Synthesis, Structure, and Molecular Dynamics of Gallium
Complexes of Schizokinen and the Amphiphilic
Evgeny A. Fadeev, Minkui Luo, and John T. Groves*
Contribution from the Department of Chemistry, Princeton UniVersity,
Princeton, New Jersey 08544
Received March 31, 2004; E-mail: email@example.com
Abstract: A new general synthesis of the citrate-based siderophores acinetoferrin (Af) and schizokinen
(Sz) and their analogues is described. The molecular structure of gallium schizokinen, GaSz, was determined
by combined1H NMR, Hartree-Fock ab initio calculations, DFT, and empirical modeling of vicinal proton
NMR spin-spin couplings. The metal-coordination geometry of GaSz was determined from NOE contacts
to be cis-cis with respect to the two chelating hydroxamates. One diaminopropane adopts a single chairlike
conformation while another is a mixture of two ring pucker arrangements. Both amide hydrogens are
internally hydrogen bonded to metal-ligating oxygen atoms. The acyl methyl groups are directed away
from each other with an average planar angle of ca. 130°. The kinetics of GaSz racemization were followed
by selective, double spin-echo inversion-recovery1H NMR spectroscopy over the temperature range of
10-45 °C. The racemization proceeds by a multistep mechanism that is proton independent between pD
5 and 12 (k0 ) 1.47 (0.15 s-1)) and acid catalyzed below pD 4 (k1 ) 2.25 (0.15) × 104M-1s-1). The
activation parameters found for the two sequential steps of the proton independent pathway were ∆Hq)
25 ( 3 kcal M-1, ∆Sq) 25 ( 7 cal M-1K-1and ∆Hq) 17.1 ( 0.2 kcal M-1, ∆Sq) 0.3 ( 2.7 cal M-1K-1.
The first step of the proton-independent mechanism was assigned to the dissociation of the carboxyl group.
The second step was assigned to complex racemization. The proton-assisted step was assigned to a
complete dissociation of the R-hydroxy carboxyl group at pD < 4. The ab initio modeling of gallium
acinetoferrin, GaAf, and analogues derived from the structure of GaSz has shown that the pendant trans-
octenoyl fragments are oriented in opposite directions with the average planar angle of ca. 130°. This
arrangement prevents GaAf from adopting a phospholipid-like structural motif. Significantly, iron siderophore
complex FeAf was found to be disruptive to phospholipid vesicles and is considerably more hydrophilic
than Af, with an eight-fold smaller partition coefficient.
Siderophores are a class of low molecular weight organic
compounds that are produced by microorganisms in response
to iron deficiency. These compounds are capable of highly
selective chelation and delivery of inorganic iron1,2to bacterial
cells via specific, receptor-mediated membrane transport mech-
anisms.3Siderophore-mediated iron uptake is an important
determinant of bacterial growth3and there is suggestive evidence
that iron proteins and siderophores are involved in cell signaling
cascades and quorum sensing.4-6Amphiphiles are a significant
and widely distributed subset of siderophores,7having been
found in pathogens,8-11terrestrial symbionts,12and marine
microorganisms.13-15Mycobactins, lipophilic siderophores of
Mycobacter smegmatis, have shown beneficial activity for the
treatment of arteriosclerosis,16breast cancer,17and postischemic
reperfusion injury.18A synthetic, amphiphilic analogue of
desferoxamine has been reported to inhibit malaria19,20and
another lipophilic tripeptide ornithine-based hydroxamate
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Published on Web 09/01/2004
10.1021/ja048145j CCC: $27.50 © 2004 American Chemical Society
J. AM. CHEM. SOC. 2004, 126, 12065-12075 9 12065
chelator was found to effectively promote growth of Mycobacter
smegmatis.21Strategies for the design of antibiotic “Trojan
Horse” siderophore conjugates have been described.22The
ubiquity of amphiphilic siderophores, the mechanisms of their
exploitation by pathogens, and their promising pharmacological
activities focus attention on the structures and functions of these
little-studied natural products.
