Biscatecholate-monohydroxamate mixed ligand siderophore-carbacephalosporin conjugates are selective sideromycin antibiotics that target Acinetobacter baumannii

Article abstract of DOI:10.1021/jm400265k

Chemical syntheses and biological evaluation of biscatecholate- monohydroxamate mixed ligand sideromycins utilizing the carbacephalosporin β-lactam antibiotic loracarbef and the fluoroquinolone antibiotic ciprofloxacin are described. The mixed ligand β-la

Full text of DOI:10.1021/jm400265k

Article
pubs.acs.org/jmc
Biscatecholate−Monohydroxamate Mixed Ligand Siderophore−
Carbacephalosporin Conjugates are Selective Sideromycin
Antibiotics that Target Acinetobacter baumannii
Timothy A. Wencewicz and Marvin J. Miller*
Department of Chemistry and Biochemistry, University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame, Indiana 46556,
United States
S
* Supporting Information
ABSTRACT: Chemical syntheses and biological evaluation of
biscatecholate−monohydroxamate mixed ligand sideromycins
utilizing the carbacephalosporin β-lactam antibiotic loracarbef
and the fluoroquinolone antibiotic ciprofloxacin are described.
The mixed ligand β-lactam sideromycin (1b) had remarkably
selective and extremely potent antibacterial activity against the
Gram-negative pathogen Acinetobacter baumannii ATCC
17961 (MIC = 0.0078 μM). The antibacterial activity of the
β-lactam sideromycin was inversely related to the iron(III)
concentration in the testing media and was antagonized by the presence of the competing parent siderophore. These data
suggested that active transport of the mixed ligand β-lactam sideromycin across the outer cell membrane of A. baumannii via
siderophore-uptake pathways was responsible for the selective and potent antibacterial activity.
carbacephalosporin conjugate (1b; Figure 1) shown previously
by our group16 to have selective antibacterial activity against A.
INTRODUCTION
■
New antibacterial chemotherapies for treating multidrug-
resistant (MDR) Gram-negative bacterial pathogens are
desperately needed.1 The ESKAPE pathogens (Enterococcus.
faecium, Staphylococcus aureus, Klebsiella pneumonia, Acineto-
bacter baumannii, Pseudomonas aeruginosa, Enterobacter aero-
genes, Escherichia coli) highlighted by the Infectious Disease
Society of America (IDSA) attest to the urgency of this public
health crisis, as many resistant infections once limited to the
hospital setting have spread to the general public.2 Among
these pathogens, Acinetobacter baumannii is being recognized as
a rising class of aggressively pathogenic Gram-negative bacteria
with the capacity to be MDR.3 New antibiotics to treat MDR A.
baumannii infections are desperately needed, as there are now
clinical strains resistant to every known antibiotic approved for
clinical use.4
Figure 1. Structures of synthetic mixed ligand siderophore (1a) and
synthetic sideromycins (1b and 1c) used in this work.
Recent efforts for developing new antibiotic agents against A.
baumannii have primarily focused on combination therapies
with known antibiotic classes.5 Other nonconventional
therapies applied toward treating MDR Gram-negative
infections have also been explored,6 including bacterial gene
transfer,7 bioengineered human tissues that produce defense
peptides,8 nitric-oxide releasing nanoparticles,9 phage therapy,10
photodynamic therapy,11 radioimmunotherapy,12 and gallium
as an iron-mimetic.13 Siderophore-mediated “Trojan Horse”
antibiotic drug delivery has been successful at treating highly
resistant Gram-negative pathogens because these antibacterial
agents overcome membrane permeability barriers14 by entering
cells through active transport mechanisms.15 The drastic need
for new antibiotics against A. baumannii inspired us to revisit a
mixed ligand biscatecholate−monohydroxamate siderophore−
baumannii. In this work, we provide insight on the mechanism
of action of conjugate 1b and show that the impressive
antibacterial potency against A. baumannii (MIC = 0.0078 μM)
results from its active transport through siderophore transport
proteins.
To date, the most successful siderophore−antibiotic
conjugates (synthetic sideromycins17) against Gram-negative
bacteria have featured β-lactam antibiotics as the warhead.18
The β-lactam antibiotics have been particularly useful in this
area because their penicillin binding protein (PBP) biological
targets are in the periplasm.19 This means that the siderophore
Received: February 20, 2013
Published: April 24, 2013
© 2013 American Chemical Society
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a
Scheme 1. Syntheses of Iron-Free (1a) and Iron-Bound (1a-Fe) Mixed Ligand Siderophores
a
Reagents and conditions: (a) 10% Pd−C, H2 (1 atm), MeOH, 26 h, 90%; (b) Fe(acac)3, MeOH, 2 h, 98%.
a
Scheme 2. Syntheses of Mixed Ligand Sideromycins 1b and 1c
a
Reagents and conditions: (a) EDC, 3, iPr2EtN, DMAP, CH2Cl2, 24 h, 52%; (b) 10% Pd−C, H2 (1 atm), HCl (3 equiv), DMF:H2O (95:5), 24 h,
68%; (c) EDC, 4, iPr2EtN, DMAP, CH2Cl2, 24 h, 52%; (d) 10% Pd−C, H2 (1 atm), MeOH, 24 h, 51%.
only needs to smuggle the antibiotic across the outer
membrane and not all the way into the cytoplasm. For this
study, we designed a siderophore−fluoroquinolone conjugate
(1c; Figure 1) with a cytoplasmic antibiotic target (DNA
gyrase19) for direct comparison with the siderophore−β-lactam
conjugate (1b).
amines of the carbacephalosporin β-lactam antibiotic, loracar-
bef, and the fluoroquinolone antibiotic, ciprofloxacin, with large
molecules, including siderophores, is well tolerated, and the
resulting conjugates still retain affinity for the protein target in
vitro.22 Carbodiimide-mediated amide formation between O-
PNB-loracarbef TFA salt 3 and protected siderophore 2 under
basic conditions provided the fully protected β-lactam
conjugate 5. Similar reaction between O-benzyl-ciprofloxacin
(4) and the protected siderophore 2 gave fully O-benzyl
protected ciprofloxacin conjugate 6.
