Hypoxia-Responsive19F MRI Probes with Improved Redox Properties and Biocompatibility

Article abstract of DOI:10.1021/acs.inorgchem.7b00500

¹⁹F magnetic resonance imaging (MRI), an emerging modality in biomedical imaging, has shown promise for in vitro and in vivo preclinical studies. Here we present a series of fluorinated Cu(II)ATSM derivatives for potential use as¹⁹F magnetic resonance agents for sensing cellular hypoxia. The synthesized complexes feature a hypoxia-targeting Cu²⁺ coordination core, nine equivalent fluorine atoms connected via a variable-length poly(ethylene glycol) linker. Introduction of the fluorine moiety maintains the planar coordination geometry of the Cu²⁺ center, while the linker length modulates the Cu^2+/+ reduction potential,¹⁹F NMR relaxation properties, and lipophilicity. In particular, the¹⁹F NMR relaxation properties were quantitatively evaluated by the Solomon-Bloembergen model, revealing a regular pattern of relaxation enhancement tuned by the distance between Cu²⁺ and F atoms. Finally, the potential utility of these complexes for sensing reductive environments was demonstrated using both¹⁹F MR phantom imaging and¹⁹F NMR, including experiments in intact live cells.

Full text of DOI:10.1021/acs.inorgchem.7b00500

Article
pubs.acs.org/IC
19
Hypoxia-Responsive F MRI Probes with Improved Redox Properties
and Biocompatibility
†
†
†
†
†
†
‡,§
́
Da Xie, Seyong Kim, Vikraant Kohli, Arnab Banerjee, Meng Yu, Jose S. Enriquez, Jeffrey J. Luci,
,
†
and Emily L. Que*
†Department of Chemistry, The University of Texas at Austin, 105 E. 24th Street Stop A5300, Austin, Texas 78712, United States
‡Department of Neuroscience, The University of Texas at Austin, Austin, Texas 78712, United States
§Imaging Research Center, The University of Texas at Austin, Austin, Texas 78712, United States
*
S Supporting Information
ABSTRACT: 19F magnetic resonance imaging (MRI), an
emerging modality in biomedical imaging, has shown promise
for in vitro and in vivo preclinical studies. Here we present a
series of fluorinated Cu(II)ATSM derivatives for potential use
19
as F magnetic resonance agents for sensing cellular hypoxia.
The synthesized complexes feature a hypoxia-targeting Cu2+
coordination core, nine equivalent fluorine atoms connected
via a variable-length poly(ethylene glycol) linker. Introduction
of the fluorine moiety maintains the planar coordination
2
+
geometry of the Cu center, while the linker length modulates
the Cu2 reduction potential, F NMR relaxation properties,
+/+
19
1
9
and lipophilicity. In particular, the F NMR relaxation
properties were quantitatively evaluated by the Solomon−
Bloembergen model, revealing a regular pattern of relaxation enhancement tuned by the distance between Cu2+ and F atoms.
1
9
Finally, the potential utility of these complexes for sensing reductive environments was demonstrated using both F MR
1
9
phantom imaging and F NMR, including experiments in intact live cells.
1
. INTRODUCTION
molecules such as perfluorocarbons (PFCs) and perfluoropo-
lyethers (PFPE) has found success for in vivo cell tracking and
measurement of tumor oxygenation through 19F MRI.
Inorganic scaffolds containing paramagnetic metal ions have
proven to be powerful for use as sensing platforms for
Hypoxia (oxygen deficiency) is a downstream effect of
accelerated cellular events such as proliferation, or an
inadequate vasculature to supply oxygen due to disease and/
or aberrant physiological environments. Hypoxia can lead to
severe pathologies including genetic instability and malignant
12,27−29
30−49
19
MRI.
In the context of F MRI, paramagnetic metal
1
−4
centers shorten the relaxation times of nearby fluorine nuclei,
progression of cells.
Decreased oxygen levels have been
1
9
and these have been used to develop responsive F MR
observed in a number of different types of cancer, ranging from5
imaging agents based on the modulation of T (transverse
brain to subcutis, compared to normal tissue or organs.
2
relaxation time) of 19F.
