Synthesis, characterization, and in vitro testing of a bacteria-targeted MR contrast agent

Article abstract of DOI:10.1002/ejic.201101362

A bacteria-targeted MR contrast agent, Zn-1, consisting of two Zn-dipicolylamine (Zn-dpa) groups conjugated to a Gd^III chelate has been synthesized and characterized. In vitro studies with S. aureus and E. coli show that Zn-1 exhibits a significant improvement in bacteria labeling efficiency vs. control. Studies with a structural analogue, Zn-2, indicate that removal of one Zn-dpa moiety dramatically reduces the agent's affinity for bacteria. The ability of Zn-1 to significantly reduce the T₁ of labeled vs. unlabeled bacteria, resulting in enhanced MR image contrast, demonstrates its potential for visualizing bacterial infections in vivo. A bacteria-targeted MR contrast agent consisting of two Zn-dipicolylamine (Zn-dpa) groups conjugated to a Gd(III) chelate has been synthesized and characterized. This contrast agent significantly reduces the T₁ of labeled versus unlabeled bacteria, resulting in enhanced MR image contrast, demonstrating its potential for visualizing bacterial infections in vivo. Copyright

Full text of DOI:10.1002/ejic.201101362

FULL PAPER
DOI: 10.1002/ejic.201101362
Synthesis, Characterization, and in vitro Testing of a Bacteria-Targeted MR
Contrast Agent
Lauren M. Matosziuk,[a][‡] Allison S. Harney,[a][‡] Keith W. MacRenaris,[a] and
Thomas J. Meade*[a]
Keywords: Medicinal chemistry / Imaging agents / Magnetic resonance imaging / Bacteria labeling / Zinc
A bacteria-targeted MR contrast agent, Zn-1, consisting of
two Zn-dipicolylamine (Zn-dpa) groups conjugated to a GdIII
chelate has been synthesized and characterized. In vitro
studies with S. aureus and E. coli show that Zn-1 exhibits
a significant improvement in bacteria labeling efficiency vs.
control. Studies with a structural analogue, Zn-2, indicate
that removal of one Zn-dpa moiety dramatically reduces the
agent’s affinity for bacteria. The ability of Zn-1 to signifi-
cantly reduce the T1 of labeled vs. unlabeled bacteria, re-
sulting in enhanced MR image contrast, demonstrates its po-
tential for visualizing bacterial infections in vivo.
monitor therapeutic response, and ultimately guide clinical
decisions. Additionally, bacteria-specific contrast agents
could aid in the study of infection pathology. Bacteria-tar-
geted imaging probes would allow for in vivo monitoring
of both infection progression and antibiotic effectiveness in
animal models. This could potentially lead to the develop-
ment of new antibiotics capable of targeting bacterial
strains that have developed resistance to current medi-
cations.
To date, several molecular imaging probes have been de-
veloped for the specific recognition of bacterial infection.
Targeting moieties that have been studied include antibod-
ies,[7] antibiotic drugs,[8] cationic peptides,[9] maltohexose
units,[10] and cationic coordination complexes such as ZnII
dipicolylamine (Zn-dpa).[11] The interaction between cat-
ionic Zn-dpa complexes and bacteria is primarily electro-
static. Zn-dpa groups have a high affinity for the anionic
phosphate groups of phospholipids and phosphorylated
amphiphiles on the bacterial cell surface.[11e,12] This affinity
is affected by the number of Zn-dpa moieties present on the
molecule, indicating that cooperative coordination of sev-
eral targeting groups is involved in binding to the bacterial
surface.[11e,12] In contrast to the near-neutral plasma mem-
brane of healthy mammalian cells, which is primarily com-
posed of zwitterionic phospholipids, the phospholipids that
compose bacterial membranes are primarily anionic.[13]
Thus, Zn-dpa complexes have a high affinity for the nega-
tively-charged bacterial membrane, rendering them selective
for bacteria over the neighboring healthy mammalian
cells.[11c,11d]
Introduction
With the advancement of medical treatments and tech-
nologies, bacterial infection has become a growing concern
in the course of patient care.[1] In the United States, approx-
imately 2 million hospital patients develop a hospital-
acquired infection each year.[1] Due to illness, organ trans-
plantation, or specific disease treatments, many patients
possess a depressed immune system that renders the indi-
vidual more susceptible to infection.[2] Further, prosthetic
materials such as stents, mesh grafts, and catheters can pro-
vide additional microenvironments for bacterial growth.[3]
Antibiotic-resistant bacteria strains increase the severity of
illness, length of hospital stay and mortality from infec-
tion.[4] Consequently, new agents and techniques to prevent,
diagnose, and treat bacterial infection are of interest.
Typically an accurate diagnosis of bacterial infection is
derived from cultures of samples obtained from the site of
suspected infection. Other clinical methods to identify in-
fection include monitoring of body temperature, white
blood cell count, erythrocyte sedimentation rate and cyto-
kine reactions – none of which are a specific response to
infection.[5] Consequently, these tests cannot differentiate
between bacterial infection and sterile inflammation, and
are prone to false positive results due to contamination.[6]
As a result, there is a need to develop molecular imaging
probes that can specifically identify bacterial infection,
[a] Departments of Chemistry, Molecular Biosciences,
Neurobiology, Biomedical Engineering, and Radiology,
Northwestern University,
2145 Sheridan Rd., Evanston, IL 60208, USA
Fax: +1-847-491-3832
Several optical agents incorporating Zn-dpa groups as
bacteria targeting domains have shown promising results
both in vitro and in mouse models.[11] However, the in-
herent limitations of optical imaging, including shallow
E-mail: tmeade@northwestern.edu
[‡] These authors contributed equally.