Universal deprotection of the O-PNB and O-benzyl
protecting groups from conjugate 5 was achieved using
optimized hydrogenolysis conditions described for this system
previously (Scheme 2).16 The deprotection proceeded in high
yield as anticipated, but the tedious purification of β-lactam
sideromycin 1b reported previously16 needed to be improved
to provide higher compound purity for biological studies. A
new purification protocol was developed using sequential
purifications by size exclusion chromatography, preparative
HPLC, and recrystallization from MeOH/Et2O to provide
analytically pure material (see the Experimental Section and
Supporting Information for complete experimental details,
characterization data, and purity analysis). Universal depro-
tection of the O-benzyl protecting groups from conjugate 6 was
achieved using conventional Pd-catalyzed hydrogenolysis
conditions (Scheme 2). The fluoroquinolone sideromycin 1c
RESULTS AND DISCUSSION
■
Syntheses of Siderophores and Sideromycins. The
synthesis of penta-O-benzyl-protected siderophore 2 was
accomplished using a literature protocol previously described
by our group.20 The iron-free (1a) and iron-complexed (1a-Fe)
siderophores were synthesized as shown in Scheme 1.
Palladium-catalyzed hydrogenolysis of protected siderophore
2 in MeOH gave the universally deprotected mixed ligand
siderophore 1a in excellent yield and purity after recrystalliza-
tion from MeOH/Et2O. Treatment of siderophore 1a with
Fe(acac)3 in MeOH gave a quantitative yield of the
siderophore−Fe(III) complex as a purple solid after recrystal-
lization from MeOH/Et2O.
The syntheses of the benzyl protected siderophore−anti-
biotic conjugates 5 and 6 were accomplished by coupling
suitably protected antibiotics 3 and 4 to the free carboxyl
terminus of protected siderophore 2 (Scheme 2). The
protected antibiotics were prepared according to previously
reported literature protocols.16,21 Derivatization of the free
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a,b
Table 1. MIC Values (μM) of Compounds 1a, 1a-Fe, 1b, and 1c against ESKAPE Panel of Bacteria
E. faecium
NCTC 7171
S. aureus
SG 511
K. pneumonia
ATCC 700603
A. baumannii
ATCC 17961
P. aeruginosa
ATCC 27853
E. aerogenes
ATCC 35029
E. coli
ATCC 25922
entry
compd
1
2
3
4
5
6
1a
>128
>128
>128
>128
32
>128
>128
32
>128
>128
>128
>128
128
>128
>128
0.125
>128
>128
0.25
>128
>128
>128
>128
>128
0.125
>128
>128
>128
>128
>128
<0.015
>128
>128
8
1a-Fe
1b
1c
>128
1
>128
2
loracarbef
ciprofloxacin
8
0.5
0.25
<0.015
a
b
MIC values were determined using the broth microdilution method using visual end point analysis according to the CLSI guidelines.23 Each
compound was tested in triplicate.
was recrystallized from MeOH/Et2O, which provided analyti-
cally pure material with no need for chromatographic
purification.
Antibacterial Activity of Siderophores and Side-
romycins against ESKAPE Pathogens. The mixed ligand
β-lactam sideromycin 1b was reported previously by our group
to have unexpected selective potency toward Acinetobacter
baumannii.16 To confirm this activity, the mixed ligand
siderophore 1a, siderophore−Fe(III) complex 1a-Fe, side-
romycin 1b, sideromycin 1c, and control antibiotics loracarbef
and ciprofloxacin were evaluated in a broth microdilution
antibacterial susceptibility assay against an ESKAPE panel of
pathogenic Gram-positive and Gram-negative bacteria (Table
1). The siderophores (1a and 1a-Fe) had no growth inhibiting
activity (MIC values >128 μM) against all of the organisms,
which was expected because the siderophores should be growth
promoting factors for the bacteria. The β-lactam sideromycin
1b gave moderate MIC values against S. aureus (32 μM) and E.
coli (8 μM) and a selectively potent MIC value of 0.125 μM
against A. baumannii, which was in agreement with the
previously reported antibacterial data.16 The parent β-lactam
antibiotic, loracarbef, displayed a much different spectrum of
antibiotic activity and had no activity against A. baumannii
(MIC value >128 μM). The fluoroquinolone sideromycin 1c
had no antibacterial activity against all the bacterial strains
tested, while the parent fluoroquinolone antibiotic, ciproflox-
acin, showed the expected broad spectrum activity.
Figure 2. Agar diffusion antagonism assay for sideromycin 1b and
siderophores 1a and 1a-Fe tested against (a) A. baumannii ATCC
17961 and (b) S. aureus SG511. Images are not shown to actual scale.
target in S. aureus, where active transport into S. aureus cells is
not required for reaching the PBP biological target.19
Antagonism of the anti-A. baumannii activity of sideromycin
1b by competing siderophores24 and Fe(III) supplementa-
tion18d,25 was also observed using a Kirby−Bauer26 agar
diffusion assay27 (Figure 3). As shown in Figure 3a,
Effect of Competing Siderophores and Fe(III) Con-
centration on Sideromycin Antibacterial Activity. The
drastic difference in the spectrum of antibacterial activity for the
β-lactam sideromycin 1b and the parent antibiotic loracarbef
was the first indication that sideromycin 1b was being
transported via siderophore-uptake pathways. If sideromycin
1b was truly entering bacterial cells via siderophore-associated
pathways, then the antibacterial activity should be antagonized
by the parent siderophore (1a) and its corresponding Fe(III)
complex (1a-Fe).24 This antagonistic effect was indeed
observed in a siderophore−sideromycin antagonism assay
performed on agar media. As shown in Figure 2a, the
antibacterial activity of sideromycin 1b against A. baumannii
ATCC 17961 was antagonized at the intersection points with
siderophore 1a and its Fe(III) complex 1a-Fe. This observation
was consistent with active transport of sideromycin 1b by A.
baumannii into the periplasm where the β-lactam could interact
with its PBP biological target and cause cell death. Interestingly,
Figure 2b shows that siderophores 1a and 1a-Fe had no
antagonistic effects on the antibiotic activity of sideromycin 1b
against S. aureus SG511, a Gram-positive strain of bacteria. This
phenomenon was consistent with an extracellular β-lactam
Figure 3. Agar diffusion antibacterial susceptibility assay for side-
romycin 1b against A. baumannii ATCC 17961. Growth inhibition
zones for 1b under Fe(III)-deficient conditions (MHII agar + 100 μM
BIPY) (a), for 1b under Fe(III)-supplemented conditions (MHII agar
+ 100 μM FeCl3) (b), for a 1:1 molar mixture of 1b and 1a under
Fe(III)-deficient conditions (c), and for a 1:1 mixture of 1b and 1a-Fe
under Fe(III)-deficient conditions (d). Images are shown at relative
but not actual scale. Measured zone diameters are the average of three
independent trials. See the Supporting Information (Table S2) for
tabulated data.