38,39,50
More importantly, judicious
Indeed, hypoxia represents an essential prognostic marker, and
1
−4
choice of the paramagnetic metals for a fluorinated system with
short T (longitudinal relaxation time) and a T /T ratio close
its identification is important for planning cancer treatment.
To date, multiple imaging modalities have been utilized for
detecting cellular hypoxia, including magnetic resonance
1
2
1
to unity enables more rapid acquisition of MR signals, thus
achieving a high signal-to-noise ratio within a certain
5
−18
19−21
imaging (MRI),
luminescence imaging,
and positron
2
2,23
acquisition time to help overcome the intrinsic sensitivity
emission tomography (PET).
Among them, an MRI-based
challenge of 19F MRI.
51−54
Recently, this has been demon-
approach is clinically desirable and practical, since it enables
whole-body imaging with high spatial resolution and anatomic
detail without exposing subjects to ionizing radiation. A number
of responsive contrast agents have been pursued for sensing
strated in a fluorinated nanoemulson incorporated with a high-
19
spin Fe(III) center, enabling sensitive cellular F MRI in
55
vivo.
Previously, we demonstrated the use of a 19F MR-based
approach to differentiate cells grown in hypoxic versus
normoxic environments. Our first generation probe was based
1
6−9
hypoxia through MR-based approaches including H,
1
9
10−12 31 13
14−16
F,
P, and hyperpolarized NMR
-based probes.
1
9
F MRI, owing to the favorable MR properties of the 19F
1
9
nucleus and negligible F signal in the human body, has shown
promise in complementing the wealth of information provided
Received: February 26, 2017
1
12,24−26
by H MRI.
In particular, use of superfluorinated
©
XXXX American Chemical Society
A
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Article
on a trifluorinated Cu(II)ATSM complex (Cu(II)ATSM-F ,
sequences with a detection limit of 0.44 mM for 4 and 0.099
mM for Cu4 and demonstrated the use of Cu4 for F NMR-
based differentiation of hypoxic cells versus normoxic cells in
live, whole cell experiments.
3
50
2+
19
ATSM = diacetyl-bis(4-methylthiosemicarbazonato)). Cu
possesses a relatively long longitudinal electronic relaxation
−
8
−10
time (T ), on the scale of 10 to 10 seconds, largely due to
1e
56−58
its well-separated electronic excited states.
significantly shortens the T values of interacting F nuclei,
This property
1
9
59
2. RESULTS AND DISCUSSION
2
such that no fluorine signal is observed in Cu(II)ATSM-F . In
3
2.1. Synthesis. Synthesis of CuATSM derivatives Cu1−4
was carried out starting from 4-methylsemicarbozone (Scheme
2). Two-step imine condensation afforded the ligand precursor
in 69% yield. Refluxing the ligand precursor with fluorinated
amines containing different ethylene glycol chain lengths
2
+
+
hypoxic environments, the reduction of Cu to Cu and ligand
−
dissociation affords the related diamagnetic [Cu(I)ATSM-F3]
complex and free ligand, effectively lengthening the T and
2
restoring a robust 1 F signal.
9
6
1
As a proof-of-concept system, Cu(II)ATSM-F3 nicely
provided the ligands, 1−4, in 59−82% yield.
Final
demonstrated that one-electron redox changes that convert
coordination was achieved using Cu(II) acetate in high yield
(82−90%), and the resulting complexes Cu1−4 were purified
using reverse-phase chromatography using acetonitrile and
water as eluents. Complex purity was supported by high-
resolution LC−MS, revealing the formation of 1:1 metal/ligand
complexes.
1
an S = / system to an S = 0 system can be exploited for
2
1
9
developing redox sensors for F MRI. CuATSM-based
complexes display a well-studied relationship between reduc-
tion potential and selectivity toward hypoxic regions, and their
60
redox properties are readily controlled by the ligand. In
addition, CuATSM derivatives have the advantage of being cell-
permeable, which enables the interrogation of the intracellular
milieu. In this paper, we report a series of new fluorinated
CuATSM derivatives (Cu1−4, Scheme 1) containing higher
2.2. Structural Characterization. Single crystals of Cu1
were grown by slow evaporation of a concentrated toluene
solution of the complex. The single crystal X-ray structure and
important structural parameters are presented in Figure 1 and
Scheme 1. Structure of Complexes Cu1-4
fluorine density and modified redox and relaxation properties
through the introduction of a variable-length linker in efforts to
improve sensitivity and selectivity of this class of probes. In
these complexes, the fluorine nuclei are further from the
Figure 1. Molecular structure of Cu1 from single crystal X-ray
diffraction (top, top view; bottom, front view). Thermal ellipsoid plot
at 50% probability level refined by a two-component disorder model.
