Linear and cyclic peptides from the entomopathogenic bacterium Xenorhabdus nematophilus

Article abstract of DOI:10.1021/np800053n

Three new peptides, xenortides A and B and xenematide, were isolated from a culture of the nematode-associated entomopathogenic bacterium Xenorhabdus nematophilus. Their structures were elucidated using NMR, MS, and chemical derivatization methods. Xenortides A and B are the iV-phenethylamide and tryptamide derivatives, respectively, of the dipeptide (NMe-L-Leu-NMe-L-Phe). The cyclodepsipeptide xenematide has the sequence (Thr-Trp-Trp-Gly), with a 2-phenylacetamide substituent at the threonine residue and one D-tryptophan. The new peptides and the two known compounds xenocoumacin II and nematophin were tested for antibacterial, antifungal, insecticidal, and anti-Artemia salina activities. Xenematide and xenocoumacin II showed moderate antibacterial activities. Xenocoumacin II, nematophin, and the two xenortides were active in the Artemia salina assay, and xenematide acted weakly insecticidal.

Full text of DOI:10.1021/np800053n

1074
J. Nat. Prod. 2008, 71, 1074–1077
Linear and Cyclic Peptides from the Entomopathogenic Bacterium Xenorhabdus nematophilus
Gerhard Lang,† Tim Kalvelage,† Arne Peters,‡ Jutta Wiese,† and Johannes F. Imhoff*,†
Kieler Wirkstoffzentrum am IFM-GEOMAR, Am Kiel-Kanal 44, 24106 Kiel, Germany, and e-nema, Gesellschaft fu¨r Biotechnologie and
Biologischen Pflanzenschutz mbH, Klausdorfer Strasse 28-36, 24223 Raisdorf, Germany
ReceiVed January 24, 2008
Three new peptides, xenortides A and B and xenematide, were isolated from a culture of the nematode-associated
entomopathogenic bacterium Xenorhabdus nematophilus. Their structures were elucidated using NMR, MS, and chemical
derivatization methods. Xenortides A and B are the N-phenethylamide and tryptamide derivatives, respectively, of the
dipeptide (NMe-L-Leu-NMe-L-Phe). The cyclodepsipeptide xenematide has the sequence (Thr-Trp-Trp-Gly), with
a 2-phenylacetamide substituent at the threonine residue and one D-tryptophan. The new peptides and the two known
compounds xenocoumacin II and nematophin were tested for antibacterial, antifungal, insecticidal, and anti-Artemia
salina activities. Xenematide and xenocoumacin II showed moderate antibacterial activities. Xenocoumacin II, nematophin,
and the two xenortides were active in the Artemia salina assay, and xenematide acted weakly insecticidal.
The genus Xenorhabdus constitutes a group of bacteria associated
with entomopathogenic nematodes. These bacteria live in a
symbiotic relationship in the gut of the nematode host, which is
able to infect insects and release bacteria into their hemolymph.
The bacteria then proliferate and contribute to the death of the insect.
There is good evidence that the insecticidal activity of Xenorhabdus
bacteria is at least partially due to the production of insecticidal
protein toxins.1,2 Besides this, a number of low molecular weight
metabolites with various biological activities have been isolated
from Xenorhabdus cultures, e.g., xenorxides, xenorhabdins, xeno-
coumacins, and nematophin.3
5-fluorophenyl)-D-valinamide],6 and HPLC analysis. In the NMR
spectra of 1 a signal set for a minor compound with only small
differences in 1H and 13C chemical shifts (for a table with data see
the Supporting Information) was discernible, although no additional
peaks or masses were detectable in the HPLC-MS analysis. The
possibility of the minor compound being a diastereomer of 1 was
ruled out by the FDVA analysis. In the NOESY spectrum the signals
of the major compound showed correlations with the respective
signals of the minor compound. These cross-peaks had the same
phase as the diagonal peaks, thus indicating chemical exchange.
Consequently, the two signal sets presumably arise from slowly
interconverting conformers of 1.