Our interest in amphiphiles23and membrane assemblies24-27
has led us to examine the citrate-based siderophore acinetoferrin
(Af) of Acinetobacter haemoliticus, an antibiotic-resistant
bacterium that causes an increasing number of difficult-to-treat
infections.28This bacterium produces two siderophores: acineto-
ferrin9(principally) and smaller amounts of acinetobactin.29,30
Acinetoferrin belongs to a class of citrate-based siderophores
(Figure 1) in which the terminal carboxyl groups of citric acid
are peptide-coupled to two hydroxamate-bearing appendages.
Other citrate-based siderophores are aerobactin,31arthrobactin,32
nannochelin,33petrobactin,34-36rhizobactin 1021,12and schizok-
Acinetoferrin is a unique member of this family, having two
lipophilic side chains resembling a phospholipid structural motif.
The amphiphilic nature of this siderophore and its unusual
structure are suggestive of a special mechanism of action,
wherein its interactions with cell membranes play an essential
role. While the coordination chemistry of ferric schizokinen
FeSz40and ferric aerobactin41have been investigated spectro-
scopically, no molecular structures of this class of citrate-based
siderophores have been reported.
Here we present an efficient and general synthesis of Af, Sz,
and their analogues. We also describe the three-dimensional
structure of the Ga3+complexes of Sz and Af derived from1H
NMR data, Hartree-Fock ab initio, and DFT molecular
modeling. The coordination geometries of the GaAf and GaSz
hydroxamate groups are unambiguously determined to be cis-
cis. The two enantiomers of GaAf and GaSz are shown by1H
NMR to equilibrate readily. A sequential, two-step mechanism
of racemization has been discerned from the kinetics of this
process. The solution structure of FeAf and the conformational
analysis of the FeAf aliphatic tails have been inferred from the
GaSz geometry. The resulting conformation of FeAf suggests
a special mode of interaction of the metal-Af complex with the
cell phospholipid membranes that is dictated by a change in
the arrangement of the lipophilic side chains upon metal binding.
Synthesis of Siderophores. Synthesis of schizokinen42and
acinetoferrin43were reported previously. We developed a general
method (Figure 2) allowing synthetic access to both compounds
and their 2-E-butenoyl and 2-E-dodecenoyl homologues without
major changes in the procedure. The key feature of this approach
is the coupling of 2-tert-butyl-1,3-di-N-(hydroxy)succinimidyl
citrate,421, with 1-N-benzoyloxy-1,3-diaminopropane dihydro-
chloride 2. Compound 2 was conveniently obtained from
propane43upon treatment with dry HCl.
Attachment of the acyl fragments to 3 was achieved in high
yield via the appropriate acyl chloride. The resulting fully
protected siderophores, 4a-d, were amenable to purification
by column chromatography under conditions dictated by the
terminal acyl chain length. This strategy has the advantage of
retaining the protected hydroxamate groups until the molecular
scaffold was fully assembled, thus lessening the iron contamina-
tion acquired from the glassware and silica that is an inherent
difficulty with these compounds.43
The benzoyl protecting groups of 4a-d were removed under
basic conditions and the resulting crude products were separated
on a short column packed with a specially prepared, iron-free
silica.43Final cleavage of the tert-butyl esters 5a-c was effected
with 95:5 TFA:H2O at 25 °C. We found that dry TFA led to
some dehydration of the citrate hydroxyl. The free siderophores
6a (Sz) and 6c were purified by gel filtration with sephadex
G-10. Compounds 6b (Af) and 6d were sufficiently lipophilic
to be separated from the residual TFA by the chloroform/water
Stoichiometry and Overall Structure of the Iron Af and
Gallium Sz Complexes. The iron complexes Fe-6(a-d) were
prepared by metalation of 6(a-d) with ferric ammonium citrate.
