Bacilotetrins A and B, Anti-Staphylococcal Cyclic-Lipotetrapeptides from a Marine-Derived Bacillus subtilis

Article abstract of DOI:10.1021/acs.jnatprod.7b00356

LC-MS and NMR spectroscopy guided metabolic profiling and dereplication of a crude extract obtained from the fermentation of a marine-derived bacterium, Bacillus subtilis, followed by chromatographic isolation yielded two new cyclic-lipotetrapeptides, bacilotetrins A (1) and B (2). Based on extensive 1D and 2D NMR and high-resolution ESIMS data analysis, the structures of 1 and 2 were elucidated, revealing the unique structures of these lipopeptides consisting of three leucines and a glutamic acid residue cyclized with a lipophilic 3-hydroxy fatty acid. The absolute stereochemistries at selected stereocenters in 1 and 2 were assigned by chemical derivatization and comparison to literature data. Compounds 1 and 2 exhibited anti-MRSA activity with MIC values of 8 to 32 μg/mL. However, these compounds showed no cytotoxicity when tested against prostate and liver cancer cell lines using the standard SRB assay.

Full text of DOI:10.1021/acs.jnatprod.7b00356

Article
Cite This: J. Nat. Prod. XXXX, XXX, XXX-XXX
pubs.acs.org/jnp
Bacilotetrins A and B, Anti-Staphylococcal Cyclic-Lipotetrapeptides
from a Marine-Derived Bacillus subtilis
Fakir Shahidullah Tareq† and Hee Jae Shin*
,‡
,‡,§
†
Department of Pharmacy, Manarat International University, Dhaka 1212, Bangladesh
‡
University of Science and Technology, 176 Gajung-dong, 217 Gajungro Yuseong-gu, Daejeon 305-333, Republic of Korea
§
Marine Natural Products Chemistry Laboratory, Korea Institute of Ocean Science and Technology (KIOST), Ansan, Republic of
Korea
*
S Supporting Information
ABSTRACT: LC-MS and NMR spectroscopy guided meta-
bolic profiling and dereplication of a crude extract obtained
from the fermentation of a marine-derived bacterium, Bacillus
subtilis, followed by chromatographic isolation yielded two new
cyclic-lipotetrapeptides, bacilotetrins A (1) and B (2). Based
on extensive 1D and 2D NMR and high-resolution ESIMS
data analysis, the structures of 1 and 2 were elucidated,
revealing the unique structures of these lipopeptides consisting
of three leucines and a glutamic acid residue cyclized with a
lipophilic 3-hydroxy fatty acid. The absolute stereochemistries
at selected stereocenters in 1 and 2 were assigned by chemical
derivatization and comparison to literature data. Compounds 1 and 2 exhibited anti-MRSA activity with MIC values of 8 to 32
μg/mL. However, these compounds showed no cytotoxicity when tested against prostate and liver cancer cell lines using the
standard SRB assay.
arine-derived Bacillus subtilis isolates have been a good
source of bioactive secondary metabolites. Several
rRNA sequencing. The bacterium B. subtilis was cultured in
large scale maintaining optimal growth conditions to obtain
maximum metabolite production. The culture broth was
filtered, and the cell-free supernatant was extracted with ethyl
acetate. The crude extract was fractionated by open column
1
M
bioactive compounds including lipopeptides, polypeptides,
macrolactones, fatty acids, polyketides, and isocoumarins have
2
been reported from this species. The production of secondary
1
metabolites is normally associated with the bacterium’s
chromatography and characterized by LC-MS and H NMR
3
1
response to a growth-limiting environment. Faced with the
data. The LC-MS and H NMR metabolic profiling data guided
depletion of essential nutrients, B. subtilis can adopt several
responses, including motility, secretion of extracellular
enzymes, competence for genetic transformation, antibiotic
production, and finally sporulation. Depending on the environ-
mental signal, one or more of these processes can be stimulated
the purification of compounds 1 and 2.
