Gageopeptins A and B, new inhibitors of zoospore motility of the phytopathogen Phytophthora capsici from a marine-derived bacterium Bacillus sp. 109GGC020

Article abstract of DOI:10.1016/j.bmcl.2015.05.070

Abstract The motility of zoospores is critical in the disease cycles of the peronosporomycetes that cause devastating diseases in plants, fishes, vertebrates, and microbes. In the course of screening for secondary metabolites regulating the motility of zoospores of Phytophthora capsici, we discovered two new inhibitors from the ethyl acetate extract of the fermentation broth of a marine-derived strain Bacillus sp. 109GGC020. The structures of these novel metabolites were elucidated as new cyclic lipopeptides and named gageopeptins A (1) and B (2) by spectroscopic analyses including high resolution MS and extensive 1D and 2D NMR. The stereoconfigurations of 1 and 2 were assigned based on the chemical derivatization studies and reviews of the literature data. Although compounds 1 and 2 impaired the motility of zoospores of P. capsici in dose- and time-dependent manners, compound 1 (IC₅₀ = 1 μg/ml) was an approximately 400-fold stronger motility inhibitor than 2 (IC₅₀ = 400 μg/ml). Interestingly, the zoospores halted by compound 1 were subsequently lysed at higher concentrations (IC₅₀ = 50 μg/ml). Compounds 1 and 2 were also tested against some bacteria and fungi by broth dilution assay, and exhibited moderate antibacterial and good antifungal activities.

Full text of DOI:10.1016/j.bmcl.2015.05.070

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Gageopeptins A and B, new inhibitors of zoospore motility of the
phytopathogen Phytophthora capsici from a marine-derived
bacterium Bacillus sp. 109GGC020
Fakir Shahidullah Tareq a,b, Choudhury M. Hasan b,c, Hyi-Seung Lee a, Yeon-Ju Lee a, Jong Seok Lee a,
Musrat Zahan Surovy d, Md. Tofazzal Islam d, Hee Jae Shin a,e,
⇑
a Marine Natural Products Chemistry Laboratory, Korea Institute of Ocean Science and Technology, Ansan, Republic of Korea
b Department of Pharmacy, Manarat International University, Dhaka, Bangladesh
c Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Bangladesh
d Department of Biotechnology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Dhaka, Bangladesh
e Department of Marine Biotechnology, University of Science and Technology, 217 Gajungro, Yuseong-gu, Daejeon, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
The motility of zoospores is critical in the disease cycles of the peronosporomycetes that cause devastat-
ing diseases in plants, fishes, vertebrates, and microbes. In the course of screening for secondary metabo-
lites regulating the motility of zoospores of Phytophthora capsici, we discovered two new inhibitors from
the ethyl acetate extract of the fermentation broth of a marine-derived strain Bacillus sp. 109GGC020. The
structures of these novel metabolites were elucidated as new cyclic lipopeptides and named gageopep-
tins A (1) and B (2) by spectroscopic analyses including high resolution MS and extensive 1D and 2D NMR.
The stereoconfigurations of 1 and 2 were assigned based on the chemical derivatization studies and
reviews of the literature data. Although compounds 1 and 2 impaired the motility of zoospores of P. cap-
Received 26 January 2015
Revised 20 May 2015
Accepted 22 May 2015
Available online xxxx
Keywords:
Zoospore motility inhibitors
Marine bacteria
Phytopathogens
Phytophthora capsici
Lytic acitivity
sici in dose- and time-dependent manners, compound 1 (IC50 = 1
stronger motility inhibitor than 2 (IC50 = 400 g/ml). Interestingly, the zoospores halted by compound 1
were subsequently lysed at higher concentrations (IC50 = 50 g/ml). Compounds 1 and 2 were also tested
lg/ml) was an approximately 400-fold
l
l
against some bacteria and fungi by broth dilution assay, and exhibited moderate antibacterial and good
antifungal activities.
Ó 2015 Elsevier Ltd. All rights reserved.
Phytophthora capsici is a peronosporomycete plant pathogen
that causes blight and fruit rot diseases in peppers and other
important commercial crops worldwide.1 This fungus-like patho-
gen has been reported to cause severe epidemics in the Central
and South America, Europe, Asia, and many states of the United
States, where vegetables are grown. The host range of P. capsici is
wide including bell pepper, cacao, cantaloupe, chayote, cucumber,
eggplant, honeydew melon, marigold, macadamia nut, papaya,
pumpkin, some bean types, squash, tomato, and watermelon.2
Different types of chemical and synthetic compounds have been
used as antimicrobial agents to inhibit plant pathogenic per-
onosporomycete and fungi for many years. Antimicrobials such
as benzimidazoles, aromatic hydrocarbons, and sterol biosynthesis
inhibitors are often used to control plant disease in agriculture.
