Synthesis of the Siderophore Coelichelin and Its Utility as a Probe in the Study of Bacterial Metal Sensing and Response

Article abstract of DOI:10.1021/acs.orglett.8b03857

A convergent total synthesis of the siderophore coelichelin is described. The synthetic route also provided access to acetyl coelichelin and other congeners of the parent siderophore. The synthetic products were evaluated for their ability to bind ferric iron and promote growth of a siderophore-deficient strain of the Gram-negative bacterium Pseudomonas aeruginosa under iron restriction conditions. The results of these studies indicate coelichelin and several derivatives serve as ferric iron delivery vehicles for P. aeruginosa.

Full text of DOI:10.1021/acs.orglett.8b03857

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Synthesis of the Siderophore Coelichelin and Its Utility as a Probe in
the Study of Bacterial Metal Sensing and Response
Jade C. Williams,† Jessica R. Sheldon,‡ Hunter D. Imlay,† Brendan F. Dutter,† Matthew M. Draelos,†
Eric P. Skaar,‡,§,∥ and Gary A. Sulikowski*,†,§,∥
†Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37232, United States
‡Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee 37232,
United States
§Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee 37232, United States
∥Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, Tennessee
37232, United States
S
* Supporting Information
ABSTRACT: A convergent total synthesis of the siderophore coelichelin is described. The
synthetic route also provided access to acetyl coelichelin and other congeners of the parent
siderophore. The synthetic products were evaluated for their ability to bind ferric iron and
promote growth of a siderophore-deficient strain of the Gram-negative bacterium Pseudomonas
aeruginosa under iron restriction conditions. The results of these studies indicate coelichelin and
several derivatives serve as ferric iron delivery vehicles for P. aeruginosa.
The genesis of coelichelin’s discovery dates back to 2000
he emergence of antibiotic-resistant strains of bacteria is a
Tsignificant public health concern necessitating the when Challis and co-workers identified a unique gene cluster in
S. coelicolor encoding a nonribosomal peptide synthetase
(NRPS).5a Coelichelin, a ferric-iron chelating peptide, was
later isolated from S. coelicolor and characterized as its
gallium(III) complex (4) by mass spectrometry and NMR
spectroscopy.5b Coelichelin was assigned the structure of a
trishydroxamate tetrapeptide (1), enabling the formation of a
proposed hexacoordinate, octahedral complex with ferric iron
(3).
The study of siderophores and their analogues have gained
attention for their potential use as novel therapeutics to
combat antibiotic resistance and treat iron metabolism
disorders.6 Notably, it has been demonstrated that side-
rophores can be employed as “Trojan horses” by conjugation
to either small- or large-molecule antimicrobial agents,
effectively harnessing the microbe’s own cellular machinery
to improve cellular uptake and consequently delivery of the
antimicrobial payload.7 In this regard, coelichelin has the
potential to serve as a probe molecule in the study of metal
acquisition pathways in select microbes as well as an
antimicrobial delivery vehicle.
discovery of novel antibiotics, and as a result, the study of
host−pathogen interactions has garnered increasing attention.1
It has been well-cited that bacteria require metals, such as iron,
for metabolism and pathogenesis, rendering metal acquisition
pathways as potential targets for the development of new
antimicrobial agents.2 In an effort to combat bacterial
pathogenesis, host organisms employ several mechanisms to
maintain low free-iron concentrations.3 Bacterial pathogens
respond to these iron-deficient conditions through the
secretion of high-affinity, small molecule iron chelators termed
siderophores.4 Coelichelin (Figure 1) is one such siderophore
produced by the bacterium Streptomyces coelicolor under iron-
deficient conditions.5
Herein, we describe the first reported total synthesis of
coelichelin (1) as well as an N-acetyl analogue (2, acetyl
coelichelin). The parent siderophore, N-acetyl analogue, and
Figure 1. Structures of coelichelin, corresponding ferric and gallium
ion chelate complexes, and acetyl coelichelin.
Received: December 4, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b03857
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Letter
derivatives were assayed for their ability to bind ferric iron and
support growth of Pseudomonas aeruginosa under iron-deficient
conditions. These studies revealed utilization of iron complexes
of coelichelin and several congeners, including N-Boc-
protected derivatives, by P. aeruginosa. The latter result is
significant as it reveals a site of structural modification without
loss of microbial uptake, implicating it as a candidate for
conjugation to an antimicrobial agent payload.
