Ferrioxamine B analogues: Targeting the FoxA uptake system in the pathogenic Yersinia enterocolitica

Article abstract of DOI:10.1021/ja035182m

A series of ferrioxamine B analogues that target the bacterium Yersinia enterocolitica were prepared. These iron carriers are composed of three hydroxamate-containing monomeric units. Two identical monomers consist of N-hydroxy-3-aminopropionic acid coupl

Full text of DOI:10.1021/ja035182m

Published on Web 01/07/2005
Ferrioxamine B Analogues: Targeting the FoxA Uptake
System in the Pathogenic Yersinia enterocolitica
Hagit Kornreich-Leshem,† Carmit Ziv,‡ Elzbieta Gumienna-Kontecka,§
Rina Arad-Yellin,† Yona Chen,‡ Mourad Elhabiri,§ Anne-Marie Albrecht-Gary,*,§
Yitzhak Hadar,*,‡ and Abraham Shanzer*,†
Contribution from the Department of Organic Chemistry, Weizmann Institute of Science,
RehoVot, Israel, Faculty of Agricultural, Food and EnVironmental Quality Sciences, The Hebrew
UniVersity of Jerusalem, RehoVot, Israel, and Laboratoire de Physico-Chimie Bioinorganique,
ULP, UMR 7509 CNRS, Strasbourg, France
Received March 16, 2003; Revised Manuscript Received November 5, 2004; E-mail: Abraham.Shanzer@weizmann.ac.il
Abstract: A series of ferrioxamine B analogues that target the bacterium Yersinia enterocolitica were
prepared. These iron carriers are composed of three hydroxamate-containing monomeric units. Two identical
monomers consist of N-hydroxy-3-aminopropionic acid coupled with â-alanine, and a third unit at the amino
terminal is composed of N-hydroxy-3-aminopropionic acid and one of the following amino acids: â-alanine
(1a), phenylalanine (1b), cyclohexylalanine (1c), or glycine (1d). Thermodynamic results for representatives
of the analogues have shown a strong destabilization (3-4 orders of magnitude) of the ferric complexes
with respect to ferrioxamine B, probably due to shorter spacers and a more strained structure around the
metal center. No significant effect of the variations at the N-terminal has been observed on the stability of
the ferric complexes. By contrast, using in vivo radioactive uptake experiments, we have found that these
modifications have a substantial effect on the mechanism of iron(III) uptake in the pathogenic bacteria
Yersinia enterocolitica. Analogues 1a and 1d were utilized by the ferrioxamine B uptake system (FoxA),
while 1b and 1c either used different uptake systems or were transported to the microbial cell nonspecifically
by diffusion via the cell membrane. Transport via the FoxA system was also confirmed by uptake experiments
with the FoxA deficient strain of Yersinia enterocolitica. A fluorescent marker, attached to 1a in a way that
did not interfere with its biological activity, provided additional means to monitor the uptake mechanism by
fluorescence techniques. Of particular interest is the observation that 1a was utilized by the uptake system
of ferrioxamine B in Yersinia enterocolitica (FoxA) but failed to use the ferrioxamine uptake route in
Pseudomonas putida. Here, we present a case in which biomimetic siderophore analogues deliberately
designed for a particular bacterium can distinguish between related uptake systems of different microorgan-
isms.
The importance of iron-acquisition processes has prompted us2-4
and others5-7 to consider artificial siderophores as structural
probes for the study of microbial iron uptake processes.
Investigation of analogues of the four natural siderophores,
enterobactin,8 ferrichrome,9,10 coprogen,11 and ferrioxamine,11
has provided the methodology in which one can reproduce the
Introduction
Iron is an essential micronutrient for all living organisms and
is involved in fundamental enzymatic reactions, such as oxygen
metabolism, electron-transfer processes, and synthesis of DNA
and RNA. To facilitate adequate iron(III) uptake, microorgan-
isms have developed low molecular weight molecules, termed
siderophores or iron carriers. Excreted into the environment,
the siderophores bind ferric ions and deliver them to the
microorganism via specific membrane receptors and transport
proteins. The receptor-regulated process guarantees meticulous
control of the intracellular iron concentration and operates
against unfavorable concentration gradients. The properties and
biological activity of the siderophores are dictated by their
structure, chirality, and the extent by which their shape fits the
binding sites of specific receptor proteins inside the membrane.1
(2) Shanzer, A.; Libman, J. In Handbook of Microbial Iron Chelates;
Winkelmann, G., Ed.; CRC: Boca Raton, FL, 1991; pp 309-338.
(3) Shanzer, A.; Libman, J.; Yakirevitch, P.; Hadar, Y.; Chen, Y.; Jurkevitch,
E. Chirality 1993, 5, 359-365.
(4) Shanzer, A.; Libman, J. Met. Ions Biol. Syst. 1998, 35, 329-354.
(5) Raymond, K. N.; Muller, G.; Matzanke, B. F. Top. Cur. Chem. 1984, 123,
49-102.
(6) Telford, J. R.; Raymond, K. N. Compr. Supramol. Chem. 1996, 1, 245-
266.
(7) Roosenberg, J. M.; Lin, Y. M.; Lu, Y.; Miller, M. J. Curr. Med. Chem.
2000, 7, 159-197.
(8) Tor, Y.; Libman, J.; Shanzer, A.; Felder, C. E.; Lifson, S. J. Am. Chem.
Soc. 1992, 114, 6661-6671.
(9) Dayan, I.; Libman, J.; Agi, Y.; Shanzer, A. Inorg. Chem. 1993, 32, 1467-
† Weizmann Institute of Science.
1475.
‡ The Hebrew University of Jerusalem.
§ University Louis Pasteur of Strasbourg.
(1) Winkelmann, G. Handbook of Microbial Iron Chelates; CRC Press: Boca
Raton, FL, 1991.
(10) Shanzer, A.; Libman, J.; Lazar, R.; Tor, Y.; Emery, T. Biochem. Biophys.
Res. Commun. 1988, 157, 389-394.
(11) Yakirevitch, P.; Rochel, N.; Albrecht-Gary, A.-M.; Libman, J.; Shanzer,
A. Inorg. Chem. 1993, 32, 1779-1787.
9
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J. AM. CHEM. SOC. 2005, 127, 1137-1145
1137

A R T I C L E S
Kornreich-Leshem et al.
Chart 1. Ferrioxamine B (A) and Its Biomimetic Analogues (B)
performance of the natural carriers, both in vitro and in vivo.
Moreover, the attachment of various fluorescent markers was
shown not to alter the microbial activity of the compounds.12-14
In this study, we have targeted the Gram-negative bacteria
Yersinia enterocolitica that have both extremely pathogenic and
nonpathogenic strains; Yersinia pestis was the cause of the Great
Plague (also called Black Death), while Yersinia enterocolitica
and Yersinia pseudotuberculosis are mainly associated with
gastroenteritis due to contaminated food or water.15 It has been
suggested that supply of iron ions and production of siderophores
may be crucial factors for infections in Yersinia pestis and in
Yersinia enterocolitica.16 Yersinia enterocolitica produces the
siderophore Yersinobactin, its major source of iron, but is
capable of utilizing iron also from exogenic siderophores, such
as ferrioxamine B (FOB). This siderophore, isolated from
Streptomyces pilosus, is utilized by Yersinia enterocolitica via
the FoxA receptor, which was cloned and sequenced. The FoxA
receptor shares a high degree of sequence conservation with
Escherichia coli ferrichrome outer-membrane receptors (FhuA)
and other TonB dependent receptors.17 The TonB protein is part
of a complex known as the Ton system. This complex is
believed to undergo conformational changes that are driven by
proton motive force (PMF), which in turn enable it to interact
with the outer-membrane receptors, thereby activating trans-
port.18
ylalanine (1b) cyclohexylalanine (1c), and glycine (1d) (Chart
1). As the N-terminal part of the molecule is considered to play
a crucial role in the recognition and uptake process,22,23 the effect
of modifications near this end in the ferrioxamine B analogues
was investigated. Using 55Fe uptake experiments, we were able
to corroborate this statement and have found that the modifica-
tions at this part of the molecule did affect the uptake mechanism
by which the compounds are transported into the microbial cell.