However, development of resistance against fungicide is a serious
problem in efficacy of these chemicals.3 Famers often use higher
concentrations of these chemicals to overcome this problem, but
this increases the risk of high-level toxic residues in the products
and environment. Therefore, there has been a growing interest in
research for the possible use of natural products, which can be safe
for the products and environment, and relatively specific for the
pest.4
As part of our ongoing research for the discovery of new inhibi-
tory compounds against the notorious phytopathogen P. capsici, we
isolated two new cyclic lipopeptides 1 and 2 from a marine-
derived bacterial strain 109GGC020. Here, we report the isolation
and characterization of 1 and 2 and their growth inhibitory activity
against P. capsici and some bacteria and fungi.
The producing strain 109GGC020 was isolated from a marine
sediment sample as described before5 and identified as Bacillus
subtilis based on the analysis of 16S rRNA gene sequencing data.
The sequence was deposited in GenBank under the accession num-
ber JQ927413. This strain is currently preserved in the Microbial
Culture Collection, KIOST, with the name Bacillus subtilis
⇑
Corresponding author. Tel.: +82 31 400 6172; fax: +82 31 400 6170.
E-mail address: shinhj@kiost.ac (H.J. Shin).
http://dx.doi.org/10.1016/j.bmcl.2015.05.070
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Tareq, F. S.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.05.070

2
F. S. Tareq et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 1
1H and 13C NMR data of compounds 1 and 2
Units
Glu-1
No.
1
2
dH,mult. (J in Hz)
dC
dH,mult. (J in Hz)
dC
1
2
3
174.5
61.8
28.2
174.3
61.9
28.1
4.04 m
2.01 m
2.11 m
2.25 m
4.03 m
2.00 m
2.11 m
2.25 m
4
30.8
30.7
COOH
176.4
176.7
Val-2
Leu-3
1
2
3
4
5
173.8
53.5
23.6
21.7
23.6
173.9
53.5
23.3
21.9
23.7
4.42 m
1.45 m
0.89 m
0.89 m
4.40 m
1.46 m
0.89 m
0.89 m
1
2
3
4
5
6
174.2
53.3
40.3
26.3
22.6
23.8
174.0
53.1
40.2
26.3
22.7
23.8
4.44 m
1.70 m
1.70 m
0.89 m
0.89 m
4.42 m
1.70 m
1.70 m
0.89 m
0.89 m
Asp-4
1
2
3
173.6
53.2
35.8
173.9
53.1
35.9
4.15 m
2.85 dd (17.0, 9.0)
2.98 dd (17.0, 4.0)
4.14 m
2.85 dd (17.0, 9.5)
2.98 dd (17.0, 4.0)
COOH
175.0
174.3
Ala-5
Leu-6
1
2
4
175.4
51.8
19.7
175.5
51.7
19.8
4.79 dd (9.5, 4.5)
1.77 m
4.79 dd (9.5, 4.5)
1.77 m
1
2
3
4
5
6
174.1
54.3
41.1
26.3
23.3
23.7
174.0
54.2
41.0
26.3
23.2
23.7
4.33 dd (11.5, 6.5)
1.70 m
1.70 m
0.93 m
0.93 m
4.32 m
1.70 m
1.70 m
0.93 m
0.93 m
Leu-7
1
174.1
174.2
2
3
4
5
6
4.22 dd (8.0, 6.0)
1.70 m
1.70 m
0.93 m
0.93 m
55.5
40.5
26.3
22.2
23.8
4.21 m
1.70 m
1.70 m
0.93 m
0.93 m
55.5
40.5
26.3
22.0
23.8
3-OH acid
1
2
176.7
43.3
176.7
43.3
2.44 dd (14.0, 8.0)
2.64 dd (14.0, 4.0)
5.30 m
2.43 dd (6.0, 3.0)
2.64 dd (15.0, 2.0)
5.29 m
3
4
73.6
26.3
73.4
26.3
1.37 m
1.37 m
1.50 m
1.50 m
5
1.55 m
1.32 br s
1.17 m
42.9
30.4–31.4
40.5
29.2
31.1
1.55 m
1.32 br s
—
—
1.17 m
42.8
30.4–31.4
—
6–10
11
12
13
14
15
1.54 m
—
1.32 br s
1.32 br s
1.12 m
40.4
29.3
38.5
35.8
37.9
1.51 m
1.32 m
1.32 m
16
17
18
0.88 m
0.88 m
0.88 m
11.9
18.4
19.8
0.88 m
0.88 m
14.6
19.8
109GGC020 under the curatorship of Shin. The bacterium was fer-
mented for 7 days and the culture broth was extracted with EtOAc.