Bacilotetrin A (1) possesses a molecular formula of
C H N O as determined from HR-(+)-ESIMS. The IR
37 66 4 8
−
1
spectra of 1 gave a broad peak at 3361 cm (NH) and 1694
(CO) cm− consistent with the presence of amide carbonyl
1
4
−1
groups, a smaller peak at 1743 cm , which was indicative for
an ester carbonyl, and a peak at 2931 cm confirming the
presence of an aliphatic chain. The H NMR data (Table 1),
or inhibited. This is evident from our studies reporting that
−
1
upon alteration of growth conditions this species produces
5
1
diverse classes of compounds. On the basis of these
recorded in CD OD and CD OH, of the chromatographically
observations, we have reported previously several classes of
metabolites including macrolatins and lipopeptides having
antibacterial and antifungal activities from the marine-derived
3
3
homogeneous material revealed the presence of a peptide
backbone by four NH signals at δ 7.33 to 9.10 together with
H
5
−7
four α-protons at δ 3.74 to 4.60 and a long aliphatic chain at
B. subtilis 109GGC020.
Furthermore, our continuous
H
δ 1.29 (SI 1 and 5). A resonance at δ 5.16 indicated the
chemical investigations on the ethyl acetate extract obtained
from the culture of the same species upon alteration of growth
conditions resulted in the isolation of two previously unknown
cyclic-lipotetrapeptides, bacilotetrins A (1) and B (2). Here, we
described their isolation, characterization, and anti-staph-
ylococcal and cytotoxic activities.
H
H
presence of a further oxygenated proton, and CH signals were
3
13
observed at δ 0.89 to 0.98. The C NMR data of 1 indicated
H
the presence of six carbonyl carbons, four of which resonated at
δC 173.3, 173.9, 174.6, and 175.9, attributed to amino acids, and
the remaining two carbonyl carbons at δ 173.0 and 173.3
C
The producing strain 109GGC020 was isolated from a
marine sediment sample collected from the Gageocho reef,
Republic of Korea, and identified as B. subtilis based on the 16S
Received: April 24, 2017
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.7b00356
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
13
Table 1. H and C NMR Data of 1 and 2 in CD OD
were elucidated for compound 1 (Figure 2). The substructure
3
1a was deduced from the intra-COSY and TOCSY correlations
bacilotetrin A (1)
bacilotetrin B (2)
δC, type δH (J in Hz)
δC, type
δH (J in Hz)
Leu-1
75.9, C
NH
1
176.0, C
a
a
8.40, d (4.5)
4.11, t (7.5)
1.65, m
NH
8.42, d (4.0)
4.07, t (7.0)
1.65, m
5
4
2
2
2
5.5, CH
55.7, CH
40.6, CH2
26.4, CH
21.8, CH3
24.1, CH3
0.7, CH2
6.4, CH
1.8, CH3
4.1, CH3
1.68, m
1.68, m
0.91, m
0.91, m
0.95, m
0.95, m
Leu-2
73.3, C
NH
Figure 1. Structures of bacilotetrins A (1) and B (2).
1
173.3, C
NH
a
a
9.10, d (6.5)
9.10, d (6.5)
5
4.9, CH
3.75, dd (11.0, 4.0) 54.9, CH
3.73, dd (11.0,
.5)
3
38.6, CH2
26.4, CH
21.3, CH3
23.9, CH3
1.65, m
1.68, m
0.91, m
0.95, m
38.6, CH2
26.3, CH
21.3, CH3
23.9, CH3
2.02, m
1.68, m
0.91, m
0.95, m
Leu-3
74.6, C
NH
1
174.6, C
NH
b
a
7.43, d (9.5)
4.44, m
7.74, d (8.5)
4.41, m
5
4
2
2
2
3.4, CH
53.3, CH
40.4, CH2
26.3, CH
21.3, CH3
23.8, CH3
0.5, CH2
5.8, CH
1.3, CH3
3.9, CH3
1.65, m
1.71, m
1.68, m
1.68, m
0.91, m
0.91, m
0.95, m
0.95, m
Glu
1
73.9, C
173.9, C
NH
b
a
Figure 2. COSY, TOCSY, HMBC, and ROESY inter- and intraresidue
correlations for 1.
