Mapping of the Modular Closthioamide Architecture Reveals Crucial Motifs of Polythioamide Antibiotics

Article abstract of DOI:10.1002/chem.201403836

Closthioamide, the first known secondary metabolite from an anaerobic microorganism (Clostridium cellulolyticum), represents a highly potent antibiotic that is active against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE) at nanomolar concentrations. To unveil structure-activity relationships of the unusual polythioamide natural product we have designed a synthetic grid to access analogues with altered terminal aromatic moieties, diverse p-phenyl substituents, different types and sizes of aliphatic spacers, varying numbers of thioamide residues, and diverse sizes and symmetries of the poly-β-thioalanyl backbone. A library of 28 closthioamide analogues was tested against a panel of human pathogenic bacteria. We found that aromatic terminal groups, the defined length of the spacer groups, the presence of all six thioamide residues and the modular arrangement of the β-thioalanyl units play essential roles for the antibiotic activity of closthioamide, yet there is a degree of freedom in the symmetry of the molecule. This study yields the first insights into pivotal structural motifs and the structural space of this new family of antibiotics, a prerequisite for the development of these promising antibiotics. Widening the scope: Structural motifs of closthioamide, the first antibiotic from strictly anaerobic microorganisms, were dissected and altered to unveil residues that are pivotal for antimicrobial activity. Using a modular approach, a library of 28 analogues was synthesized. First insights were gained into the structural space of this new family of antibiotics (see diagram).

Full text of DOI:10.1002/chem.201403836

DOI: 10.1002/chem.201403836
Full Paper
&
Natural Products
Mapping of the Modular Closthioamide Architecture Reveals
Crucial Motifs of Polythioamide Antibiotics
Florian Kloss,[a] Alina Iulia Chiriac,[c] and Christian Hertweck*[a, b]
Abstract: Closthioamide, the first known secondary metabo-
lite from an anaerobic microorganism (Clostridium cellulolyti-
cum), represents a highly potent antibiotic that is active
against methicillin-resistant Staphylococcus aureus (MRSA)
and vancomycin-resistant Enterococcus faecalis (VRE) at
nanomolar concentrations. To unveil structure–activity rela-
tionships of the unusual polythioamide natural product we
have designed a synthetic grid to access analogues with al-
tered terminal aromatic moieties, diverse p-phenyl substitu-
ents, different types and sizes of aliphatic spacers, varying
numbers of thioamide residues, and diverse sizes and sym-
metries of the poly-b-thioalanyl backbone. A library of 28
closthioamide analogues was tested against a panel of
human pathogenic bacteria. We found that aromatic termi-
nal groups, the defined length of the spacer groups, the
presence of all six thioamide residues and the modular ar-
rangement of the b-thioalanyl units play essential roles for
the antibiotic activity of closthioamide, yet there is a degree
of freedom in the symmetry of the molecule. This study
yields the first insights into pivotal structural motifs and the
structural space of this new family of antibiotics, a prerequi-
site for the development of these promising antibiotics.
Introduction
sequencing the overlooked biosynthetic potential of strictly
anaerobic bacteria, for example, Clostridium species, has
become evident.[4c] However, all biosynthesis genes investigat-
ed to date were silent under laboratory conditions and appa-
rently need to be triggered by specific signals.[5] Addition of an
aqueous soil extract to C. cellulolyticum provides the cues for
pathway activation and led to the discovery of the first antibi-
otic, closthioamide (1a, CTA, see Figure 1) from an anaerobe.
The dramatic emergence and dissemination of resistant bacte-
rial pathogens during the past decades is one of the most
challenging problems in current healthcare.[1] Indeed, alarming
numbers of nosocomial and community-acquired infections
caused by multidrug-resistant bacteria and the lack of novel
antimicrobial agents in the drug discovery pipelines could indi-
cate a beginning second pre-antibiotic era.[2] Despite the
urgent need for new antibiotics, only a small number of anti-
bacterials has been approved as therapeutics, and practically
all of them share their scaffolds with established pharmaceuti-
cals.[3] This is particularly worrying as in the battle against the
rapid resistance development, new antibiotics with novel archi-
tectures and different modes of action are urgently needed.
Whereas natural products are still the major source of inspira-
tion,[3] screening for new leads is hampered by frustratingly
high rediscovery rates. A promising approach to bypass this
problem is the genomics-guided search for natural products in
yet unexplored organisms.[4] With the advent of full genome
Figure 1. Structure of closthioamide (CTA, 1a).