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sideromycin 1b produced a growth inhibition zone with a
diameter of 20.6 mm in media made deficient in Fe(III) by
addition of 100 μM 2,2′-bipyridine (BIPY). As shown in Figure
3b, addition of 100 μM FeCl3 to the testing media reduced the
diameter of the growth inhibition zone to 16.2 mm. The inverse
relationship between Fe(III) availability and antibacterial
potency of sideromycin 1b against A. baumannii was consistent
with siderophore-related active transport across the outer
membrane.25 Additionally, the antagonistic effects of side-
rophore 1a and its Fe(III) complex 1a-Fe were confirmed in
the agar diffusion assay. As shown in parts c and d of Figure 3,
1:1 molar ratios of sideromycin 1b and siderophore 1a or
Fe(III) complex 1a-Fe, respectively, gave statistically reduced
diameters of growth inhibition in Fe(III)-deficient media
relative to 1b alone (Figure 3a).
Table 2. MIC Values (μM) of Sideromycin (1b) against A.
baumannii and S. aureus under Varying Concentrations of
Fe(III) and in the Presence of Competing Siderophores (1a
a,b
and 1a-Fe)
test organism
A. baumannii ATCC
17961
S.aureus SG511
c
d
entry
compd
1b
MHII+Fe
0.5
MHII−Fe
MHII+Fe MHII−Fe
1
2
3
0.0078
0.0312
0.25
64
nt
nt
32
16
32
e
1b + 1a
nt
1b + 1a-Fe
nt
a
MIC values were determined using the broth microdilution method
using visual end point analysis according to the CLSI guidelines.23
b
c
Each compound was tested in triplicate. MHII+Fe: Mueller−Hinton
d
broth no. 2 + 100 μM FeCl3. MHII−Fe: Mueller−Hinton broth no. 2
+ 100 μM BIPY. nt: not tested.
The results from Figure 3 are summarized graphically in
Figure 4 by plotting the diameter of the growth inhibition zone
e
while in Fe(III)-deficient media (MHII−Fe), the MIC was
0.0078 μM. This represents a 64-fold increase in activity when
going from Fe(III)-supplemented to Fe(III)-deficient con-
ditions. When 1:1 molar mixtures of sideromycin:siderophore,
1b:1a and 1b:1a-Fe, were tested, MIC values of 0.031 and 0.25
μM, respectively, were recorded. These results were in
agreement with data from the agar diffusion assays (Figures 3
and 4) where the siderophore Fe(III)-complex 1a-Fe
antagonized the antibacterial activity of sideromycin 1b to a
greater extent than the iron-free siderophore 1a. As expected,
the antibacterial activity of sideromycin 1b against S. aureus was
not influenced to any great extent by the addition Fe(III) or
siderophores (1a and 1a-Fe) to the media.
FURTHER DISCUSSION AND PERSPECTIVE
■
To better understand the highly potent and selective activity of
sideromycin 1b against A. baumannii, it was important to
consider what is known about the organism’s natural use of
siderophores. The complete genome sequences of six A.
baumannii strains have recently been reported, and genome
analysis revealed that siderophore biosynthetic genes were
present in 5 out of the 6 sequenced strains.28 The strain lacking
the siderophore biosynthetic genes was the only nonclinical
isolate of A. baumannii sequenced, which suggested that
siderophores are an important virulence factor for A. baumannii
during infection. So far, three biosynthetic gene clusters for
unique siderophores have been identified in A. baumannii,29
and the structures of two classes of these siderophores,
(pre)acinetobactin30 and fimsbactins31 (Figure 5), have been
elucidated.
Figure 4. Effect of varying Fe(III) concentrations and competing
siderophores (1a and 1a-Fe) on the antibacterial activity of
sideromycin 1b against S. aureus SG511 and A. baumannii ATCC
17961 in the agar diffusion assay. The vertical axis indicates the
diameter of the growth inhibition zone in millimeters (average of three
independent trials), and the horizontal axis indicates the test organism.
See the Supporting Information (Table S2) for tabulated data.
b
aMHII−Fe: Mueller−Hinton agar no. 2 + 100 μM BIPY. MHII+Fe:
Mueller−Hinton agar no. 2 + 100 μM FeCl3.
on the vertical axis under the assay conditions specified by the
color coded legend. Analogous experiments were performed
using 1b against a Gram-positive pathogen, S. aureus SG511.
The activity against S. aureus was not significantly perturbed by
varying the Fe(III) concentration in the testing media or under
competition conditions with an equimolar amount of parent
siderophore (1a) or Fe(III) complex (1a-Fe). These results are
consistent with the lack of antagonism observed in Figure 2 and
again suggest that active transport is not necessary to reach the
S. aureus extracellular PBP target of the β-lactam warhead.19 In
contrast, the testing results against A. baumannii (discussed
previously when referring to Figure 3) show a clear dependence
on Fe(III) concentration and antagonism by 1a and 1a-Fe
consistent with active transport of 1b.
The influence of Fe(III) concentration and competing
siderophores on the antibacterial activity of sideromycin 1b
was further quantified by determining MIC values using a broth
microdilution assay (Table 2). In Fe(III)-supplemented
Mueller−Hinton no. 2 broth media (MHII+Fe), sideromycin
1b produced an MIC value of 0.5 μM against A. baumannii,
Interestingly, both (pre)acinetobactin and the fimsbactins are
mixed ligand siderophores containing catecholate and hydrox-
amate iron(III)-chelating ligands (Figure 5). Much is known
about acinetobactin assembly30d,e and trafficking,30b,c but it is
still unclear whether preacinetobactin or acinetobactin is the
biologically active siderophore. The structural similarity of
(pre)acinetobactin to the siderophores anguibactin, pseudo-
monine, vibriobactin, pyochelin, and yersiniabactin suggests
that it might also form a 1:1 siderophore:iron complex with
oxygen and nitrogen atoms from the catecholate, hydroxamate,
oxazoline, and imidazole ring participating in iron(III)
chelation.32 With this unique structural type of metal complex,
it is unlikely that the synthetic mixed-ligand siderophore 1a and
sideromycin 1b (which presumably utilize two catecholates and
one hydroxamate to make a 1:1 complex with iron) from this
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relationships of the fluoroquionlone antibiotics, it is unlikely
that covalent modification of the ciprofloxacin warhead in
conjugate 1c diminished all ability to inhibit DNA gyrase. It is
well-known that the C-7 position of the fluoroquinolone
scaffold is the most forgiving in terms of bulky modifications.33
In the case of ciprofloxacin, the presence of a secondary or
tertiary amine is optimal, but the distal nitrogen of the
piperazine unit can be alkylated or acylated and still inhibit
DNA gyrase with great potency; even when derivatized with
large/siderophores such as pyoverdine from Pseudomonas
aeruginosa as proven by Abdallah and co-workers.22b On the
basis of these arguments, it is more likely that mixed ligand
synthetic sideromycin 1c was not trafficked to the cytoplasm in
A. baumannii. This suggests that sideromycins built around
mixed ligand siderophore 1a that target Gram-negative bacteria
should feature antibiotics with periplasmic targets (Figure 6).
