Xentrivalpeptides A-Q: Depsipeptide diversification in xenorhabdus

Article abstract of DOI:10.1021/np300279g

Seventeen depsipeptides, xentrivalpeptides A-Q (1-17), have been identified from an entomopathogenic Xenorhabdus sp. Whereas the structure of xentrivalpeptide A (1) was determined after its isolation by NMR spectroscopy and the advanced Marfey's method, the structures of all other derivatives were determined using a combination of stable isotope labeling and detailed MS analysis.

Full text of DOI:10.1021/np300279g

Article
pubs.acs.org/jnp
Xentrivalpeptides A−Q: Depsipeptide Diversification in Xenorhabdus
Qiuqin Zhou,† Andrea Dowling,‡ Heinrich Heide,§ Jens Wohnert,† Ulrich Brandt,§ James Baum,⊥
̈
Richard ffrench-Constant,‡ and Helge B. Bode*,†
†Institut fur Molekulare Biowissenschaften, Goethe Universitat Frankfurt, 60438 Frankfurt am Main, Germany
̈
̈
‡Biosciences, University of Exeter in Cornwall, Tremough Campus, Penryn, Cornwall TR10 9EZ, U.K.
§Zentrum fur Biologische Chemie, Molekulare Bioenergetik, Klinikum der Goethe Universitat Frankfurt, 60590 Frankfurt am Main,
̈
̈
Germany
⊥Monsanto Company, Chesterfield, Missouri 63017, United States
S
* Supporting Information
ABSTRACT: Seventeen depsipeptides, xentrivalpeptides A−Q (1−17), have
been identified from an entomopathogenic Xenorhabdus sp. Whereas the
structure of xentrivalpeptide A (1) was determined after its isolation by NMR
spectroscopy and the advanced Marfey's method, the structures of all other
derivatives were determined using a combination of stable isotope labeling
and detailed MS analysis.
ntomopathogenic bacteria of the genera Xenorhabdus and
Photorhabdus live in symbiosis with nematodes of the
Monsanto Company when grown in LB medium with 2%
Amberlite XAD-16. From a 5 L culture grown under the same
conditions, 22 mg of 1 was isolated from the XAD extract using
preparative HPLC/MS. The molecular formula of 1 was
determined from HRESIMS analysis (m/z 860.4880) as
C46H65N7O9 (Table S1, Figure S1), and the structure of 1
was determined by detailed 1D (1H,13C) and 2D (COSY,
HSQC, HMBC, TOCSY) NMR experiments (Table 1). Eight
different spin systems were identified on the basis of coupling
E
genera Steinernema and Heterorhabditis, respectively, and
together with them are able to infect and kill different insect
larvae.1−4 Although protein toxins are mainly responsible for
larval mortality,5,6 small molecules that are toxic to insect cells
have also been identified.7 It has been postulated that these
bacteria produce antibiotics to protect the insect cadaver from
competitors living in the soil, and compounds showing these
activities have been identified recently.7−9 Besides several small
molecules such as isopropylstilbenes,10,11 anthraquinones,11,12
and xenofuranones,13 recent work has shown that several
peptides are produced by these bacteria. Among them are the
highly polar PAX peptides,14 the GameXPeptides,15 and the
depsipeptides xenematide16,17 and szentiamide.18 Of these, the
PAX peptides,14,19 GameXPeptides,15 and xenematides16,17 are
produced as mixtures of several derivatives. Thus, Xenorhabdus
and Photorhabdus have proven to be a rich source of bioactive
natural products, and our goal is to identify additional bioactive
natural products from them. Here we describe the identification
and structure elucidation of the xentrivalpeptides, depsipeptides
that show a much higher chemical diversity than the
xentiamides16,17 and szentiamide,18 the two known depsipep-
tide classes from Xenorhabdus.