Coelichelin (1) is a tetrapeptide belonging to the
hydroxamate class of siderophores, which we envision could
be assembled through two key peptide couplings leading to
N,O-protected derivatives of coelichelin (5, Figure 2). Taking
Synthesis of N-benzyloxy-L-ornithine (8) began with
conversion of commercially available L-pyroglutamic acid to
the corresponding benzyl ester, followed by N-Boc protection
and reduction of the derived N-Boc amide to afford alcohol 9
(Scheme 1).9 Parikh−Doering oxidation of 9 yielded an
intermediate aldehyde that was condensed in situ with O-
benzylhydroxylamine hydrochloride, leading to a mixture of
oximes (10). Following chromatographic purification, reduc-
tion of 10 with sodium cyanoborohydride at low pH provided
N-benzyloxy-L-ornithine (8).10 Similarly, N-benzyloxy-D-orni-
thine (6a) was prepared from D-pyroglutamic acid starting with
esterification using allyl alcohol.11 Once again, N-Boc
protection of the derived ester was followed by reduction to
afford alcohol 12.12 The latter was subjected to the previously
described one-pot oxidation−oxime formation to yield oxime
13 in 71−82% yield. In this case, reductive amination of
benzyloxime 13 was followed by N-formylation and allyl ester
group removal to afford N-benzyloxy-D-ornithine (6a). In
anticipation of preparing acetyl coelichelin (2), N-acetylation
replaced N-formylation to ultimately yield acetamide 6b.
The remaining central amino acid, D-allo-threonine (7,
Figure 2), was prepared employing a known synthetic route
starting from D-threonine in five steps8 and the amino
protected as its tert-butyl carbamate to give 7. Coupling of
N-benzyloxyamine-L-ornithine (8) and D-allo-threonine (7)
using HATU reagent followed by TFA-mediated removal of
the Boc protecting groups afforded the bis-ammonium salt 11.
Gratifyingly, the latter was coupled with 2 equiv of ornithine
6a or 6b to complete protected tetrapeptide 5a or 5b,
respectively. Hydrogenolysis of 5a in methanol resulted in
removal of benzyl ether and ester groups to provide N-Boc-
protected coelichelin (12a). Likewise, hydrogenolysis of 5b
yielded N-Boc-protected acetyl coelichelin (12b). Upon
treatment with TFA in dichloromethane, carbamates 12a and
12b afforded coelichelin (1) and acetyl coelichelin (2),
respectively. For the purpose of structural correlation, the
Figure 2. Overview of synthetic strategy toward coelichelin and acetyl
coelichelin.
advantage of the pseudosymmetry of 5, first coupling of D-allo-
threonine (7) and N-benzyloxy-L-ornithine (8) would be
followed by removal of Boc protecting groups to reveal
peripheral amino groups. A second amide coupling of the
diamino intermediate with 2 equiv of D-ornithine 6 would
deliver protected coelichelins (5) and following global
deprotection yield coelichelin (1) and acetyl coelichelin (2).
A synthesis of N-Boc-D-allo-threonine (7) from D-threonine in
five steps has been previously reported.8 N-Benzyloxy-D- and
-L-ornithines (6) and (8) were derived from D- and L-
pyroglutamic acid, respectively.
Scheme 1. Total Synthesis of Coelichelin (1) and Acetyl Coelichelin (2)
B
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gallium(III) complex of synthetic coelichelin was prepared,
and its mass spectral and 1H NMR data compared favorably to
data reported for the gallium(III) complex of natural
coelichelin as reported by Challis.5
Affinity testing for ferric iron using CAS assays was
performed on coelichelin, acetyl coelichelin, and various
protected derivatives (12a, N-Boc; 12b, acetyl N-Boc, N,O-
protected 5a, and acetyl N,O-protected 5b). The commercial
siderophore desferrioxamine (DFO) served as a positive
control and doubly distilled water (dd-H2O) as a negative
control. The results of the CAS assays are presented in Figure
3 and reveal that desferrioxamine (DFO) is a slightly superior
grown under iron restriction. In the absence of endogenously
synthesized siderophores, growth of P. aeruginosa is wholly
dependent upon the uptake and utilization of the side-
rophore(s) provided (i.e., coelichelin). To this end, a
siderophore-deficient strain of P. aeruginosa14 was grown
under iron restriction, and apo- and holo-forms of coelichelin,
acetyl coelichelin, and its synthetic congeners were provided as
the sole iron source and evaluated for their ability to support
growth of the bacteria. The results of these studies are
summarized in Figure 4. Apo- (iron loaded) forms were
Figure 4. Coelichelin and congeners can facilitate growth of P.
aeruginosa under iron restriction. Agar plate bioassays were performed
to assess the ability of coelichelin (1, red bars) and its various
synthetic congeners (white bars) to promote the iron-dependent
growth of siderophore-deficient P. aeruginosa. Apo- (clear bars) and
holo- (checkered bars) forms of the substrates were assayed. 2,5-
Dihydroxybenzoic acid (DHBA; green) and ddH2O (blue) served as
positive and negative controls, respectively. Free ferric chloride was
provided at the same concentrations as used to iron-load the
substrates (black). Statistical differences relative to apo-coelichelin
(apo-1) were determined by one-way ANOVA with Dunnett’s
multiple comparison test where *p < 0.05 and ****p < 0.0001.