NH
7.33, d (8.5)
4.58, m
7.77, d (10.0)
4.56, m
5
3
3
1
1.6, CH
51.6, CH
35.5, CH2
31.4, CH2
173.3, C
5.5, CH2
1.5, CH2
73.3, C
1.81, m
1.96, m
2.43, m
2.43, m
in each individual amino acid and inter-HMBC correlations
among α-protons, NH protons, and carbonyl carbons of four
amino acids (Figure 2). The substructure 1b was deduced from
the TOCSY and HMBC correlations of the oxygenated proton
resonating at δ 5.16 with the carbonyl carbon at δ 173.0 and
COOH
COOH
β-OH acid
1
4
73.0, C
173.0, C
1.6, CH2
2.29, dd (14.0, 8.5) 41.5, CH2
2.26, dd (14.0,
.0)
8
H
C
13
2
.73, dd (13.5, 4.5)
2.70, dd (13.5,
.5)
a long aliphatic chain at δ 1.29. The C NMR spectrum of 1
H
4
contained eight methyl carbons; among them, six methyl
carbons were attributed to three Leu residues, and the
remaining two methyl groups were assumed to be present in
7
4
2
3
3
3.8, CH
5.16, m
1.58, m
73.9, CH
5.12, m
1.58, m
0.4, CH2
40.4, CH2
5.8−30.9, CH2 1.29, brds
25.8−33.2, CH2 1.29, brds
the fatty acid chain. One methyl group at δ 14.6 was assigned
C
0.6, CH
1.50, m
29.3, CH
1.52, m
as a terminal methyl, and another methyl group at δ 23.9 was
C
3.2, CH2
1.29, brds
30.8, CH2
1.29, brds
1.29, brds
1.12, m
located at C-11 in the fatty acid unit by the analysis of TOCSY
and HMBC correlations. Based on the molecular weight of 1,
the fatty acid chain was determined to be a C14 fatty acid
amidated to the N-terminal amine of the peptide. The chain
length of the fatty acid was confirmed by the LC-(−)-APCIMS
data of the acid hydrolysate of 1 (SI 22). Thus, the fatty acid
was determined to be the same as the previously described fatty
3
5.8, CH
1
4.6, CH3
3.9, CH3
0.89, m
0.90, m
37.9, CH2
1
.29, brds
2
11.9, CH3
0.87, m
0.86, m
0.87, m
1
2
9.8, CH3
3.2, CH3
a,b
8
Chemical shifts and coupling constants of 1 and 2 were determined
acid 3-hydroxy-11-methyltridecanoic acid. The cyclic-lipote-
13
1
in CD OH and DMSO, respectively. The C and H NMR data of 1
3
trapeptide structure of 1 was then deduced by connecting the
substructures 1a and 1b on the basis of ROESY and HMBC
experiments showing long-range correlations of an NH proton
at δ 8.40 and an α-proton at δ 4.11 of Leu to the methylene
and 2 were measured at 125 and 500 MHz, respectively.
(
overlapped) were attributable to ester and carboxylic
H
H
1
functionalities. These detailed IR absorbances together with
the H and C NMR data analysis revealed the lip-
otetrapeptidic nature of 1.
On the basis of COSY, TOCSY, and HMBC correlations,
two partial substructures, 1a, a tetrapeptide consisting of four
amino acids, and 1b, a lipophilic unit (3-hydroxy fatty acid),
signals at δ 2.29/2.73 and a carbonyl carbon at δ 173.0 of the
H
C
1
13
fatty acid, resulting in the sequence Leu-Leu-Leu-Glu-fatty acid
(Figure 2). The point of cyclization of 1 was deduced from the
long-range HMBC correlation of an oxygenated proton at δH
5.16 of the fatty acid with a carbonyl carbon at δ 173.9 of the
C
carboxyl group of the C-terminal Glu residue.