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejic.201101362.
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depth of penetration and surface weighting of images, limit overall charge of the ZnII-binding domains incorporated in
the clinical applicability of such agents.[14] Here we describe the complexes was varied to examine the effect of the deriv-
the extension of Zn-dpa optical probes for the detection of atives on bacterial cell labeling.
bacterial infection into the more clinically relevant field of
magnetic resonance imaging (MRI). The use of targeted
MR contrast agents increases the local concentration of
GdIII at the site of interest, enhancing the sensitivity of the
probe.[15]
Complexes 2 and 3 were synthesized and characterized
as described previously.[16] Both 2 and 3 contain two flexible
pendant arms capable of coordinating one ZnII ion. Zn-3
was used as a control for low-affinity binding to bacteria.
The binding domain of 3 is composed of two anionic acet-
ate groups, making the ZnII-bound complex (Zn-3) charge
neutral. This complex does not possess any electrostatic af-
finity for the anionic bacterial membrane. In contrast, the
binding domain of 2 consists of two neutral picolyl arms,
giving the ZnII-bound complex, Zn-2, an overall charge of
2+ (Figure 2). The positive charge of the Zn-2 complex is
expected to moderately enhance its affinity for bacteria over
Zn-3. Previous studies by Smith et al.[11e] have suggested
that addition of a second Zn-dpa moiety significantly im-
proves bacteria labeling, leading us to synthesize 1. This
agent contains two dpa groups, each of which is capable of
binding one ZnII ion to give the final ZnII-bound complex,
Zn-1, an overall charge of 4+ (Figure 2).
The synthesis of 1 begins with methylation of the carbox-
ylic acids of 5-hydroxyisophthalic acid to give the diester 4,
in accordance with literature procedures (Scheme 1).[17] The
phenol was protected with ethoxymethyl chloride and the
esters reduced with lithium aluminum hydride to produce
the diol 6. The alcohol groups were converted to chlorides
using trichlorotriazine and DMF to form the dichloride
7.[18] Displacement of the chlorides with dipicolylamine
gave the protected, bis-dpa compound 8. The phenol was
deprotected with trifluoroacetic acid (TFA) in dichloro-
methane to give 9 in nearly quantitative yield. The bis-dpa
phenol 9 was allowed to react with a bromoalkane deriva-
tive of tris-tert-butyl-protected DO3A to give the final, pro-
tected ligand 10. Deprotection of the tert-butyl groups with
TFA and subsequent metallation with Gd(OAc)3, yielded
the final compound 1, which was purified by semi-prepara-
tive HPLC.
To specifically identify bacterial infection by MRI, two
Zn-dpa moieties were conjugated to a GdIII chelate to form
the bacteria-targeted MR contrast agent Zn-1 which has an
overall charge of 4+ (see Figures 1 and 2). Previous studies
with bacteria-targeted optical agents have indicated that the
number of Zn-dpa units affects the labeling efficiency of the
compound.[11e] To explore whether this effect is observed
for MR agents, two previously published ZnII-binding con-
trast agents, Zn-2 and Zn-3 (Figure 2), were synthesized
and examined as control probes.[16] As expected, Zn-1 was
found to bind to Staphylococcus aureus (S. aureus) to a
much greater extent than either Zn-2 or Zn-3. The high
affinity of Zn-1 for bacterial cells permits MR imaging of
cells in vitro and suggests the use of Zn-1 for in vivo detec-
tion of bacterial infection.
Figure 1. Two bacteria-targeted Zn-dpa domains were conjugated
to a macrocyclic GdIII chelate to develop a bacteria-targeted MR
probe. The probe’s affinity for bacteria is due to the electrostatic
attraction between the anionic bacteria membrane and the positive
charge of the Zn-dpa moieties.
Results and Discussion
Characterization of Zn-1
Synthesis of Bacteria-Targeted MRI Contrast Agents
Relaxation times (T1 and T2) of Zn-1 suspended in 1.0%
A series of ZnII-binding MR contrast agents composed (w/v) agarose (all subsequent bacterial samples were sus-
of a bacteria-targeted ZnII-binding domain conjugated to a pended in agarose) were measured at 1.41 (37 °C) and 7 T
GdIII chelate were synthesized (Figure 2). The number and (25 °C) in order to provide data at both clinical and re-
Figure 2. Structures of complexes used for bacteria labeling studies. The ZnII-binding domains of the complexes are shown in blue, the
GdIII chelate is shown in red. After the addition of ZnII, Zn-1 and Zn-2 have charges of 4+ and 2+, respectively, while Zn-3 is charge
neutral.[16] The positive charge of Zn-1 and Zn-2 is expected to enhance the complex’s affinity for anionic bacterial cell membranes. Zn-
3 was used as a control for low-affinity binding.
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Bacteria-Targeted MR Contrast Agent
Scheme 1. Synthesis of the bacteria-targeted MR contrast agent 1 that contains two dipicolyl moieties, each of which is capable of binding
one ZnII ion to give the final ZnII-bound complex an overall charge of 4+. The europium and terbium analogues were synthesized in a
similar manner, using EuCl3 and Tb(OAc)3 in place of Gd(OAc)3.
search field strengths. The r1 increases from 4.7 mm–1 s–1 to
5.6 mm–1 s–1 and the r2 increases from 9.0 mm–1 s–1 to
21.5 mm–1 s–1 at 1.41 T and 7 T respectively.
