimicrobial activity results of 1–3 indicated their possibilities to
be drug candidates too. In particular, these LPs or their modified form would be utilized as antifungal
agents as well as biosurfactants.
Acknowledgments
The authors express gratitude to Kun Cho, Korea Basic Science Institute, Ochang, Korea, for
providing mass data. This research was supported in part by Korea Institute of Ocean Science and

Mar. Drugs 2014, 12
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Technology (Grant PE99121 to Hee Jae Shin) and the Ministry of Oceans and Fisheries (Grant
PM57420 to Jae Seol Shim), Korea.
Conflicts of Interest
The authors declare no conflict of interest.
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