Synthesis and bioactivity of analogues of the marine antibiotic tropodithietic acid

Article abstract of DOI:10.3762/bjoc.10.188

Tropodithietic acid (TDA) is a structurally unique sulfur-containing antibiotic from the Roseobacter clade bacterium Phaeobacter inhibens DSM 17395 and a few other related species. We have synthesised several structural analogues of TDA and used them in b

Full text of DOI:10.3762/bjoc.10.188

Synthesis and bioactivity of analogues of the marine
antibiotic tropodithietic acid
Patrick Rabe1, Tim A. Klapschinski1, Nelson L. Brock1, Christian A. Citron1,
Paul D’Alvise2, Lone Gram2 and Jeroen S. Dickschat*1
Letter
Open Access
Address:
Beilstein J. Org. Chem. 2014, 10, 1796–1801.
1Kekulé-Institut für Organische Chemie, Rheinische
Friedrich-Wilhelms-Universität Bonn, Gerhard-Domagk-Straße 1,
53121 Bonn, Germany and 2Department of Systems Biology,
Technical University of Denmark, Matematiktorvet bldg. 301, 2800
Kongens Lyngby, Denmark
doi:10.3762/bjoc.10.188
Received: 17 April 2014
Accepted: 22 July 2014
Published: 06 August 2014
Email:
This article is part of the Thematic Series "Natural products in synthesis
and biosynthesis".
Jeroen S. Dickschat* - dickschat@uni-bonn.de
* Corresponding author
Associate Editor: K. N. Ganesh
Keywords:
© 2014 Rabe et al; licensee Beilstein-Institut.
License and terms: see end of document.
antibiotics; natural products; Roseobacter; SAR study; tropodithietic
acid; tropone
Abstract
Tropodithietic acid (TDA) is a structurally unique sulfur-containing antibiotic from the Roseobacter clade bacterium Phaeobacter
inhibens DSM 17395 and a few other related species. We have synthesised several structural analogues of TDA and used them in
bioactivity tests against Staphylococcus aureus and Vibrio anguillarum for a structure–activity relationship (SAR) study, revealing
that the sulfur-free analogue of TDA, tropone-2-carboxylic acid, has an antibiotic activity that is even stronger than the bioactivity
of the natural product. The synthesis of this compound and of several analogues is presented and the bioactivity of the synthetic
compounds is discussed.
Introduction
Tropodithietic acid (TDA, 1a) is an antibiotic produced by the that are located on a plasmid [6,7], and the adjacent paaZ2 gene
marine bacterium Phaeobacter inhibens. It has an unusual [8], a mutated copy of paaZ from the PAA catabolon. Particu-
structure that is made up by a dithiet moiety fused to tropone-2- larly interesting is the mechanism of sulfur introduction that we
carboxylic acid (Figure 1) [1]. In Phaeobacter the compound is have recently investigated in a study combining gene knock-
accompanied by hydroxy-TDA 2, while its tautomer outs and feeding experiments with 34S-labelled amino acids [8].
thiotropocin (1b) was previously reported from Pseudomonas In this study we could show that S-thiocysteine is the direct
[2,3]. The biosynthesis of 1 starts from phenylalanine and sulfur precursor of TDA. The introduction of sulfur proceeds
requires the paaABCDE and paaG genes of the upper phenyl- via nucleophilic attack of S-thiocysteine to the Michael acceptor
acetic acid (PAA) catabolon [4,5], the six genes tdaABCDEF of tropone-2-carboxylic acid coenzyme A ester and oxidative
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Beilstein J. Org. Chem. 2014, 10, 1796–1801.
elimination of cysteine. The second sulfur atom is introduced by several targets [13]. Here we report on the synthesis and bioac-
analogous attack to the vinylogous Michael acceptor. The tivity of several TDA analogues for investigating the
volatiles tropone (3) and tropone hydrate 4 can also be detected structure–activity relationship (SAR) of this unique marine
in P. inhibens headspace extracts and are shunt products of the antibiotic.
TDA biosynthetic pathway [9]. Clardy and coworkers have
Results and Discussion
recently reported on a series of structurally related compounds
with algicidal activity, represented by roseobacticide A (5), but Synthesis of analogues of tropodithietic acid
their biosynthesis is unknown [10].