Uptake experiments using the fluorescently labeled molecule
2 demonstrated the ability of this siderophore to serve as a carrier
of the fluorophore into the cells, with no impairment to its
properties as an iron carrier. Furthermore, the efficient fluores-
cence quenching of 2 by Fe(III) ions has provided an additional
tool for following the pathway of the parent compound 1a. The
observation that 1a did not use the ferrioxamine B uptake system
in Pseudomonas putida indicated the selectivity of this com-
pound and is of major importance since 1a can be envisioned
as a specific synthetic siderophore to the pathogenic Yersinia
enterocolitica via the ferrioxamine B uptake system.
Past experiments from our laboratory have shown that while
a number of chiral ferrioxamine analogues promoted growth in
different microbial systems by using both the ferrioxamine B
and the coprogen receptors, thus acting as broad-range carriers,19
others, such as glutamic acid derivatives, were found to
differentiate between analogous receptors of different micro-
organisms, thus acting as narrow-range carriers.19,20 These
findings together with results concerning species-specific fer-
richrome analogues10,21 have challenged us to design narrow-
range analogues of ferrioxamine B.
The major goal of this work was to prepare synthetic
siderophores that would be taken up by the bacterium Yersinia
enterocolitica mainly via the ferrioxamine B uptake system. We
have synthesized a retro-hydroxamate family of compounds that
mimic the essential structural features of ferrioxamine B. They
are composed of three monomeric units, based on variable amino
acids and N-hydroxy-3-aminopropionic acid, linked to form a
linear structure containing three hydroxamic acids. Two of the
units are common for all of the derivatives and are built of
â-alanine coupled with N-hydroxy-3-aminopropionic acid, while
the third monomeric unit, next to the N-terminal was altered
by using different amino acids, namely, â-alanine (1a), phen-
Results
To understand the structural requirements needed for a
compound to be transported into Yersinia enterocolitica via the
FoxA receptor, a set of biomimetic analogues of the natural
siderophore ferrioxamine B were synthesized. Ferrioxamine B
is known to form five enantiomeric pairs of isomers when
binding trivalent metal ions, as was established by studying its
kinetically inert Cr(III) complexes.24 To minimize the number
of stereoisomers and confer mainly cisoid complexes, the
distances between the hydroxamate binding groups in our new
compounds were shortened.11 Interestingly, a racemic mixture
of cisoidal isomers was identified in the crystal structures of
ferrioxamine D125 and ferrioxamine E26 and also in the recently
reported structure of ferrioxamine B.27 In our design, the order
of the hydroxamate group was inverted relative to that in the
natural compound, thus forming retro-hydroxamate derivatives.
This inversion was done to facilitate the synthesis by using
(12) Weizman, H.; Ardon, O.; Mester, B.; Libman, J.; Dwir, O.; Hadar, Y.;
Chen, Y.; Shanzer, A. J. Am. Chem. Soc. 1996, 118, 12368-12375.
(13) Nudelman, R.; Ardon, O.; Hadar, Y.; Chen, Y.; Libman, J.; Shanzer, A. J.
Med. Chem. 1998, 41, 1671-1678.
(14) Meijler, M. M.; Arad-Yellin, R.; Cabantchik, Z. I.; Shanzer, A. J. Am.
Chem. Soc. 2002, 124, 12666-12667.
(15) Bartlett, C. L. R. East. Mediterr. Health J. 1996, 2, 51-60.
(16) Baumler, A.; Hantke, K. In Transition Metals in Biology and their
Coordination Chemistry; Wiley-VCH: Weinheim, Germany, 1997; pp 51-
55.
(22) Muller, G.; Matzanke, B. F.; Raymond, K. N. J. Bacteriol. 1984, 160, 313-
318.
(17) Baumler, A. J.; Hantke, K. Mol. Microbiol. 1992, 6, 1309-1321.
(18) Faraldo-Gomez, J. D.; Sansom, M. S. Nat. ReV. Mol. Cell. Biol. 2003, 4,
105-116.
(23) Dhungana, S.; White, P. S.; Crumbliss, A. L. J. Am. Chem. Soc. 2003,
125, 14760-14767.
(19) Jurkevitch, E.; Hadar, Y.; Chen, Y.; Yakirevitch, P.; Libman, J.; Shanzer,
A. Microbiology (UK) 1994, 140, 1697-1703.
(24) Leong, J.; Raymond, K. N. J. Am. Chem. Soc. 1975, 97, 293-296.
(25) Hossain, M. B.; Jalal, M. A. F.; van der Helm, D. Acta Crystallogr., Sect.
C 1986, 1305-1310.
(20) Berner, I.; Yakirevitch, P.; Libman, J.; Shanzer, A.; Winkelmann, G. Biol.
Met. 1991, 4, 186-191.
(26) van der Helm, D.; Poling, M. J. Am. Chem. Soc. 1976, 98, 82-86.
(27) Dhungana, S.; White, P. S.; Crumbliss, A. L. J. Biol. Inorg. Chem. 2001,
6, 810-818.
(21) Jurkevitch, E.; Hadar, Y.; Chen, Y.; Libman, J.; Shanzer, A. J. Bacteriol.
1992, 174, 78-83.
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1138 J. AM. CHEM. SOC. VOL. 127, NO. 4, 2005

Targeting the FoxA Uptake System in Y. enterocolitica
A R T I C L E S
Scheme 1. Synthesis of Ferrioxamine B Analoguesa
a Reagents: (a) DIC, HOBt, ACN; (b) 0.1 N NaOH, MeOH; (c) DIC, HOBt, NMP; (d) 55% TFA in DCM, 5% TEA in DCM; (e) 10% TEA in MeOH,
48 h; (f) H2, 10% Pd/C, EtOH; (g) 50% TFA, DCM.
natural amino acids as building blocks, while being expected
not to affect the iron-binding capabilities nor the biological
activities of the retro-compounds.28 Introduction of a variety of
amino acids as part of the N-terminus of the “monomers”
enabled insertion of chiral centers, thus directing the complex
configuration and modification of the hydrophobicity, bulkiness,
and length of this part of the molecule (Scheme 1).
Synthesis and Characterization of the Siderophore Ana-
logues and their Iron(III) Complexes. The monomeric units
(4a-d) were prepared in solution11 and were assembled to yield
the final compounds following the protocols of solid-phase
peptide synthesis (Scheme 1).29 The structures of the products
were confirmed by spectroscopic measurements (IR, NMR, UV,
Figure 1. CD spectra of 1b and 1c. Solvent: methanol/0.1 N aqueous
sodium acetate (4:1). Complex concentration: 0.3 mM.
and ESI-MS), and the Fe(III) complexes were prepared. This
was done by titrations with FeCl3 and was monitored by UV-
vis spectroscopy following the increase of the absorption at λ
≈ 420 nm, which is characteristic of iron(III) trishydroxamate.5
The stoichiometry, which was found to be 1:1, was further
confirmed by ESI-MS.
a Λ-cis configuration,30 differing from the achiral compounds,
ferrioxamine B, 1a, and 1d, which exhibit no Cotton signal.
This observation indicates that even a single chiral center in
the vicinity of the amino terminus can direct the configuration
of the complex.