1
constants in the H NMR spectrum and COSY data (Figure
1a). Several signals around 1 ppm were assigned to methyl
groups from four valines and one threonine, and in addition,
two phenyl groups were identified on the basis of the typical
chemical shifts of δH 7.20−7.30 with total integration of 10
protons and typical δC shifts between 127 and 138 in the 13C
NMR spectrum. Each spin system could be connected with one
of eight quaternary carbons in the range δC 169.5−174.9 by
HMBC correlations (Figure 1b). In this way, the linear
sequence derived from the connections of α-H with quaternary
carbons and the connection between the β-H in Thr (3) and
the quaternary carbon in Val (7) established the structure of
the depsipeptide with a six-membered ring (Figure 1b).
The absolute configurations of the amino acids were
determined using the advanced Marfey's method,20 showing
that only phenylalanine has the D-configuration (Table S2). We
RESULTS AND DISCUSSION
■
During our search for new secondary metabolites from
Xenorhabdus bacteria, we identified compound 1 with m/z
860.5 in extracts of Xenorhabdus sp. 85816 obtained from the
Received: April 17, 2012
Published: October 1, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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Table 1. NMR Spectroscopic Data (500 MHz (1H), 125
a
MHz (13C) in CD3OD) of 1, δ in ppm
subunit
PA
position
δC
δH, mult (J in Hz)
1
174.2
43.6
2
3.60, d (1.0)
3
137.0
130.3
129.7
128.2
172.8
60.2
b
c
4
7.29, m
7.30, m
7.25, m
5
6
L-Val(1)
CO
α
4.25, d (7.3)
2.10, m
β
31.7
γ
19.8
0.94, d (6.7)
0.92, d (6.8)
d
δ
18.6
L-Thr(2)
D-Phe(3)
CO
169.5
55.0
α
4.79, d (3.8)
5.09, m
β
69.9
γ
14.6
1.14, d (2.4)
CO
171.3
55.3
α
4.94, dd (5.2, 9.9)
2.91, dd (9.8, 12.6)
3.24, dd (5.0, 12.5)
β1
40.5
β2
40.5
γ
137.0
130.6
129.7
127.9
174.9
62.4
b
c
δ
7.27−7.29
7.27−7.29
7.20−7.24
Figure 1. Subunits (a) with selected COSY (bold lines) and HMBC
correlations (arrows) in xentrivalpeptide A (1) (b). Amino acid
numbering from N- to C-terminus.
ε
ζ
L-Pro(4)
CO
Table 2. Data for Xentrivalpeptides A−Q (1−17)
α
4.09, dd (4.9, 8.7)
1.79, m
β1
30.7
a
b
no.
compound
tR/min
amount
fragment B (m/z)
β2
30.7
1.89, m
1
A
B
C
D
E
10.4
6.4
100.0
1.1
625.3
γ1
25.5
1.47, m
2
γ2
25.5
1.79, m
3
7.6
<1.0
3.0
δ1
48.6
2.68, m
4
8.8
δ2
48.6
3.44, m
5
9.8
2.1
L-Val(5)
L-Val(6)
L-Val(7)
CO
173.6
65.5
6
F
12.7
11.5
9.8
<1.0
<0.1
<1.0
3.5
α
3.64, d (11.3)
2.31, m
7
G
H
I
β
30.8
8
γ
20.3
0.89, d (6.4)
0.98, d (6.4)
9
11.3
8.2
δ
20.1
10
11
12
13
14
15
16
17
J
<1.0
<1.0
2.1
591.3
CO
173.5
58.0
K
L
9.2
α
4.42, d (5.9)
2.04, m
9.8
β
33.6
d
M
N
O
P
12.2
12.3
9.8
<0.1
3.7
γ
18.7
0.93, d (7.0)
0.99, d (7.0)
639.3
611.3
δ
19.7
<1.0
<0.1
<0.1
CO
172.0
62.4
7.6
α
β
γ
3.94, d (7.7)
2.07, m
c
Q
8.5
526.3
30.2
a
b
Amount relative to 1 calculated from peak areas in HPLC/MS. For
19.8
1.13, d (6.7)
1.04, d (6.8)
b-d
c
details on fragment B see Figure 2. 17 has a smaller ring than the
other derivatives (see structure below and Figure S7). Retention time
(tR).