The hydration number, q, of Zn-1 was determined by
comparing the fluorescence lifetimes of the TbIII analogue,
Zn-1-Tb, in D2O and H2O. The fluorescence lifetimes were
measured and fit to an exponential curve. The decay times,
1.06 ms in H2O and 1.89 ms in D2O, were related to the
hydration number using Equation (1), giving a q value of
1.5.[19] The q value is an average measure of how many
water molecules are directly bound to the lanthanide at a
given time. The non-integer value of 1.5 indicates that that
in solution, the complex coordinates either one or two inner
sphere water molecules.
(1)
1H NMR spectroscopy was used to confirm that 1 binds
two ZnII ions per complex. The EuIII analogue of 1 (1-Eu)
1
was synthesized and the H NMR chemical shifts of the
dpa groups were monitored as ZnII was titrated into solu-
Figure 3. Aromatic region of 1-Eu COSY spectrum (60 °C, DMSO)
used to assign peaks of the ZnII binding domains of 1. The addition
1
tion. The H peaks of the macrocycle and the propylene
linker could not be assigned because the peaks are signifi-
of ZnII produces significant changes in the chemical shift of the
protons on the ZnII binding domain until a ZnII:1-Eu ratio of 2 is
cantly broadened and shifted due to their proximity to the reached.
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EuIII. However, there is enough separation between the lan-
thanide and the protons of the ZnII-binding dpa groups to
make assignment possible, and a COSY spectrum was ac-
quired to assign the aromatic dpa peaks (Figure 3).
ZnCl2 was added in 0.16 equiv. aliquots until a ZnII:1-Eu
ratio of 3.82 was obtained. During the titration significant
changes in the chemical shift of the dpa protons were ob-
served until a ZnII:1-Eu ratio of 2 was reached. The ad-
dition of excess ZnII beyond this stoichiometry produced
only negligible changes in chemical shift, confirming that
1-Eu binds two ZnII ions per complex (Figure 4).
Figure 5. In vitro cellular labeling of S. aureus and E. coli with Zn-
1, Zn-2, and Zn-3. S. aureus (top) or E. coli (bottom) were incu-
bated with increasing concentrations of Zn-1 (black bars), Zn-2
(gray bars), or Zn-3 (white bars) at room temperature in LB broth.
GdIII content was analyzed by ICP-MS and is represented as the
total amount of GdIII per sample. Data are represented as the me-
ansϮSEM.
GdIII content with increasing incubation concentrations,
likely due to non-specific binding of the agents to the cell
surface. At all incubation concentrations the GdIII content
of Zn-2 and Zn-3 incubated S. aureus is significantly less
than cells incubated with Zn-1. Specifically, the GdIII con-
tent of S. aureus incubated with 100 μm Zn-1 is approxi-
mately 16-fold higher than bacteria incubated with 100 μm
Zn-2 and 18-fold higher than bacteria incubated with Zn-3
(Figure 5).
Interestingly, Zn-2 does not show an increased affinity
for the anionic bacterial membrane despite its positive
charge. This suggests that for bacteria-targeted MR con-
trast agents, the mechanism of interaction between the Zn-
dpa groups and the membrane phospholipids requires the
presence of two Zn-dpa moieties, possibly due to a coopera-
tive-binding effect of multiple Zn-dpa moieties on the same
molecule coordinating to anionic phosphates on the bacte-
rial surface.[11e,12] We speculate that this cooperative inter-
action, in addition to the greater charge of the complex, is
responsible for the increased affinity of Zn-1 for S. aureus.
In vitro labeling experiments with E. coli indicate that
Figure 4. Top: aromatic region of 1H NMR spectrum of Eu-1 with
increasing amounts of ZnII. Bottom: Chemical shift of peaks c, e,
f, and d (see Figure 3) as a function of ZnII/Eu-1 ratio. It is clear
that the peaks of the ZnII binding domain shift only until a ZnII/
Eu-1 ratio of 2 is obtained, confirming that each Eu-1 binds two
ZnII ions.
In vitro Labeling of Bacteria with MRI Contrast Agents
The relative affinity of Zn-1, Zn-2, and Zn-3 for bacteria Zn-1 exhibits an affinity for Gram-negative strains; how-
was determined by quantifying the amount of GdIII bound ever, the extent of non-specific binding exhibited by Zn-2
to bacterial cells after incubation with various concentra- and Zn-3 is much greater for E. coli than S. aureus. For
tions of each contrast agent. The binding affinity was exam- lower concentrations of contrast agent (Յ 100 μm), Zn-1
ined for both a Gram-positive strain, S. aureus, and a incubated E. coli retains more GdIII than Zn-2 or Zn-3
Gram-negative strain, E. coli (Figure 5).
treated cells (Figure 5). Similar to S. aureus, the GdIII con-
Zn-1 binds readily to S. aureus in a dose-dependent man- tent of Zn-1 incubated E. coli increases in a concentration-
ner, with maximum binding occurring at an incubation con- dependent manner and plateaus at 50 μm (contrast agent
centration of 100 μm (Figure 5). However, S. aureus incu- incubation concentration) whereas the GdIII content of Zn-
bated with Zn-2 and Zn-3 show only minor increases in 2 and Zn-3 incubated E. coli continues to increase even
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Bacteria-Targeted MR Contrast Agent
above 50 μm incubation concentrations. This supports the
The pronounced reduction in T1 relaxation observed at
hypothesis that these increases are due to non-specific bind- 1.41 T as compared to 7 T may be explained by the field
ing to the surface of E. coli.
strength dependence of the relationship between rotational
correlation time (τR) and relaxivity.[20] Binding of MR con-
trast agents to large macromolecules is known to increase
the τR, and subsequently the r1, of contrast agents.[20–21]
Zn-1 is presumed to adhere to the bacterial surface, so it is
reasonable to expect both the τR and r1 of the agent to
increase upon binding. However, the influence of τR on re-
laxivity is dependent on the field strength; at lower field
strengths (i.e. 1.41 T), the effect of τR enhancement on re-
laxivity is more pronounced.[20] Consequently, it is reason-
able that bacteria labeled with Zn-1 exhibit greater decrease
in T1 at lower field strengths.