From the CD spectra (Figure 1), it was possible to conclude
that the iron complexes of the chiral derivatives 1b and 1c adopt
The EPR signals of the iron complexes of ferrioxamine B
and its analogues (1a, 1b, 1c, 1d, 6a, and 2) were recorded at
(28) Emery, T.; Emery, L.; Olsen, R. K. Biochem. Biophys. Res. Commun. 1984,
119, 1191-1197.
(29) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149-2154.
(30) Abv-Dari, K.; Raymond, K. N. J. Am. Chem. Soc. 1977, 99, 2003-2005.
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A R T I C L E S
Kornreich-Leshem et al.
Scheme 2. Protocol for the Synthesis of the Fluorescently
Labeled Compound 2a
Figure 2. Changes in the fluorescence of 2 on titration with Fe(III) ions.
Table 1. Stability Constants of the Ferric Complexes Formed with
Ferrioxamine B, 1a, 1b, and 2 (I ) 0.1 M, T ) 25.0 ( 0.2 °C)
ligand L
log KLFe(3σ)
ferrioxamine B
1a (â-alanine)
1b (phenylalanine)
ferrioxamine B
2
30.99a,b
26.8(3)b
26.1(1)b
30.0(1)c
27.2(4)c
a From refs 35 and 36. b With water. c With methanol/water (80/20 by
weight).
a Reagents: (a) H2, 10% Pd/C, EtOH; (b) DHP, TsOH; (c) 0.1 N NaOH,
MeOH; (d) DIC, HOBt, dry THF; (e) 2.5% TFA in DCM.
and secretion of the fluorescent ligand to the medium, and the
accumulation of radioactivity in the cells resulting from 55Fe
uptake, we were able to track the path of the iron from the
environment to the cells.
Binding Properties. The stability constants of three biomi-
metic analogues of ferrioxamine B (1a, 1b, and 2) were
determined using a fruitful combination of UV-vis absorption
spectrophotometry and potentiometry (Table 1). For solubility
reasons, a mixed solvent of methanol/water (80/20 by weight)
was used for 2. For the sake of comparison, ferrioxamine B
was examined in the same solvent (Table 1).
The comparison of the stability constants (Table 1) shows
that analogues 1a, 1b, and 2 are about 3-4 orders of magnitude
less stable than ferrioxamine B in water and in methanol/water.
If the shortening of the spacers between the binding units in
the synthetic derivatives does not significantly affect their acido-
basic properties,37 it is of importance in the stability of the
corresponding ferric complexes. The decrease in flexibility of
the chain is indeed crucial for the ligands to self-organize around
the ferric center and to fulfill the octahedral coordination
requirements of the metal. Moreover, similar binding constants
for 1a, 1b, and 2 (Table 1) clearly show that the substitution of
the C- or N-terminal by bulky aromatic moieties, namely, benzyl
for 1b and 6-aminonaphthalimide for 2, does not influence the
stability of the ferric complexes.
Microbial Studies. 55Fe uptake studies have shown that the
bacteria Yersinia enterocolitica sequester iron from all of the
current biomimetic analogues of ferrioxamine B at similar
efficiencies (Table 2). To establish which of the compounds
use the ferrioxamine B uptake system (FoxA), we have used a
variety of methods, namely, (i) uptake of 55Fe in the presence
110 °K, and all revealed EPR signals at g ) 4.32 and similar
line shapes, which is characteristic of Fe(III) in high spin state
(S ) 5/2) in an environment of low symmetry.31,32 Since it has
been shown that the EPR signal of Fe(III)-ferrioxamine B
complexes have g ) 4.3,33 like that obtained in our experiments,
we assume that the iron(III) complexes of the analogues, which
differ mainly at the terminal domains, have geometry similar
to that of the natural siderophore ferrioxamine B. Since we do
find differences in the biological behavior among the analogues,
we assume that the terminal domains play an important role in
the recognition events.
The synthetic protocol for the preparation of the fluorescent
derivative 2 is described in Scheme 2. Following hydrogenation
of 5a, THP protection (7), and hydrolysis (8), the doubly
protected 1a (8) was attached via the carboxyl terminal to
6-(ethylamino)-N-butyl-1,8-naphthalimides 9 and yielded the
fluorescently labeled compound 2 (Scheme 2). Since we have
found that by blocking the terminal amine with Boc group, as
in 6a, the inhibition of the compound by ferrioxamine B was
decreased, we assume that the free amine at the terminus has a
role in the uptake process of ferrioxamine B analogues.
Moreover, fluorescent labeling at the carboxyl terminal was
expected not to obstruct the recognition of 2 by the ferrioxamine
B uptake system since the recognition occurs at the iron(III)-
binding domain.12,21,34 By formation of the iron complex of 2,
the fluorescence of 2 (λem ) 533 nm) is quenched to 10% of
its original intensity (Figure 2).
Following the time-dependent fluorescence accumulation in
the growth medium, which results from iron release in the cells
(31) Ecker, D.; Lancaster, J., Jr.; Emery, T. J. Biol. Chem. 1982, 257, 8623-
8626.
(35) Evers, A.; Hancock, R. D.; Martell, A. E.; Motekaitis, R. J. Inorg. Chem.
1989, 28, 2189-2195.
(32) Butovich, L. A.; Reddy, C. C. Biochem. J. 2002, 365, 865-871.
(33) Yegorov, D.; Kozlov, A. V.; Azizova, O. A.; Vladimirov, Y. A. Free
Radical Biol. Med. 1993, 15, 565-574.
(36) Schwarzenbach, G.; Schwarzenbach, K. HelV. Chim. Acta 1963, 46, 1390-
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(34) Ferguson, A. D.; Hofmann, E.; Coulton, J. W.; Diederichs, K.; Welte, W.
Science 1998, 282, 2215-2220.
(37) Gumienna-Kontecka, E.; Kornreich-Leshem, H.; Elhabiri, M.; Shanzer, A.;
Albrecht-Gary, A.-M. Unpublished results.
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Targeting the FoxA Uptake System in Y. enterocolitica
A R T I C L E S
Table 2. 55Fe Uptake in Yersinia enterocolitica WA-C Mediated by
the Analogues 1a-1d: Inhibition by the Respiratory Inhibitor,
NaN3, and by Ferrioxamine B (FOB)
Table 3. 55Fe Uptake in Pseudomonas putida JM218 Mediated by
the Biomimetic Analogues 1a, 1b, 1c, and Ferrioxamine B:
Inhibition by NaN3, Ferrioxamine B, Coprogen, and Ferrichrome
Inhibitor
Inhibitor
b
Ksid
NaN3
(%)
FOB
(%)
coprogen
(%)
ferrichrome
(%)
b,c
(pmol Fe/min for
5 ×
108 bacteria)
siderophore
Ksid
siderophore
NaN3 (%)a
FOB (%)a
ferrioxamine B
1a (â-alanine)
1b (phenylalanine)
81
18
35
0.43 ( 0.06
0.32 ( 0.05
0.73 ( 0.23
0.68 ( 0.12
43 N.I.a
82 43
N.I.a
33
N.I.a
31
ferrioxamine B
1a (â-alanine)
1b (phenylalanine)
1c (cyclohexylalanine)
1d (glycine)
70
60
26
56
64
71
81
0.46 ( 0.05
0.46 ( 0.05
0.41 ( 0.09
0.45 ( 0.10
0.54 ( 0.15
0.35 ( 0.05
0.43 ( 0.04
39
10
6
23
14
35
1c (cyclohexylalanine) 77 N.I.a
18
26
a N.I. is no inhibition. b The concentration of Fe(III)-siderophore in the
culture medium was 1 µM. c With pmol Fe/min for 5 × 108 bacteria.
