Highly efficient total synthesis of the clostridium-derived anti-MRSA antibiotic closthioamide

Article abstract of DOI:10.1002/ejoc.201001695

The antibiotic closthioamide from Clostridium cellulolyticum, the first example of a secondary metabolite from strictly anaerobic bacteria, was synthesized by a versatile and highly efficient synthetic route. This starts from simple building blocks and involves convergent peptide coupling and polythionation. The antibiotic closthioamide from Clostridium cellulolyticum, the first example of a secondary metabolite from strictly anaerobic bacteria, was synthesized by a versatile and highly efficient route starting from simple building blocks, involving convergent peptide coupling and polythionation. Copyright

Full text of DOI:10.1002/ejoc.201001695

SHORT COMMUNICATION
DOI: 10.1002/ejoc.201001695
Highly Efficient Total Synthesis of the Clostridium-Derived anti-MRSA
Antibiotic Closthioamide
Florian Kloss,[a] Thorger Lincke,[a] and Christian Hertweck*[a]
Keywords: Thioamides / Polythioamides / Antibiotics / Total synthesis / Natural products
The antibiotic closthioamide from Clostridium cellulolyticum,
efficient synthetic route. This starts from simple building
the first example of a secondary metabolite from strictly an- blocks and involves convergent peptide coupling and poly-
aerobic bacteria, was synthesized by a versatile and highly
thionation.
While less than a handful naturally occurring thioamides
are known,[3,4] this class of compounds has played a role
in synthetic medicinical chemistry. For example, thioamides
have been developed as therapeutics against Mycobacterium
tuberculosis,[5,6] as broad-band anthelminics,[7] antifun-
gals,[8] and have been discussed in support of inhibition of
the anthrax lethal factor.[9]
More importantly, closthioamide represents a highly
active antibacterial agent, in particular against methicillin-
resistant Staphylococcus aureus (MRSA), and vancomycin-
resistant Enterococcus faecalis (VRE), with only moderate
cytotoxicity.[3] Yet, its availability is limited by low pro-
duction rates in C. cellulolyticum cultures. To provide con-
venient access to ample amounts of the antibiotic, we have
developed an efficient synthetic strategy.
Introduction
Natural products of microbial origin represent the lead-
ing source for novel antiinfectives. It is remarkable that the
majority of antibiotics in clinical use have been derived or
inspired from the scaffolds of natural products.[1] Even so,
with the emergence and rapid distribution of multiresistant,
nosocomial pathogens, there is an urgent demand for the
discovery and development of novel antibiotic substances.
Often, however, screening for new leads is frustrating due
to high rediscovery rates. Thus, alternative strategies are
needed to tap the vast biosynthetic potential of microbes,
which is often hidden because biosynthesis genes are silent
or downregulated. We have recently triggered a cryptic bio-
synthetic pathway in Clostridium cellulolyticum, a strictly
anaerobic, cellulose-degrading bacterium.[2] Through ad-
dition of aqueous soil extracts, we succeeded in stimulating
the culture to produce a novel antibiotic, named clos-
thioamide (1a).[3] Notably, this is the first known secondary
metabolite from a strictly anaerobic bacterium. Our finding
not only pointed to an entirely new class of antibiotic pro-
ducers, but also revealed a novel drug scaffold: Closthio-
amide represents a fully unprecedented, linear hexathio-
amide (Figure 1).
Results and Discussion
Initially, we synthesized the corresponding hexamide
(closamide, 2a) as a potential precursor of closthioamide,
since conversions of amides to thioamides are well-estab-
lished reactions.[10,11] However, this approach was ham-
pered by unwanted side reactions and decreased solubility
of intermediates bearing multiple amide moieties. Thus, we
decided to employ a convergent strategy to assemble the
hexamide backbone (Scheme 1). Moreover, this strategy
would grant a higher degree of flexibility with regard to
prospective structural modifications.
The initial steps of the synthetic approach of closamide
(2a) were designed on the basis of readily available, inexpen-
sive building blocks, p-hydroxybenzoic acid (3a), β-alanine,
and 1,3-diaminopropane (7) by conventional peptide cou-
pling using carbodiimides (EDC, DCC) and hydroxybenzo-
triazole (HOBt) (Scheme 1). The four β-alanine moieties
were either introduced as benzyl ester 4 for coupling reac-
tions with benzoates 3–5, or as carbamate 8 for the coupling
to amines, e.g. 1,3-diaminopropane (7). In the latter case,
both amino groups were converted simultaneously to the