B
DOI: 10.1021/acs.jnatprod.7b00356
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Journal of Natural Products
Article
a
Table 2. Anti-Staphylococcal Activity of Bacilotetrin A (1) and Bacilotetrin B (2)
MRSA strain (MIC, μg/mL)
compound
ATCC25923
XU2120
SA1199B
RN4220
EMRSA15
bacilotetrin A
bacilotetrin B
norfloxacin
8
16
16
16
16
16
8
32
64
32
NA
2
NA
NA
4
a
Norfloxacin: positive control; NA: not active.
Bacilotetrin B (2) possesses a molecular formula of
C H N O deduced by HR-(+)-ESIMS. Comparison of the
H NMR chemical shifts of 2 with those of 1 implied that they
conducted with a PrimeLine binary pump with RI-101 (Shodex) and
variable UV detector (M 525). Semipreparative HPLC was performed
using ODS (YMC-Pack-ODS-A, 250 × 10 mm i.d. 5 μm) and silica
3
8
68
4
8
1
(
YMC-Pack-SIL, 250 × 10 mm i.d, 5 μm) columns. Analytical HPLC
shared the same planar structure except for the presence of an
additional methylene group as indicated by the molecular
formula. The methylene group might be located either on the
alkyl chain or on the peptide chain. To determine the exact
location of the methylene group, a detailed investigation on the
structure of 2 was undertaken by a series of 1D and 2D NMR
experiments (SI 15 to 20). These experiments showed the
presence of the same amino acid sequence Leu-Leu-Leu-Glu in
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 seawater was collected from the East Sea of South
Korea at depths of 20 m.
Isolation and Taxonomy of the Strain 109GGC020. The strain
109GGC020 was isolated from a sediment sample collected from the
Gageocho reef, Republic of Korea, in 2010 and cultured on a modified
Bennett’s media agar plate (media composition: 1 L of 100% natural
seawater with a salt concentration of 18.5 g/L, 1% dextrose, 0.1% yeast
extract, 0.1% beef extract, 0.2% tryptone, and 1.8% agar with the pH
adjusted to 7.1) and 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 B. subtilis
109GGC020 under the curatorship of Hee Jae Shin.
1
and 2. Therefore, the additional methylene group must be
located in the alkyl chain. This observation was confirmed by
the LC-(−)-APCIMS peak at m/z 257.05 [M − H] of the
hexane phase of the acid hydrolysate of 2 (SI 23) and identified
−
the lipophilic part as a 3-hydroxytetradecanoic acid. The careful
1
3
interpretation of the C NMR data of 2 revealed nine methyl
carbons at δ 11.9 to 23.9 in 2, six of them attributed to three
Culture Conditions. The seed culture of the strain 109GGC020
was performed in triplicate into 100 mL flasks, containing 50 mL of
medium (composition as above). An aliquot (5 mL) from the seed
culture was inoculated aseptically into 2 L flasks (total 50 flasks)
containing 1.2 L of sterilized culture medium with the same
composition. The growth conditions for the strain was optimized to
obtain maximum production prior to large-scale culture by varying salt
concentrations in the medium and monitoring the metabolite
production by H NMR analysis as described before. The production
culture was incubated with constant shaking at 28 °C for 7 days.
Extraction and Isolation of Compounds. The culture broth was
filtered by high-speed centrifugation, and the cell-free filtrate was
extracted with EtOAc (two times). The EtOAc layer was evaporated to
dryness under reduced pressure using a chilled rotary evaporator and
subjected to ODS (C18) open column chromatography followed by a
C
Leu residues by 2D data analysis and the remaining three
methyl groups located in the fatty acid chain based on the
TOCSY and HMBC correlations. The methyl carbon
resonating at δ 11.9 was located as a terminal methyl, and
C
the remaining methyl carbons resonating at δ 19.8 and 23.2
C
were located at C-9 and C-11 in the fatty acid unit, respectively.