For a detailed SAR study we first aimed at a series of com-
pounds with a seven-membered carbocyclic core and an overall
simplified structure as compared to the antibiotic TDA. There-
fore, tropone-2-carboxylic acid (13), lacking the unprecedented
dithiet moiety of TDA, was synthesised according to Scheme 1.
Cyclohexenone 6 was first converted with NaHMDS and
TMSCl into the corresponding silylenol ether that upon oxi-
dation with m-CPBA under migration of the TMS group yielded
the protected hydroxy ketone 7. Aldol reaction with tert-butyl
acetate to 8a and deprotection with TBAF gave 9a in 24% yield
over four steps. Oxidative cleavage of the glycol with NaIO4
resulted in a β-ketoester aldehyde that upon treatment with
silica gel underwent an intramolecular aldol condensation to a
mixture of 10a and 11a that were separable by column chroma-
tography. Oxidation with DDQ gave tert-butyl tropone-2-
carboxylate (12a) that was efficiently converted into 13 by stir-
ring in TFA. The synthesis of 12a followed a previously
published route for ethyl tropone-2-carboxylate (12b) [14], but
Figure 1: TDA and related natural products from Phaeobacter
inhibens.
TDA exhibits a strong antibiotic activity against a broad spec- saponification of this ester led to decomposition, and therefore,
trum of bacteria including alpha- and gammaproteobacteria, the tert-butyl ester 12a was prepared that allowed for a conver-
flavobacteria and actinobacteria [11], but not against the sion into 13 under acidic conditions.
eukaryotic model organisms Artemia sp. and Caenorhabditis
elegans, suggesting P. inhibens or other TDA-producing Furthermore, a series of TDA analogues all with a seven-
bacteria from the Roseobacter clade as promising candidates to membered carbocyclic core bearing a carbonyl function, a
be used as probiotics in aquacultures [12]. The mode of action carboxylic acid or ester function in 2-position, and various func-
of TDA is unknown, but it is difficult to select resistant and tional groups such as halogen atoms and methoxy groups was
tolerant strains from long-term exposures to sub inhibitory synthesised (Scheme 2). Acrolein (14) was reacted with tert-
concentrations of TDA, suggesting that TDA may interact with butyl acetate (15) and LDA to yield the aldol product 16 that
Scheme 1: Synthesis of tropone-2-carboxylic acid (13).
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Beilstein J. Org. Chem. 2014, 10, 1796–1801.
Scheme 2: Synthesis of halogenated TDA analogues.
was subsequently oxidised to the β-keto ester 17 in a Jones oxi- Two structurally related halogenated compounds lacking the
dation. Treatment of the latter with TMSCl, imidazole and carboxylic acid function as in TDA, 2,3,4-trichlorotropone (24)
DMAP resulted in 18 as a 3:2 mixture of two diastereomers that and 3,4,5-trichloro-6,7-dihydrotropone (25), were prepared
proved to be unstable. However, the crude product was suffi- through a known route (Scheme 3) [15]. Finally, a few cyclo-
ciently pure for its direct usage in a Diels–Alder reaction with heptanone derivatives with rigorously simplified structures as
tetrachlorocyclopropene, resulting in the adduct 19 as a single compared to TDA were included in this study. The β-ketoester
stereoisomer. The formation of only one stereoisomer is 27 containing a Michael acceptor was synthesised from methyl
explainable by an E/Z isomerisation of 18 and a Diels–Alder cycloheptanone-2-carboxylate (26) by oxidation with Cu(OAc)2
reaction that only proceeds from (Z)-18, but not from (E)-18. and Pb(OAc)2 according to a known procedure [16]. The com-
Treatment of 19 with TBAF resulted in cleavage of the TMS pound cyclohept-2-en-1-one (28) is commercially available,
protecting group followed by instantaneous elimination of HCl while we have previously reported the synthesis of cyclohept-4-
under cyclopropane ring opening to 20 with 16% yield. Further- en-1-one (29) that was identified in headspace extracts from
more, similar amounts of the fluorinated derivative 21 (17%) streptomycetes [17].