In our search for novel metabolites from nematode-associated
bacteria we performed a chemical screening of an extract of X.
nematophilus. This screening led to the identification of three new
peptides, xenortide A (1), xenortide B (2), and xenematide (3), as
well as the known compounds xenocoumacin II4 and nematophin.5
HPLC-MS analysis of an XAD extract of a liquid culture of X.
nematophilus showed the presence of several compounds, with the
most prominent one being readily attributed by its molecular mass
and UV spectrum to nematophin, an antibacterial compound
commonly found in cultures of X. nematophilus. Nematophin and
four further components (1-3 and xenocoumacin II) were purified
using a combination of liquid-liquid and gel chromatography.
The 1H and 13C NMR spectra of xenortide A (1; Table 1) showed
signals for two phenyl groups. By evaluation of the COSY and
HMBC data these phenyl groups were attributed to one phenyla-
A related compound, xenortide B (2), coeluted with 1 in reversed-
phase HPLC, but was readily separated from it by employing gel
chromatography on Sephadex LH-20. The ESIMS indicated a mass
of 448.3 for 2. The NMR spectra suggested a structural relationship
to 1, with significant differences apparent only in the aromatic
region of the spectra (Table 1). Instead of signals of a second phenyl
residue, as in the spectra of 1, an ABCD system and a singlet were
discernible, indicative of an indole residue. Analysis of the NMR
data revealed 2 to be an analogue of 1 with a tryptamide group in
place of the N-phenethylamide at the C-terminus. The amino acid
residues in 2 both have the L-configuration, as determined by an
FDVA analysis.
Xenematide (3), which was isolated by fast centrifugal partition-
ing chromatography (FCPC), had a molecular mass of 662.3. The
1H NMR spectrum of 3 in DMSO-d6 displayed signals for two
indole moieties and one phenyl group. The resonances of four amide
protons between 7.4 and 8.8 ppm coupling with signals between
2.5 and 4.7 ppm indicated a peptidic structure. By analyzing the
COSY and HMBC spectra, ꢀ-alanine, two tryptophan residues,
threonine, and a phenylacetyl (PAA) group were identified as the
building blocks of 3. The sequence of the amino acids was
determined in a straightforward manner as PAA-Thr-Trp-Trp-ꢀ-
1
lanine and one phenethylamine residue. A third H spin system
was characteristic of a leucine residue. The chemical shifts of the
remaining signals from two methyl groups (δH 1.83 and 1.97; δC
33.6 and 32.2) indicated that they were attached to nitrogen atoms.
Their positions at the amino groups of the leucine and the
phenylalanine residue were evident from long-range H,C-correla-
tions from the methyl protons to the R-carbon of the respective
amino acids. HMBC correlations from the N-methyl group of
phenylalanine to the carbonyl carbon of leucine and from one of
the methylene groups of phenethylamine to the carbonyl carbon
of phenylalanine showed the overall structure of 1 to be that of a
doubly N-methylated leucyl-phenylalanine dipeptide with an N-
phenethylamide C-terminus. The accurate mass of m/z 410.2794
measured for the [M + H]+ ion was consistent with this structure.
Both amino acids were found to have the L-configuration by acid
hydrolysis, subsequent derivatization with D-FDVA [NR-(2,4-dinitro-
2
Ala by using the JHC correlations of the amide protons with the
carbonyl carbons of the respective adjoining amino acids. The
HMBC correlation from the ꢀ-proton of threonine to the carbonyl
carbon of ꢀ-alanine indicated a ring closure with an ester bond
between these residues. After mild acid hydrolysis, with phenol
and dithioerythritol added to prevent oxidation of tryptophan, the
configurations of the amino acid residues were determined by the
FDVA method. The threonine residue was found to have the normal
(2S,3R)-configuration. However, the HPLC analysis of the FDVA
derivatives revealed that both L- and D-tryptophan were present.
An assignment of the correct configurations to the two tryptophan
* To whom correspondence should be addressed. Tel: +49-431-6004450.
Fax: +49-431-6004452. E-mail: jimhoff@ifm-geomar.de.
† Kieler Wirkstoffzentrum.
‡ e-nema.