Polyacrylamide gel electrophoresis showed that Fe-6(a-d) were
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Figure 1. Structure of acinetoferrin, Af (R ) trans-1-heptenyl) and Sz (R
A R T I C L E S
Fadeev et al.
12066 J. AM. CHEM. SOC.9VOL. 126, NO. 38, 2004
13C NMR (CD3OD) δ: 20.43, 27.72, 37.76, 46.28, 46.64, 75.36,
172.17, 173.85, 177.21.
CH2), 1.48 (m, 4H, CH2), 1.82 (m, 4H, CH2), 2.14 (m, 4H, CH2), 2.69
(ab-quartet, 4H, citrate CH2), 3.20 (t, 4H, CH2), 3.73 (t, 4H, CH2), 6.62
(broad d, 2H, CH), 6.83 (m, 2H, CH).
13C NMR (CD3OD) δ: 14.42, 23.55, 27.35, 29.07, 29.32, 32.65,
33.53, 37.77, 45.23, 47.04, 75.28, 120.54, 148.42, 168.58, 172.31,
Anal. Calcd for C28H48N4O9: C, 57.52; H, 8.27; N, 9.58; O, 24.63.
Found: C, 57.60; H, 8.42; N, 9.34.
2.70 (ab-quartet, 4H, citrate CH2), 3.20 (t, 4H, CH2), 3.61 (t, 4H, CH2),
6.63 (d, 2H, CH), 6.85 (m, 2H, CH).
13C NMR (CD3OD) δ: 18.43, 27.65, 37.78, 45.24, 46.87, 75.25,
121.98, 143.32, 168.67, 172.24, 177.25.
ESI-MS: m/z 473 (MH+), 495 (MNa+).
1.47 (m, 4H, CH2), 1.82 (m, 4H, CH2), 2.24 (m, 4H, CH2), 2.68 (ab-
quartet, 4H, citrate CH2), 3.20 (t, 4H, CH2), 3.70 (t, 4H, CH2), 6.62
(broad d, 2H, CH), 6.84 (m, 2H, CH).
13C NMR (CD3OD) δ: 14.62, 23.85, 27.76, 30.44, 30.55, 30.65,
30.78, 33.16, 33.84, 45.26, 47.31, 75.25, 120.53, 148.34, 168.62, 172.28,
Anal. Calcd for C36H64N4O9: C, 62.04; H, 9.26; N, 8.04; O, 20.66.
Found: C, 62.30; H, 9.55; N, 7.70.
Iron Acinetoferrin Complex (FeAf). Acinetoferrin 6c, 1 equiv from
2 mM solution in 100 mM pH 7.4 HEPES (aqueous + 20% vol. of
DMF or methanol to solubilize the acinetoferrin) buffer was mixed
with 1 equiv of 2 mM (in Fe concentration) solution of ferric ammonia
citrate in 100 mM pH 7.4 HEPES buffer and kept in a dark place at 25
°C for 24 h to allow for slow iron acquisition from ferric citrate clusters
Iron and Gallium Schizokinen Complexes (FeSz and GaSz).
Schizokinen 6a, 1 equiv from 2 mM aqueous solution was mixed with
1 equiv of an aqueous solution of ferric nitrate (or GaBr3). The resulting
solution was slowly neutralized by aqueous NaOH.