The crude extract (11.8 g) was subjected to column chromatogra-
phy and HPLCs to yield pure compounds 1 (8.9 mg) and 2
(4.5 mg) (Fig. 1).6
Gageopeptin A (1) was isolated as an amorphous solid and its
molecular formula was determined to be C53H93N7O13 based on
the HRESI mass at m/z 1034.6758 [MÀH]À. Preliminary, the 1H
NMR and 13C NMR resonances (Table 1) of 1 were found quite sim-
ilar to the previously reported cyclic lipopeptides.7 Later, the
detailed analysis of COSY and HMBC correlations enabled us to
determine the sequence of amino acids in 1, Glu-Val-Leu-Ala-
Asp-Leu-Leu-3-hydroxy fatty acid, which was same as the pub-
lished compounds (Fig. 2).7 However, in the 13C NMR spectra of 1
few more 13C resonances were observed which guided us to care-
fully investigate the structure of 1. It was found that among the 12
methyl carbons in 1, 9 were attributed to five amino acids and
therefore, the remaining 3 methyl carbons must be present in
the fatty acid chain. Based on the HMBC correlations, the methyl
carbon resonated at dC 11.9 was assigned as a terminal methyl,
Please cite this article in press as: Tareq, F. S.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.05.070

F. S. Tareq et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
O
HO
O
O
O
O
HO
O
O
O
NH
N
O
H
N
H
NH
O
NH
NH
O
(
)
7
NH
NH
O
(
)
9
O
HN
O
HN
H
O
NH
O
N
H
N
H
N
O
O
O
O
O
OH
O
OH
1
2
Figure 1. Structures of gageopeptins A (1) and B (2).
O
O
HO
O
HO
O
O
O
O
O
N
H
NH
N
H
NH
O
O
NH
NH
NH
NH
O
(
)
7
O
(
)
7
O
O
COSY
TOCSY
HMBC
HN
O
HN
O
H
N
H
N
O
O
NH
NH
O
O
O
O
OH
OH
Figure 2. Key COSY, TOCSY and HMBC correlations in gageopeptin A (1).
whereas the methyl carbons resonated at dC 18.4 and dC 19.8 were
located at C-12 and C-14 of the fatty acid, respectively. Primarily,
the fatty acid chain was determined to be a C18 fatty acid based
on the composition of the peptide moiety and the molecular
weight of the molecule amidated to the N-terminal amine of the
peptide. The fatty acid was then confirmed as a 3-hydroxy-12,14-
dimethylhexadecanoic acid from the acid hydrolysis of 1 followed
structure of compound 2 was constructed by the detailed analysis of
COSY, TOCSY, and HMBC correlations.
The absolute configuration of the amino acid residues and the
stereocenter at C-3 of the fatty acid moiety in 1 and 2 was deter-
mined by acid hydrolysis8 followed by Mosher’s MTPA and
Marfey’s methods sequentially.9,10 Mosher’s method lead to the
assignment of absolute configuration at C-3 of the fatty acid in 1
as R-form (Fig. 4, SI-1).11 Additionally, Marfey’s method unambigu-
ously established the configuration of each of the amino acids to be
L-form (Fig. 5, SI-1). This result was consistent with the absolute
stereochemistry of the previously reported (R)-3-hydroxy fatty
acid and also supported by the literature reviews, as analogues of
this fatty acid display negative specific rotation in MeOH.12,13 The
absolute stereochemistry at the 3-hydroxy position of the fatty
acid in 2 was determined to possess R configuration by the com-
parison of optical rotation value with that of the fatty acid in 1.
To infect host plants, pathogens like P. capsici asexually produce
characteristic biflagellate (heterokont) motile spores, called
zoospores.14 Several lines of evidence indicate that these wall-less
zoospores locate their hosts guided by the specific chemical signals
secreted from the hosts.15 When zoospores find potential infection
sites of the hosts, they stop motility, attach to the host surface
using posterior flagella, and rapidly transform to round-shaped
cystospores after shedding flagella.15 The cystospores then
germinate to form germ tubes within 30–40 min to invade plant
tissues and initiate infection. The successful infection of this obli-
gate notorious phytopathogen can be attributed in part to the
speed of asexual differentiation to generate biflagellate motile
zoospores that target potential infection site of the plant through
water films. Any disruption of zoospore release from sporangia
by APCI-MS analysis (m/z at 300.26 [M+H]+) with a molecular rota-
25
tion value [
a
]
D
À22 (0.5, MeOH). Collectively, the data obtained
allowed determination of the chemical structure of 1.