δ
18.9
a
PA: phenyl acetyl; d: doublet; m: multiplet.
interchangeable.
Assignments are
applied a combination of labeling and detailed MS analysis to
strain 85816, as previously described, to differentiate isobaric
building blocks such as leucine and isoleucine.15 From the
results of labeling experiments using deuterated or fluorinated
building blocks in LB medium, and an inverse labeling
experiment adding nonlabeled 12C building blocks to a culture
grown in fully 13C-labeled medium, the building blocks of 1
could be confirmed (Figure 3). For example, labeling with p-
fluorophenylalanine or p-fluorophenylacetic acid in LB medium
showed the expected +18 Da shift to m/z 878.5 (Figure 3b and
c), and labeling with phenylalanine in a 13C-labeled culture
analyzed strain 85816 for other derivatives of this class of
depsipeptides and could indeed identify several, which we
named xentrivalpeptides because they were isolated from a
Xenorhabdus strain and all contained at least three (tri) valines.
All xentrivalpeptides showed similarity to 1 based on the MS
fragmentation patterns (Table 2, Table S1). We performed
extensive MS fragmentation experiments with 1 (Table S1,
Figure S1), which revealed a general fragmentation pattern for
this class of peptides (Figure 2) used for the structure
elucidation of the other xentrivalpeptides. Additionally, we
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Figure 2. General fragmentation pathway of xentrivalpeptides A−P (1−16) and the proposed fragment structures. For fragment data see Table S1.
showed the expected shift to a lower mass due to the
incorporation of nine and eight carbons from incorporated
phenylalanine and phenylacetic acid, respectively (Figure 3j).
Moreover, from cultivation in 13C and 15N medium the number
of carbon and nitrogen atoms of 1 could be determined easily,
thus affording the correct molecular formula as shown
previously15 (Figure 3f and g).
The analysis of these labeling experiments followed by MS
fragmentation experiments (Figures S2−S5) allowed the
structure elucidation of xentrivalpeptides B−P (2−16). The
absolute configurations of the amino acids were not determined
experimentally due to their low production titer (Table 2), but
are assumed to follow that of 1.
The differences between 1−9 were only in the side chain, as
shown by an identical ring fragment B of m/z 625.3 (Figure 2,
Table S1) and confirmed by labeling experiments showing the
presence of the same ring amino acids as in 1 (Figure S2).
Xentrivalpeptides A−G (1−7) differed from each other at R1
(acyl moiety) only. The nature of R1 in these derivatives was
unambiguously determined from labeling experiments: D6-
propionic acid was incorporated into 3, as detected from a mass
shift of +5 Da from m/z 798.5 to 803.5, 12C-butyric acid was
incorporated in a 13C-labeled culture into 4, as shown by a shift
of −4 Da from m/z 854.5 (U-13C-labeled 4) to m/z 850.5, and
five carbons from the 12C-leucine-derived isovaleryl unit were
incorporated into 5 and 6 in 13C medium due to the presence
of isovaleryl (in 5) or 5-methylhexyl acyl groups (in 6),
resulting in a −5 Da shift in both compounds. Additionally, a
sufficient amount of 4 was isolated allowing full characterization
via NMR spectroscopy (Table S3, Figure S6). The presence of
an acetyl moiety in 2 was deduced from the HRESIMS data,
and the valine labeling results showed that one valine is present
in the side chain (amino acid two), thus securing the acetyl
group as the acyl moiety. The presence of a hexanoyl moiety in
7, which was produced only in very minor amounts (Table 2),
was proposed, as neither four carbons from valine (as
isobutyryl) nor leucine were incorporated into the acyl chain
and assuming that the acyl moiety is not branched (data not
shown). For 8 and 9 the incorporations of α-aminobutyric acid
and isoleucine in 13C medium were detected at amino acid 1
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instead of the usual valine due to the mass shifts of 4 and 6 Da,
respectively, and an unchanged fragment B for both
compounds (Figure S2). As isoleucine is incorporated into 9,
the configuration in the isoleucine side chain is proposed to be
S, as for the natural amino acid. The R1 group was determined
to be phenylacetyl in 8 and 9 from the 8 Da mass shifts from
12C-phenylalanine in the 13C medium and incorporation of p-
fluorophenylacetic acid (Figure S2).