were isolated. Its formation is explained by the nucleophilic
attack of fluoride to the Michael acceptor in 20 followed by Bioactivity tests
elimination of chloride. In contrast, treatment of 19 with potas- All synthetic compounds were screened in bioactivity tests
sium carbonate in MeOH yielded only minor amounts of 20 and towards two bacterial strains, the Gram-positive Staphylo-
the substitution product, methoxy derivative 22, as main prod- coccus aureus 8325 and the Gram-negative Vibrio anguillarum
uct. All three compounds 20–22 were treated under various oxi- 90-11-287 (Table 1). These organisms were selected for our
dation conditions (including DDQ, IBX, SeO2, and MnO2) to bioactivity tests, because S. aureus is a clinically important
install the tropone moiety by dehydrogenation, but unfortu- human pathogen and strains with resistances against multiple
nately all these reaction conditions failed. Compound 22 was antibiotic drugs cause severe problems with nosocomial infec-
subsequently converted into the free acid 23 by treatment with tions. The bioactivity against V. anguillarum is interesting from
TFA, while similar conversions of 20 and 21 were unsuccessful an ecological point of view, because P. inhibens was originally
due to the instability of the products.
isolated from scallop rearings [18-20], suggesting that the
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Beilstein J. Org. Chem. 2014, 10, 1796–1801.
pounds were of equal or even better antibiotic activity. Com-
pound 25 was as effective, and tropone-2-carboxylic acid (13),
closely resembling the structure of TDA, but lacking its sulfur
atoms, was even more effective with a MIC corresponding to
half the MIC of TDA (MIC against S. aureus was 26 μM versus
39 μM, while the MIC against V. anguillarum was 6.5 μM
versus 19 μM). This indicates that the dithiet moiety of TDA is
not essential for the direct antibacterial effect of the compound,
and may have other physiological or ecological functions. This
result is particularly surprising, because the coenzyme A ester
of 13 is an intermediate along the biosynthetic pathway to TDA,
so the question arises what may have been the evolutionary
advantages of extending the biosynthetic pathway from the
coenzyme A ester of 13 to TDA by the introduction of two
sulfur atoms. A possible answer may be that TDA has more or
other targets than 13 which may have the consequence that the
development of resistances against TDA is prevented, thus
offering an evolutionary advantage.
Conclusion
Scheme 3: Further compounds included in this SAR study.
We have synthesised a series of compounds with seven-
membered carbocyclic rings. Their structures were inspired by
the antibiotic TDA from the marine bacterium P. inhibens. One
compound, 3,4,5-trichloro-5,6-dihydrotropone (25), showed a
strong antibiotic activity against S. aureus and V. anguillarum
natural function of P. inhibens may be to prevent molluscs from
Vibrio infections.
Most of the compounds tested had minimal inhibitory concen- that is similar to the activity of TDA, while tropone-2-
trations (MICs) exceeding that of TDA, however, two com- carboxylic acid (13) had an even stronger antibiotic effect. This
Table 1: Bioactivity tests with TDA and TDA analogues.
Compounda
Staphylococcus aureus 8325
Vibrio anguillarum 90-11-287
MIC/mg L−1
MIC/μM
MIC/mg L−1
MIC/μM
1
7.8
39
3.9
19
10a
10b
11a
12a
12b
13
inactive
125
125
600
700
600
600
700
6.5
700
125
inactive
125
125
600
700
26
125
125
125
3.9
1.0
20
inactive
125
inactive
125
21
430
430
22
inactive
inactive
inactive
7.8
inactive
inactive
125
23
24
720
89
25
45
15.6
inactive
46.9
31.3
31.3
26
inactive
62.5
27
370
570
570
280
280
570
28
62.5
29
62.5
aMICs were determined using the microdilution method according to guidelines of the Clinical and Laboratory Standards Institute [21]. Compounds
with MICs > 125 mg L−1 were regarded as inactive.
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II Broth (Cat. No. 297963, Becton, Dickinson and Company,
Sparks, MD, USA). The minimum inhibitory concentration
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Supporting Information
Supporting Information File 1
Synthetic procedures, compound characterisation data and
copies of NMR spectra.
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Acknowledgements
This work was funded by the Deutsche Forschungsgemein-
schaft as part of the Transregional Collaborative Research
Centre SFB-TR 51 (“Roseobacter”), by a PhD scholarship of
the Beilstein-Institut zur Förderung der Chemischen
Wissenschaften (to P.R.), and by a PhD scholarship of the
Fonds der Chemischen Industrie (to N.L.B.).
21.Wikler, M. A.; Cockerill, F. A.; Craig, W. A.; Dudley, M. N.;
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