Synthesis and structure confirmation of fuscachelins A and B, structurally unique natural product siderophores from Thermobifida fusca

Article abstract of DOI:10.1039/c2ob26010g

The fuscachelin siderophores have been prepared synthetically as have their metal chelation complexes. The heterodimeric nature of the fuscachelin decamer lends itself to a convergent synthetic strategy. Synthetic access to the natural products and intermediates will provide readily adaptable tools in future studies examining iron-sequestration and the biosynthetic machinery.

Full text of DOI:10.1039/c2ob26010g

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COMMUNICATION
Synthesis and structure confirmation of fuscachelins A and B, structurally
unique natural product siderophores from Thermobifida fusca†
Eric J. Dimise,a Heather L. Condurso,a,b Geoffrey E. Stokera and Steven D. Bruner*b
Received 29th March 2012, Accepted 24th May 2012
DOI: 10.1039/c2ob26010g
The fuscachelin siderophores have been prepared syntheti-
cally as have their metal chelation complexes. The hetero-
dimeric nature of the fuscachelin decamer lends itself to a
convergent synthetic strategy. Synthetic access to the natural
products and intermediates will provide readily adaptable
tools in future studies examining iron-sequestration and the
biosynthetic machinery.
The acquisition of the essential nutrient iron constitutes a serious
challenge to microorganisms due to the virtual insolubility of
ferric hydroxide complexes that form under aerobic growth con-
ditions.1 Siderophores are natural product secondary metabolites
that are capable of solubilizing iron(III) via the formation of
high-affinity chelation complexes.2
Fig. 1 The fuscachelins.
Several classes of siderophores are known with structurally
diverse scaffolds that evolved to sequester iron. The natural pro-
ducts are assembled by both nonribosomal peptide synthetase
(NRPS)-dependent and independent biosynthetic pathways.3
in future experiments aimed at probing the biosynthetic machinery
Genome mining for orphan biosynthetic gene clusters has
recently led to the discovery of novel natural products, including
siderophores.4,5 We recently described the structure elucidation
of the fuscachelins (1–3, Fig. 1) using a genome mining
approach.6 The fuscachelins are produced by the moderately
thermophilic actinomycete Thermobifida fusca and represent
some of the only known secondary metabolites isolated from
thermophilic bacteria.
and the chemistry and biology of iron acquisition.
The fuscachelins are decameric peptide siderophores with a
scaffold consisting of eight peptide bonds, one hydroxamic acid
linkage and one depsipeptide bond that serves to form the
10-membered lactone of fuscachelin A (1). The N-δ-hydroxy-
ornithine/serine macrocycle provides a framework for the cis-
hydroxamate necessary for metal binding and has no direct
precedent.
The work revealed a novel structural scaffold for iron
chelation along with the description of a unique biosynthetic
gene cluster. The predicted parent natural product is the macro-
cyclic lactone, fuscachelin A (1). Two additional isolated pro-
ducts, fuscachelins B and C (2, 3), were believed to be the result
of hydrolysis and aminolysis, respectively, of 1. Described here
is the first total synthesis of this family of natural products.7 The
synthesis confirms the structure assignment of the fuscachelins
in addition to providing a source of small molecule tools for use
The core dipeptide 6 contains hydroxamate functionality that
is utilized in the natural product as a bidentate ligand in the
chelation of ferric iron. The synthesis begins with activation of
N-Boc-L-Ser(O-Bn) using DCC–HOBt, followed by addition of
O-benzylhydroxylamine to afford the benzyl protected hydroxa-
mate 4. N-Boc-δ-bromo-L-norvaline benzyl ester 5 is prepared in
two steps from N-Boc-L-Glu-OBn, serving as the precursor to
the N-δ-hydroxyornithine (HOOrn) residue.8 N-Alkylation of 4
is achieved via nucleophilic displacement of bromide 5 under
basic conditions using potassium iodide as a catalyst to give a
protected version of the Ser-N-δ-hydroxyornithine core dipep-
tide. Alkylation of protected hydroxamates frequently results in a
mixture of N- and O-alkylation products. We screened various
conditions and electrophiles (tosyl, iodo) and found that the use
of KI with an alkyl bromide gave the desired N-alkylated
product as the major regioisomer (∼1.2 : 1), consistent with pre-
vious observations.7d,9 Brief treatment of this intermediate with
aDepartment of Chemistry, Boston College, Chestnut Hill, MA 02467,
USA
bDepartment of Chemistry, University of Florida, Gainesville, FL 32611,
USA. E-mail: bruner@chem.ufl.edu; Tel: +1 (352) 392-0525
†Electronic supplementary information (ESI) available: Full experimen-
tal procedures, 1H/13C NMR, mass spectra, comparison of authentic and
synthetic 1 and 2 and HPLC chromatographic purification. See DOI:
10.1039/c2ob26010g
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neat TFA affords the O-benzyl protected Ser-HOOrn building
block (6, Scheme 1).
The core-flanking tetramer (7) is comprised of an D-Arg-Gly-
Gly sequence N-capped with 2,3-dihydroxybenzoic acid (DHB).
The four catecholate oxygen atoms serve as ligands for iron(III)
in the natural product, and taken with the core-hydroxamate,
provide the hexadentate coordination sphere for metal chelation.
The synthesis of 7 utilizes solution phase peptide coupling
methodology.
A two step coupling–deprotection sequence
provides the N-terminal deprotected D-Arg(Pmc)-Gly-Gly(OBn)
tripeptide from N-Fmoc-D-Arg(Pmc) and Gly-Gly(OBn) starting
materials. In an additional DCC mediated coupling reaction, 2,3-
bis(dibenzyloxy)benzoic acid10 is appended to the N-terminus to
afford the fully protected tetrapeptide benzyl ester. Saponifica-