Solvent molecules and hydrogen atoms omitted for clarity.
2+
paramagnetic Cu relative to our previous complexes, resulting
19
2+
in F relaxation times that are attenuated yet NMR and MRI
signals that are not fully quenched. By taking Cu4 and its
ligand, 4, as representative compounds, we collected 19F MR
phantom images that demonstrate selective imaging of the
Tables S2−S4. In this complex, the Cu sits in a pseudo-
square-planar [N S ] pocket, and no intra- or intermolecular
2
2
interactions are observed between the metal center and the
ligand ether oxygen. The geometry of the Cu2+ coordination
site well mimics the parent CuATSM complex, indicating a
1
9
complex versus the ligand through different F MR pulse
a
Scheme 2. Synthesis of Cu1−4
a
Conditions: (a) 2,3-butanedione, conc HCl, H O, 0−5 °C, 2.5 h (95%); (b) 4,4-dimethylsemicarbazone, acetic acid, DMF, room temperature (rt),
2
4
8 h (73%); (c) H N(CH CH O) C(CF ) (n = 1, 2, 3, 4), CH CN, 90 °C, 16 h (59−82%); (d) cupric acetate hydrate, DMF, rt, 16 h (82-90%).
2
2
2
n
3
3
3
B
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Inorganic Chemistry
similar chemical environment of the Cu . The average
Article
2+
62
P values from 1.65 to 2.87, which reflects the hydrophobic
nature of the C−F bonds. On the other hand, a progressive
decrease in log P values was observed with Cu1, Cu2, Cu3, and
Cu4 having values of 2.87, 2.53, 2.23, and 1.65, respectively.
This validates that incorporation of hydrophilic ethylene glycol
linkers significantly attenuates the lipophilicity of these
complexes.
2+
Cu −F distance is 8.87 Å, longer than in first generation
compounds CuATSM-F (6.48 Å) and CuATSM-F , (6.66
3
6
50
Å). Likely due to the flexible nature of the extended ethylene
glycol linkers, single crystals of Cu2−4 did not form.
Optimized structures of Cu1−4 were obtained using density
functional theory (DFT) calculations at the uB3LYP/6-
3
1G(d,p) level (Figure S2). The optimized structure of Cu1
2.5. Cyclic Voltammetry. Hypoxia selectivity in this class
of compounds is in part governed by their Cu2 reduction
potential, with optimal selectivity demonstrated for CuATSM
+/+
well resembles its single crystal structure, and all four
complexes share the same square planar coordination
configuration. As expected, extending the ethylene glycol
chain increases the distance between the paramagnetic Cu2+
center and the fluorine tags, with average Cu−F distances in
Cu1−4 of 8.98, 11.74, 15.12, and 18.18 Å, respectively. The
above results suggest analogous coordination environments of
Cu in Cu1−4 and CuATSM, and no interactions were
observed between the oxygen atoms contained in the linker and
Cu(II).
with an E1/2 = −0.63 V versus SCE (measured in DMF), and
60
no selectivity for compounds with E1 ≥ −0.50 V. In our
/2
previous work, incorporation of fluorine atoms five atoms away
from the coordinating S atoms resulted in cathodic shifts in
reduction potential, so much so that incorporation of two −CF
3
2+
50
groups in this scaffold yielded a complex with E1 = −0.49 V.
/2
We hypothesized that increasing the distance between the
fluorine tag and the Cu center should cause an anodic shift in
reduction potential, rendering complexes with reduction
potentials better tuned for hypoxia selectivity. As shown in
Figure 3, in DMF solution, all four complexes displayed a
2
.3. EPR Spectroscopy. EPR spectra for all four complexes
were recorded in DMF at room temperature (Figure 2). Cu1−
Figure 3. Cyclic voltammograms of Cu1−4 (vs SCE) in DMF
solution at 298 K.