MR Imaging of Bacterial Cells Labeled with Zn-1, Zn-2,
Zn-3
To evaluate the potential of Zn-1 as a molecular imaging
probe, T1 and T2 relaxation times of S. aureus labeled with
Zn-1, Zn-2, and Zn-3 were acquired at 1.41 T and 7 T. (T2
data can be found in the Supporting Information). Bacteria
were labeled as described above, using an incubation con-
centration of 300 μm contrast agent. The samples were
washed with LB media and diluted in agarose to create a
uniform suspension of bacteria. The T1-weighted image ac-
quired at 7 T shows significant contrast enhancement in the
Zn-1 labeled sample compared to untreated bacteria; con-
versely, Zn-2 and Zn-3 labeled bacteria show no such con-
trast enhancement (Figure 6).
Equation (1) used to calculate q of Zn-1-Tb. τH20 and
τD20 are the time constants for the exponential fluorescence
decay in H2O and D2O, respectively.
Conclusions
We have developed a MR contrast agent (Zn-1) that is
capable of labeling bacteria cells in vitro. The agent is com-
posed of two cationic, bacteria-targeting ZnII-dpa groups
conjugated to a macrocyclic GdIII complex. NMR studies
confirm that each dpa group on the complex binds one ZnII
ion, giving the final Zn-1 complex an overall charge of 4+.
Zn-1 shows a dose-dependent affinity for S. aureus nearly
20 times higher than the charge neutral control compound
Zn-3. The low labeling efficiency of Zn-2, a contrast agent
containing only one Zn-dpa group, indicates that the inter-
action between the Zn-dpa groups of Zn-1 and the anionic
phosphate groups of the bacterial membrane require the
presence of two Zn-dpa groups for effective binding. The
reduction in the T1 relaxation time of bacteria labeled with
Zn-1 corresponds to a significant contrast enhancement at
7 T, though as expected is more pronounced at lower field
strengths, verifying the potential of Zn-1 as an in vivo MR
probe for detection of bacterial infection.
Figure 6. S. aureus cultures were incubated with 300 μm Zn-1, Zn-
2 or Zn-3. Cells were washed 3 times with LB-broth and resus-
pended in 1% agarose. Scale bar represents 300 μm. Top: T1-
weighted map was acquired at 7 T at 25 °C (TR/TE = 500/11 ms).
Cells labeled with Zn-1 are clearly brighter than both unlabeled
bacteria and bacteria treated with Zn-2 or Zn-3. Bottom: T1 relax-
ation times of agarose suspensions of S. aureus were measured
using a saturation recovery pulse sequence with an echo time (TE)
of 11 ms and repetition times (TR) as indicated in the Experimental
Procedures at 7 T and 1.41 T. Data are presented as the percent
reduction in T1 vs. bacteria incubated with LB media, washed, and
suspended in agarose.
Experimental Section
Bacterial Culture: Staphylococcus aureus (29213) and Escherichia
coli K-12 (29425) were obtained from ATCC (Rockville, MD). Bac-
teria were grown from glycerol stocks, cultured in Luria–Bertani
(LB) growth media with agitation overnight at 37 °C. All experi-
ments were performed after the bacteria had reached stationary
phase to ensure that bacterial growth did not affect labeling effi-
ciency. The optical density at 600 nm (OD600) was measured before
and after labeling to confirm that the number of bacteria did not
change significantly during the course of the experiment.
At 7 T, the T1 relaxation time of S. aureus cultures lab-
eled with Zn-1 is approximately 21% lower than the T1 of
untreated cultures. At 1.41 T the reduction in T1 increases
to 44% (Figure 6). Conversely, the T1 of Zn-2 and Zn-3
labeled bacteria is reduced by only 10 and 28% vs. control
at 7 T and 1.41 T, respectively (Figure 6).
Synthesis of Zn-1: Chloromethyl ethyl ether and 2,2Ј-dipicolylam-
ine were obtained from TCI America. All other chemicals were
obtained from Sigma Aldrich and used without further purifica-
tion. EMD 60F 254 silica gel plates were used for thin layer
chromatography and visualized using UV light, iodoplatinate stain,
or cerium ammonium molybdate (CAM) stain. Column
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chromatography was performed using standard grade 60 Å 230–
400 mesh silica gel (Sorbent Technologies). Unless otherwise noted,
1H and 13C NMR were obtained on a Bruker Avance III 500 MHz
NMR Spectrometer. A Varian 1200 L single-quadrupole mass
spectrometer was used to acquire electrospray ionization mass spec-
tra (ESI-MS). Semi-preparative HPLC was performed on a Waters
19ϫ250 mm Atlantis C18 Column. Analytical HPLC-MS was per-
formed using a Waters 4.6ϫ250 mm 5 μm Atlantis C18 column
using the Varian Prostar 500 system equipped with a Varian 380
LC ELSD system, a Varian 363 fluorescence detector, and a Varian
2 H, CH2-CH3), 1.23 (t, J = 7.1 Hz, 3 H, CH2-CH3) ppm. 13C
NMR (126 MHz, CDCl3): δ = 157.85 (Car-OCH2), 139.38 (Car-
CH2Cl), 121.96 (Car), 116.37 (Car), 93.15 (O-CH2-O), 64.46
(CH2CH3), 45.76 (CH2Cl), 15.13 (CH2CH3) ppm.