Gageopeptin B (2) was isolated as an amorphous solid and its
molecular formula was determined to be C52H91N7O13 based on
the high-resolution ESIMS peak at m/z 1020.6595 [MÀH]À. The
1H and 13C NMR data of 2 showed a close similarity with 1, indicat-
ing a lipopeptide nature of 2. A careful investigation of the 13C NMR
data of 2 revealed the absence of a methine and a methyl carbon
resonated at dC 11.9 and 29.2, which were observed in 1. This
observation lead to further detailed investigation of the structure
of compound 2 by running a series of 1D and 2D NMR experiments.
The detailed NMR data analysis revealed that 1 and 2 had the same
amino acids sequence, indicating that 2 had a different type of fatty
acid than that of 1. Based on HMBC correlations, a methyl carbon
resonated at dC 14.6 was found to be a terminal methyl and
another methyl carbon resonated at dC 19.8 constituted side chain
locating at C-14 in the fatty acid. The LC-MS analysis (m/z at
286.2508 [M+H]+) of the hexane phase of the acid hydrolysate of
2 confirmed the chain length of the fatty acid. These extensive
NMR data along with LC-MS results confirmed the presence of a
3-hydroxy-14-methylhexadecanoic acid in 2 with a molecular
25
rotation value [
a]
À30 (0.2, MeOH). Finally, the complete chemical
D
Please cite this article in press as: Tareq, F. S.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.05.070

4
F. S. Tareq et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
B
A
Motility inhibition Lysis
Compound 1 (50 µg/ml)
100
80
60
40
20
0
Compound 1 (5 µg/ml)
100
80
60
40
20
0
15
30
45
Time (min)
60
15
30
45
Time (min)
60
D
C
100
80
60
40
20
0
Compound 2 (500 µg/ml)
100
80
60
40
20
0
Compound 1 (60 min)
15
30
45
Time (min)
60
5
10
50
100
200
300
Dose (µg/ml)
Figure 3. Time-course and dose-dependent motility inhibitory and lytic activities of compounds 1 and 2 against P. capsici zoospores. (A) Motility inhibitory activity of
compound 1 at 5 g/ml; (B) Motility inhibitory and lytic activity of compound 1 at 50 g/ml; (C) Lytic activity of compound 1 at 5 g/ml; (D) Motility inhibitory activity of
g/ml.
l
l
l
compound 2 at 500
l
found to display motility inhibition at 500 lg/ml but did not show
Table 2
Minimum inhibitory concentrations (MICs) of 1 and 2
lytic activity at this concentration. These results suggested that the
methyl group at C-12 of the fatty acid in 1 is critical for activity.
Furthermore, minimum growth inhibitory activity of compounds
1 and 2 was evaluated against both Gram positive and Gram neg-
ative bacteria and fungi by broth dilution assay.17 Different growth
conditions of bacteria and fungi were maintained while culturing
these microorganisms.18,19 Compounds 1 and 2 showed good inhi-
bitory activity against fungi compared to bacteria (Table 2). These
results suggested the new compounds 1 and 2 could be good bio-
probes for antimicrobial agents. In particular, compound 1 can be
used to develop fungicides targeting the economically important
pathogenic peronosporomycete P. capsici.
Microorganisms
MICs (lg/ml)
1
2
P.C.
Fungi
R. solani
C. acutatum
B. cinera
4
8
4
8
8
8
1
1
1
Gram positive bacteria
S. aureus
B. subtilis
16
16
16
32
2
2
Gram negative bacteria
S. Typhi
P. aeruginosa
16
16
32
16
2
2
P.C.: Positive control (Azithromycin for Bacteria and Amphotericin B for Fungi).
Acknowledgments
The authors express gratitude to Dr. Y.H. Kim, Korea Basic
Science Institute, Ochang, Korea, for providing high resolution
mass data. This research was supported in part by Korea Institute
of Ocean Science and Technology (Grant PE99273 to H.J.S.), the
project titled ‘Construction of Ocean Research Station and their
Application Studies’ funded by the Ministry of Oceans and
Fisheries, Korea and the World Bank through a Higher Education
Quality Enhancement Project (CP # 2071 to MTI).