Fragmentation of xentrivalpeptides J−M (10−13) revealed a
fragment B with m/z 591.3 (Table S1). The difference of −34
Da compared to fragment B of 1 points to a leucine/isoleucine
instead of a phenylalanine, which was confirmed by the
incorporation of D10-leucine into 10−13 (Figure S3) and no
incorporation of phenylalanine into fragment B in these
compounds. Moreover, MS fragmentation proved that 10−12
showed an identical fragmentation of fragment B with
fragments D and E showing the mass shift of 34 Da. As valine
incorporation could be observed in the side chain for 10−13,
these compounds again differed only in the acyl moiety, whose
nature was assigned from the incorporation of butyric acid in
10, leucine-derived isovalerate incorporation in 11 and 13, and
the incorporation of 4-fluorophenylacetic acid or phenylalanine
in 12 (Figure S3), as described above for 4, 5, and 1,
respectively.
Figure 3. MS spectra from labeling experiments of xentrivalpeptide A
(1). LB medium (12C, a), LB medium with p-fluoro-DL-phenylalanine
(12C + p-F-Phe, b), with p-fluorophenylacetic acid (12C + p-F-PAA, c),
2
with additional L-[2,3,3,5,5′,6,6′,7-2H]phenylalanine (12C + H8-Phe,
d), with DL-[2,3,4,4,4,5,5,5-2H8]valine (12C + 2H8-Val, e), 15N medium
(15N, f), 13C medium (13C, g), 13C medium with L-proline (13C + Pro,
h), with L-valine (13C + Val, i), with L-phenylalanine (13C + Phe, j),
and with L-threonine (13C + Thr, k), respectively.
Xentrivalpeptide N (14, C47H67N7O9) showed a fragment B
(Table S1) with m/z 639.3, indicating the presence of leucine/
isoleucine instead of valine in the ring, as was confirmed by
labeling with isoleucine in fully 13C-labeled medium and by the
expected mass shift of 6 Da in fragment B (Figure S4). The
position of the isoleucine was readily identified from MS
fragmentation experiments that confirmed the 14 Da mass shift
only in fragments C, E, and F (Table S1, Figure 2), which is
consistent with isoleucine (2S,3S configuration as in 9) as
amino acid 6. All other feeding experiments confirmed that 14
is otherwise identical to 1.
Fragment B of xentrivalpeptides O (15) and P (16) showed
m/z 611.3 (Table S1), indicating the loss of a methyl group
compared to 1. Thus the presence of α-aminobutyric acid
instead of valine was proposed. This was confirmed in the
respective feeding experiment (Figure S5). Fragments C, E, and
F showed the −14 Da mass shift compared to 1, thus again
confirming amino acid 6 as the variable position (Table S1).
Whereas the structure of 15 could be confirmed from labeling
experiments and MS fragmentation experiments (Figure S5),
labeling of 16 was very weak. However, as no labeling of leucine
or isoleucine was observed and butyrate is an abundant starting
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Therefore, current efforts in our group are directed toward
synthesizing the xentrivalpeptides to verify the proposed
absolute configuration and to broaden the scope of biological
testing.
EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were
determined with a MPM-H2 melting point meter by Schropp
■
Geratetechnik and are uncorrected. Optical rotations were measured
̈
on a Perkin-Elmer 341 polarimeter. UV spectra were recorded with a
GE NanoVue Plus photometer. IR spectra were obtained with a
Perkin-Elmer Spectrum Two spectrometer. NMR spectra were
recorded with a Bruker AV 400 spectrometer using deuterated
methanol as solvent. Collision-induced dissociation (CID) was
performed on the ion trap in the amaZon X in positive ion mode,
and HRESIMS analysis was carried out using an LTQ Orbitrap
(Thermo Fisher). Preparative HPLC was performed using an Waters
autopurification system.
unit for xentrivalpeptides (Table 2), we postulate that 16 has a
butyryl unit as found in 4.
Strain Cultivation and Extract Preparation. Xenorhabdus sp.
85816 was identified as X. stockiae based on its recA sequence
(Genbank accession number JX485977) and was cultivated in LB
medium at 30 °C. For extract analysis, 20 mL of LB medium was
inoculated with 1% overnight culture, and 2% Amberlite XAD-16 was
added. After cultivation for 72 h, the XAD beads were collected,
washed once with 5 mL of H2O, and extracted with 20 mL of MeOH.
To isolate the peptides, XAD beads from a 5 L culture were extracted
with 200 mL of MeOH three times. The extract was fractionated on
silica gel with CHCl3 and MeOH with a linear gradient of 5% to 40%
MeOH. The fractions were analyzed by HPLC-MS, and fractions
eluted with ∼20% MeOH contained the xentrivalpeptides. The
enriched fraction containing xentrivalpeptides was used for HPLC
purification.
The molecular formula of C41H56N6O8 for xentrivalpeptide
Q (17) as determined by HRESIMS indicated the loss of one
valine moiety compared to 1. Fragmentation for 17 differed
significantly from that of 1−16. Fragments A′ and B′ with m/z
544.3 and 526.3, which correspond to fragments A and B in 1−
16, showed the expected loss of one valine moiety due to the
loss of 99 Da. Additional fragments confirmed that only one
valine in the ring is missing (Figure S7). Thus, 17 was
confirmed to be a ring-contracted derivative of 1.
Feeding Experiments in Nonlabeled Medium. The cell pellet
from a 1 mL overnight Xenorhabdus sp. 85816 culture was washed
once and resuspended with 1 mL of LB medium. The 5 mL feeding
culture was inoculated with a 1% (50 μL) solution of washed cells.
After incubation for 6 h at 30 °C, 200 rpm, 50 μL of a stock solution
(100 mM) of substrate (p-fluoro-DL-phenylalanine, p-fluorophenyl-
acetic acid, L-[2,3,3,5,5′,6,6′,7-2 H8 ]phenylalanine, DL-
[2,3,4,4,4,5,5,5-2H8]valine, L-[2,3,3,4,5,5,5,5′,5′,5′-2H10]leucine, or
[U-2H6]propionic acid) was added. Two further feedings of substrate
were carried out after 24 and 48 h to a final concentration of 3 mM.
Cultures were harvested after 72 h of incubation by extraction with 5
mL of EtOAc. The extracts were evaporated to dryness and dissolved
in 500 μL of MeOH. Diluted solutions were analyzed by HPLC-MS,
and control cultivation was carried out without feeding.