6a (N-Boc â-alanine)
2
FoxA system, while the iron accumulation in the FoxA deficient
strain was considerably lower. As a control experiment, it was
shown that the uptake of the ferrichrome-55Fe(III) complex by
the FoxA deficient mutant was similar to that of the wild type,
indicating that this bacterial strain is deficient only in the FoxA
uptake system.39
a For % inhibition ) [1 - (Ksid
inhibitor/Ksid)] × 100, K )
+
competitor or
uptake rate. b The concentration of Fe(III)-siderophore in the culture
medium was 1 µM.
When the Fe(III) complex of 2 was added to the culture
growth medium of Yersinia enterocolitica WA-C, emergence
of fluorescence was observed (Figure 4a) as a result of
accumulation of the free ligand 2 in the culture media that
follows the iron release inside the cells and excretion of the
free ligand to the medium. Incubation of the FoxA- strain
Yersinia enterocolitica 12-8 with the Fe(III) complex of 2
resulted, as expected, with the appearance of only weak
fluorescence in the culture growth medium. It should be noted
that the fluorescence of the labeled ferrichrome analogue B9-
NBD13 was not changed, which is further indication that this
siderophore utilizes a different receptor and not the FoxA, in
contrast to compounds 2 and 1a that enter the microbial cell
via the FoxA receptor, thus mimicking ferrioxamine B. The
similar performances of 1a and 2 as iron carriers (Table 2)
indicate that the fluorescent marker at the carboxyl terminus
does not modify the ability of the molecule to interact with the
receptor, in other words, that the carboxyl terminus may have
a lesser role in the uptake process. Additional 55Fe uptake
experiments with 2 further support the bioactivity of this
analogue and demonstrate the role of the FoxA system (Figure
4b).
55Fe(III) uptake experiments were conducted with the non-
siderophore-producing mutant Pseudomonas putida JM218 in
order to check the selectivity of 1a (Table 3). This microorgan-
ism was used as an indicator strain since it possesses a range
of siderophore uptake systems, including those for ferrichrome,
ferrioxamine, and coprogen. It was found that while the uptake
of 55Fe(III) from 1b and 1c was decreased by NaN3, that from
1a was significantly less inhibited, suggesting that the transport
of 1a is less dependent on an energy-driven process whereas
1b and 1c are dependent on such a process. Corroboration of
this finding was achieved by a competition experiment between
the 55Fe(III) uptake using 1a and ferrioxamine B, which has
indicated that 1a could not be transported by the ferrioxamine
B uptake system in this bacterium. It is noteworthy that in a
competition experiment with ferrichrome or coprogen, known
to be utilized by Pseudomonas putida, the 55Fe(III) uptake from
1a was not inhibited, indicating that this process in Pseudomonas
putida JM218 is not an energy-driven, nonspecific process, but
Figure 3. Iron uptake in Yersinia enterocolitica WA-C (blue) from 55Fe-
FOB (b), 55Fe-1a (2), 55Fe-ferrichrome (9), and in Yersinia enterocolitica
12-8 (FoxA-, red) from 55Fe-FOB (b), 55Fe-1a (2), and 55Fe-ferrichrome
(9). Fe(III)-siderophore concentration in the culture medium was 2.5 µM.
of NaN3, which is known for its ability to inhibit energy-
dependent processes, (ii) uptake of 55Fe in competition with
natural siderophores, (iii) comparison between the uptake rates
in the wild-type bacteria Yersinia enterocolitica WA-C and the
FoxA deficient strain, Yersinia enterocolitica 12-8,38 and (iv)
labeling of the bioactive compound with a fluorescent probe
and following the time-dependent accumulation of fluorescence
in the medium. The results of the inhibition experiments with
NaN3 and ferrioxamine B are summarized in Table 2. As seen
from the results, compounds 1a, 1c, and 1d are significantly
affected by NaN3, indicative of the participation of an energy-
driven processes in the uptake, while 1b was less affected. The
competition experiments, by incubation of Yersinia entero-
colitica WA-C with the synthetic analogues loaded with 55Fe-
(III) ions, have resulted in iron accumulation inside the bacterial
cells, which was reduced by addition of the natural siderophore
ferrioxamine B as a competitor (Table 2). Significant inhibition
was obtained for 1a (39 ( 7%), whereas 1d and 1c were
inhibited to a lesser extent (23 ( 2% and 6 ( 1%, respectively).
To further confirm the assumption that the FoxA uptake
system is being utilized, we have carried out another 55Fe uptake
experiment using both the wild-type strain and the FoxA
deficient strain (Figure 3). It was found that the 55Fe(III)
complexes of ferrioxamine B and 1a have led to accumulation
of similar amounts of iron ions in the bacterial cells having the
(39) Ferrichrome is utilized by Yersinia enterocolitica via the FcuA receptor:
Koebnik, R.; Hantke, K.; Braun, V. Mol. Microbiol. 1993, 7, 383-393.
(38) Baumler, A. J.; Hantke, K. J. Bacteriol. 1992, 174, 1029-1035.
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A R T I C L E S
Kornreich-Leshem et al.
Figure 4. (a) Fluorescence accumulation in cultures of Yersinia enterocolitica WA-C (blue) facilitated with Fe(III)-2 ([), Fe(III)-B9-NBD (9), and in
cultures of Yersinia enterocolitica 12-8 (FoxA-, red) facilitated with Fe(III)-2 ([) and Fe(III)-B9-NBD (9). (b) Uptake of 55Fe(III)-2 inhibited by FOB
and NaN3 in Yersinia enterocolitica WA-C.
rather based on diffusion. In contrast, 1b and 1c enter the cell
via an active uptake processes, similar to those of the natural
siderophores coprogen, ferrioxamine B, and ferrichrome (Table
3). The same observation was reported by Jurkevitch et al. who
have shown that ferrioxamine B and coprogen use different
receptors in Pseudomonas putida but share common transport
elements.19
1c, has resulted in greater utilization of an active uptake system,
different from that of the ferrioxamine B. A shorter chain
between the hydroxamate and the terminal amine (1d versus
1a) has decreased the active uptake via the FoxA system.
Blocking the terminal amine of 1a by a Boc group has led to a
considerable decrease in the inhibitory effect of ferrioxamine
B on the uptake rate (Table 2). However, this effect might be
attributed to the bulkiness of the Boc group.
Discussion
Attaching a fluorescent group at the carboxyl terminus of 1a
did not affect its uptake (2, Table 2), indicating that modification
at the C-terminus is well tolerated by the receptor with respect
to both recognition and uptake. However, inhibition experiments
against ferrioxamine B indicated that bulky substituents at the
N-terminus decreased susceptibility to competition (namely, 1b,
1c, and 6a), whereas substitution at the C-terminus, as in
compound 2, did not significantly change the susceptibility to
competition by ferrioxamine B.
A new family of biomimetic analogues that target the
ferrioxamine B uptake system in Yersinia enterocolitica was
prepared. The analogues possess three main features: (i) short
bridges between the hydroxamate’s binding groups that allow
the formation of mainly cisoidal complexes with Fe(III) ions,
(ii) inversion of the hydroxamate directionality relative to that
in the natural compound, and (iii) modifications at the N-
terminus. The structure and the conformation of the chain that
holds the terminal amine are believed to play a major role in
determining the uptake properties of the various ferrioxam-
ines.22,23,27,40 Indeed, we have found that altering this part of
the molecule affects neither the configuration of the metal center,
as shown by EPR, nor the stability of the ferric complexes, as
illustrated by similar binding constants (Table 1). By contrast,
these modifications drastically affect the uptake mechanisms
in Yersinia enterocolitica. Even if the ferric species with 1a,
1b, and 2 are strongly destabilized with respect to ferrioxamine
B, due to the shortening of the bridges between the hydroxam-
ate’s binding units of about 30%, the specific chelation remains
strong along the receptor binding and uptake process. Moreover,
it might facilitate the intracellular release mechanism.