Closthioamide features a symmetric linear backbone linked
by six thioamide moieties, an entity that is, although well es-
tablished in diverse fields of medicinal chemistry,[6] extremely
scarce among natural products.[7] Moreover, CTA shows potent
antimicrobial activity against infamous pathogens such as me-
thicillin-resistant Staphylococcus aureus (MRSA) and vancomy-
cin-resistant Enterococcus faecalis (VRE) at nanomolar concen-
trations.[7,8] Thus, the chemical structure of CTA is not only fully
unprecedented, but also represents a novel, yet unexplored
class of antibiotics.
[a] F. Kloss, Prof. Dr. C. Hertweck
Department of Biomolecular Chemistry
Leibniz Institute for Natural Product Research and Infection Biology
HKI, Beutenbergstr. 11a, 07745 Jena (Germany)
E-mail: Christian.Hertweck@hki-jena.de
[b] Prof. Dr. C. Hertweck
Chair of Natural Product Chemistry
Friedrich Schiller University, Jena (Germany)
Using convergent peptide coupling and polythionation tech-
niques we designed a highly efficient synthetic route towards
CTA, which confirmed the potent antibiotic activity.[8] Further-
more, genetic engineering of the producer strain allowed for
the isolation of seven congeners of CTA that enabled first in-
sights into the importance of the number of thioamide moiet-
[c] Dr. A. I. Chiriac
Department of Pharmaceutical Microbiology
Institute for Microbiology and Biotechnology, 53115 Bonn (Germany)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201403836.
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ies and the overall size of the molecule for the antibacterial ac-
tivity.[9] We also found that CTA specifically chelates CuI ions in
a compact and symmetric manner involving all six thioamide
moieties in the metal complex.[10] Yet, insights into the structur-
al requirements for antibacterial activity have remained elusive.
Here we report the first systematic study to unveil structural
features of polythioamides that are crucial for antibacterial
activity.
derivatives 3b–h and 3n–t (for selected precursor pairs see
Table 1). Thionation with Lawesson’s reagent[11] in pyridine
gave the corresponding thioamides (2b–h, n–t). Deprotection
of the O-cyclohexylated products (2n, o, p, s) with trifluorome-
thanesulfonic acid (TFMSA) in trifluoroacetic acid (TFA)[8] and of
the O-tert-butylated derivatives (2 f, r, t) with cerium(III) chlo-
ride and sodium iodide[12] yielded phenolic closthioamide de-
rivatives (1n–t) and the aliphatic alcohol 1 f. The benzylamine
derivative 1g was synthesized through deprotection of the
corresponding tert-butyl carbamate 2g with hydrogen chloride
in dry dioxane. All derivatives were tested in vitro for antibac-
terial activities and antiproliferative and cytotoxic effects ac-
cording to standardized procedures.
Results and Discussion
With the modular structure and
the convergent synthetic ap-
proach at hand, we mapped the
Table 1. Library of synthesized symmetrically substituted closthioamide derivatives, analogues and precursors
structure of CTA to reveal resi-
dues that could have an impact
on antibacterial activity. Specifi-
cally, we aimed to elucidate the
role of: 1) the terminal aromatic
substituents, 2) the p-phenyl
substituents, 3) the type and size
of aliphatic spacers, 4) the
number of thioamide residues,
and 5) the size and symmetry of
the poly-b-thioalanyl backbone
(Figure 2). To tackle this ambi-
tious goal we synthesized 28
closthioamide derivatives, ana-
logues and homologues using
a modular approach (Scheme 1,
Table 1).
(see Scheme 1).
Series
Precursor
pair
A
R[a]
m
n
r
s
2,3
2,3
2,3
2,3
1–3
1–3
2,3
1–3
1–3
1–3
1–3
1–3
1–3
b
c
d
e
f
g
h
n
o
p
r
9a
9a
9a
8
8
8
5a
-(CH2)3-
-(CH2)3-
-(CH2)3-
-(CH2)3-
-(CH2)3-
-(CH2)3-
-(CH2)3-
-(CH2)2-
-(CH2)3-
-(CH2)3-
-(CH2)3-
-(CH2)3-
-(CH2)3-
phenyl
3-pyridyl
4-pyridyl
4-oxazolyl
-(CH2)2-OH
4-(NH2-CH2)-ph·HCl
4-MeO-ph
4-OH-ph
4-OH-ph
4-OH-ph
4-OH-ph
4-OH-ph
2
2
2
2
2
2
2
2
3
2
–
2
2
2
2
2
2
2
2
2
2
2
3
–
–
2
1
1
1
1
1
1
1
1
1
1
0
0
2
1
1
1
1
1
1
1
1
1
1
0
1
1
5b
5c
4a
4d
4c
4b
5d
5d
5e
7a
5d
5 f
8
9b
9c
9a
11a
11a
8
s
t
4-OH-ph
[a] Target residue; ph=phenylene; for experimental details see the Supporting Information.