Feeding Experiments in Labeled Medium. The cell pellet from
a 1 mL overnight Xenorhabdus sp. 85816 culture was washed once and
resuspended with 1 mL of ISOGRO-13C medium. ISOGRO-13C
medium was prepared with 1 g of ISOGRO-13C powder (Sigma-
Aldrich), 1.8 g/L K2HPO4, 1.4 g/L KH2PO4, and 11.1 mg/L
CaCl2·H2O in 100 mL of H2O. The feeding culture was started by
inoculation of a 1% (50 μL) solution of washed cells in 5 mL of
ISOGRO-13C medium. After incubation for 6 h at 30 °C, 200 rpm, 50
μL of a stock solution (100 mM) of substrate (L-α-aminobutyric acid,
butyric acid, L-α-valine, L-α-leucine, L-α-proline, L-α-threonine, L-α-
phenylalanine, or L-α-isoleucine) was added. Two further feedings of
every substrate were carried out after 24 and 48 h of incubation to a
final concentration of 3 mM. Cultures were harvested after 72 h of
incubation by extraction with 5 mL of EtOAc. The extracts were
evaporated to dryness and dissolved in 500 μL of MeOH. Diluted
solutions were analyzed by HPLC-MS. Control cultivation was carried
out without feeding. Xenorhabdus sp. 85816 was also cultivated in 15N-
labeled medium without feeding of substrate. The 15N-labeled medium
was prepared in the same manner as the 13C-labeled medium.
HPLC and Mass Spectrometry. Analysis of the extracts was
carried out on an Ultimate 3000 LC system from Dionex, coupled to
an amaZon X electrospray ionization mass spectrometer from Bruker
Daltonics. Peptides were separated on a C18 column (Acquity UPLC
The xentrivalpeptides are the third and by far the most
diverse class of depsipeptides isolated from Xenorhabdus8,16−18
and thus a nice example of natural combinatorial biochemistry,
which in addition to the N-acyl variability is in part due to
relaxed amino acid specificities in the corresponding
adenylation (A) and condensation (C) domains of the
nonribosomal peptide synthetase (NRPS) responsible for
biosynthesis.21−23 Whereas this was sometimes interpreted as
a “mistake” of the respective enzymes in the past, it might in
fact be beneficial for the producing organism to generate a large
chemical diversity with a minimal set of building blocks and
enzymes.24 As these derivatives might also have different
biological activities, the resulting compounds might allow
Xenorhabdus to kill several different insect larvae as well as to
protect them against different food competitors. However, so
far no biological activity has been detected for 1 in standard
bioactivity tests (antibiotic Gram-negative or Gram-positive,
antifungal, cytotoxic against eukaryotic cells). Whereas bio-
logical activity for other Xenorhabdus-derived compounds has
been detected at low concentrations,7 only a high concentration
(100 μg/mL) of 1 led to actin ruffling, pointing to the
cytoskeleton as the target for the xentrivalpeptides (Figure S8),
as will be investigated in the future in more detail.
Because the xentrivalpeptides lack any unusual amino acids,
their synthesis should be facile using solid-phase methods.8
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BEH, 1.7 μm 2.1 × 50 mm, flow rate 0.6 mL/min, Waters).
Acetonitrile−H2O containing 0.1% HCOOH was used as the mobile
phase under a linear gradient from 35% to 55% CH3CN over 11 min.
The relative amount of derivatives was obtained by comparison of the
peak areas to the peak area of 1. For preparative purification an
Xbridge column (Waters, OBD, 5 μm, 19 × 150 mm) was used with
CH3CN−H2O containing 0.1% HCOOH as the mobile phase with a
linear gradient from 35% to 55% CH3CN over 22 min for the
separation.
(4) Waterfield, N. R.; Ciche, T.; Clarke, D. Annu. Rev. Microbiol.
2009, 63, 557−574.
(5) Bowen, D.; Rocheleau, T. A.; Blackburn, M.; Andreev, O.;
Golubeva, E.; Bhartia, R.; ffrench-Constant, R. H. Science 1998, 280,
2129−2132.
(6) ffrench-Constant, R. H.; Dowling, A.; Waterfield, N. R. Toxicon
2007, 49, 436−451.
(7) Proschak, A.; Schultz, K.; Herrmann, J.; Dowling, A. J.;
Brachmann, A. O.; ffrench-Constant, R.; Muller, R.; Bode, H. B.