10.1021/np800053n CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/21/2008

Notes
Journal of Natural Products, 2008, Vol. 71, No. 6 1075
Table 1. 1H and 13C NMR Data (600 MHz, CH3OD) for Xenortides A (1) and B (2)
1
2
position
Leu
δC, mult.
δH (J in Hz)
δC, mult.
δH (J in Hz)
1
2
3
4
5
6
175.3, qC
59.0, CH
42.7, CH2
25.6, CH
23.8, CH3
22.3, CH3
33.6, CH3
171.9, qC
59.5, CH
35.6, CH2
175.3, qC
59.0, CH
42.7, CH2
25.6, CH
23.8, CH3
22.3, CH3
33.6, CH3
171.9, qC
59.5, CH
35.6, CH2
3.57, dd (8.0, 5.9)
1.30-1.40, m
1.71, m
0.93, d (6.7)
0.91, d (6.7)
1.83, s
3.57, dd (8.0, 5.9)
1.30-1.40, m
1.71, m
0.93, d (6.7)
0.91, d (6.7)
1.83, s
NMe
Phe
1
2
3
5.41, dd (11.0, 5.6)
3.19, dd (14.5, 5.6)
3.00, dd (14.4, 11.0)
5.41, dd (11.0, 5.6)
3.19, dd (14.5, 5.6)
3.00, dd (14.4, 11.0)
4
138.5, qC
129.8b, CH
130.2b, CH
127.4c, CH
32.2, CH3
41.9, CH2
36.4, CH2
140.4, qC
129.5b, CH
129.6b, CH
127.8c, CH
138.5, qC
129.8a, CH
130.2a, CH
127.4b, CH
32.2, CH3
5/9
6/8
7
NMe
1
2
3
4/8
5/7
6
1
2
3
4
5
6
7
8
7.25, m
7.25, m
7.15-7.30, m
7.15-7.30, m
2.97, s
3.43, t (7.5)
2.78, t (7.3)
7.15-7.30, m
7.15-7.30, m
2.97, s
PEAa
7.19, m
7.15-7.30, m
7.15-7.30, m
tryptamine
41.3, CH2
26.2, CH2
113.1, qC
128.8, qC
119.3, CH
119.7, CH
122.4, CH
112.3, CH
138.2, qC
123.4, CH
3.47 - 3.77, m
2.95, m
7.56, d (8.0)
7.02, t (7.5)
7.08, t (7.5)
7.33, d (8.0)
9
11
7.04, s
a PEA ) phenethylamine. b Chemical shifts with same superscripts may be interchanged. c Chemical shifts with same superscripts may be inter-
changed.
Table 2. 1H and 13C NMR Data (600 MHz, DMSO-d6) for Xenematide (3)
position
δC, mult.
δH (J in Hz)
position
Trp2
δC, mult.
δH (J in Hz)
ꢀ-Ala
1
2
169.2, qC
33.9, CH2
4
5
6
7
8
9
10
11
12
NH
1
2
3
4
NH
1
109.3, qC
127.0b, qC
118.2, CH
118.2, CH
121.0, CH
111.34, CH
136.0c, qC
2.52, m
2.40, m
3.39, m
3.32, m
7.55, d (7.8)
7.00, td (7.8, 1.0)
7.08, m
3
34.6, CH2
NH
1
2
7.39, t (6.1)
7.36, bd (8.0)
Trp1
171.0, qC
54.5, CH
25.7, CH2
4.19, ddd (10.0, 7.9,3.7)
3.18, m
10.7, s
7.09, m
8.81, d (6.7)
3
123.4, CH
2.87, m
4
5
6
7
8
9
10
11
12
NH
1
110.6, qC
126.9b, qC
117.9, CH
118.2, CH
120.8, CH
111.28, CH
135.9c, qC
Thr
170.2, qC
54.02, CH
72.0, CH
16.2, CH3
4.65, dd (9.2, 2.2)
5.11, qd (6.3, 2.3)
1.05, d (6.2)
7.49, d (7.8)
6.96, m
7.05, td (7.8, 1.0)
7.33, bd (8.0)
8.09, d (9.5)
PAAa
170.6, qC
41.7, CH2
2
3.65, d (14.1)
3.55, d (14.1)
10.6, s
6.96, m
123.1, CH
3
136.3, qC
129.0, CH
128.1, CH
126.1, CH
8.72, d (6.7)
4/8
5/7
6
7.26, m
7.29, m
7.19, m
Trp2
172.0, qC
54.0, CH
25.7, CH2
2
3
4.53, q (7.3)
2.82 - 2.92, m
a PAA ) 2-phenylacetic acid. b Chemical shifts with same superscripts may be interchanged. c Chemical shifts with same superscripts may be inter-
changed.