Figure 1. Schematic representation of the structure of closthio-
amide (1a).
[a] Leibniz Institute for Natural Product Research and Infection
Biology (HKI), Department of Biomolecular Chemistry,
Beutenbergstr. 11a, 07745 Jena, Germany
Fax: +49-3641-5320804
E-mail: christian.hertweck@hki-jena.de
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201001695.
Eur. J. Org. Chem. 2011, 1429–1431
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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F. Kloss, T. Lincke, C. Hertweck
SHORT COMMUNICATION
Scheme 1. Schematic presentation of the total synthesis of closthioamide (1a). Reagents and conditions: (i) 5a: EDC, HOBt, triethylamine
(TEA), DCM, room temp., 42 h, 68%; 5b: DCC, HOBt, Hünig’s base, DCM, room temp., 18 h, 97%; (ii) 6a: H2, Pd/C (1 mol-%), MeOH,
room temp., 75 min, Ͼ99%; 6b: H2, Pd/C (1 mol-%), MeOH, room temp., 3.5 h, Ͼ99%; (iii) EDC, HOBt, DMF, room temp., 96 h, 88%;
(iv) (1) dioxane, HCl (1.7 m), 50 °C, sonication; (2) MeOH, 50 °C, sonication; directly converted; (v) 2a: DCC, HOBt, TEA, DMF, room
temp., 21 h, 61%; 2b: EDC, HOBt, Hünig’s base, DMF, 30 °C, 68 h, 89%; (vi) 1a: Lawesson’s reagent, pyridine, 100 °C, 50 h, 12%; 1b:
Lawesson’s reagent, pyridine, closed vessel, 100 °C, 23 h, 96%; (vii) TFMSA in TFA (1 m), 0 °C, 12 min, 93%. For details see Supporting
Information.
corresponding bis(amide) 9 (Scheme 1). Subsequently, the (12%, ca. 5% over steps 3a Ǟ 1a). While the efficiency of
benzyl groups of 5a were removed by Pd/C-mediated hydro- this reaction proved to be unsatisfactory, we found that in
genolysis in a methanolic suspension, yielding the corre- principle a one-pot hexathionation would be feasible. En-
sponding acid 6a in nearly quantitative yield. Both Boc couraged by these findings, we next installed protective
groups of 9 were readily cleaved in HCl/MeOH to afford groups for both phenolic hydroxy groups. We assumed that
the bis(ammonium chloride) 10, which could be used for cyclohexyl ether (OChx) groups would tolerate thionation
the following steps without further purification.[12] The conditions and might even increase the solubility of the re-
hexamide backbone of closamide (2a) was assembled spective polyamides. Since the phenolic hydroxy group is
through the fusion of 2 equiv. of acid 6a and 1 equiv. of also responsible for side reactions observed in amide syn-
bis(hydrochloride) 10. In this reaction the conversion of the theses, we decided to introduce the OChx groups at an early
starting materials crucially depends on the amount of sol- stage, as p-cyclohexyloxybenzoic acid (3b).[13] In this modi-
vent. Due to the poor solubility of closamide (2a), its purifi- fied route, the amide synthesis of 5b and deprotection to 6b
cation was only achieved by washing with suitable solvents proved to be highly efficient (97%), and eventually the Chx-
and reprecipitation from methanol. Although thionation protected hexamide 2b could be isolated in excellent yield
methods are well established, the transformation of the (89%). According to our optimized thionation reaction, 2b
hexamide 2a to the corresponding hexathioamide 1a proved was completely converted to thioamide 1b by Lawesson’s
to be challenging. Hexamide 2a was found to be insoluble reagent in pyridine (closed vessel) after 23 h. This reaction
in solvents that were commonly used for thionation. How- proved to be remarkably selective. Pure 1b could be ob-
ever, we found that the use of pyridine with a high excess tained in 96% yield by a simple aqueous workup of the
of Lawesson’s reagent circumvented these problems. HPLC reaction mixture.
monitoring of the reaction showed that the hexamide was
Yet, the removal of the cyclohexyl group initially caused
fully consumed after 50 h. Unfortunately though, apart some difficulties. Several strong acidic media [HBr/acetic
from the desired thioamide 1a, the formation of two other acid, TFA, trifluoromethanesulfonic acid (TFMSA), etc.]
main products was observed. These could be tentatively as- led to decomposition and/or dethionation of 1b. However,
signed to (thio)phosphoric esters that are presumably we were able to efficiently and selectively cleave the Chx
formed in a side reaction with the phenolic hydroxy groups. ethers in a 1 m solution of TFMSA in TFA[14] at 0 °C within
In this way, closthioamide could be purified from the mix- 12 min without any side reactions. After aqueous workup,
ture by open column chromatography in only very low yield the target molecule was obtained in very good yield [93%,
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Eur. J. Org. Chem. 2011, 1429–1431