Importance of the terminal aromatic substituents
To evaluate the impact of the terminal aromatic substituents,
a focused library of derivatives bearing phenyl, heterocyclic
and aliphatic moieties was synthesized (Table 2, entries 2–7).
All derivatives were less potent than CTA. However, the least
affected candidate was the phenyl derivative (2b) with submi-
cromolar activities against the Gram-positive test strains. The
pyridyl substituted derivatives 2c and 2d, showed reduced ac-
tivities, and the 4-oxazolyl (2e) and aliphatic analogues (2 f,
1 f) were nearly inactive in the assays. These findings indicate
that the terminal six-membered aromatic rings are crucial. In
particular a phenolic group confers stronger antibacterial activ-
ities than heterocycles or aliphatics. However, we found that
the p-hydroxyl substituent in CTA is not totally essential for an-
tibacterial activity.
Figure 2. General synthetic approach to access CTA derivatives and sites of
structural diversification (boxed areas).
Taking advantage of the established polythionation ap-
proach, we designed a synthetic grid that allows the flexible
design of polythioamides. In brief, using peptide synthesis
techniques, diamines 11 a, b were coupled with N-Boc protect-
ed amino acids (10a, b) to afford dicarbamates 9a–c. Terminal
groups were introduced as carboxylic acids 4a–d (Scheme 1,
right) or as 3-amidopropanoic/4-amidobutanoic acids (5a–f,
Scheme 1, middle), obtained from coupling of b-alanine benzy-
lester or benzyl g-aminobutyrate with carboxylic acids and sub-
sequent hydrogenolysis (see Supporting Information). After de-
protection, coupling of diamides 9a’–c’ with 5a–f or of the ho-
mologue tetraamide 8’ with 4a–d or 5 f afforded closamide
The impact of the p-phenyl substituent
Since the p-hydroxyl moiety of CTA strongly contributes to the
antimicrobial activity, we reasoned that either steric, polar or
electronic effects are responsible for the increased activity. In
order to pinpoint the type of effects, we introduced a series of
substituents including ethers and esters into the CTA architec-
ture (Table 2, entries 8–18). We either incorporated the sub-
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Scheme 1. Modular approach for the synthesis of symmetric closthioamide derivatives and analogues. Reagents: i) EDC, HOBt, DMF; ii) dioxane/HCl; iii) EDC,
HOBt, Hꢁnig’s base, DMF; iv) Lawesson’s reagent, pyridine; v) CeCl3, NaI, acetonitrile; vi) TFMSA, TFA; ph=phenylene; see Table 1 (for more details see Sup-
porting Information).
stituents before thionation or employed formal semisynthetic
strategies starting from CTA (1a), taking advantage of the
moderate reactivity of both phenolic groups. Derivatives 12a–
d were readily available using modified standard procedures
for the derivatization of phenols to afford esters 12a, b or silyl
ether 12d (Scheme 2).
When sterically demanding substituents (tert-butyl, cyclohex-
yl, tbdms, tbdps) were installed, we observed a complete loss
of activity (Table 2, compounds 2g, k, m, 12c, d). A plausible
explanation would be reduced membrane permeability. Mono-
deprotection of the tert-butyl analogue 2k afforded asymmet-
ric mono-tert-butyl derivative 1k, which showed good activity
against S. aureus and E. faecium similar to the phenyl derivative
2b. To further evaluate the effect of polarity and steric
demand, the methylether (2h), fluoro-analogue (2i) and ace-
tate (12a) were synthesized and tested. Even in the sub-micro-
molar range, all three derivatives were active against the
Gram-positive test strains. The methyl ether (2h) showed very
good activity against S. aureus and E. faecium and even better
activity against E. coli than CTA. Furthermore, the methyl ether
showed drastically increased antiproliferative activity. In con-
trast, the more polar benzylamine derivative 1g was only mod-
erately active. We therefore concluded that small, electron-do-
nating p-substituents promote the antimicrobial activity, and
that a hydroxyl group is not strictly required.