[5-(Ethoxymethoxy)benzene-1,3-diyl]bis[N,N-bis(pyridin-2-ylmeth-
yl)methanamine] (8): DIEA (1.74 g, 13.5 mmol) and 2,2Ј-dipicolyl-
amine were stirred in DCM (10 mL) for 10 min. A solution of 7 in
DCM (10 mL) was added and the reaction allowed to stir at room
temperature. After 4 d, completion of the reaction was confirmed
by MS. The solvent was evaporated and the residue purified on a
silica column eluting with 4% methanol/chloroform to give 3.5 g of
product as a yellow oil (99% yield). 1H NMR (500 MHz, CDCl3): δ
= 8.48 (dd, J = 3.8, 2.3 Hz, 4 H, o-Hpy), 7.66–7.54 (m, 8 H, Hpy),
7.11 [td, J = 5.1, 3.3 Hz, 5 H, Hpy (4 H), Har (1 H)], 7.02 (d, J =
1.4 Hz, 2 H, Har), 5.20 (s, 2 H, O-CH2-O), 3.78 (s, 8 H, N-CH2-
1
335 UV/Vis detector. H NMR of 1-Eu as a function of ZnII con-
centration was performed on a Bruker Avance III 600 MHz spec-
trometer at 60 °C in d6-DMSO.
Dimethyl 5-hydroxyisophthalate (4) was synthesized according to
literature procedure.[17]
C
py), 3.71 (q, J = 7.1 Hz, 2 H, CH2-CH3), 3.63 (s, 4 H, Car-CH2-
Dimethyl 5-(Ethoxymethoxy)isophthalate (5): To a solution of 4
(4.42 g, 21 mmol) in acetone (50 mL) was added K2CO3 (11.5 g,
84 mmol). The reaction was cooled to 0 °C, and chloromethylethyl
ether (2.74 g, 29 mmol) was added. After 12 h, TLC (30% ethyl
acetate/hexanes) confirmed completion of the reaction. The reac-
tion mixture was filtered and the solvent evaporated. The residue
was purified on a silica gel column, eluting with 15% ethyl acetate/
N), 1.19 (t, J = 7.1 Hz, 3 H, CH2CH3) ppm. 13C NMR (126 MHz,
CDCl3): δ = 159.88 (CH2-o-Cpy-Npy), 157.74 (Car-OCH2), 149.02
(o-Cpy-Npy), 140.85 (Car-CH2N), 136.53 (c-Cpy), 122.78 (m-Cpy),
122.44 (Car), 122.04 (m-Cpy), 115.12 (Car), 93.32 (O-CH2-O), 64.27
(CH2CH3), 60.16 (N-CH2-Cpy), 58.57 (Car-CH2-N), 15.26
(CH2CH3) ppm. MS (ESI-positive): m/z = 575.3 [M+], 597.3 [M +
Na+].
1
hexanes, to give 4.72 g of product as a white solid (84% yield). H
NMR (500 MHz, CDCl3): δ = 8.29 (t, J = 1.5 Hz, 1 H, Haryl), 7.84
(d, J = 1.5 Hz, 2 H, Haryl), 5.27 (s, 2 H, O-CH2-O), 3.91 (s, 6 H,
O-CH3), 3.71 (q, J = 7.0 Hz, 2 H, CH2CH3), 1.19 (t, J = 7.1 Hz,
3 H, CH2CH3) ppm. 13C NMR (126 MHz, CDCl3): δ = 166.08
(COOMe), 157.38 (Car-OCH2), 131.83 (Car-COOMe), 124.00 (Car),
121.55 (Car), 93.14 (O-CH2-O), 64.68 (CH2-CH3), 52.48 (COO-
CH3), 15.12 (CH3) ppm.
3,5-Bis{[bis(pyridin-2-ylmethyl)amino]methyl}phenol (9): A solution
of 8 (2.21 g, 3.84 mmol) in DCM was heated to 40 °C. Trifluoro-
acetic acid (2.38 mL, 23 mmol) was added and the reaction allowed
to stir for 12 h (monitored by TLC: 10% methanol/DCM). The
protonated product was extracted from DCM with water. The
aqueous layer was neutralized and the product extracted into
DCM. The organic layers were combined, dried with Na2SO4, fil-
tered and the solvent evaporated to give 1.73 g of product as a
yellow oil. 1H NMR (500 MHz, CDCl3): δ = 8.52–8.40 (m, 4 H, o-
Hpy), 7.65–7.54 (m, 8 H, Hpy), 7.12 (td, J = 5.7, 2.4 Hz, 4 H, Hpy),
7.02 (s, 1 H, Har), 6.79 (d, J = 1.2 Hz, 2 H, Har), 3.77 (s, 8 H, CH2-
[5-(Ethoxymethoxy)-1,3-phenylene]dimethanol (6): Lithium alumi-
num hydride (1.22 g, 32.8 mmol) was suspended in THF (50 mL),
and the mixture cooled to 0 °C. A solution of 5 (4 g, 14.9 mmol)
in THF (40 mL) was added dropwise to the LAH suspension. After
2 h, completion of the reaction was confirmed by TLC (5% MeOH/
DCM). The reaction was quenched with 15 mL of water. The reac-
tion mixture was filtered and the solvent evaporated. The residue
was brought up in ethyl acetate and washed with water and brine.