(zoosporogenesis) or zoospore motility remarkably decreases the
potential for pathogenesis.16 We tested the activity of compounds
1 and 2 on the motility and viability of P. capsici zoospores using
homogeneous solution method.5 Interestingly, compounds 1 and
2
significantly inhibited the motility of zoospores (Fig. 3).
Moreover, in the presence of compounds 1 and 2, zoospores moved
very slowly in their axis or spun in tight circles instead of display-
ing straight swimming in a helical fashion. Compound 1 was very
potent to arrest the motility of zoospores at the concentrations
Supplementary data
starting from 1
displayed lytic activity after increasing the concentration at
50 g/ml. It might be noted that compound 1 showed lytic activity
after 30 min. However, this compound displayed strong lytic activ-
ity after 1 h at 10 g/ml. On the other hand, compound 2 was
lg/ml (Table 3, SI-2). Nevertheless, compound 1
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.05.
070.
l
l
Please cite this article in press as: Tareq, F. S.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.05.070

F. S. Tareq et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
(MeOH) mmax 3310, 2945, 1742–1656 cmÀ1
;
1H and 13C NMR data (CD3OD),
References and notes
Table 1; HRESIMS m/z 1020.6595 [MÀH]À
.
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1. Islam, M. T.; Tiedemann, A. V.; Laatsch, H. MPMI 2011, 24, 938.
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8. Acid hydrolysis. Compound 1 (2.5 mg) was dissolved in 6 N HCl (0.60 ml) and
stirred at 125 °C for 23 h. The solution was cooled, diluted with water, and
extracted with chloroform. The chloroform extract was concentrated by a
stream of N2 and the aqueous part was evaporated to dryness under reduced
pressure. The fatty acid obtained from the chloroform extract was derivatized
with Mosher’s reagent to determine the absolute configuration at the
stereocenter C-3 of the 3-hydroxy fatty acid. The aqueous part was subjected
for the assignment of absolute stereochemistry of amino acids by Marfey’s
method. In an analogous way, compound 2 (1.8 mg) was hydrolyzed and
analyzed. Compound 1a (1.5 mg): colorless oil; 1H NMR data (CD3OD) dH 2.38
(H-2a, dd, J = 15.0, 8.5), 2.54 (H-2b, dd, J = 15.5, 5.0), 3.98 (H-3, m), 1.35 (H-4a,
m, overlapped), 1.47 (H-4b, m, overlapped), 1.49 (H2-5, m, overlapped), 1.29
(H2-6-H2-11, brds), 1.16 (H-12, m), 1.29 (H-13, m, overlapped), 1.49 (H-14, m,
overlapped), 1.29 (H-15, m, overlapped), 0.86 (H3-16, m, overlapped), 0.87
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6. Strain culture, extraction and isolation of compounds. The seed culture of the
strain 109GGC020 was carried out into 100 ml flasks (total 3 flasks) containing
50 ml modified Bennett’s culture medium (for 1 L, the medium composition
was 1% dextrose, 0.1% yeast extract, 0.1% beef extract, 0.2% tryptone, 1.8% agar,
100% natural sea water, and pH 7.1). The seed culture (0.2% v/v) was inoculated
aseptically into 2 L flasks (total 50 flasks) containing 1.2 L sterilized culture
medium (same composition as above). The culture was incubated at 28 °C for
7 days. The culture was harvested (cells were separated from the broth by
centrifugation) and the broth was extracted with EtOAc (2 times). The solvent
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 MeOHÀH2O (v/v) (1:4, 2:3, 3:2,
4:1 and 100:0) as eluent. The 100% MeOH fraction was fractionated again and
purified by C18 semi-preparative and analytical HPLCs using a solvent system
(2H3-17,18, m, overlapped); APCI-MS m/z 300.26 [M+H]+; molecular rotation
27
[a]
D
À22 (c 0.5, MeOH).
9. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113,
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(95% MeOH in H2O) to yield pure compounds 1 and 2. Gageopeptin A (1):
27
amorphous solid; [
a
]
À152 (c 0.05, MeOH); IR (MeOH) mmax 3290, 2930, 1718–
1635 cmÀ1
;
1H and 1D3C NMR data (CD3OD), Table 1; HRESIMS m/z 1034.6758
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[MÀH]À. Gageopeptin B (2): amorphous solid; [
a
]
À123 (c 0.05, MeOH); IR
27
D
Please cite this article in press as: Tareq, F. S.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.05.070