1
5
The fatty acid was then confirmed as a previously reported 3-
9
hydroxy-9,11-dimethyltridecanoic acid. Finally, the structure of
2
was elucidated based on the same tetrapeptide chain Leu-
Leu-Leu-Glu in 1, cyclized at the C-terminal of Glu by the 3-
hydroxy-9,11-dimethyltridecanoic acid.
The absolute stereochemistry of the amino acid residues
Leu1−3 and Glu) in 1 and 2 was determined to be L by acid
(
stepwise gradient program of MeOH in H O (1:4, 2:3, 3:2, 4:1, and
2
hydrolysis and derivatization with Marfey’s reagent followed by
1
00:0) as eluent. The 100% MeOH fraction, which showed interesting
10
chiral HPLC analysis. In addition, the 3R-hydroxy config-
uration of the fatty acids in 1 and 2 was assigned by direct
characteristic peaks of peptides, was subjected to further fractionations
and purification by C18 semipreparative and analytical HPLC using
2
7
comparison of their specific rotation values [α] −6.2 (c 0.05,
isocratic elution with 90% MeOH in H O to yield pure compounds 1
D
2
MeOH) and [α]27 −29.0 (c 0.05, MeOH), respectively, with
and 2.
D
7
,8,11
Bacilotetrin A (1) (5.8 mg): amorphous solid; [α]27 −22.1 (c 0.05,
those of previously reported 3-hydroxy fatty acids.
D
−
1
MeOH); IR (MeOH) ν 3366 (NH), 1694 (CO), 1743, 2931 cm
Anti-staphylococcal activity of compounds 1 and 2 was
evaluated against clinically isolated MRSA strains
max
1
13
(
aliphatic chain); H and C NMR data (CD OD and CD OH), see
3 3
+
Table 1; HR-(+)-ESIMS m/z 695.4953 [M + H] (calcd for
(
ATCC25923, XU212, SA1199B, RN4220, and EMRSA15)
+
C H N O , 695.4959 [M + H] ).
3
7
66
4
8
by the broth dilution assay according to the guidelines of the
BSAC (Table 2). Compounds 1 and 2 were found to be
27
Bacilotetrin B (2) (3.6 mg): amorphous solid; [α] −18.6 (c 0.05,
1
2
D
−1
MeOH); IR (MeOH) ν 3260 (NH), 1575 (CO), 1734, 2925 cm
max
1
13
noncytotoxic when tested against two human cancer cell lines
(
aliphatic chain); H and C NMR data (CD OD and CD OH),
3 3
+
(
prostate and liver cancer cell lines) by the sulforhodamine B
Table 1; HR-(+)-ESIMS m/z 731.4935 [M + Na] (calcd for
1
3
+
(SRB) assay.
C
38
H
68
N
4
O
8
, 731.4935 [M + Na] ).
Acid Hydrolysis of 1 and 2. Compound 1 (1.5 mg) was heated at
23 °C for 23 h with 6 N HCl (500 μL) under constant stirring. The
1
EXPERIMENTAL SECTION
■
reaction was monitored from time to time by LC-MS analysis. The
reaction mixture was cooled, mixed with water, and extracted with
hexane (Hx). The Hx extract was concentrated under a stream of N2,
and the aqueous part was evaporated to dryness under reduced
pressure. The Hx extract containing a fatty acid (1a) was subjected to
recording specific rotation values to assign the stereochemistry at C-3
of the fatty acid, and the aqueous part was used for the determination
of the stereochemistry of the amino acids by Marfey’s method. In an
General Experimental Procedures. NMR spectroscopic data
were acquired on a Varian Unity 500 spectrometer. 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.
High-resolution ESIMS data were recorded on a hybrid ion-trap time-
of-flight mass spectrometer (Shimadzu LC/MS-IT-TOF). HPLC was
C
DOI: 10.1021/acs.jnatprod.7b00356
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
analogous way, the absolute stereochemistry at C-3 of the fatty acid
and amino acids in 2 was assigned.