Uptake experiments with NaN3 have enabled us to determine
which of the compounds enter the cells of Yersinia enterocolitica
by an active transport route. We have found that compound 1b
enters in a passive pathway, whereas compounds 1a, 1c, and
1d make greater use of active routes. Elucidation of this route
by means of competition uptake experiments with ferrioxamine
B has produced the following bioactivity order: 1a > 1d >
1c. Uptake experiments with the FoxA deficient strain have
further confirmed this mechanism. The results may also imply
that the structure of the chain between the final hydroxamate
group and the terminal amine has an important role in the uptake
process. Incorporation of an aromatic side chain, as in 1b, has
led to greater utilization of a nonactive uptake route, while
insertion of a bulky side chain, as in the cyclohexyl group in
The comparison between mechanisms of uptake in Yersinia
enterocolitica and in Pseudomonas putida, from results of the
competition experiments, has indicated that 1a can target the
bacteria Yersinia enterocolitica via the ferrioxamine B uptake
system (39% inhibition), but it is immune to competition by
FOB in P. putida. By contrast, 1b is less susceptible to
competition by FOB in Y. enterocolitica and more susceptible
in P. putida. These opposite relative sensitivities to competition
show that 1a and 1b can differentially target a specific bacterial
strain’s FOB uptake systems.
We have used Empirical force field calculations41 for model-
ing the conformations of the different iron complexes. As can
be seen in Figure 5, the differences between the energetically
most stable conformations of the iron complexes of the
analogues originate mainly from the chain that links the
hydroxamate group to the terminal amine. The fact that the
cyclic ferrioxamine E efficiently utilizes the FoxA receptor in
Yersinia enterocolitica42 may help in elucidating the structural
and conformational factors that control the recognition and
transport events. The crystal structure of ferrioxamine E, as
obtained by van der Helm et al., is essentially a planar structure
with two dissimilar faces, one polar and one nonpolar.26 On
the basis of this information, we suggest that forming a
“coplanar” conformation similar to that of ferrioxamine E may
be the guideline for the analogues’ bioactivity. This correlates
well with Dhungana and Crumbliss who have suggested that
(41) Felder, C. E.; Shanzer, A. Biopolymers 2003, 68, 407-421.
(42) Deiss, K.; Hantke, K.; Winkelmann, G. BioMetals 1998, 11, 131-137.
(40) Muller, G.; Raymond, K. N. J. Bacteriol. 1984, 160, 304-312.
9
1142 J. AM. CHEM. SOC. VOL. 127, NO. 4, 2005

Targeting the FoxA Uptake System in Y. enterocolitica
A R T I C L E S
1
was measured with an SLM Instrument (Model 4800). H NMR and
COSY spectra were measured on a Bruker AMX-400 MHz spectrom-
eter or on a Bruker DPX-250 MHz spectrometer using the solvent
deuterium signal as an internal reference. All J values are given in
hertz. Unless otherwise stated, the spectra were recorded in CDCl3.
EPR spectra were recorded using 1 mM solutions of the iron
complexes in methanol, which contained about 10% (v/v) 0.1 N sodium
acetate solution. The spectra were recorded at 110 Κ using an ER 200
D-SRC spectrometer (Bruker).
Mass spectroscopy (ESI-MS) was performed with an LCZ 4000
instrument (Micromass, Manchester, UK), and MALDI-TOF was
performed with a 2E instrument (Micromass, Manchester, UK). CD
spectra were recorded on an Aviv-202 circular dichroism spectrometer
(Lakewood, NJ). Flash chromatography was performed using Merck
230-400 mesh silica gel. The purity of all compounds was controlled
Figure 5. Empirical force field-minimized conformations of 1a, 1b, 1d,
and ferrioxamine E: (A) Upper view and (B) side view.
1
by H NMR spectra and by thin-layer chromatography (TLC) using
the iron(III) complex octahedron face containing the carbonyl
oxygens might be the determining factor for recognition.27 We
assume that the substantial activity of 1a results from its ability
to form an unshielded planar conformation and the appropriate
structure of the pendant amine group that contributes an
additional favorable interaction with the receptor. Several
suggestions as to the nature of this interaction have been
postulated, including intramolecular hydrogen bonding, inter-
molecular second-coordination-sphere for host-guest com-
plexation,43-45 or a solubilization factor. The decreased inhibi-
tion by FOB of 1d probably results from the chain at the
N-terminus, which is too short for such an interaction. Since
the length of the methylene chain near the N-terminus is
identical in 1b and 1d, decreased inhibition by FOB of 1b might
stem from interference of the phenyl at the side chain.
silica gel 60 F-254 on aluminum plates and at least two different solvent
systems and was visualized by UV light, ninhydrin, iodine, or FeCl3/
EtOH.
Potentiometric titrations were performed using an automatic titrator
system, DMS 716 Titrino (Metrohm), with a combined glass electrode
(Metrohm 6.0234.100, Long Life) connected to a microcomputer.
Solvents and Materials. Acetonitrile and CH2Cl2 were dried by
filtration through basic alumina. THF was distilled from Na/benzophe-
none under Ar. Protected amino acids were purchased from Novabio-
chem (Laufelfingen, Switzerland). Unless otherwise mentioned, all
reagents were purchased from Sigma and were used without further
purification. All solvents (HPLC grade) for synthesis and purification
were obtained from Labscan (Dublin, Ireland). The following abbrevia-
tions were used: DIC ) diisopropylcarbodiimide, TsOH ) p-toluene-
sulfonic acid, TEA ) triethylamine, NMP ) N-methyl-2-pyrrolidone,
DDW ) double-distilled water, Cha ) 3-cyclohexyl-L-alanine, Gly )
glycine, â-Ala ) â-alanine, Phe ) L-phenylalanine, HOBt ) 1-hy-
droxybenzotriazole, DCM ) dichloromethane, MeOH ) methanol,
ACN ) acetonitrile, TFA ) trifluoroacetic acid, Bzl ) benzyl, Boc )
tert-butyloxycarbonyl, Ar ) aryl, Ph ) phenyl, and Et ) ethyl,
EtOAc ) ethyl acetate.
Conclusions
Design and synthesis of “tailor-made” siderophore analogues,
which target narrow-range microbial uptake systems, is a very
important goal due to their prospective role in therapy and
diagnostics. Siderophore-drug conjugates46 may provide a
complementary treatment for systematic infections that com-
plicate the therapy by Desferal,47 for example, which promotes
bacterial growth in Yersinia enterocolitica and prevents, there-
fore, the administration of the drug as long as the infection
persists. They may also help in the fight against antibiotic-
resistant microorganisms.7,48
General Procedures. Compounds 3a-d, 4a-d, and N-(benzyloxy)-
3-aminopropionic acid ethyl ester were prepared according to published
procedures.11 6-Ethylamino-N-butyl-1,8-naphthalimides (9) was pre-
pared according to Chang et al.49 and de Silva et al.50
General Protocol for the Synthesis of the Trimers. The “trimer”
was synthesized by solid-phase peptide synthesis procedures using
Merrifield resin (chloromethylated polystyrene, 2% divinylbenzene
resin). Cesium salts of the first monomeric units were prepared by
dissolving the monomers (4a-d) (1.1 equiv, 1.2 mmol) in MeOH (50
mL) and DDW (5 mL) and were titrated to pH 7 with 20% Cs2CO3.