The organic layer was dried with Na2SO4, filtered, and the solvent
evaporated to give 2.63 g of product as a white, oily solid (83%
yield). 1H NMR (500 MHz, CDCl3): δ = 6.99 (s, 1 H, Har), 6.94
(d, J = 1.4 Hz, 2 H, Har), 5.22 (s, 2 H, O-CH2-O), 4.64 (d, J =
3.7 Hz, 4 H, CH2-OH), 3.72 (q, J = 7.0 Hz, 2 H, CH2CH3), 2.15
(s, 2 H, OH), 1.22 (t, J = 7.1 Hz, 3 H, CH2CH3) ppm. 13C NMR
(126 MHz, CDCl3): δ = 157.85 (Car-OCH2), 142.97 (Car-CH2OH),
118.73 (Car), 113.92 (Car), 93.15 (O-CH2-O), 65.15 (CH2-OH),
64.51 (CH2CH3), 15.23 (CH2-CH3) ppm.
C
py), 3.56 (s, 4 H, Car-CH2-N), 2.60–2.20 (m, 1 H, OH) ppm. 13C
NMR (126 MHz, CDCl3): δ = 159.88 (CH2-Cpy-Npy), 157.57 (Car-
OH), 148.80 (o-Cpy), 140.74 (Car-CH2N), 136.86 (p-Cpy), 123.03 (m-
Cpy), 122.24 (m-Cpy), 120.07 (Car), 114.86 (Car), 60.05 (N-CH2-Cpy),
58.77 (Car-CH2-N) ppm. MS (ESI-positive): m/z = 517.2 [M+],
539.2 [M + Na+].
Tri-tert-butyl 10-[3-(3,5-Bis{[bis(pyridin-2-ylmethyl)amino]methyl}-
phenoxy)propyl]-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxyl-
ate (10): Tri-tert-butyl 10-(3-bromopropyl)-1,4,7,10-tetraazacyclo-
dodecane-1,4,7-tricarboxylate (tris-tert-butyl-DO3A) was synthe-
sized by reacting tris-tert-butyl-DO3A with a large excess of 1,3-
dibromopropane. K2CO3 (1.85 g, 13.48 mmol) and dibromopro-
pane (3.4 mL, 30.36 mmol) were dissolved in acetonitrile (400 mL)
and cooled to 0 °C. Tris-tert-butyl-DO3A (2 g, 3.36 mmol), synthe-
sized according to literature procedure, was dissolved in acetonitrile
(50 mL) and added to the above solution via dropping funnel. Re-
action progress was monitored by MS. After three days the reaction
was filtered, the solvent evaporated, and the crude product run on
a silica gel column, eluting with 2–4% methanol in DCM. The
bromopropyl-DO3A was added to a solution of K2CO3 (1.25 g,
9.05 mmol) and 9 (937 mg, 1.81 mmol) in acetonitrile (60 mL). The
reaction was heated to 70 °C and allowed to reflux under nitrogen
for 48 h. The reaction mixture was filtered and the solvent evapo-
rated. The crude product was purified on a silica gel column, elut-
ing with 7–10% methanol in chloroform to give 460 mg of yellow
oil (25% yield). 1H NMR (500 MHz, CDCl3): δ = 8.51 (dt, J = 4.9,
1.4 Hz, 4 H, o-Hpy), 7.75–7.52 (m, 8 H, Hpy), 7.15 [ddd, J = 6.9,
1,3-Bis(chloromethyl)-5-(ethoxymethoxy)benzene (7): Trichlorotri-
azine (4.35 g, 23.58 mmol) was dissolved in DMF (10 mL) at room
temperature and allowed to stir for one hour until the formation
of a yellow precipitate was observed. A solution of 6 (2 g,
9.43 mmol) in DCM (30 mL) was added and the reaction allowed
to stir overnight at room temperature. After confirming completion
of the reaction by TLC (15% ethyl acetate/hexanes), the reaction
mixture was transferred to a separatory funnel and washed success-
ively with water, a saturated solution of Na2CO3, and brine. The
organics were dried with Na2SO4, filtered and the solvent evapo-
rated. The residue was adsorbed to silica and purified on a silica
gel column, eluting with 5% ethyl acetate/hexanes to give 1.29 g of
1
product as a clear oil (55% yield). H NMR (500 MHz, CDCl3): δ
= 7.06 (d, J = 1.5 Hz, 1 H, Har), 7.03 (d, J = 1.6 Hz, 2 H, Har),
5.24 (s, 2 H, O-CH2-O), 4.54 (s, 4 H, CH2-Cl), 3.73 (q, J = 7.1 Hz, 4.8, 1.5 Hz, 5 H, 4 Hpy (4 H), Har (1 H)], 6.75 (d, J = 1.5 Hz, 2 H,
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Bacteria-Targeted MR Contrast Agent
Har), 3.92 (s, 2 H, CH2-CH2-OH), 3.79 (s, 8 H, N-CH2-Cpy), 3.64 After washing, quantification of gadolinium was accomplished
(s, 4 H, Car-CH2-N), 3.41–1.80 [m, 26 H, N-CH2-CH2-N (16 H), using inductively coupled plasma mass spectrometry (ICP-MS) of
N-CH2-COOH (6 H), N-CH2-CH2-CH2 (4 H)], 1.44 (d, J = 9.8 Hz,
27 H) ppm. 13C NMR (126 MHz, CDCl3): δ = 172.68 (COO-tBu),
171.77 (COO-tBu), 158.68 (CH2-Cpy-Npy), 157.96 (Car-OCH2),
acid-digested samples. Specifically, bacterial cell pellets were di-
gested in 100 μL of concentrated nitric acid (Ͼ 69%, Sigma, St.