Importance of the aliphatic spacers
Derivatives with a modified aliphatic backbone were synthe-
sized using the corresponding diamides 9b, c and carboxylic
precursors 5d, e as described above. b-Thioalanyl moieties
were replaced for g-thioaminobutyryl moieties, and the diami-
nopropane bridge was substituted by ethylenediamine. These
modifications (see derivatives 1n–p) resulted in a dramatic de-
crease or even complete loss of antibacterial activity. Thus, we
concluded that the length of each aliphatic spacer group plays
a pivotal role for antibiotic activity (Table 3).
Scheme 2. Semisynthetic approaches for phenolic CTA derivatives. Reagents:
i) Ac2O, DMAP, pyridine; ii) 3,5-dinitrobenzoyl chloride, Hꢁnig’s base, acetoni-
trile; iii) tbdpsCl, imidazole, DMF; dnb=3,5-dinitrobenzoyl (for more details
see the Supporting Information).
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Table 2. Influence of the terminal moieties on the corresponding bioactivity values.
EntrCyomCpomunpdound
R
R
Antimicrobial activity
Antiproliferative/cytotoxic activity
(MIC [mm])
(GI50/CC50 [mm])
[a] Drugs efflux system deficient strain; [b] mono-deprotected (for experimental details see the Supporting Information); Cip=ciprofloxacin.
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Table 3. Influence of the aliphatic spacer moieties on the corresponding bioactivity values.
Compound
A
m
n
Antimicrobial activity
Antiproliferative/cytotoxic activity
m
n
(MIC [mm])
(GI50/CC50 [mm])
[a] Drugs efflux system deficient strain.
Influence of the number of thioamide residues
Partial hydrolysis of CTA in C. cellulolyticum cultures provided
two derivatives (closthioamide B and C) each with one internal
b-thioalanyl sulfur replaced by oxygen.[9] These analogues
showed drastically reduced antibacterial activity compared to
CTA (i.e., one order of magnitude per thioamide moiety).[9] To
investigate whether the terminal (aromatic) thioamide func-
tionalities equally contribute to the antibacterial bioactivity, we
synthesized the symmetric partially thionated closthioamide
analogue 1w (Scheme 3).
Tetraamide 8 was efficiently transformed into the polythioa-
mide 2v using our established general thionation protocol.
As for derivative 2g, careful handling of the acid-sensitive
thioamides was crucial for the selective removal of Boc resi-
dues. After successful deprotection in dry dioxane/hydrogen
chloride, 2v’ was coupled with p-hydroxybenzoic acid (16) to
afford closthioamide analogue 1w. Like closthioamides B and
C, analogue 1w showed only weak antibiotic activity. Taken to-
gether, these data suggest a cooperative effect of all six thioa-
mide functionalities of CTA (Table 4).
Scheme 3. Synthesis of partially thionated closthioamide 1w. Reagents:
i) Lawesson’s reagent, pyridine; ii) dioxane/HCl; iii) EDC, HOBt, Hꢁnig’s base,
DMF (for more details see Supporting Information).
Impact of the size of the poly-b-thioalanyl backbone
The presence of b-thioalanyl moieties and six intact thioamides
proved to be a prerequisite for submicromolar antimicrobial
activities of CTA. However, it remained to be clarified whether
or not the size and symmetry of the poly-b-thioalanyl back-
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Table 4. Influence of the position of thioamide functionalities on the corresponding bioactivity values.
Compound
SC=S
SC=O
Antimicrobial activity
Antiproliferative/cytotoxic activity
(MIC [mm])
(GI50/CC50 [mm])
[a] Drugs efflux system deficient strain.
bone have impact on the antibiotic potency. We first synthe-
sized symmetric homologues 1r–t (with 0, 2 or 6 b-thioalanyl
residues) employing the modular fusion approach (Scheme 2
and Table 1). Compounds 1r and 1s were prepared from dia-
mine 11 a and carboxylic acids 7a or 5d (Scheme 2, left),
whereas octathioamide 1t was obtained by fusing tetraamide
8 and acid 5 f (Scheme 2, right).
We therefore concluded that a trithioamide residue (two b-
thioalanyl moieties and an aromatic thioamide) installed at the
diaminopropane bridge could be the key to highly potent anti-
microbial polythioamides.
