Identification and isolation of insecticidal oxazoles from Pseudomonas spp.

Article abstract of DOI:10.3762/bjoc.8.85

Two new and five known oxazoles were identified from two different Pseudomonas strains in addition to the known pyrones pseudopyronine A and B. Labeling experiments confirmed their structures and gave initial evidence for a novel biosynthesis pathway of t

Full text of DOI:10.3762/bjoc.8.85

Identification and isolation of insecticidal oxazoles
from Pseudomonas spp.
Florian Grundmann1, Veronika Dill1, Andrea Dowling2,
Aunchalee Thanwisai3, Edna Bode1, Narisara Chantratita3,
Richard ffrench-Constant2 and Helge B. Bode*1
Letter
Open Access
Address:
Beilstein J. Org. Chem. 2012, 8, 749–752.
1Department of Molecular Biotechnology, Goethe-Universität
Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany,
2Biosciences, University of Exeter, Penryn, Cornwall, TR10 9EZ, UK
and 3Faculty of Tropical Medicine, Mahidol University, 420/6
Ratchawithi Road, Ratchathewi, Bangkok 10400, Thailand
doi:10.3762/bjoc.8.85
Received: 18 March 2012
Accepted: 02 May 2012
Published: 18 May 2012
Email:
This article is part of the Thematic Series "Biosynthesis and function of
secondary metabolites".
Helge B. Bode* - h.bode@bio.uni-frankfurt.de
* Corresponding author
Guest Editor: J. S. Dickschat
Keywords:
© 2012 Grundmann et al; licensee Beilstein-Institut.
License and terms: see end of document.
insecticidal activity; labradorin; oxazole; Pseudomonas; secondary
metabolite
Abstract
Two new and five known oxazoles were identified from two different Pseudomonas strains in addition to the known pyrones
pseudopyronine A and B. Labeling experiments confirmed their structures and gave initial evidence for a novel biosynthesis
pathway of these natural oxazoles. In order to confirm their structure, they were synthesized, which also allowed tests of their
bioactivity. Additionally, the bioactivities of the synthesis intermediates were also investigated revealing interesting biological
activities for several compounds despite their overall simple structures.
Findings
During our search for novel natural products from entomo- not shown). Subsequent large scale cultivation and purification
pathogenic bacteria, strain PB22.5 was isolated from a soil of the main components led to the isolation of five major com-
sample collected in Thailand by using the baiting technique for pounds, which were subjected to structure elucidation by MS
the isolation of entomopathogenic bacteria and/or entomopatho- (Supporting Information File 1, Figure S2) and NMR analysis
genic nematodes, as described previously [1-4]. HPLC–MS (Supporting Information File 1, Tables S1–S6). Whereas the
analysis of extracts from strain PB22.5 grown in LB showed structures 1 and 2 could be identified as pseudopyronines A (1,
several peaks (Supporting Information File 1, Figure S1) that 267.4 m/z [M + H]+, C16H26O3) and B (2, 295.4 m/z [M + H]+,
have not been detected in other entomopathogenic bacteria (data C18H30O3) [5-7], three other compounds were identified as the
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Beilstein J. Org. Chem. 2012, 8, 749–752.
known oxazole derivatives labradorin 1 (3, 241.1 m/z [M + H]+, In order to determine the genus of the producing strain PB22.5,
C15H16N2O), labradorin 2 (4, 255.2 m/z [M + H]+, C16H18N2O) we sequenced its 16S-rRNA gene revealing it to be a
and pimprinaphine (5, 275.1 m/z [M + H]+, C18H14N2O) Pseudomonas sp. with closest homology to P. putida (100%
(Figure 1) [8,9].
similarity: Supporting Information File 1, Figure S4) [11-14].
As judged on the basis of high-resolution MALDI–MS and
LC–ESIMS/MS data the well-known entomopathogenic
P. entomophila [15,16] also produces compounds 3–6 but not 1
and 2. Furthermore two novel oxazole derivatives named
labradorin 3 (7, 268.4 m/z [M + H]+, C17H20N2O) and
labradorin 4 (8, 283.2 m/z [M + H]+, C18H22N2O) were
detected, but they could not be isolated due to their very low
production.
Labeling experiments were performed in order to confirm the
oxazole structures and to reveal their biosynthesis. Therefore,
both strains were cultivated in fully labeled 13C or 15N media
and 12C precursors were added (Figure 2).
Five carbons of leucine are incorporated in compound 3, while
eight carbons of compound 5 originate from phenylalanine,
which confirms these moieties to be amino-acid derived
(Figure 3).
Ten carbons and one nitrogen of tryptophan were incorporated
in all oxazoles, confirming the indole moiety to be derived from
tryptophan. Surprisingly, no incorporation of carbon from trypt-
amine was observed, and the fact that nitrogen labeling also
occurs when 14N-tryptamine, leucine or phenylalanine is fed
indicates an efficient transaminase activity in both strains, as
one would indeed expect for a bacterial strain living on
proteinogenic substrates such as insects. Indole acetaldehyde
was also fed in 13C-labeled medium, but, in this case as well, no
incorporation was observed (data not shown).
Figure 1: Structures of pseudopyronines A (1) and B (2) and natural
oxazoles (3–8) as well as synthetic oxazole derivatives (9–11). All
natural and synthetic oxazoles show the characteristic MS fragment
130 m/z [M + H]+. The intermediates of the oxazole syntheses (12–27)
are also shown as they were also tested for their biological activity.
Thus, indolepyruvate or an unknown degradation product
thereof is proposed as a precursor for the indole moiety.
Unfortunately, the production of 7 and 8 was insufficient for
analysis in the labeled media, but the nature of the side chains
was concluded from a fatty-acid analysis of the producing