Louis, MO, USA) and placed at 70 °C for at least 12 h to allow for
148.01 (o-Cpy), 139.57 (Car-CH2-N), 135.56 (p-Cpy), 121.76 (m-Cpy), complete sample digestion. Ultra pure H2O (18.2 MΩ·cm) and
121.10 (m-Cpy), 120.50 (Car), 112.46 (Car), 81.76 (O-C-CH3), 81.45 multi-element internal standard containing Bi, Ho, In, Li(6), Sc,
(O-C-CH3), 80.91 (O-C-CH3), 65.29 (O-CH2-CH2), 59.03 (N-CH2- Tb, and Y (CLISS-1, Spex Certiprep, Metuchen, NJ, USA) were
Cpy), 57.65 (Car-CH2-N), 55.64 (N-CH2-CH2-N), 54.86 (N-CH2- then added to produce a final solution of 3.0% nitric acid (v/v) and
CH2-N), 49.48 (N-CH2-COOH), 27.17 (C-CH3), 27.03 (C-CH3), 5.0 ng/mL internal standard up to a total sample volume of 10 mL.
26.87 (C-CH3), 25.18 (CH2-CH2-CH2) ppm. MS (ESI-positive): Samples were then syringe filtered using 0.2 μm polyamide filters
m/z = 1071 [M+], 1093 [M + Na+], 547.6 [(M+ + Na+)/2], 558.3 (Macherey–Nagel, Germany) into new 15 mL conical tubes. Indi-
[(M + 2Na+)/2], 573.4 [(M + Na+ + K+)/2], 595.3 [(M + 3K+)/2].
vidual Gd elemental standards were prepared at 1.00, 5.00, 10.0,
25.0, 50.0, 100, and 250 ng/mL concentrations with 3.0% nitric
acid (v/v) and 5.0 ng/mL internal standards up to a total sample
volume of 25 mL (using volumetric flasks).
Bis-dpa-Gd (1): The protected ligand 10 (338 mg, 0.315 mmol) was
dissolved in a mixture of 95:2.5:2.5 TFA/triisopropylsilane/water
and allowed to stir for four hours. The acid was evaporated under
nitrogen. Removal of the tert-butyl protecting groups was con-
firmed by ESI-MS. The residue was brought up in water (5 mL)
and the pH adjusted to 6.5 with NaOH (1 m). Gd(OAc)3 6 H2O
(148 mg, 0.47 mmol) was added, and the solution allowed to stir at
50 °C for 4 d, adjusting the pH back to 6.5 with NaOH (1 m) as
needed. The product was purified with reverse phase semi-prepara-
tive HPLC using a C18 column and eluting with a gradient of 0–
100% acetonitrile in water over 35 min, tr = 23 min. The purity and
identity of the product was confirmed using analytical HPLC-MS
on a C18 column, eluting with a gradient of 20–100% acetonitrile
in water over 35 min, tr = 19.5 min. The solvent was evaporated
from pure fractions, and the residue brought up in DMSO and
freeze dried. MS (ESI-positive) m/z = 1058.3 [M+], 530.6 [M2+/2].
ICP-MS was performed on a computer-controlled (Plasmalab soft-
ware) Thermo X series II ICP-MS (Thermo Fisher Scientific, Wal-
tham, MA, USA) equipped with a CETAC 260 autosampler (Om-
aha, NE, USA). Each sample was acquired using 1 survey run (10
sweeps) and 3 main (peak jumping) runs (100 sweeps). The isotopes
selected for analysis were 157,158Gd, and 115In and 165Ho (chosen
as internal standards for data interpolation and machine stability).
Relaxation Time Measurements at 1.41 T: T1 and T2 relaxation
times were measured on a Bruker mq60 NMR analyzer equipped
with Minispec v. 2.51 Rev.00/NT software (Billerica, MA, USA)
operating at 1.41 T (60 MHz) and 37 °C. T1 relaxation times were
measured using an inversion recovery pulse sequence (t1_ir_mb)
with the following parameters: four scans per point, 10 data points
for fitting, mono-exponential curve fitting, phase cycling, 10 ms
first pulse separation, and a recycle delay and final pulse separation
Ն 5T1. T2 relaxation times were measured using a Carr–Purcell–
Meiboom–Gill (CPMG) pulse sequence (t2_cp_mb) with the fol-
lowing parameters: four scans per point, mono-exponential curve
fitting, phase cycling, 10 ms first pulse separation, 15 second recy-
cle delay, 1 ms 90°–180° pulse separation (tau), while altering the
number of data points to ensure accurate mono-exponential curve
fitting (500–10000 data points for fitting). Relaxivities were deter-
mined by taking the slope of a plot of 1/T1 (s–1) or 1/T2 (s–1) vs.
gadolinium concentration (mm) of each compound in either LB
broth or in 1% (w/v) agarose.
Bis-dpa-Eu (1-Eu): The same procedure was followed as for 1, using
EuCl3·6H2O for metallation. Retention times were the same as for
1. MS (ESI-positive): m/z = 1053.2 [M+] 527.2 [M2+/2].
Bis-dpa-Tb (1-Tb): The same procedure was followed as for 1, using
Tb(OAc)3·6H2O for metallation. Retention times were the same as
for 1. MS (ESI-positive): m/z = 1059.2 [M+] 530.2 [M2+/2].