The biological assays revealed that antimicrobial activities of
these analogues increase with the number of b-thioalanyl units
(Table 5). Interestingly, despite its smaller overall size the tetra-
thioamide 1s has an even higher activity than the partially
thionated derivative 1w. Among these derivatives octathioa-
mide 1t is the most potent compound, but has slightly lower
activity against S. aureus and substantially reduced activity
against E. coli compared to CTA. These results indicate that
four b-thioalanyl residues (=CTA, 1a) confer maximal activity
to the molecule. Next, we investigated asymmetric analogues
with an odd number (three and five) of b-thioalanyl units.
These derivatives (1x, y) were isolated in trace amounts as by-
products in the total synthesis of CTA when using crude pep-
tide precursors including incorrect peptide coupling products
(that is, traces of unprotected b-alanine or incomplete cou-
pling; see the Supporting Information).[8] Notably, both com-
pounds proved to be highly active against all Gram-positive
bacteria tested. The heptathioamide 1y showed similar poten-
cy against E. coli as CTA. This finding was quite unexpected, as
the octathioamide 1t is nearly inactive against E. coli (Table 5).
Scheme 4. Synthesis of asymmetrically substituted closthioamide 1z. Re-
agents: i) EDC, HOBt, Hꢁnig’s base, DMF; ii) H2, Pd(OH)2 (C), MeOH; iii) diox-
ane/HCl; iv) Lawesson’s reagent, pyridine; v) CeCl3, NaI, acetonitrile; p-
tosÀ =p-toluenesulfonate (for more details see Supporting Information).
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Table 5. Influence of the size and symmetry of the poly-b-thioalanyl backbone on bioactivity.
Compound
Sulfur atoms
Antimicrobial activity
Antiproliferative/cytotoxic activity
(MIC [mm])
(GI50/CC50 [mm])
[a] Drugs efflux system deficient strain; [b] MTBA=4-hydroxy-N-methylbenzothioamide (synthesized).[13]
To test this hypothesis, we developed a synthetic route for
the asymmetric octathioamide isomer 1z (Scheme 4). Starting
with N-Boc-protected b-alanine we prepared dipeptide 17.
After deprotection, coupling to mono-N-Boc-protected diami-
nopropane (19) afforded the asymmetric dicarbamate 20. Boc
cleavage and coupling with diamide precursor 22 (see the
Supporting Information) yielded the asymmetric closamide ho-
mologue 3z. Thionation and tert-butyl cleavage eventually
gave the asymmetric closthioamide homologue 1z. This com-
pound proved to be particularly active against the Gram-posi-
tive test strains (Table 5).
Table 3, 1n) results in a complete loss of bioactivity. We there-
fore conclude that b-thioalanyl moieties are the major feature
of this kind of polythioamide antibiotics, yet a symmetric archi-
tecture is not a prerequisite for antibacterial action.
Conclusion
The synthesis and biological evaluation of 28 closthioamide
derivatives, analogues and homologues of the highly potent
Clostridium-derived antibiotic closthioamide was reported in
this study. Our results provide the first insight into structure–
activity relationships of this novel type of antibiotic. Aromatic
terminal groups equipped with small p-substituents and the
length of each spacer group in the polythioamide chain play
pivotal roles for antibiotic activity of closthioamide. Further-
These results illustrate that the number of b-thioalanyl units
in the polythioamide chain can be modified to some degree
without impairing the antibiotic potency. This is remarkable as
already the removal of one methylene group in the chain (e.g.,
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more, we have shown that all thioamide sulfurs of CTA contrib-
ute to the antimicrobial activity. Finally, we have unequivocally
shown the importance of the modular arrangement of b-thioa-
lanyl units and that the symmetric structure is not a strict re-
quirement in this class of antibiotics (Figure 3).
Keywords: antibacterial agents
products · structure–activity relationships · thioamides
·
antibiotics
·
natural
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Experimental Section
For experimental details see the Supporting Information.
Acknowledgements
We thank A. Perner for MS analysis, H. Heinecke for NMR meas-
urements, Jonas Krꢂmer for help with MIC determination, H.-M.
Dahse for cytotoxicity and antiproliferative testing and T. Neu-
wirth and E. J. N. Helfrich for assistance in synthesis. This work
was supported by the “Pakt fꢁr Forschung und Innovation” of
the Free State of Thuringia and the Federal Ministry of Science
and Technology (BMBF, Germany).
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Received: June 6, 2014
Published online on October 3, 2014
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