Figure 5. Structures of mixed ligand siderophores (preacinetobactin,
acinetobactin, and fimsbactin A) produced by A. buamannii. Catechol
and hydroxamic acid iron(III)-chelating groups are shown in red.
work share the same membrane transport system as (pre)-
acinetobactin in A. baumannii, but at this point it cannot be
ruled out.
Figure 6. A generic trihydroxamate sideromycin selective for Gram-
positive pathogens35a,b and a generic mixed ligand sideromycin
selective for Gram-negative pathogens (this work).
A recent publication reported the structures, biosynthetic
genes, and membrane transport genes of the fimsbactins, a new
class of siderophores from A. baumannii.31 The structure of the
fimsbactins are remarkably similar to the synthetic mixed ligand
siderophore 1a used in this study. Both structures contain two
catechols and one hydroxamic acid ligand arranged in a similar
order and architecture. While no structural information is
currently available, it is possible that the synthetic siderophore
1a and sideromycin 1b mixed ligand iron(III) complexes mimic
the fimsbactin−iron(III) complexes. As a result, these side-
rophore−iron(III) complexes might share the same uptake
pathway in A. baumannii, which could explain the high
sensitivity of A. baumannii to the mixed ligand biscatecho-
late−monohydroxamate−β-lactam sideromycin 1b. Addition-
ally, the transcription of all the siderophore biosynthetic and
transport genes in A. baumannii are under the genetic control of
a ferric uptake regulator (Fur) protein, which helps rationalize
the inverse correlation of anti-A. baumannii activity and Fe(III)
concentration observed for sideromycin 1b in this study.29b
Further investigation to see if the synthetic mixed ligand
biscatecholate−monohydroxamate siderophore 1a and side-
romycin 1b utilize fimsbactin or acinetobactin transport
systems in A. baumannii would be of great value for optimizing
the therapeutic approach of siderophore-mediated antibiotic
delivery described in this work.
The mixed ligand−fluoroquinolone sideromycin 1c showed
no antibacterial activity against all the strains tested (Table 1).
As discussed previously, the mixed ligand−β-lactam side-
romycin 1b was transported by Gram-negative bacteria, such
as A. baumannii, across the outer membrane into the periplasm
where its PBP biological target is located. The lack of
antibacterial activity of the mixed ligand−fluoroquinolone
sideromycin 1c suggested that it was never transported across
the inner membrane of A. baumannii into the cytoplasm where
the fluoroquinolone’s biological target, DNA gyrase, is located.
On the basis of the well established structure−activity
We previously reported siderophore−fluoroquinolone con-
jugates based on trihydroxamate siderophores from the
ferrioxamine family with selectively potent activity against
Gram-positive bacteria, including S. aureus, able to transport
ferrioxamine siderophores to the cytoplasm.34 Therefore,
antibiotics with cytoplasmic targets might be reserved for
sideromycins that target Gram-positive bacteria (Figure 6). The
problem of delivering antibacterial drugs to the cytoplasm of
Gram-negative bacteria with siderophore delivery vectors
remains to be solved. One general method to expand the
scope of antibacterial agents available for use in siderophore-
mediated drug delivery is to design new microbe-triggered
linker systems that selectively release the antibiotic warhead
near the desired site of action.35
SUMMARY AND CONCLUSIONS
■
New antibacterial treatments for MDR A. baumannii are
desperately needed as strains resistant to every known
antibiotic are beginning to spread.3,4 The mixed ligand
siderophore−β-lactam conjugate 1b described here represents
an important new agent that kills A. baumannii with exceptional
nanomolar potency (MIC = 0.0078 μM) and selectivity in
whole cell antibacterial assays. To evaluate the usefulness in
continuing the development of sideromycin 1b as a potential
chemotherapeutic treatment for A. baumannii infections, the
antibacterial activity against clinical MDR isolates of A.
baumannii and efficacy in mouse infection models are currently
being pursued.
EXPERIMENTAL SECTION
Materials and Instrumentation. Reactions were conducted
under an atmosphere of dry argon unless otherwise stated. All
solvents and reagents were obtained from commercial sources and
■
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used without further purification unless otherwise stated. Dichloro-
methane (CH2Cl2) was distilled from calcium hydride. Dimethylfor-
mamide (DMF) and diisopropylethylamine (iPr2EtN) were used from
Acros Seal anhydrous bottles. Pentabenzyl-biscatechol−monohydrox-
amate siderophore 2 was prepared according to a previously described
method.20 Protected antibiotics 3 and 4 were prepared as described
previously.16,21,34 Sorbent Technologies silica gel 60 (32−63 μm) was
used for all silica gel column chromatography purifications.
All microbiological media and liquids were sterilized before use by
autoclaving at 121 °C for at least 15 min unless otherwise stated.
Distilled, deionized, and filtered water (Millipore Milli-Q Advantage
A10 water purification system) was used to prepare all aqueous
solutions and media. Luria broth (LB) was purchased from VWR.
Mueller−Hinton no. 2 broth (MHII broth; cation adjusted) was
purchased from Sigma-Aldrich (St. Louis, MO). Mueller-Hinton no. 2
agar (MHII agar; HiMedia Laboratories) was purchased from VWR.
of a freshly prepared 1 mg/mL filter-sterilized aqueous solution of
FeCl3 to 34 mL of melted MHII agar with gentle mixing.
MIC Determination by the Broth Microdilution Assay.
Minimum inhibitory concentrations (MICs) were determined using
the broth microdilution method according to the Clinical and
Laboratory Standards Institute (CLSI, formerly the NCCLS) guide-
lines23 and as described previously by our group.27,34 The specific
details of this assay are provided in the Supporting Information.
Paper Strip Agar Diffusion Siderophore−Sideromycin
Competition Assay. A general version of this assay has been
described previously by our group,34 and the specific procedure used
in this work is provided. Cultures of S. aureus SG511 and A. baumannii
ATCC 17961 were grown in LB broth for 18−24 h, and standard cell
suspensions of 1.5 × 106 CFU/mL were prepared in saline solution
(0.9% NaCl) according to a 0.5 BaSO4 McFarland Standard.36 Each
standardized suspension (0.1 mL) was added to 34 mL of sterile,
melted MHII−Fe agar tempered to 47 °C. After gentle mixing, the
inoculated agar media was poured into a sterile plastic Petri dish (145
mm × 20 mm) and allowed to solidify near a flame with the lid
cracked for ∼30 min. Solutions of the test compounds dissolved in a
1:10 mixture of DMSO:MeOH (1a, 1a-Fe, and 1b) or sterile, distilled,
and deionized H2O (loracarbef) were prepared at the desired
concentration (2.0 mM for 1a and 1a-Fe, 0.5 mM for 1b, 1.0 mM
and 0.1 mM for loracarbef against A. baumannii and S. aureus,
respectively). Sterile filter paper strips (Whatman no. 1 standard grade,
cut to ∼1 cm × 8 cm) were soaked in the test compound and laid on
the surface of the inoculated agar media as shown in Figure 2. The
Petri dishes were incubated at 37 °C for 18−24 h and then stored at rt
until being photographed at the 48 h time point.