units by using NOE correlations was impossible because of the
flexibility of the molecule.
ematide (3) and xenocoumacin II, the latter showing an MIC of 40
µg/mL against the phytopathogens Xanthomonas campestris, Ral-
stonia solanacearum, and Pseudomonas syringae, and an MIC of
20 µg/mL against Bacillus subtilis. At a concentration of 20 µg/
mL xenocoumacin II inhibited the growth of S. lentus by 70%.
Xenematide (3) had an MIC value of 10 µg/mL against Bacillus
subtilis, Pseudomonas fluorescens, Pseudomonas syringae, Erwinia
amyloVora, and Ralstonia solanacearum. At a maximum concentra-
Besides the three new peptides 1-3 the two known Xenorhabdus
metabolites xenocoumacin II and nematophin were also isolated.
Compounds 1-3, xenocoumacin II, and nematophin were tested
for antimicrobial activity against various Gram-positive and Gram-
negative bacteria as well as the yeast Candida glabrata. The only
compounds exhibiting a noticeable antibacterial effect were xen-

1076 Journal of Natural Products, 2008, Vol. 71, No. 6
Notes
Chart 1
are available as Supporting Information; HRESIMS m/z 663.2923 [M
+ H]+ (calc for C37H39N6O6 663.2931).
tion of 40 µg/mL the other three compounds did not inhibit
microbial growth. It is noteworthy that we were not able to
reproduce the previously described strong antistaphylococcal effect
of nematophin.5,7
Xenocoumacin II also was notably active in the Artemia salina
assay (IC50 ) 9 µg/mL). In the same assay nematophin showed an
IC50 value of 50 µg/mL, while 1 and 2 were weakly active, with
an IC50 of about 200 µg/mL. Among the isolated compounds, only
xenematide (3) exhibited insecticidal activity against Galleria
mellonella, with two out of five larvae dead after a six-day period.
Preparation and Analysis of D- and L-FDVA Derivatives.
Compounds 1 and 2 (3 mg each) were hydrolyzed by heating in HCl
(6 M; 1 mL) for 20 h at 110 °C. The solutions were then evaporated
to dryness and redissolved in H2O (250 µL). A 1% (w/v) solution (100
µL) of D-FDVA6 in acetone was added to an aliquot (50 µL) of the
acid hydrolysate solution (or to 50 µL of a 50 mM solution of the
respective amino acid). After addition of a NaHCO3 solution (1 M; 40
µL) and DMSO (70 µL) the mixture was incubated at 60 °C (2 h). The
reactions were quenched by addition of HCl (2 M; 30 µL). The reaction
mixtures were then diluted 100-fold with MeOH-H2O (1:1) and
analyzed by HPLC (Phenomenex Onyx monolithic C18, 100 × 3.0;
solvents: A water + 0.1% HCOOH, B MeCN + 0.1% HCOOH; linear
gradient: 0 min 5% B, 4 min 60% B; 6 min 100% B; 40 °C; 2 mL
min-1). The amino acid standards were derivatized with both D- and
L-FDVA. Retention times (min) of the amino acid derivatives were as
follows: D-FDVA-L-NMe-Leu (3.85), L-FDVA-L-NMe-Leu (3.62),
D-FDVA-L-NMe-Phe (3.61), and L-FDVA-L-NMe-Phe (3.52). HPLC
analyses of the peptide hydrolysates derivatized with D-FDVA both
showed peaks for L-NMe-Leu and L-NMe-Phe at 3.85 and 3.61 min,
respectively. Xenematide (3; 200 µg) was hydrolyzed with HCl (400
µL; 110 °C; 1 h) containing phenol (3% w/v) and dithioerythritol (1%
w/v). The hydrolysate was derivatized with L-FDVA as described above.