tion with NaOH gives 7, the liberated C-terminus of which is
now available for coupling to core dipeptide 6 (Scheme 2).
With 6 and 7 in hand, the stage was set for the convergent
assembly of the fully protected fuscachelin B decapeptide. This
was achieved in one step via a DCC–HOBt mediated peptide
coupling reaction, which gave the full length, fully protected
peptide in 49% yield for the two couplings. The Pmc protecting
groups were quantitatively removed from the arginine side
chains via treatment with a solution of 95 : 5 : 0.1 of TFA : H2O :
thioanisole (Scheme 3).
Removal of the seven benzyl groups posed an unexpected
challenge. A one step, global deprotection of the peptide was
envisioned, based on well established precedent, employing
Scheme 3 Convergent assembly of fuscachelin A.
commonly utilized hydrogenolysis conditions.7a,c,d,11 Unfortu-
nately, the benzyl protected peptide proved to be insoluble in
common organic solvents. In addition, a screen of typical carbon
supported metal hydrogenolysis catalysts failed to provided any
product at both atmospheric and increased hydrogen pressures.
Use of catalytic amounts of perchloric acid provided a small
amount of the desired product, but resulted primarily in product
decomposition.7b Ultimately, hydrogenolytic conditions (1 atm)
employing Pd black as a catalyst in a 1 : 3 methanol : acetic acid
solution yielded the fully deprotected fuscachelin
B (2)
(Scheme 3). The observed yield was not a result of turnover,
but a consequence of high affinity of the product for the metal
catalyst and low recovery from reverse phase preparative HPLC
in the final purification.
Macrolactonization of 2 provides the parent compound, fusca-
chelin A (1). The acyl coupling reagents PyBOP, DCC, DMAP
and HOBt were screened in varying combinations, as were
the Yamaguchi–Yonemitsu macrolactonization conditions.12
However, a combination of the reagents EDC and DMAP in
DMF proved to be the highest yielding, giving the lactone in
75% yield after HPLC purification (Scheme 3). The synthetic
samples of fuscachelins A and B match the natural samples iso-
lated from T. fusca (see ESI†). Formation of a seven-membered
lactone is a possible competing reaction in the condensation.
However, a single product is observed and the final product is
identical to the natural product, extensively characterized as the
ten-membered seryl lactone.6
Scheme 1 Synthesis of core dipeptide 6.
As a demonstration of the utility of the synthetic scheme, we
undertook the preparation of an analog of the natural product
for use as a probe into siderophore biosynthesis. We have a
longstanding interest in the biosynthetic mechanisms of macro-
cyclization catalyzed by thioesterase domains of nonribosomal
Scheme 2 Synthesis of core-flanking tetramer 7.
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Scheme 5 Preparation of Ga+3-fuscachelin A.
Completion of their total synthesis confirms the recently de-
termined structures of the isolated natural products. The biomi-
metic route provides useful small molecule tools in our ongoing
studies of peptide biosynthesis. In addition, the synthetic route
provides facile access to the siderophores and analogues for the
future study of high affinity metal chelation and biological iron
acquisition.
Scheme 4 Synthesis of a fuscachelin analog for use as a probe into the
biosynthetic pathway.
The authors gratefully acknowledge John Beckley for experi-
mental assistance. This work was supported by the NSF
(CAREER-0645653).
peptide synthetases and have recently demonstrated the utility of
synthetic probes to provide a structural basis of enzyme func-
tion.13 To probe the mechanism of enzyme catalyzed cyclization
of fuscachelin A, a non-cyclizable analog was designed lacking
the nucleophilic serine hydroxyl. In addition, the hydroxamate
functionality was removed to decrease metal binding and ease
handling. The common intermediate 7 is coupled to L-Ala-OBn
then N-Boc-L-Orn-OtBu using standard conditions. A second
equivalent of 7 is then added to the free amine and global depro-
tection provides the desired compound. For this analog, iterative
coupling of 7 was favored as standard peptide coupling can be
used for each building block (Scheme 4).
Siderophores have evolved to selectively bind, solubilize and
transport ferric iron into iron-starved cells. Once charged with
metal, highly conserved protein pathways are employed in the
processes of ferri-siderophore recognition, uptake, iron release
and metal trafficking.2,14 Several well characterized systems
have been studied using Ga3+-siderophore complexes as ferric-
siderophore mimicks.14 Unlike their paramagnetic, ferric
counterparts, these complexes offer the possibility of examin-
ation using NMR spectroscopy.15a,b,e,16 In addition, gallic-
siderophore complexes have proven their worth in the
crystallographic analysis of protein-siderophore co-complexes.14c
Due to the versatility of these complexes in numerous studies
and the importance of confirming the metal-chelating ability of
the synthetic molecules, we prepared gallic complexes of the
fuscachelins. This can be done in a straightforward manner by
treating fuscachelin A or B with an excess of aqueous GaBr3
(Scheme 5).15a The complexes were purified using reverse phase
HPLC and characterized by mass spectrometry (see ESI†). Syn-
thetic access to pure Ga3+-fuscachelin complexes will facilitate
the study of proteins implicated in iron metabolism in actinomy-
cetes, an ongoing effort in our laboratory.
Notes and references
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Here we have outlined a facile synthetic route to the
fuscachelins, novel siderophore natural products isolated from
the actinomycete T. fusca. The fuscachelins represent an unprece-
dented scaffold for siderophore-based acquisition of iron.
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