́
McFarland BaSO4 turbidity standards were purchased from bioMer-
ieux, Inc. Sterile plastic Petri dishes (145 mm × 20 mm; Greiner Bio-
One) and sterile polystyrene 96-well plates (BD Falcon) used for
antibacterial susceptibility testing were purchased from VWR. Ferric
chloride (anhydrous, reagent grade), and 2,2′-bipyridine (reagent plus
grade) were purchased from Sigma-Aldrich.
1H NMR and 13C NMR spectra were recorded on a 600 MHz
Varian DirectDrive spectrometer, and FID data was processed using
ACD/ChemSketch version 10.04. Chemical shifts (δ) are reported in
parts per million (ppm) and are referenced to residual solvent.
Coupling constants (J) are reported in hertz (Hz).
High resolution, accurate mass measurements were obtained with a
Bruker micrOTOF II electrospray ionization time-of-flight mass
spectrometer in positive ion mode using direct sample injection.
Sample was introduced via flow injection at a rate of 4 μL/min, and
mass spectra were accumulated from 50 to 3000 m/z for 2 min.
HPLC-MS was performed on a Waters ZQ instrument consisting of
a chromatography module Alliance HT, photodiode array detector
2996, and mass spectrometer Micromass ZQ with an MS electrospray
source operated at a capillary voltage of 3.5 kV and a desolvation
temperature of 300 °C. A YMC Pro C18 reverse phase column (3.0
mm × 50 mm) fit with a 0.5 μm precolumn frit and a YMC Pro C18
guard column (2.0 mm × 10 mm) was used for all analyses. Mobile
phases used were 0.1% TFA in H2O (A) and 0.1% TFA in CH3CN
(B). A gradient was formed from 5%−80% of B in 10 min, then 80%−
95% of B in 2 min, and then 95%−5% of B in 3 min at a flow rate of
0.7 mL/min (total run time of 15 min).
Preparative HPLC purifications were performed on a Waters
preparative binary pump system at a flow rate of 15 mL/min with UV
detection at 280 nm using a YMC-Pack Pro C18 column (150 mm ×
20 mm; 5 μm particle size) fit with a guard column. Mobile phases
used were 0.1% TFA in H2O (A) and 0.1% TFA in CH3CN (B). A
gradient was formed from 30%−60% of B over 4 min, then 60%−90%
of B over 0.25 min, and was held at 90% of B for 0.75 min.
Thin layer chromatography (TLC) was performed with Al-backed
Merck 60-F254 or Al-backed Merck RP-C18 F256 silica gel plates using a
254 nm lamp and aqueous FeCl3 for visualization. Melting points were
determined in capillary tubes using a Thomas−Hoover melting point
apparatus and are uncorrected. The purity of compounds tested in
biological assays was evaluated by analytical HPLC and verified to be
≥95%, unless otherwise noted.
Modified Kirby−Bauer Agar Diffusion Antibiotic Suscepti-
bility Assay. A modified Kirby−Bauer agar diffusion assay was used
to assess compound antibacterial activity.26 Details of this assay have
been reported previously by our group and can be found in the
Supporting Information.27,34
Procedures for the Syntheses of Siderophores and Side-
romycins. Biscatechol−Monohydroxamate Siderophore (1a).
Compound 2 (145.0 mg, 0.12 mmol) was dissolved in 15 mL of
MeOH in an HCl-washed 25 mL round-bottom flask sealed under
argon. The flask was charged with 10% Pd−C (30.0 mg) and exposed
to a balloon of hydrogen gas (∼1 atm). Reaction progress was
monitored by RP-C18 TLC (1.5:1 CH3CN:H2O; FeCl3 stain), and
after 26 h there was no remaining starting material (2). The flask was
flushed with argon, and the mixture was vacuum filtered through
Celite. Evaporation of the MeOH gave a white solid that was
recrystallized from MeOH/Et2O to provide the desired siderophore
(1a) in 90% yield (80.5 mg, 0.11 mmol); mp 71−73 °C (color
1
change), 150−160 °C (dec). H NMR (600 MHz, CD3OD) δ 7.23−
7.19 (m, 2 H), 6.94−6.90 (m, 2 H), 6.73−6.68 (m, 2 H), 3.61−3.54
(m, 2 H), 3.48−3.40 (m, 4 H), 3.38 (t, J = 6.2 Hz, 2 H), 3.35 (t, J =
6.5 Hz, 2 H), 3.12 (t, J = 6.7 Hz, 2 H), 2.74 (t, J = 6.6 Hz, 2 H), 2.67
(dt, J = 17.2, 6.6 Hz, 2 H), 2.58−2.53 (m, 2 H), 2.52−2.44 (m, 2 H),
1.96 (dt, J = 14.6, 7.2 Hz, 1 H), 1.82 (ddd, J = 13.2, 6.5, 6.2 Hz, 1 H),
1.75−1.54 (m, 6 H), 1.51−1.43 (m, 2 H), 1.34−1.25 (m, 2 H).
HRMS-ESI (m/z): [M + H]+ calcd for C34H48N5O12, 718.3294; found,
718.3278. Note: The 13C NMR spectrum in CD3OD and DMSO-d6
shows rotamers resulting in a complex mixture of 13C-signals.
Biscatechol−Monohydroxamate Siderophore−Iron(III) Complex
(1a-Fe). Siderophore 1a (10.0 mg, 0.015 mmol) and Fe(acac)3 (5.9
mg, 0.017 mmol) were dissolved in 5 mL of MeOH. The purple
solution was heated at 40 °C (oil bath temperature) for 2 h. The
MeOH was removed under reduced pressure, and the purple residue
was dissolved in a minimal amount of MeOH and then precipitated by
addition of Et2O. The desired siderophore−iron(III) complex (1a-Fe)
was isolated in 98% yield as a purple solid (11.5 mg, 0.015 mmol); mp
>260 °C. HRMS-ESI (m/z): [M + 3H]+ calcd for C33H45FeN5O12,
771.2409; found, 771.2416. HPLC-MS retention time of 1.65 min.