Highly Efficient Total Synthesis of Closthioamide
ca. 73% (3b Ǟ 1a), ca. 70% (7 Ǟ 1a) over all steps]. It hexathionation, but also provides the basis for a large vari-
should be highlighted that the entire route to 1a afforded ety of prospective modifications. Bioactivity tests have con-
only a single flash-chromatographic purification (5b).
firmed the impressive activity of 1a against multiresistant
The two hexathioamides 1a and 1b were subjected to bacterial pathogens. Our protocol provides efficient access
standardized antimicrobial assays to evaluate the antimicro- to a variety of closthioamides and thus sets the stage for
bial activity.[15] The antibiotic activity of synthetic clos- studying their mode of action and structure–activity rela-
thioamide (1a) was verified in comparison to the natural tionships – important prerequisites for the further develop-
product (Table 1).[3] The activity of synthetic closthioamide ment of this novel class of antibiotics.
was shown to be equivalent to the activity of the bacterial
metabolite, thus giving direct evidence for the potency of
the hexathioamide. In contrast, monothioamides, exam-
plarily represented by 4-hydroxythiobenzamide, exhibit neg-
ligible antimicrobial activities (Table 1). Surprisingly, de-
spite the common hexathioamide backbone, the Chx ether
protected derivative 1b is nearly inactive against the test or-
ganisms. It appears that the phenyl substituent (OH, ChxO)
plays an eminent role for the antibiotic potency and bioac-
tivity profiles of the thioamides. Ongoing studies will pro-
vide more information about structure–activity relation-
ships and address the biological target of the closthioam-
ides.
Supporting Information (see footnote on the first page of this arti-
cle): Detailed experimental procedures and product characteriza-
tion.
Acknowledgments
We thank A. Perner and F. Rhein for MS and NMR measurements
and C. Weigel for performing biological assays. This work was fin-
ancially supported by the Pakt für Forschung und Innovation.
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[12] The use of trifluoroacetic acid for deprotection showed also
good applicability since the reaction proceeds faster (within
minutes). However, the corresponding trifluoroacetates require
a more careful workup (anion exchange, removal of excess
acid).
[13] This reagent was synthesized according to a reported method,
but is also commercially available from various suppliers (Sup-
porting Information).
[14] Y. Nishiyama, K. Kurita, Tetrahedron Lett. 1999, 40, 927–930.
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[a] Ec: Escherichia coli; MRSA: methicillin-resistant Staphylococcus
aureus; VRE: vancomycin-resistant Enterococcus faecalis; Mv: My-
cobacterium vaccae; nat.: natural isolate.
Conclusions
We have developed a highly efficient and simple pro-
cedure for the first de novo synthesis of closthioamide start-
ing from readily available starting material. The convergent
synthetic route not only allows the multigram synthesis of
the unique polythioamide antibiotic involving a one-pot
Received: December 16, 2010
Published Online: February 3, 2011
Eur. J. Org. Chem. 2011, 1429–1431
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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