The solution was evaporated to dryness in vacuo and dried over P2O5
for 5 h. Then, the monomers (1.1 mmol) were shaken with the resin
(3.0 g, 0.7 mmol/g) in NMP (25 mL) overnight at 50 °C. The Boc
group was removed with 55% TFA in DCM (1 × 2 min, 1 × 30 min),
washed with DCM (1 × 2 min), and neutralized with 5% TEA in NMP
(2 × 2 min). The second monomer was mixed with HOBt (1.5 equiv,
3.15 mmol), DIC (1.5 equiv, 3.15 mmol), and TEA (3 equiv, 6.3 mmol)
in NMP (15 mL) for 10 min and was added to the reaction vessel. The
reaction proceeded overnight. After the removal of the Boc group, the
coupling of the third monomer was carried out as described for the
second one. Each reaction was followed by washes with NMP (5 × 2
min) and DCM (2 × 2min). To ensure completion, the reactions were
monitored by ninhydrin.51 The resin was filtered, dried in vacuo
overnight, and suspended in 10% TEA in MeOH. The suspension was
The ability to prepare siderophore analogues that utilize the
ferrioxamine B uptake system in Yersinia enterocolitica, which
is the major result of this study, is an additional step toward
the achievement of these goals.
Experimental Section
General Methods. IR spectra were recorded on a Nicolet 510 FTIR
spectrometer. Absorption frequencies are given in cm-1. UV-vis
spectra were measured either with a Hewlett-Packard model 8450A
diode array or with Varian CARY 50 and CARY 300 spectrophotom-
eters. Molar extinction coefficients are given in M-1 cm-1 units.
Fluorescence spectra were recorded on an SLM-AMINCO MC200
fluorescence spectrophotometer. In biological experiments, fluorescence
(43) Trzaska, S. M.; Toone, E. J.; Crumbliss, A. L. Inorg. Chem. 2000, 39,
1071-1075.
(44) Caldwell, C. D.; Crumbliss, A. L. Inorg. Chem. 1998, 37, 1906-1912.
(45) Spasojevic, I.; Armstrong, S. K.; Brickman, T. J.; Crumbliss, A. L. Inorg.
Chem. 1999, 38, 449-454.
(49) Chang, S. C.; Utecht, R. E.; Lewis, D. E. Dyes Pigm. 1999, 43, 83-94.
(50) deSilva, A. P.; Nimal-Gunaratne, H. Q.; Habib-Jiwan, J. L.; McCoy, C.
P.; Rice, T. E.; Soumillion, J. P. Angew. Chem., Int. Ed. Engl. 1995, 34,
1728-1731.
(51) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal. Biochem.
1970, 34, 595-598.
(46) Braun, V. Drug Resist. Updates 1999, 2, 363-369.
(47) Melby, K.; Slordahl, S.; Gutteberg, T. J.; Nordbo, S. A. Br. Med. J. (Clin.
Res. Ed.) 1982, 285, 467-468.
(48) Braun, V.; Braun, M. Curr. Opin. Microbiol. 2002, 5, 194-201.
9
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A R T I C L E S
Kornreich-Leshem et al.
stirred for 48 h, and the resin was filtered out, washed with CHCl3 and
MeOH, and concentrated to dryness. The cleavage reaction was repeated
in order to obtain additional product. Chromatography on silica gel
afforded the methyl esters of the trimers 5a, 5b, 5c, and 5d.
(250 MHz, CD3OD): δ 5.39 (s, 1H, NHCO), 3.78 (m, 6H, CH2NOH),
3.57 (s, 3H, CH3OOC), 3.32 (t, 4H, CH2NH), 3.10 (t, 2H, CH2NH),
2.77 (t, 2H, NH2CH2CH2CO), 2.56 (m, 6H, CH2CON(OH) and CH2-
CONH), 2.41 (m, 4H, CH2COOMe and CH2CONH). ESI-MS: 507.4
[M + H]+. Fe(III) complex, UV-vis: λmax 421 nm, ꢀ ) 2736. ESI-
MS: 560.29 [M + H]+.
Trimer 5a. Purification by chromatography, using CHCl3/MeOH
1
(95:5). H NMR (400 MHz, CDCl3): δ 7.32 (m, 15H, ArH), 6.60 (b,
1H, NHCO), 5.31 (b, 1H, NHBoc), 4.74 (s, 6H, CH2Ph), 3.92 (t, 6H,
CH2NOBzl), 3.58 (s, 3H, COOCH3), 3.41 (m, 4H, NHCH2), 3.33 (m,
2H, BocNHCH2), 2.52 (m, 6H, CH2CONO and CH2COOMe), 2.48 (m,
2H, CH2CONO), 2.38 (m, 4H, CH2CONH), 1.38 (s, 9H, t-Bu); ESI-
MS: 899.5 [M + Na]+.
Trimer 1b. The compound was prepared by using the procedure
described for compound 1a. Yield: 100%. 1H NMR (250 MHz, CD3-
OD): δ 7.25 (m, 5H, ArH), 4.51 (m, 1H, CHCH2Ph), 3.70 (m, 6H,
CH2NOH), 3.54 (s, 3H, CH3OOC), 3.32 (m, 4H, NHCH2CH2), 3.87
(m, 2H, CHCH2Ph), 2.53 (t, 6H, J ) 7.5, CH2CO), 2.40 (t, 4H, J ) 5,
CH2CO). Fe(III) complex, UV-vis: λmax 410 nm, ꢀ ) 2156. ESI-MS:
636.27 [M + H]+.
Trimer 1c. The compound was prepared by using the procedure
described for compound 1a. 1H NMR (250 MHz, CD3OD): δ 5.38 (s,
2H, NHCO), 4.30 (dd, 1H, CR H), 3.47 (m, 6H, CH2NOH), 3.56 (s,
3H, CH2OOC), 3.30 (m, 4H, CONHCH2), 2.55 (m, 6H, CH2CO), 1.65
(m, 8H, C6H11), 1.18 (m, 5H, CâH2 and C6H11). Fe(III) complex, UV-
vis: λmax 415 nm, ꢀ ) 2058. ESI-MS: 642.37 [M + H]+.
Trimer 5b. Purification by chromatography, using CHCl3/MeOH
(96:4). 1H NMR (400 MHz, CDCl3): δ 7.35 (m, 15H, ArH), 7.19 (m,
3H, C6H5), 7.06 (m, 2H, C6H5), 6.85 (b, 1H, BocNH), 6.48 (b, 1H,
NH), 5.57 (b, 1H, NH), 4.99 (dt, 1H, CR of Phe), 4.85 (q, 2H, J ) 2.5,
OCH2Ph), 4.76 (s, 2H, OCH2Ph), 4.72 (s, 2H, OCH2Ph), 4.07, 3.70
(m, 1H, CH2NOBzl), 3.96 (m, 4H, CH2NOBzl), 3.59 (s, 3H, COOCH3),
3.47 (m, 3H, CONHCH2), 3.40 (b, 1H, CONHCH2), 2.86 (Abq, 2H,
J1 ) 8.75, J2 ) 5, Câ of Phe), 2.56 (t, 6H, CH2CO), 2.43 (t, 4H, J )
4.25, CH2CO), 2.27 (m, 2H, CH2CO of first Boc-monomer), 1.37
(s, 9H, t-Bu).
Trimer 1d. The compound was prepared by using the procedure
described for compound 1a. 1H NMR (250 MHz, CD3OD): δ 4.14 (s,
2H, CRH), 3.90 (m, 6H, CH2NOH), 3.47 (s, 3H, CH3OOC), 3.20 (t,
2H, CH2NH), 2.87 (t, 2H, CH2NH), 2.58 (m, 10H, CH2CO). Fe(III)
complex, UV-vis: λmax 415 nm, ꢀ ) 2309.
1
Trimer 5c. H NMR (400 MHz, CDCl3): δ 7.37 (m, 15H, ArH),
7.01 (b, 1H, NH), 6.57 (b, 1H, NH), 5.59 (b, 1H, BocNH), 4.94 (s,
1H, CRH), 4.80 (m, 5H, CH2Ph), 4.11 (m, 1H, CH2NOBzl), 3.99 (m,
4H, CH2NOBzl), 3.48 (m, 4H, CONHCH2), 2.60 (m, 10H, CH2CO),
1.75 (b, 1H, C6H11), 1.58 (m, 4H, C6H11), 1.38 (m, 2H, CHCH2C6H11),
1.14 (m, 4H, C6H11), 0.9 (m, 1H, C6H11), 0.7 (m, 1H, C6H11).