Note: The iron(III)-complex 1a-Fe was insoluble in pure water, many
common organic solvents (EtOAc, CH2Cl2, CHCl3, Et2O, CH3CN,
MeOH), and mixed water/organic solvent systems (H2O/MeOH,
H2O/CH3CN). The iron(III)-complex 1a-Fe was highly soluble in
Preparation of Fe(III)-Supplemented and Fe(III)-Depleted
Media. Iron-deficient and iron-supplemented media prep has been
described previously by our group34 and is summarized again here.
Iron-deficient MHII broth (MHII−Fe) was prepared by adding 0.8
mL of a 1 mg/mL filter-sterilized aqueous solution of 2,2′-bipyridine
to 49.2 mL of sterile MHII broth. Iron-supplemented MHII broth
(MHII+Fe) was prepared by adding 0.8 mL of a freshly prepared 1
mg/mL filter-sterilized aqueous solution of FeCl3 to 49.2 mL of sterile
MHII broth. Iron-deficient MHII agar (MHII−Fe) was prepared by
adding 0.5 mL of a 1 mg/mL filter-sterilized aqueous solution of 2,2′-
biypridine to 34 mL of melted MHII agar with gentle mixing. Iron-
supplemented MHII agar (MHII+Fe) was prepared by adding 0.5 mL
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DMSO, DMF, and weakly basic aqueous solutions (5% NaHCO3 in
H2O).
H), 3.25 (td, J = 13.4, 6.6 Hz, 1 H), 3.16−3.09 (m, 2 H), 2.85−2.81
(m, 2 H), 2.71−2.47 (m, 8 H), 2.43−2.36 (m, 1 H), 1.98−1.92 (m, 1
H), 1.79 (ddd, J = 13.6, 6.7, 6.6 Hz, 1 H), 1.76−1.68 (m, 2 H), 1.68−
1.52 (m, 6 H), 1.52−1.42 (m, 2 H), 1.32−1.23 (m, 2 H). 13C NMR
(150 MHz, CD3OD) δ 175.6, 175.6, 174.9, 174.9, 174.6, 174.6, 174.1,
173.9, 171.8, 171.7, 171.6, 171.6, 166.4, 163.6, 150.6, 150.5, 150.4,
150.4, 147.5, 147.5, 147.5, 147.5, 138.3, 138.2, 130.8, 130.7, 130.6,
130.2, 130.0, 129.7, 129.5, 129.4, 129.4, 129.0, 125.6, 124.7, 119.8,
119.8, 119.7, 119.7, 119.7, 118.8, 118.7, 118.7, 118.6, 116.9, 116.9,
116.8, 116.8, 74.0, 59.9, 59.7, 59.6, 54.1, 46.9, 46.9, 44.6, 40.5, 40.2,
40.0, 38.1, 37.7, 32.5, 32.2, 32.2, 31.3, 29.8, 29.8, 29.7, 29.5, 29.5, 28.9,
28.6, 27.9, 27.8, 27.2, 27.1, 26.2, 24.8, 24.7, 23.1. HRMS-ESI (m/z):
[M + H]+ calcd for C50H62ClN8O15, 1049.4018; found, 1049.4010.
HPLC-MS retention time of 5.75 min.
Penta-O-benzyl-biscatechol−Monohydroxamate−O-PNB-Lora-
carbef Conjugate (5). The synthesis of this compound has been
reported previously by a slightly different synthetic approach.16 O-
PNB-loracarbef TFA salt (3; 208.0 mg, 0.35 mmol) was dissolved in 5
mL of anhydrous CH2Cl2, and the solution was cooled to 0 °C (ice
bath temp). An excess of iPr2EtN (0.40 mL, 2.3 mmol) was slowly
added under argon, followed directly by a catalytic amount of DMAP
(7.0 mg, 0.06 mmol), a solution of benzyl-protected siderophore 2
(450.0 mg, 0.38 mmol) in 10 mL of CH2Cl2, and EDC-HCl (136.0
mg, 0.71 mmol), respectively. The mixture was warmed to rt and
stirred overnight under dry argon. After 24 h, TLC (3% MeOH in
CH2Cl2; FeCl3 stain) showed no remaining starting material (3). The
CH2Cl2 was evaporated under reduced pressure, and the resulting oil
was partitioned between 50 mL of EtOAc and 50 mL of 1 N HCl. The
layers were separated, and the EtOAc was washed with 25 mL of H2O.
All the aqueous layers were combined and extracted with 50 mL of
EtOAc. The EtOAc layers were combined and washed with 50 mL of
brine, dried over MgSO4, gravity filtered, and concentrated under
reduced pressure. This gave 650 mg of a viscous oil that was purified
via silica gel column chromatography (1.25 in. × 4 in. silica gel; 3−5%
MeOH in EtOAc). Pure benzyl-protected mixed ligand siderophore−
loracarbef conjugate (5) was obtained in 52% yield as an off-white,
Penta-O-benzyl-biscatechol−Monohydroxamate−O-Benzyl-ci-
profloxacin Conjugate (6). O-Benzyl-ciprofloxacin hydrochloride salt
(4) was free-based using Amberlite IR400(OH−) resin in CHCl3 for 4
h. The resulting O-benzyl-ciprofloxacin amine (45.0 mg, 0.11 mmol),
penta-O-benzyl-biscatechol-monohydroxamate 2 (117.0 mg, 0.10
mmol), iPr2EtN (0.04 mL, 0.23 mmol), DMAP (3.0 mg, 0.025
mmol), and EDC-HCl (31.0 mg, 0.16 mmol) were dissolved in 5 mL
of anhydrous CH2Cl2, respectively. After 24 h at rt, TLC (6% MeOH
in CH2Cl2; FeCl3 stain) showed no remaining starting material 2. The
mixture was diluted with CH2Cl2 (35 mL), washed with H2O (30
mL), saturated aqueous NaHCO3 (30 mL), and brine (30 mL), dried
over anhydrous MgSO4, filtered, and concentrated. The crude product
was purified by silica gel column chromatography (1 in. × 5 in. silica
gel; 3−5% MeOH in CHCl3) to give the desired product (6) in 52%
1
waxy solid (302.5 mg, 0.18 mmol); mp 70−72 °C. H NMR (600
MHz, DMSO-d6) δ 9.32 (br s, 1 H), 9.11 (d, J = 8.5 Hz, 1 H), 8.88 (br
s, 1 H), 8.82 (br s, 1 H), 8.63 (d, J = 7.6 Hz, 1 H), 8.27−8.21 (m, 2
H), 7.77 (br s, 1 H), 7.69 (d, J = 8.8 Hz, 1 H), 7.51−7.03 (m, 35 H),
6.75 (d, J = 10.3 Hz, 1 H), 6.66 (s, 1 H), 5.48 (d, J = 7.6 Hz, 1 H),
5.46−5.35 (m, 5 H), 5.20−5.08 (m, 2 H), 5.07−4.98 (m, 2 H), 4.92−
4.82 (m, 2 H), 3.95−3.73 (m, 4 H), 3.59−3.47 (m, 2 H), 3.33−3.07
(m, 8 H), 2.99−2.91 (m, 2 H), 2.62 (ddd, J = 18.8, 12.1, 6.3 Hz, 4 H),
2.55−2.44 (m, 4 H), 2.34−2.23 (m, 2 H), 1.85−0.98 (m, 12 H).