NMR Spectroscopy. All NMR spectra were recorded on Varian,
Inc. 300, 400, and 500 MHz instruments. Deuterated solvents were
purchased from Cambridge Isotope Laboratories, Inc. The
COSY and NOESY spectra of GaSz were recorded in D2O using
conventional pulse sequences, and in a 9:1 H2O:D2O mixture using
the corresponding pulse sequences modified with a WET water
suppression pulse train.83GaSz racemization rates were measured with
a NOESY-1D experiment in H2O:D2O 1:1 without solvent suppression
using a double pulsed field gradient spin-echo (DPFGSE) selective
multiplet excitation technique.45The methylene proton signal at δ 1.5
was selectively inverted and integral intensities of that multiplet and
of a signal at δ 2.2 were recorded as a function of mixing time. The
mixing time was within a range of 0.025-0.5 s. The sample pH was
adjusted by adding small volumes (∼10 µL) of 0.1M DCl or NaOD
solutions into the sample NMR tube and the pD was determined with
an Aldrich NMR pH electrode. A 0.2 pH unit correction was applied
to a pH meter reading to get the sample acidity instead of the suggested
0.4 since the latter value was determined for the pure D2O solutions.84
1H NMR (CD3OD) δ: 0.94 (t, 6H, CH3), 1.35 (m, 8H,
1H NMR (CD3OD) δ: 1.81 (m, 4H, CH2), 1.93 (d, 6H, CH3),
1H NMR (CD3OD) δ: 0.90 (t, 6H, CH3), 1.29 (m, 24 H, CH2),
The racemization rate constant was determined by fitting the temporal
buildup of the exchange peak and the parent peak integral intensity
ratio to a quadratic polynomial a t2+ b t. The fitting parameter b was
used for the rate constant value. The quadratic polynomial produced
better fitting results than linear because the initial linear buildup period
was too short. The ∆Gqfor the dynamic racemization of GaSz was
calculated by the Eiring equation using the determined rate constants
in the temperature range of 10-45 °C at pD ) 8.5.
Computations. The Gaussian 9856package was used for all
computations. All torsional angle energy profiles and structural
geometry optimizations were performed with RHF/6-31G(d) and
B3LYP/6-31G(d)85-87methods. Solution-phase computations were done
with the Onsager dielectric continuum SCRF model54,55at the continuum
dielectric constant value of 78.39. The cavity radius a0) 5.80 Å was
used as determined by the molecular volume calculation in Gaussian98.
The molecular structure figures were prepared with the MolMol
Transmission Electron Microscopy. TEM images (Figure S22)
were obtained with a JEOL 100C electron microscope using uranyl
acetate (1.5 wt %) as the negative staining reagent. Freshly prepared
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) SUV (0.05 mM,
final concentration) was incubated for 12 h at 25 °C with blank buffer,
Af (1:20, final molar ratio between apo-Af and DMPC) and ferric-
acinetoferrin, FeAf (1:20, final molar ratio between FeAf and DMPC).
A thin layer of mixed solution was then deposited onto a gold-coated
grid. After being stained by aqueous uranyl acetate (1.5 wt %), the
grid was dried in the air at room temperature for 15 min and then
subjected to TEM image analysis. DMPC SUV in the absence of either
Af or FeAf could be distributed homogeneously on the gold-coated
grid, while DMPC SUV incubated with either Af or FeAf exhibited a
heterogeneous distribution. Therefore, the images of the latter were
recorded by selecting vesicle-aggregated regions. Partition coefficients
were determined at 25 °C by the standard octanol-water procedure.
Acknowledgment. We thank Prof. Robert Pascal and Dr.
Adrzej Jarzecki for helpful discussions on quantum calculations,
Dr. Istvan Pelczer and Dr. Carlos Pacheco for assistance with
the NMR experiments, and Dr. Joseph Goodhouse for assistance
with TEM imaging. Financial support of this research by the
National Science Foundation (CHE-0221978), through the
Environmental Molecular Science Institute (CEBIC) at Princeton
University is gratefully acknowledged.
Supporting Information Available: Mass and NMR spectra,
charts with calculated torsion energy profiles, TEM images,
images and coordinate tables of calculated structures, table with
calculation parameters, and a scheme of the GaSz epimerization
mechanism. This material is available free of charge via the
Internet at http://pubs.acs.org.
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Structures of Ga3+Siderophore Complexes
A R T I C L E S
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