For the HPLC analysis of the tryptophan derivatives the HPLC
conditions were the same as above. The threonine derivatives were
analyzed using a different gradient (0 min 10% B, 5.5 min 30% B).
The retention times of the derivatized amino acid standards were as
follows: L-FDVA-L-Thr (2.87), L-FDVA-L-allo-Thr (3.10), D-FDVA-
L-Thr (4.38), D-FDVA-L-allo-Thr (3.77), L-FDVA-L-Trp (3.24), and
D-FDVA-L-Trp (3.51). The derivatized hydrolysate showed peaks
corresponding to L-Thr, L-Trp, and D-Trp.
Experimental Section
General Experimental Procedures. Optical rotations were measured
with a Perkin-Elmer 341 polarimeter. NMR spectra were recorded on
a 600 MHz spectrometer (Bruker AV600). For calibration of 13C and
1H NMR chemical shifts the carbon signals and the residual proton
signals of the solvent were used (CD3OD: δH 3.31 and δC 49.0; DMSO-
d6: δH 2.50 and δC 39.52). HPLC analyses were performed using a
C18 column (Phenomenex Luna C18(2), 4.6 × 250 mm, 5 µm) applying
a H2O/MeCN gradient with 0.1% (v/v) HCOOH added to both solvents.
For MS detection the HPLC was coupled to an ESI-ion trap system
(Esquire 4000, Bruker). HRMS were acquired on a FT-ICR spectrom-
eter (7 T Apex-Qe, Bruker) using positive electrospray ionization.
Cultivation and Extraction. The strain Xenorhabdus nematophilus
was isolated from the nematode Steinernema carpocapsae according
to Akhurst et al. (1980).10 The identity of this isolate was confirmed
by the 16S rDNA sequence analysis. The strain was cultivated in a
previously described medium (20 L).8 After 30 h at 30 °C XAD-16N
resin (200 g) was added to the culture, which was left shaking for
another 20 h, before washing the resin with H2O (800 mL) and eluting
with MeOH (400 mL).
Isolation of Metabolites. The extract was dried, redissolved in H2O
(400 mL), and consecutively extracted with EtOAc (2 × 400 mL) and
n-BuOH (2 × 400 mL). After drying, the BuOH extract was subjected
to gel chromatography on Sephadex LH-20 (2.5 × 110 cm; MeOH;
2.5 mL/min). Xenortide A (1; 35 mg), xenortide B (2; 7.8 mg), and
xenocoumacin II (7.6 mg) were eluted from 102 to 132 min, from 138
to 150 min, and from 88 to 95 min, respectively. The EtOAc fraction
was separated by liquid-liquid chromatography on a fast centrifugal
partitioning chromatography system (FCPC; Kromaton) using the
solvent system H2O/MeOH/EtOAc/n-heptane (4.5:5.5:4.5:5.5) with the
upper layer as the stationary phase (7 mL min-1; 1400 rpm).
Xenematide (3; 6.7 mg) and nematophin (39 mg) eluted after 18 and
129 min, respectively.
Antimicrobial Assays. The antimicrobial assays were performed
as described before9 with the following strains: Bacillus subtilis (DSM
347), Escherichia coli K12 (DSM 498), Staphylococcus lentus (DSM
6672), Pseudomonas syringae var. aptata (DSM 50252), P. fluorescens
(NCIMB 10586), Xanthomonas campestris (DSM 2405), Ralstonia
solanacearum (DSM 9544), and Candida glabrata (DSM 6425).
Insecticidal Assay. The isolated compounds, dissolved at 1 mg/mL
in a 50% aqueous EtOH solution, were tested for oral toxicity to larvae
of the greater wax moth (Galleria mellonella). Five larvae were placed
in a plastic box (2 cm diameter) and fed with 1 g of a diet composed
of 0.8 mL of the substance, 0.2 mL of glycerol (86%), and 1 g of
wheat bran. An untreated control group were fed with 1 g of a mixture
of 0.4 mL of distilled H2O, 0.4 mL of EtOH, 0.2 mL of glycerol, and
1 g of wheat bran. The larvae were incubated at 35 °C for 6 days, and
the mortality and the ability to spin silk were recorded. The experiment
was done in four replicates except for nematophin (two replicates).