HRMS-ESI (m/z): [M + Na]+ calcd for C92H96ClN9NaO17,
1656.6505; found, 1656.6538. Note: The 13C NMR spectrum in
DMSO-d6 shows rotamers resulting in a complex mixture of 13C-
signals.
1
yield as a clear wax (80.9 mg, 0.05 mmol). H NMR (600 MHz,
CDCl3) δ 8.53−8.46 (m, 1 H), 8.36−8.18 (m, 1 H), 8.09−7.85 (m, 3
H), 7.73−7.57 (m, 1 H), 7.54−7.00 (m, 32 H), 6.97−6.51 (m, 4 H),
6.41 (d, J = 4.1 Hz, 1 H), 6.31 (br s, 1 H), 5.40−5.36 (m, 2 H), 5.17−
4.96 (m, 8 H), 4.91−4.86 (m, 2 H), 3.86−3.75 (m, 2 H), 3.75−3.55
(m, 4 H), 3.51−3.35 (m, 3 H), 3.34−3.03 (m, 10 H), 2.87−2.75 (m, 2
H), 2.71 - 2.39 (m, 6 H), 2.16−1.90 (m, 2 H), 1.75−1.31 (m, 12 H),
1.13−1.00 (m, 2 H). 13C NMR (150 MHz, CDCl3) δ 173.7, 173.2,
173.0, 172.3, 172.2, 172.2, 172.0, 171.7, 171.6, 171.3, 170.4, 169.7,
165.4, 165.3, 165.2, 165.1, 165.1, 165.0, 154.1, 152.4, 151.6, 151.6,
151.6, 148.3, 147.8, 146.8, 146.7, 146.5, 144.0, 137.9, 136.5, 136.3,
136.3, 136.3, 136.3, 136.2, 129.1, 129.0, 128.8, 128.8, 128.8, 128.7,
128.7, 128.6, 128.6, 128.6, 128.6, 128.5, 128.5, 128.5, 128.4, 128.4,
128.4, 128.2, 128.2, 128.2, 128.1, 127.9, 127.9, 127.8, 127.8, 127.8,
127.6, 127.6, 127.5, 127.3, 127.3, 127.3, 127.3, 127.0, 126.0, 124.4,
124.4, 124.3, 124.2, 123.3, 123.1, 123.1, 123.0, 122.8, 117.9, 117.7,
116.8, 116.5, 115.1, 113.4, 113.2, 110.0, 105.1, 76.4, 76.4, 76.3, 76.2,
76.1, 71.2, 71.1, 71.0, 71.0, 66.3, 50.0, 49.5, 47.2, 45.1, 41.4, 39.2, 38.9,
38.7, 38.5, 37.0, 36.9, 34.5, 31.5, 28.6, 27.2, 26.6, 26.6, 26.3, 25.9, 24.9,
23.8, 22.6, 8.1. HRMS-ESI (m/z): [M + Na]+ calcd for
C93H99FN8NaO14, 1593.7157; found, 1593.7155.
Biscatechol−Monohydroxamate−Ciprofloxacin Conjugate (1c).
Penta-O-benzyl-biscatechol−monohydroxamate−O-benzyl-ciprofloxa-
cin conjugate (6; 75.0 mg, 0.05 mmol) was dissolved in 8 mL of
MeOH:EtOAc (3:1) in an HCl-washed, 10 mL round-bottom flask
sealed under argon. The flask was charged with 10% Pd−C (18.5 mg)
and exposed to a balloon of hydrogen gas (∼1 atm). Reaction progress
was monitored by RP-C18 TLC (1.5:1 CH3CN:H2O; FeCl3 stain),
and after 24 h there was no remaining starting material (6) and a new
product appeared giving a strong FeCl3 positive test was present (Rf
0.44; purple with FeCl3 stain). The flask was flushed with argon, and
the mixture was diluted with MeOH, vacuum filtered through Celite,
and concentrated under reduced pressure to give a tan film. The film
was dissolved in MeOH and the product (1c) precipitated as a faint
purple solid after addition of cold Et2O. The desired conjugate (1c)
was obtained in 51% yield as a faint purple solid (25.0 mg, 0.025
mmol); mp 131−134 °C (dec). 1H NMR (600 MHz, CD3OD) δ 8.74
(s, 1 H), 7.95 (d, J = 13.8 Hz, 1 H), 7.54 (br s, 1 H), 7.22−7.17 (m, 2
H), 6.89−6.85 (m, 2 H), 6.68−6.61 (m, 2 H), 3.83−3.77 (m, 4 H),
3.71 (br s, 1 H), 3.63−3.56 (m, 2 H), 3.48−3.32 (m, 12 H), 3.13 (t, J
= 6.9 Hz, 2 H), 2.82 (t, J = 5.7 Hz, 2 H), 2.74−2.63 (m, 4 H), 2.48 (dt,
J = 13.7, 6.8 Hz, 2 H), 1.98−1.91 (m, 2 H), 1.81 (dt, J = 14.1, 7.0 Hz,
Biscatechol−Monohydroxamate−Loracarbef Conjugate (1b).
The synthesis of this compound has been reported previously by a
slightly different synthetic approach.16 Benzyl-protected conjugate 5
(209.5 mg, 0.13 mmol) was dissolved in 2.50 mL of DMF:H2O (95/5;
v/v) in an HCl-washed round-bottom flask. Concentrated HCl (33.4
μL, 0.38 mmol) and 10% Pd−C (41.9 mg) were added, respectively,
and the flask was sealed under argon. The flask was then flushed
several times with hydrogen gas using intermediate vacuum
evacuations, and the mixture was left stirring at rt under a balloon
of hydrogen gas (∼1 atm). Reaction progress was monitored by RP-
C18 TLC (1.5:1 CH3CN:H2O; FeCl3 stain), and after 24 h there was
no remaining starting material (5) and a new product appeared giving
a strong FeCl3 positive test was present (Rf 0.78; purple with FeCl3
stain). The flask was flushed with argon, and the mixture was vacuum
filtered through glass filter paper. The DMF and H2O were removed
using high vacuum rotary evaporation (∼1 mmHg), which gave 250
mg of a viscous oil. This material was purified by size exclusion
chromatography (Sephadex LH20, 10.0 g; 10% MeOH in EtOAc).