Figure 3. Chrome azurol S (CAS) assay for the detection of iron-
binding capabilities. (A) CAS activity of coelichelin (1), acetyl
coelichelin (2), 5a, 5b, 12a, 12b, dd-water (ddH2O; negative
control), and desferrioxamine (DFO; positive control) at the
concentrations indicated, where a decrease in absorbance at 630 nm
is reflective of an increase in iron-binding activity. (B) Visual
comparison at 100 μM concentration of each chelator. Data are
reflective of three independent experiments.
superior in promoting bacterial growth across all forms of
natural (1, coelichelin) and unnatural (2, acetyl coelichelin; N-
Boc derivatives 12a/12b). Notably, acetyl coelichelin (2)
promoted P. aeruginosa growth to the greatest extent relative to
all chelators assayed. Interestingly, N-Boc-protected congeners
(12a/12b) promoted P. aeruginosa growth, suggesting that the
peripheral amino groups present in coelichelin (1) can tolerate
additional functionality without perturbing siderophore
activity. This flexibility in uptake of structurally modified
siderophores is likely facilitated by the siderophore receptor
FoxA, which in other pseudomonads has been linked to piracy
of ferrioxamine and coelichelin.15 To confirm the bioactivity of
coelichelin (1), a fluorescence assay was employed to assess
iron-restriction of siderophore-proficient P. aeruginosa PAO1
through detection of its fluorescent siderophores, pyochelin
and pyoverdine (Supporting Figure S1).16 In the presence of
coelichelin (1), a dose-dependent decrease in pyochelin and
pyoverdine fluorescence is observed, indicating that this
siderophore is being utilized by P. aeruginosa as an iron
source. In contrast, no dose-dependent changes to fluorescence
were observed when P. aeruginosa was provided with the fully
protected tetrapeptide (5a), indicating that is not utilized as an
iron source by the bacterium. The results of these studies
iron chelator in comparison to coelichelin (1) and N,O-
protected coelichelin (5a) and N,O-protected acetyl coeliche-
lin (5b) are effectively inactive. N-Boc-protected coelichelin
(12a), N-Boc-protected acetyl coelichelin (12b), and acetyl
coelichelin (2) were also capable of chelating ferric iron. This
result has positive implications for the potential development
of coelichelin probes and/or antibiotic conjugates, and
siderophore activity of coelicehlin and its congeners was
further investigated in the following assays.
Coelichelin and its various synthetic congeners were then
assayed for their ability to serve as the sole iron source for a
bacterial reporter strain that is devoid of endogenous
siderophore production. Under iron restriction, many bacteria
express not only the biosynthetic and uptake machinery for
endogenously produced siderophores but also receptor
proteins for pirating siderophores produced by heterologous
microorganisms.13 To induce expression of both endogenous
and “xenosiderophore” uptake systems, P. aeruginosa was
C
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ment of coelichelin probes and/or antibiotic conjugates.
In conclusion, we describe a highly convergent synthesis to
coelichelin and various congeners and demonstrate the ability
of several to deliver iron(III) to a siderophore-deficient strain
of P. aeruginosa, a known opportunistic Gram-negative human
pathogen. Also revealed are sites tolerant of modification,
implying the potential for development of siderophore−
antibiotic conjugates. Preliminary results indicate that
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of interest, and efforts are underway to probe this metal
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.8b03857.
Experimental procedures, characterization data, bio-
logical activity assays, and NMR spectra for new
compounds (PDF)
(10) Kishimoto, S.; Nishimura, S.; Hatano, M.; Igarashi, M.; Kakeya,
H. J. Org. Chem. 2015, 80, 6076−6082.
(11) Rigo, B.; Lespagnol, C.; Pauly, M. J. J. Heterocycl. Chem. 1988,
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AUTHOR INFORMATION
(12) Qu, S.; Chen, Y.; Wang, X.; Chen, S.; Xu, Z.; Ye, T. Chem.
Commun. 2015, 51, 2510−2513.
■
Corresponding Author
*E-mail: gary.a.sulikowski@vanderbilt.edu.
ORCID
(13) Sheldon, J. R.; Laaskso, H. A.; Heinrichs, D. E. Iron Acquisition
Strategies for Bacterial Pathogens. In Virulence Mechanisms of Bacterial
Pathogens, 5th ed.; Cornick, N. A., Ed.; ASM Press: Washington, DC,
2016; p 43.
Gary A. Sulikowski: 0000-0002-1067-0767
Notes
(14) Ankenbauer, R. G.; Cox, C. D. J. Bacteriol. 1988, 170, 5364−
5367.
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Aigle, B. Appl. Environ. Microbiol. 2015, 81, 3132−3141. (b) Llamas,
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(16) Dumas, Z.; Ross-Gillespie, A.; Ku
London, Ser. B 2013, 280, 20131055.
The authors declare no competing financial interest.
́
ACKNOWLEDGMENTS
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̈
mmerli, R. Proc. R. Soc.
J.C.W. acknowledges the support of the Vanderbilt Chemical
Biology of Infectious Diseases (CBID) training program (T32
AI112541). M.M.D. acknowledges the support of the
Vanderbilt NSF-REU program (CHE 0850976). E.P.S. is
supported by R01 AI101171.
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