Luminesence Lifetime Measurements: The luminescence lifetime of
a 200 mm solution of Zn-1-Tb was measured H2O and D2O on a
Hitachi (San Francisco, CA) F4500 fluorimeter, using a λx
=
254 nm and λem = 544 nm. Twenty-five scans were acquired,
averaged and fit to a monoexponential decay function.
MR Imaging and Relaxation Time Measurements at 7.05 T: All MR
imaging was performed on an 89 mm bore size PharmaScan 7.05 T
MR imaging spectrometer fitted with shielded gradient coils
(Bruker BioSpin, Billerica, MA, USA) using a RF RES 300 1H
089/023 quadrature transmit/receive mouse brain volume coil
(Bruker BioSpin, Billerica, MA, USA). All MR images were ac-
quired using Paravision 5.0.1 software (Bruker BioSpin, Billerica,
MA, USA).
Quantification of Bacterial Cell Labeling by Inductively Coupled
Plasma Mass Spectrometry (ICP-MS): Stock solutions (1 mm) of
1, 2, and 3 were prepared by dissolving each contrast agent in a
solution of 2% DMSO in LB media. One molar equivalent of
ZnCl2 was added to 2 and 3, while two molar equivalents of ZnCl2
were added to 1. The solutions were incubated at room temperature
for 20 min (to form the ZnII-bound complexes), and were placed
in a sonicating water bath (Branson 5510; Branson Ultrasonics,
Danbury, CT) for an additional 10 min to ensure that all material
was dissolved. The stock solutions were diluted to the desired con-
centrations with LB media.
S. aureus cultures were labeled with 300 µm Zn-1, Zn-2, and Zn-3
and then washed three times the LB broth as described previously.
After the final wash samples were centrifuged at 6000 g for 3 min
to pellet the bacteria, which were then suspended in 1.0% (w/v)
low melting point agarose (Sigma, St. Louis, MO) in 7.5 mm outer
diameter NMR tubes and incubated on ice to allow for gelation of
the agarose. Samples were then positioned in a 23 mm mouse brain
volume transmit/receive coil prior to imaging.
S. aureus or E. coli cells were grown from a glycerol stock in LB
media overnight. One mL aliquots of bacteria were taken and cen-
trifuged at 6000 g for 3 min. The bacterial cell pellets were re-sus-
pended in various concentrations of Zn-1, Zn-2 or Zn-3 and al-
lowed to incubate at room temperature for 1 h while rotating. Bac-
teria were then centrifuged at 6000 g for 3 min, the supernatant was
decanted, and the bacterial cell pellets were re-suspended in 1 mL
LB media. This was repeated two more times for a total of three
washes to remove any unbound contrast agent and decrease non-
specific binding.
T1-weighted images were acquired using a rapid-acquisition rapid-
echo variable-repetition time (RAREVTR) pulse sequence using
the following parameters: RARE factor: 1, echo time (TE): 11 ms,
averages: 3, matrix size (MTX): 128ϫ128, field of view (FOV):
25ϫ25 mm2, 6 slices, slice thickness: 1.5 mm, interslice distance:
Eur. J. Inorg. Chem. 2012, 2099–2107
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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T. J. Meade et al.
FULL PAPER
2.0 mm, repetition times, TR = 15000, 10000, 8000, 6000, 3000,
1500, 1000, 750, 500, 300, 200, and 150 ms, and a total scan time
of ca. 3 h 45 min. T1 values of selected regions of interest (ROIs)
of 5 out of 6 slices were calculated using the T1 saturation recovery
mono-exponential curve fitting formula provided by the image se-
quence analysis (ISA) tool in Paravision 5.0.1 software (Bruker
BioSpin, Billerica, MA, USA).
closure can be found at http://pyrite.chem.northwestern.edu/analyt-
icalserviceslab/asl.htm. Metal analysis was performed at the North-
western University Quantitative Bioelemental Imaging Center gen-
erously supported by the NASA Ames Research Center (grant
number NNA04CC36G). L. M. M. acknowledges support from the
National Science Foundation Graduate Research Fellowship.
A. S. H. acknowledges support from the Natural Sciences and
Engineering Research Council of Canada for a post-graduate fel-
lowship.
Color T1 maps were generated using using Jim v. 6.0 software (Xin-
apse Systems Ltd., Aldwincle, UK). Briefly, the desired ROIs were
masked using the contour ROI function T1 maps were then gener-
ated using the saturation-recovery T1 fit function in the image-
least-squares fitter entering TRs in the single input image configu-
ration. Color maps were generated using an inverted rainbow color
lookup table setting the maximum T1 to 3000 ms. Images were pro-
cessed using the image resampler by resizing pixels to smooth the
image by converting a 128ϫ128 MTX to 384ϫ384 MTX (chang-
ing the number of columns and rows under pixel resizing specifica-
tions).
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Color T2 maps were generated using using Jim v. 6.0 software (Xin-
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Supporting Information (see footnote on the first page of this arti-
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Acknowledgments
The authors gratefully acknowledge Daniel Mastarone for assist-
ance with NMR experiments. This work was generously supported
by the National Institutes of Health (NIH) (grant nuber
R01EB005866). Imaging work was performed at the Northwestern
University Center for Advanced Molecular Imaging generously
supported by the National Cancer Institute Cancer Center Support
Grant (NCI CCSG P30 CA060553 awarded to the Robert H. Lurie
Comprehensive Cancer Center). MRI was performed on the 7 T
Bruker Pharmascan system purchased with the support of the
National Center for Research Resources (NCRR) (grant number
1S10RR025624-01). NMR studies were completed at the North-
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Received: December 5, 2011
Published Online: March 28, 2012
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