Several fractions of varying purity (70−90% pure by analytical HPLC)
were isolated from the size exclusion column, and each fraction (Fr)
was recrystallized from MeOH/Et2O to give the desired conjugate in
68% yield as off-white solids: Fr 1 (17.5 mg), Fr 2 (44.8 mg), Fr 3
(19.5 mg), Fr 4 (10.0 mg). A portion of the largest fraction (Fr 2; 30
mg) was subjected to purification by preparative HPLC (see Materials
and Instrumentation for exact details of purification) where the desired
compound 1b elutes at 4.15 min. Pure fractions were lyophilized, and
the obtained solid was recrystallized from MeOH/Et2O to give 15 mg
of an analytically pure sample of conjugate 1b as a white-solid used for
spectral characterization and biological testing; mp 123−125 °C (color
1
change), 186−188 °C (dec). H NMR (600 MHz, CD3OD) δ 7.44−
7.28 (m, 5 H), 7.23−7.18 (m, 2 H), 6.94−6.90 (m, 2 H), 6.73−6.68
(m, 2 H), 5.41 (d, J = 8.8 Hz, 1 H), 5.36 (d, J = 4.7 Hz, 1 H), 3.89−
3.84 (m, 1 H), 3.81 (ddd, J = 13.9, 7.2, 7.0 Hz, 1 H), 3.47−3.32 (m, 8
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2 H), 1.75−1.69 (m, 2 H), 1.69−1.54 (m, 4 H), 1.53−1.44 (m, 2 H),
1.40−1.34 (m, 2 H), 1.34−1.27 (m, 2 H). HRMS-ESI (m/z): [M +
H]+ calcd for C51H64FN8O14, 1031.4521; found, 1031.4522. HPLC-
MS retention time of 7.76 min. Note: The 13C NMR spectrum in
CD3OD and DMSO-d6 shows rotamers resulting in a complex mixture
of 13C-signals.
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* Supporting Information
Complete list of strains, markers, and origins for micro-
organisms used in this work. Complete list of antibiotic
susceptibility testing data from the agar diffusion assay. Copies
of 1H NMR and 13C NMR spectra for compounds 1a, 1b, 1c, 2,
5, and 6. Copies of HPLC-MS chromatograms for compounds
subjected to biological testing (1a-Fe, 1b, and 1c). This
material is available free of charge via the Internet at http://
pubs.acs.org.
(6) Radu Mihu, M.; Martinez, L. R. Novel therapies for treatment of
multidrug-resistant Acinetobacter baumannii skin infections. Virulence
2011, 2, 97−102.
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Filutowicz, M.; Wendt, J. L.; Suzuki, H.; Dominguez, M. A novel
antibacterial gene transfer treatment for multidrug-resistant Acineto-
bacter baumannii-induced burn sepsis. J. Burn Care Res. 2007, 28, 6−
12.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 574-631-7571. Fax: 574-631-6652. E-mail: mmiller1@
nd.edu.
Notes
The authors declare no competing financial interest.
(8) Thomas-Virnig, C. L.; Centanni, J. M.; Johnston, C. E.; He, L. K.;
Schlosser, S. J.; Van Winkle, K. F.; Chen, R.; Gibson, A. L.; Szilagy, A.;
Li, L.; Shankar, R.; Allen-Hoffmann, B. L. Inhibition of multidrug-
resistant Acinetobacter baumannii by nonviral expression of hCAP-18 in
a bioengineered human skin tissue. Mol. Ther. 2009, 17, 562−569.
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Alfieri, A.; Friedman, J. M. Sustained release nitric oxide releasing
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Isolation and characterization of phi AB2: a novel bacteriophage of
Acinetobacter baumannii. Res. Microbiol. 2010, 161, 308−314.
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Franklin, M. J.; Baer, D. G.; Hamblin, M. R. Photodynamic therapy for
Acinetobacter baumannii burn infections in mice. Antimcrob. Agents
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ACKNOWLEDGMENTS
■
We gratefully acknowledge the use of NMR facilities provided
by the Lizzadro Magnetic Resonance Research Center at The
University of Notre Dame (UND) and the mass spectrometry
services provided by The UND Mass Spectrometry &
Proteomics Facility (N. Sevova, Dr. W. Boggess, and Dr. M.
V. Joyce; supported by the National Science Foundation under
CHE-0741793). We thank Prof. Shariar Mobashery (UND)
and Dr. Sergei Vakulenko (UND) for providing the ESKAPE
panel of bacteria and Dr. Nuno Tiago Giao Antunes for some
initial antibiotic susceptibility testing. Partial funding for this
work was provided by NIH grant AI054193. T.A.W. gratefully
acknowledges The UND Chemistry−Biochemistry−Biology
(CBBI) Interface Program funded by NIH (T32GM075762)
for three years of fellowship support. T.A.W. thanks UND
Department of Chemistry and Biochemistry (Grace Fellow-
ship), the Center for Environmental Science and Technology
(CEST), and Bayer for additional support.
(12) Dadachova, E.; Casadevall, A. Antibodies as delivery vehicles for
radioimmunotherapy of infectious diseases. Expert Opin. Drug Delivery
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5961−5970.
ABBREVIATIONS USED
■
acac, acetylacetonate; ATCC, American Type Culture
Collection; BIPY, 2,2′-bipyridine; Bn, benzyl; Boc, tert-
butoxycarbonyl; DMAP, 4-dimethylaminopyridine; DMF,
dimethylformamide; DMSO, dimethylsulfoxide; EDC, 1-ethyl-
3-(3-dimethylaminopropyl)carbodiimide; Fur, ferric uptake
regulator; HPLC, high-performance liquid chromatography;
HRMS, high-resolution mass spectrometry; MDR, multidrug-
resistant; MHII, Mueller−Hinton media no. II; MIC, minimum
inhibitory concentration; NMR, nuclear magnetic resonance;
PBP, penicillin binding protein; PNB, para-nitrobenzyl; TFA,
trifluoroacetic acid
́ ́
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multidrug resistance in Acinetobacter baumannii. J. Antimicrob.
Chemother. 2007, 59, 1210−1215.
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2012, 4, 297−313. (b) Mollmann, U.; Heinisch, L.; Bauernfeind, A.;
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Kohler, T.; Ankel-Fuchs, D. Siderophores as drug delivery agents:
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application of the “Trojan Horse” strategy. Biometals 2009, 22, 615−
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