Boc-[NHCH2CH2CON(OTHP)CH2CH2CO]3-OMe (7). The free
trishydroxamate trimer 6b (140 mg, 0.23 mmol) and 3,4 dihydro-2H-
pyran (DHP, 0.315 mL, 3.45 mmol) were dissolved in dry CH2Cl2 (45
mL), and p-toluenesulfonic acid (13.3 mg, 0.069 mmol) was added.
The solution was stirred for 5 h at rt. After DHP (0.315 mL, 3.45 mL)
was added again, the solution was stirred overnight. The organic mixture
was washed with water, dried over Na2SO4, and concentrated in vacuo.
The crude product was used in the next reaction without further
1
Trimer 5d. H NMR (250 MHz, CDCl3): δ 7.36 (m, 15H, ArH),
6.60 (b, 1H, NHCO), 5.30 (b, 1H, NHCO), 4.78 (m, 6H, CH2Ph), 4.20
(d, 2H, CR), 3.94 (m, 6H, CH2NO), 3.61 (s, 3H, CH3OCO), 3.45 (m,
2H, CH2NH), 3.35 (m, 2H, CH2NH), 2.57 (m, 6H, CH2CO), 2.40 (m,
4H, CH2CO), 1.41 (s, 9H, t-Bu). ESI-MS: 885.23 [M + Na]+.
Trimer 6a. Hydrogenation of the protected trimer (225 mg, 0.26
mmol) in absolute EtOH (30 mL) with 10% Pd/C (70 mg) proceeded
for 4.5 h at room temperature and atmospheric pressure of H2. Filtration
of the catalysts and evaporation of the solvent afforded the free
1
purification. Yield: 61%. H NMR (400 MHz, CDCl3): δ 6.50 (bs,
1H, NHCO), 4.88 (bs, 3H, OCHOCH2), 4.03 (m, 7H, NCHCH2 and
OCH2CH2), 3.86 (m, 1H, OCH2CH2), 3.75 (m, 1H, OCH2CH2), 3.68
(s, 3H, CH3OOC), 3.60 (m, 4H, OCH2CH2 and NHCH2), 3.52 (m, 4H,
OCH2CH2 and NHCH2), 3.40 (m, 1H, OCH2CH2 and NHCH2), 1.78
(m, 4H, CH2CO), 1.59 (m, 2H, CH2CO), 1.45 (m, 6H, CH2CO), 1.55
(m, 18H, CH2 of THP), 1.45 (m, 9H, t-Bu).
1
trihydroxamate ligand (151 mg, 0.254 mmol). Yield: 97%. H NMR
(250 MHz, CDCl3 + CD3OD): δ 3.59 (m, 6H, J ) 4.5, CH2NOH),
3.39 (s, 3H, CH3OCO), 3.1 (m, 4H, J ) 7. 5, CH2NH), 3.0 (m, 2H,
CH2NH), 2.37 (m, 4H, CH2CO), 2.19 (m, 4H, CH2CO), 1.26 (s, 9H,
t-Bu). ESI-MS: 629.5 [M + Na]+.
Boc-[NHCH2CH2CON(OTHP)CH2CH2CO]3-OH (8). The THP-
protected trimer 7 (120 mg, 0.14 mmol) was dissolved in MeOH (7
mL), and the mixture was treated with 1 M aqueous sodium hydroxide
solution (2.3 mL) at room temperature. The reaction mixture was
monitored by TLC every 1 h, and additional portions of aqueous sodium
hydroxide solutions were added until all starting material had been
consumed. The mixture was washed with EtOAc/hexane (1:1) to remove
THP polymer impurities and was cooled in an ice bath and acidified
with KHSO4 to pH 2. Methanol was evaporated, and the residue was
extracted with ethyl acetate. The organic fraction was washed with
water, dried on MgSO4, filtered, and concentrated to afford the trimer
acid 8 (50 mg, 0.06 mmol). Yield: 45%. 1H NMR (250 MHz,
CDCl3): δ 7.9, 6.75 (b, 1H, NHCO), 4.88 (b, 3H, OCHOCH2), 3.96
(m, 8H, NCH2CH2 and OCH2CH2), 3.50 (m, 10H, NCH2CH2 and OCH2-
CH2), 2.60 (m, 12H, CH2CO), 1.66 (m, 18H, CH2 of THP), 1.46 (m,
9H, t-Bu).
Trimer 6b. The compound was prepared by using the procedure
1
described for compound 6a. Yield: 86%. H NMR (250 MHz, CD3-
OD): δ 7.14 (s, 5H, ArH), 4.90 (b, 1H, CR), 3.75 (m, 6H, CH2NOH),
3.56 (s, 3H, CH3OCO), 3.21 (m, 6H, CONHCH2), 2.56 (m, 8H, CH2-
CO), 2.40 (m, 4H, CH2CO), 1.25 (s, 9H, t-Bu). ESI-MS: 977.0 [M +
Na]+.
Trimer 6c. The compound was prepared by using the procedure
1
described for compound 19. Yield: 90%. H NMR (250 MHz, CD3-
OD): δ 3.75 (m, 6H, J ) 3.75, CH2NO), 3.57 (s, 3H, CH3OOC), 3.33
(t, 4H, CONHCH2), 2.56 (m, 6H, CH2CO), 2.41 (m, 4H, CH2CO), 1.81,
1.61, 1.19, 0.82 (m, 13H, C6H11 and CâH2), 1.3 (9H, t-Bu). ESI-MS:
712.9 [M + Na]+.
Trimer 6d. The compound was prepared by using the procedure
described for compound 6a, but 5d was dissolved in EtOH/EtOAc
(1:1). 1H NMR (250 MHz, CD3OD): δ 4.14 (s, 2H, CR), 3.80 (m, 6H,
CH2NOH), 3.67 (s, 3H, CH3OOC), 3.44 (t, 2H, CH2NH), 3.30 (t, 2H,
CH2NH), 2.60 (m, 10H, CH2CO), 1.42 (s, 9H, t-Bu).
Trimer 1a. 6a (50 mg, 0.08 mmol) was dissolved in dry CH2Cl2
(4 mL) and TFA (2 mL), and the reaction was stirred for 30 min at
room temperature. The solution was evaporated, and the oily product
was redissolved in CH2Cl2 and evaporated. The product was then
dissolved in MeOH (3 mL), and basic polymer (diethylaminoethylpoly-
styrene, Fluka, 20 mg) was added to remove the TFA salt. Filtration
of the polymer and evaporation of the solvent afforded the free amine
trihydroxamate trimer 1a (40 mg, 0.08 mmol). Yield: 100%. 1H NMR
Boc-[NHCH2CH2CON(OTHP)CH2CH2CO]3-6-(ethylamino)-N-
butyl-1,8-naphthalimide (10). Trimer 8 (50 mg, 0.059 mmol) was
dissolved in dry THF (4 mL). The solution was cooled to 0 °C, and
HOBt (8 mg, 0.059 mmol), DIC (9µL, 59 mmol), and 6-(ethylamino)-
N-butyl-1,8-naphthalimide (18.5 mg, 0.059 mmol) were added. The
solution was stirred for 1 h at 0 °C and then at room temperature
overnight. The THF was evaporated, and the crude product was purified
by flash chromatography (2-5% MeOH/CHCl3) to give 10 (18 mg,
1
0.016 mmol). Yield: 27%. H NMR (250 MHz, CDCl3): δ 8.51 (d,
1H, J ) 7.5, naphthyl), 8.34 (m, 2H, J ) 7, naphthyl), 7.89 (b, 1H,
NHCO), 7.58 (t, 1H, J ) 7, naphthyl), 7.34 (b, 1H, NHCO), 6.99 (b,
9
1144 J. AM. CHEM. SOC. VOL. 127, NO. 4, 2005

Targeting the FoxA Uptake System in Y. enterocolitica
A R T I C L E S
1H, NHCO), 6.56 (d, 1H, J ) 7, naphthyl), 4.83 (b, 3H, OCHOCH2),
4.11 (t, 2H, J ) 7, CH2N of imide), 3.74 (m, 9H, CH2CH2O and CH2-
NO), 3.49 (m, 13H, CH2NH and CH2CH2O), 2.69, 2.71 (m, 12H, CH2-
CO), 1.65 (m, 22H, CH2 of n-butyl and THP), 1.42 (m, 9H, t-Bu).