Amino Acid Analysis by Marfey’s Method in 1 and 2. The
Mohd. Hanafi, School of Pharmacy, University College
London, United Kingdom, for the cytotoxicity test.
aqueous hydrolysate of 1 was resuspended in H O (100 μL) and
treated with 0.1% 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide solution
2
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■
(
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in acetone (Marfey’s reagent, 20 μL) and 1 N NaHCO (10 μL) at 40
3
°
C for 2 h. The solution was cooled to room temperature, neutralized
with 1 N HCl (4 μL), and evaporated to dryness. The residue was
(
2
(
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reversed-phase HPLC (YMC-Pack ODS, 250 × 4.6 mm, 5 μm, flow
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3
2
(
containing 0.1% acetic acid (AcOH) for 60 min. Similarly, standard
amino acids (both L and D) were derivatized with Marfey’s reagent and
analyzed by HPLC. The derivatized Leu and Glu residues in the
hydrolysate of 1 were obtained at the same retention time as the
derivatized standard L-Leu (39.0 min) and L-Glu (29.6 min). In a
similar manner, the hydrolysate of 2 was analyzed and had the same
retention time as the derivatized standard L-Leu (39.0 min) and L-Glu
(
Development. In Bacillus subtilis and Other Gram-Positive Bacteria;
Sonenshein, A, Hoch, J., Losick, R., Eds.; ASM Press: WA, 1993; pp
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(
85−800.
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9) Tareq, F. S.; Lee, M.; Lee, H. S.; Lee, Y. J.; Lee, J. S.; Hasan, C.
(
(
29.6 min).
Anti-Staphylococcal Activity Test of 1 and 2. The anti-MRSA
(
activity of compounds 1 and 2 was investigated with ATCC 25923,
SA-1199B, RN4220, XU212, EMRSA-15, and EMRSA-16 by the broth
dilution assay. These strains were cultured on nutrient agar (Oxoid)5
plates and incubated for 24 h at 37 °C. An inoculum density of 5 × 10
colony-forming units of each bacterial strain was prepared in normal
saline (9 g/L) by comparison with a 0.5 MacFarland turbidity
standard. The inoculum (125 μL) was added to all wells, and the
microtiter plate was incubated at 37 °C for the corresponding
incubation time. For MIC determination, 20 μL of a 5 mg/mL
methanolic solution of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra-
zolium bromide (MTT) was added to each of the wells and incubated
for 20 min. Bacterial growth was indicated by a color change from
yellow to dark blue. The MIC was recorded as the lowest
(
(
M.; Islam, M. T.; Shin, H. J. J. Agric. Food Chem. 2014, 62, 5565−5572.
(
(
1
10) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591−596.
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2
(12) Andrews, J. M. J. Antimicrob. Chemother. 2001, 48, 29−42.
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Natl. Cancer Inst. 1990, 82, 1107−1112.
5
concentration at which no growth was observed.
Cytotoxicity Test by SRB Assay. Cancer cell growth inhibitory
activity of compounds 1 and 2 was determined according to an SRB
assay. In brief, a 96-well plate was loaded with selected cell lines (liver
cancer and prostate cancer), and test samples (30, 10, and 3 μg/mL)
were added. After incubation for 48 h, anchorage-dependent cells were
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 was dissolved in 10 mM Tris
base solution, and absorbance was measured at 510 nm using a
microplate reader.
ASSOCIATED CONTENT
Supporting Information
■
*
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.7b00356.
1D and 2D NMR spectra of 1 and 2, amino acid analysis
data, and HRESIMS data (PDF)
AUTHOR INFORMATION
Corresponding Author
Tel: +82-31-400-6172. Fax: +82-31-400-6170. E-mail:
■
*
shinhj@kiost.ac.kr.
ORCID
Hee Jae Shin: 0000-0002-5952-5325
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported in part by the Ministry of Oceans
and Fisheries, Korea (Grants PM59880 to J.S.S. and PM60300
to H.J.S.). The authors would like to thank Mohd. Mukrish
D
DOI: 10.1021/acs.jnatprod.7b00356
J. Nat. Prod. XXXX, XXX, XXX−XXX