̈
Xentrivalpeptide A (1): colorless solid; mp 140 °C; [α]20 −33 (c
ChemBioChem 2011, 12, 2011−2015.
D
0.36; CHCl3); UV (MeOH/H2O) λmax (log ε) 218 (4.34), 258 (3.82);
IR νmax 3280, 2967, 1756, 1633, 1531, 1454, 1157 cm−1; for NMR data
see Table 1; HRESIMS m/z 860.4880 [M + H]+ (calcd for
C46H66N7O9, 860.4922).
(8) Nollmann, F. I.; Dowling, A.; Kaiser, M.; Deckmann, K.; Grosch,
S.; ffrench-Constant, R.; Bode, H. B. Beilstein J. Org Chem 2012, 8,
528−533.
(9) Park, D.; Ciezki, K.; van der, H. R.; Singh, S.; Reimer, D.; Bode,
H. B.; Forst, S. Mol. Microbiol. 2009, 73, 938−949.
(10) Joyce, S. A.; Lango, L.; Clarke, D. J. Adv. Appl. Microbiol. 2011,
76, 1−25.
Determination of the Absolute Amino Acid Configurations
(ref 20). Approximately 0.5 mg of 1 was hydrolyzed with 0.8 mL of 6
M HCl in an ACE high-pressure tube at 110 °C for 16 h. The
hydrolysate was evaporated to dryness and resuspended in 100 μL of
H2O. To each half-portion (50 μL) were added 10 μL of 1 M
NaHCO3 and 100 μL of 1% FDLA Nα-(5-fluoro-2,4-dinitrophenyl)-L-
leucinamide or D-leucinamide (L-FDLA or D-FDLA, solution in
acetone), respectively. The reaction vials were closed and placed in a
water bath at 40 °C for 1 h. After that, the reactions were cooled to
room temperature, quenched with 10 μL of 1 M HCl, and evaporated
to dryness. The residue was dissolved in 400 μL of MeOH. The
analyses of L- and LD-FDLA-derivatized amino acids were carried out
with LC-MS. Acetonitrile−H2O containing 0.1% HCOOH was used
as solvent with a linear gradient from 20% to 60% CH3CN over 34
min.
Bioactivity Tests. Xentrivalpeptide A (1) was tested against Gram-
negative (Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis)
and Gram-positive bacteria (Staphylococcus aureus, S. epidermidis,
Micrococcus luteus, Bacillus subtilis), the yeast Saccharomyces cerevisiae,
the eukaryotic cell lines L-929 (mouse connective tissue fibroblast;
ACC 2) and HL-60 (human acute myeloid leukemia; ACC 3), and
Galleria mellonella hemocytes as described previously.7
(11) Richardson, W. H.; Schmidt, T. M.; Nealson, K. H. Appl.
Environ. Microbiol. 1988, 54, 1602−1605.
(12) Brachmann, A. O.; Joyce, S. A.; Jenke-Kodama, H.; Schwar, G.;
̈
Clarke, D. J.; Bode, H. B. ChemBioChem 2007, 8, 1721−1728.
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ASSOCIATED CONTENT
* Supporting Information
■
(21) Finking, R.; Marahiel, M. A. Annu. Rev. Microbiol. 2004, 58,
453−488.
S
HRMS and fragmentation data for all compounds, NMR data
for 1 and 4, and activity of 1 against G. mellonella hemocytes.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +49 69 798 29557. Fax: +49 69 798 29527. E-mail: h.
bode@bio.uni-frankfurt.de.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Work in the Bode lab was supported by the Monsanto
Company, the Deutsche Forschungs-gemeinschaft (DFG), and
the European Community’s Seventh Framework Program
(FP7/2007-2013) under grant agreement no. 223328; the
latter also supported work in the ffrench-Constant lab. The
LTQ Orbitrap was funded in part by the DFG within SFB-815
(Project Z).
REFERENCES
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