H-[NHCH2CH2CON(OH)CH2CH2CO]3-6-(ethylamino)-N-butyl-
1,8-naphthalimide (2). 10 (25 mg, 0.022 mmol) was dissolved in dry
CH2Cl2 (2 mL) and TFA (20 µL). The reaction was stirred for 4 h at
room temperature. The solution was evaporated, and small impurities
were removed by MeOH/ether precipitation. The product was then
dissolved in MeOH, and basic polymer (N,N-diethylaminoethylpoly-
styrene, Fluka, 20 mg) was added to remove the TFA salt. Filtration
of the polymer and evaporation of the solvent afforded the free amine
trihydroxamate trimer 2 (14 mg, 0.018 mmol). Yield: 82%. 1H NMR
(250 MHz, CD3OD): δ 8.47 (m, 2H, naphthyl), 8.35 (d, 1H, J ) 7,
naphthyl), 7.75 (b, 1H, NHCO), 7.60 (t, 1H, J ) 7, naphthyl), 7.35 (b,
1H, NHCO), 6.80 (d, 1H, J ) 7, naphthyl), 4.11 (t, 2H, J ) 5, CH2N
of imide), 3.83 (m, 6H, CH2NO), 3.56 (m, 6H, CH2NH), 3.14 (t, 2H,
CH2NH), 2.84 (t, 2H, J ) 7, CH2NH), 2.62, 2.47 (m, 12H, CH2CO),
1.67 (m, 2H, CH3CH2CH2CH2), 1.40 (m, 2H, CH3CH2CH2), 0.97 (m,
3H, CH3CH2). UV-vis: λmax 425 nm, ꢀ ) 12 460. ESI-MS: 808.7
[M + Na]+.
General Protocol for the Determination of Stability Constants.
For solubility reasons, solutions of analogues 1a and 1b of ferrioxamine
B were prepared in distilled water, while that of ligand 2 was prepared
in solvent made of 80% spectrophotometric grade methanol (Merck)
and 20% water. The ionic strength was fixed at I ) 0.1 M. As a
reference for the iron(III) binding properties by a trihydroxamate ligand,
the protonation and stability constants of the methanesulfonate salt of
ferrioxamine B (Desferal, Ciba-Geigy), FOB, were also determined
under the same experimental conditions.
grown in LMKB medium53 overnight at 28 °C on a rotary shaker at
180 rpm to a final absorbance of 0.3-0.5 at 620 nm. The bacterial
cultures were then centrifuged for 15 min at 5000 rpm, resuspended in
fresh half-strength standard succinate medium (SSM)54 to a final OD620
of 0.6, and incubated for 60 min in a water bath at 28 °C. Half-strength
SSM medium was unable to support Yersinia growth (data not shown),
which eliminates possible changes in bacterial cell numbers during the
iron uptake studies. The 55Fe-siderophore complex was added to the
bacterial culture at a final concentration of 1 µM, unless mentioned
otherwise. When appropriate, NaN3 was added to a final concentration
of 5 mM 30 min prior to the addition of the siderophores. For
competition tests, the nonlabeled competing Fe-siderophore complex
was added to the bacterial culture together with the tested 55Fe-
siderophore at the same final concentration. For each time interval,
aliquots (0.5 mL) were taken in duplicates, layered onto a mixture of
dibutyl/octyl phthalate (1:1 v/v, Sigma), and immediately centrifuged.
The supernatant was removed, and the Eppendorf tube tips were cut.
Radioactivity in the tips of the tubes containing the bacterial cells was
counted by a â-scintillation counter (TR-1900, Packard). Each experi-
ment was performed three times in duplicates.
In Vivo Fluorescence Studies. Bacterial culture was grown as
described for the 55Fe uptake studies. The Fe-siderophore complex
was added to the bacterial suspension to a final concentration of 5 µM.
Aliquots of 0.5 mL were collected and centrifuged in each time interval,
and the supernatant was divided into triplicates of 150 µL. The
fluorescence of the samples was measured in a 96 wells polystyrene
microtiter plate by an ELISA fluorimeter (Fluostar, SLT Salzburg
Austria). The samples were excited at 485 nm, and the emission was
measured at 538 nm. Results are given in arbitrary fluorescence intensity
units. Each experiment was performed three times in triplicate.
Water was further purified by passing it through a mixed bed of an
ion-exchanger (Bioblock Scientific R3-83002, M3-83006) and activated
carbon (Bioblock Scientific ORC-83005). Both methanol and water
were deoxygenated by CO2- and O2-free argon (Sigma Oxiclear
cartridge). All stock solutions were prepared using an AG 245 Mettler
Toledo analytical balance (precision 0.01 mg).
The potentiometric and UV-vis titrations were used to determine
the stability constants of iron(III) complexes of studied ferrioxamine
B analogues.37
Circular Dichroism Measurements. Methanolic solutions of ligands
1b and 1c were mixed with a methanolic solution of FeCl3 (5mM) in
1:1 stoichiometry and diluted with methanol/0.1 N aqueous NaOAc
(4:1) to a final concentration of 0.3 mM. Measurements were preformed
after equilibration for 24 h at room temperature. Previously studied
compound 411 was used as a reference, demonstrating a relatively lower
chiral preference of 1b and 1c.
Bacterial Strains. The plasmidless strain of Yersinia enterocolitica
WA-C and its FoxA- mutant, WA-C 12-8, which lacks the ferrioxamine
receptor FoxA-, were kindly provided by K. Hantke (Tubingen,
Germany).38 The sid- mutant, Pseudomonas putida JM218, was kindly
provided by L. C. van Loon (Utrecht, The Netherlands).52
Microbial 55Fe Uptake Studies. 55Fe uptake studies were conducted
according to Weizman et al.12 with minor modifications. Bacteria were
Acknowledgment. The authors express their thankfulness to
Mrs. Rachel Lazar for her skillful assistance in synthesis, Dr.
Clifford Felder for his help in modeling, Dr. Lev Weiner for
the EPR measurements, and Dr. Fabien Fratajak for providing
compound 9. Financial support from the Martin and Helen
Kimmel Center for Molecular Design, the Angel Faivovitch
Foundation for Ecological Studies, and the G. M. J. Schmidt
Minerva Center on Supramolecular Architectures are greatly
acknowledged. A.S. holds the Siegfried and Irma Ullman
Professorial Chair.
Supporting Information Available: Spectral data for 3a-d
and 4a-d. This material is available free of charge via the
Internet at http://pubs.acs.org.
JA035182M
(52) Marugg, J. D.; van Spanje, M.; Hoekstra, W. P. M.; Schippers, B.;
Weisbeek, P. J. J. Bacteriol. 1985, 164, 563-570.
(53) Hofte, M.; Seong, K. Y.; Jurkevitch, E.; Verstraete, W. Plant Soil 1991,
130, 249-257.
(54) Meyer, J. M.; Abdallah, M. A. J. Gen. Microbiol. 1978, 107, 319-328.
9
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