Detailed analysis of the HPLC–MS data showed WS-30581 A strain, which showed only straight-chain fatty acids (data not
(6, 227.1 m/z [M + H]+, C14H14N2O) [10] as an additional shown).
oxazole derivative, but which was only produced in minute
amounts. WS-30581 A (6) was identified by comparing the In order to confirm the structure of all oxazole derivatives, to
retention time and the MS fragmentation (Supporting Informa- compare their retention times, and to provide enough material
tion File 1, Figure S2) of the product contained in the extract, for bioactivity tests, oxazoles 3–6 and 8 were synthesized.
with a synthesized compound. All oxazoles showed a character- Briefly, the respective tryptamine derivatives were formed,
istic fragment ion of 130 m/z [M + H]+ (Supporting Informa- which were then oxidized at the alpha-position with the help of
tion File 1, Figure S2), and labeling experiments (Supporting 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and
Information File 1, Figure S3) enabled the elucidation of this cyclized to give the oxazoles by using phosphorylchloride as
ion as 3-methylidene-3H-indolium (Figure 1).
described [9]. Additionally, three nonnatural derivatives were
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Beilstein J. Org. Chem. 2012, 8, 749–752.
Figure 2: MS data from strain PB22.5, which was cultivated in [U-13C] and [U-15N] medium background and LB medium as a control. Feeding experi-
ments with 12C and 14N amino acids confirmed structures 3 and 5 and gave initial insights into the biosynthesis.
Figure 3: All incorporated biosynthetic precursors of the oxazoles are shown in color. The nitrogen shown in green is derived from transamination as
part of the amino acid metabolism. SX = activated ester, which may be coenzyme A or enzyme (acyl carrier protein or polyketide synthase or nonribo-
somal peptide synthetase) bound.
synthesized to get a more diverse set of oxazole derivatives experiments are needed to fully elucidate the biosynthesis of
differing from the natural compounds by unusual (compound 9 these simple heterocyclic compounds.
and 10) or very long (compound 11) acyl chains.
All synthesized compounds and intermediates thereof
Additionally, the synthetic intermediates were also tested as were tested for their biological activity, as a broad range of
precursors in labeling experiments as described above, but activities has already been described for these oxazoles,
again no incorporation could be observed. Thus, the biosyn- including anticonvulsant and antithrombotic activity as well as
thesis via tryptamine amides, which are subsequently oxidized activity against human lung and pancreas cancer cell lines
and cyclized to give the oxazoles, can be excluded and more [9,10,17].
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Beilstein J. Org. Chem. 2012, 8, 749–752.
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cytes from the greater wax moth Galleria melonella according
to the method described by Proschak et al. [19]. Three oxazole
compounds show LD50 [µg ml−1] values against Galleria
hemocytes of 30 (compound 3), 1.7 (compound 6), and 103
(compound 9) (Table S7). Compounds 3, 5, 6, and 9 are active
in the MCF-7 assay with EC50 [µg ml−1] values of 58, 363, 26,
and 34, respectively. Similar to previous results [19], several
tryptamine amide derivatives (20–24, 26) showed cytotoxic
activity against the MCF-7 cells with 24 being the most potent
compound (1.02 µg ml−1). Interestingly, 26 also showed
activity against Galleria hemocytes, although its oxazole
derivative 10 did not, which may point to different targets for
both compound classes.
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The class of oxazole compounds, which were identified in this
article, are not only prevalent in Pseudomonas but also in other
bacteria such as Streptomyces [20] or Streptoverticillium [8,10],
suggesting a biological relevance also in these bacteria.
However, as concluded from the observed activity against
insect cells, they could significantly add to the overall insecti-
cidal activity of the investigated Pseudomonas strains.
15.Vodovar, N.; Vinals, M.; Liehl, P.; Basset, A.; Degrouard, J.;
Spellman, P.; Boccard, F.; Lemaitre, B. Proc. Natl. Acad. Sci. U. S. A.
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Supporting Information
Supporting Information File 1
19.Proschak, A.; Schultz, K.; Herrmann, J.; Dowling, A. J.;
Brachmann, A. O.; ffrench-Constant, R.; Müller, R.; Bode, H. B.
ChemBioChem 2011, 12, 2011–2015. doi:10.1002/cbic.201100223
20.Joshi, B. S.; Taylor, W. I.; Bhate, D. S.; Karmarkar, S. S. Tetrahedron
1963, 19, 1437–1439. doi:10.1016/S0040-4020(01)98569-2
General experimental procedures, isolation of the strain and
taxonomic identification, cultivation and extraction,
isolation, labeling experiments, synthesis, bioactivity
results and compound characterization.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-8-85-S1.pdf]
License and Terms
Acknowledgements
This is an Open Access article under the terms of the
Creative Commons Attribution License
Work in the Bode lab was supported by the LOEWE Schwer-
punkt Insektenbiotechnologie, the European Community's
Seventh Framework Programme (FP7/2007-2013) under grant
agreement no. 223328, which also supported work in the
ffrench-Constant and Chantratita labs, and the Deutsche For-
schungsgemeinschaft (DFG).
(http://creativecommons.org/licenses/by/2.0), which
permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
The license is subject to the Beilstein Journal of Organic
Chemistry terms and conditions:
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