Angewandte
Chemie
DOI: 10.1002/anie.201304714
Natural Products
Formation of a Dinuclear Copper(I) Complex from the Clostridium-
Derived Antibiotic Closthioamide**
Florian Kloss, Sacha Pidot, Helmar Goerls, Thorsten Friedrich, and Christian Hertweck*
Strictly anaerobic microorganisms, which represent the oldest
living beings on earth, are ubiquitous in the environment and
in the intestines of higher organisms. Despite their huge
impact on human health, ecology, and industrial biotech-
nology, surprisingly little is known about the natural product
Figure 1. Structure of closthioamide (CTA).
[
1]
chemistry of the oxygen-free world. Until recently, no
antibiotic or any other secondary metabolite was known from
anaerobes. Bioinformatics analyses of genome sequences of
anaerobes, however, have shown a broad biosynthetic poten-
tial, in particular for polyketides and nonribosomal pep-
[
6,7]
VRE) at nanomolar concentrations.
Genetic manipulation
of the producing strain allowed the isolation and character-
ization of seven additional thioamide congeners, which
appeared to be shunt metabolites and degradation products
of CTA. However, the chemical function of the thioamide-
rich molecule has remained a mystery. Here we report that
CTA acts as a selective metal chelator and disclose the
unparalleled architecture of the complex.
[
2–4]
tides.
A plausible explanation for the meager metabolic
[8]
profiles is that the limited energy supply under strictly
anaerobic conditions causes down-regulation or even silenc-
ing of the respective genes in the absence of a particular
[5]
trigger. For the soil-derived bacterium Clostridium cellulo-
lyticum, the cues were contained in an aqueous soil extract.
Only in the presence of this complex additive, C. cellulolyti-
cum cultures produced a highly unusual metabolite, named
During our efforts to optimize CTA production rates
(typically less than 0.2 mgL ), we obtained increased yields
À1
of isolated CTA when adding potassium cyanide to the
cultures prior to organic workup. Thus we suspected that
a portion of CTA is bound in metal complexes and that the
ligand is displaceable by cyanide. Yet, a metal complex of
CTA could not be detected in the broth, likely because of the
low production rates, poor chromatographic performance,
and strong background signals.
[6]
closthioamide (CTA, 1; Figure 1). CTA has a unique sym-
metrical structure featuring six thioamide moieties. Apart
from its highly unusual structural characteristics, CTA exerts
a remarkable antimicrobial potential, in particular against
problematic nosocomial pathogens (including MRSA and
The structure of the symmetric polythioamide 1 strongly
suggests that this compound plays a role as a metal ion ligand,
in particular because the overall architecture is reminiscent of
biogenetic chelators such as the siderophore desferrioxami-
[
*] Dipl.-Chem. F. Kloss, Dr. S. Pidot, Prof. Dr. C. Hertweck
Leibniz Institute for Natural Product Research and Infection
Biology, HKI, Dept. of Biomolecular Chemistry
Beutenbergstr. 11a, 07745 Jena (Germany)
[9]
E-mail: Christian.Hertweck@hki-jena.de
ne B. In stark contrast to siderophores that bind hard Lewis
3
+
acids (Fe ), the occurrence of the high number of sulfur
atoms in CTA indicates a preference for soft metal ions. To
identify potential metal ions that could bind to CTA, we
screened mixtures of CTA and metal salts in methanol by
reverse-phase thin-layer chromatography (RP-TLC), taking
advantage of the strong UV absorbance of thioamides near
Prof. Dr. C. Hertweck
Chair of Natural Product Chemistry
Friedrich Schiller University
Jena (Germany)
Dr. H. Goerls
Institute for Inorganic and Analytical Chemistry
Friedrich Schiller University
2
70 nm. By means of this assay we could narrow down the
Humboldtstr. 8, 07743 Jena (Germany)
+
2+
number of potential candidates, with Cu /Cu being the most
Prof. Dr. T. Friedrich
Institute for Biochemistry, University of Freiburg
Albertstr. 21, 79104 Freiburg i. Br. (Germany)
+
2+
promising (Figure 2A, series a). Only with Cu /Cu was
a quantitative consumption of CTA observed and recovery of
the ligand upon cyanide addition could only be observed for
Cu (partial) and Cu (complete). In contrast, the bands for
Fe /Fe remained unchanged, thus indicating that the
thioamide has degraded (Figure 2A, series b).
[
**] We thank S. Behnken for initial biological studies, A. Perner for MS
analyses, H. Heinecke for NMR measurements, Uwe Knꢀpfer for
support in fermentations, and Dr. T. Wichard (FSU) for initial UPLC
method development. This work was supported by the “Pakt fꢀr
Forschung und Innovation” of the Free State of Thuringia and the
Federal Ministry of Science and Technology (BMBF (Germany)),
and the International Leibniz Research School for Biomolecular and
Microbial Interactions (ILRS), as part of the excellence graduate
school Jena School for Microbial Communication (JSMC).
2
+
+
2
+
3+
This pre-selection was confirmed by electrospray ioniza-
tion (ESI-MS) and high-resolution (HR) mass spectrometry
(Orbitrap) measurements. Ions of CTA–metal complexes
could only be detected for the copper-containing samples.
+
2+
Interestingly, for both Cu and Cu we observed two
2
+
dominant peaks, m/z = 410 [CTA + 2Cu] and m/z = 757
[CTA + Cu] , each showing the characteristic isotope pattern
+
Angew. Chem. Int. Ed. 2013, 52, 10745 –10748
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
10745