Artemia salina Assay. A series of 2-fold dilutions of the test
compounds with amounts from 50 to 0.781 µg/well were prepared in
96-well microplates, which were then dried. To each well were added
between 10 and 20 freshly hatched A. salina nauplii in artificial seawater
(300 µL). After 24 h the number of immotile nauplii per well was
counted.
Xenortide A (1): amorphous, white solid; [R]20D -54 (c 0.2, MeOH);
1
for H and 13C NMR data, see Table 1; COSY and HMBC data are
available as Supporting Information; HRESIMS m/z 410.2794 [M +
H]+ (calc for C25H36N3O2 410.2807).
Xenortide B (2): amorphous, yellowish solid; [R]20 -58 (c 0.3,
D
MeOH); for H and 13C NMR data, see Table 1; COSY and HMBC
1
data are available as Supporting Information; HRESIMS m/z 449.2908
[M + H]+ (calc for C27H37N4O2 449.2916).
Xenematide (3): amorphous, yellowish solid; [R]20 +45 (c 0.2,
D
MeOH for 1H and 13C NMR data, see Table 2; COSY and HMBC data

Notes
Acknowledgment. This is a publication from the Kieler Wirk-
stoffzentrum KiWiZ at IFM-GEOMAR funded by the Ministerium fu¨r
Wissenschaft, Wirtschaft and Verkehr of the state of Schleswig-
Holstein, Germany. The authors are grateful to R. Wicher for perform-
ing the extractions, A. Erhard for running the activity tests, Dr. B.
Lindner (Forschungszentrum Borstel) for measuring HRMS, and Ms.
G. Kohlmeyer-Yilmaz and Ms. M. Ho¨ftmann for running and processing
NMR experiments.
Journal of Natural Products, 2008, Vol. 71, No. 6 1077
(2) Brown, S. E.; Cao, A. T.; Hines, E. R.; Akhurst, R. J.; East, P. D.
J. Biol. Chem. 2004, 279, 14595–14601.
(3) Webster, J. M.; Chen, G.; Hu, K.; Li, J. In Entomopathogenic
Nematology; Gaugler, R., Ed.; CAB International, 2002; Chapter 5,
pp 99-114..
(4) McInerney, B. V.; Taylor, W. C.; Lacey, M. J.; Akhurst, R. J.; Gregson,
R. P. J. Nat. Prod. 1991, 54, 785–795.
(5) Li, J.; Chen, G.; Webster, J. M. Can. J. Microbiol. 1997, 43, 770–
773.
(6) Bru¨ckner, H.; Keller-Hoehl, C. Chromatographia 1990, 30, 621–629.
(7) Kennedy, G.; Viziano, M.; Winders, J. A.; Cavallini, P.; Gevi, M.;
Micheli, F.; Rodegher, P.; Seneci, P.; Zumerle, A. Bioorg. Med. Chem.
Lett. 2000, 10, 1751–1754.
Supporting Information Available: 1H and 13C NMR spectra and
tables of data of xenortides A (1) and B (2) as well as xenematide (3)
are available free of charge via the Internet at http://pubs.acs.org.
(8) Johnigk, S. A.; Ecke, F.; Poehling, M.; Ehlers, R. U. Appl. Microbiol.
Biotechnol. 2004, 64, 651–658.
(9) Lang, G.; Wiese, J.; Schmaljohann, R.; Imhoff, J. F. Tetrahedron 2007,
63, 11844–11849.
(10) Akhurst, R. J. J. Gen. Microbiol. 1980, 121, 303–309.
References and Notes
(1) Ryu, K. G.; Bae, J. S.; Yu, Y. S.; Park, S. H. Biotechnol. Bioprocess
Eng. 2000, 5, 141–145.
NP800053N