(
quasi)reversible peak for the Cu2+/+ redox couple, with peak
potential separations ranging from 80 to 127 mV. Incorporation
Figure 2. X-band EPR spectrum of CuATSM and Cu1−4 in DMF
of electron-withdrawing perfluorinated tert-butyl ether groups
solution at 298 K.
+
2+
induced a slight cathodic shift of E1/2 for Cu /Cu , as seen in
Cu1 (−0.60 V) and Cu2 (−0.61 V), but the effect was
attenuated with an increase of the ethylene glycol chain length
(−0.63 V for both Cu3 and Cu4). The above reduction
potentials indicate that complexes Cu3 and Cu4, with sufficient
4
displayed nearly identical EPR spectra at room temperature,
with giso = 2.062 and ACu = 97 G. These values fall well within
the expected range for a square planar [CuN S ] coordination
2
2
6
3,64
2+
environment,
and are similar to those measured for the
parent CuATSM complex (also shown). In particular, a
separation between Cu and fluorinated moieties, have optimal
65
redox properties for hypoxia selectivity.
1
9
19
quintet superhyperfine splitting pattern is observed in the
2.6. F NMR properties. The F NMR spectra of the four
2
+
3
1
400−3650 G region, giving a superhyperfine constant of A =
ligands and corresponding Cu complexes in d -DMSO
N
6
6 G. This feature, also supported by the g value, reveals a
revealed significant relaxation effects for all ethylene glycol
chain lengths. A singlet peak at around −70.0 ppm was seen for
all of the eight compounds, indicating that all appended
fluorine atoms are in equivalent environments. Compared to
the sharp peaks of free ligands 1−4 (half-height peak widths
<10 Hz), the corresponding Cu1−4 complexes displayed
broader peaks with half-height peak widths ranging from 61 Hz
iso
2
+
66
significant covalent character for the Cu −ligand interaction.
The incorporated ethylene glycol moieties have little effect on
the symmetry of the CuN S coordination core, leaving the two
2
2
nitrogen-donating ligands indistinguishable as is true with the
65
parent symmetric CuATSM complex. These results further
certify that our modifications to the CuATSM core do not
perturb the copper coordination environment.
2
+
for Cu1 to 16 Hz for Cu4 (Figure 4A). The fact that the Cu
2
.4. Lipophilicity. We evaluated the n-octanol/water
center was able to strongly relax distant fluorine spins even in
the Cu4 is consistent with the long electronic relaxation time of
the Cu2+ center, which leads to dramatic decreases in T2
relaxation times of interacting nuclei. A table summarizing
relevant NMR parameters for 1−4 and Cu1−Cu4 is provided
in the Supporting Information (Table S1).
partition coefficient log P (log(Coctanol/Cwater), where C =
concentration) for Cu1−4 as well as the nonfluorinated
67
CuATSM according to Leo’s method. While the introduction
of the perfluoro-t-butyl moiety increases the lipophilicity of the
complexes, ethylene glycol linkers will have the opposite effect.
Consistent with previous reports, the nonfluorinated CuATSM
showed a mild lipophilicity (log P = 1.50). All of the
synthesized CuATSM derivatives, Cu1−4, showed higher log
The longitudinal (T ) and transverse (T ) relaxation times of
1
2
both complexes and ligands were determined in DMSO (Figure
4B). For both the ligands and the complexes, increases in both
C
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Figure 4. (A) 19F NMR spectra of 20 mM Cu1−4. (B) Relaxation times (T , T ) of 20 mM Cu1−4. (C) Linear fitting of longitudinal PRE rates
1
2
6
2
(
Γ ) and transverse PRE rates (Γ ) to the 1/r for Cu1−4. The R value for the fitting are 0.999 and 0.985 for longitudinal PRE rates (Γ ) and
transverse PRE rates (Γ ), respectively. NMR spectra and relaxation times were acquired at 298 K in d -DMSO at a field strength of 9.4 T.
1
2
1
2
6
Figure 5. (A) UV−vis absorption changes by chemical reduction using Na S O . (B) 19F NMR spectra illustrating the one-electron reduction of Cu3
2
2
4
in a mixed solvent of 70% DMSO/20% HEPES buffer (pH 7.0, 20 mM)/10% D O using Na S O .
2
2
2
4
4
−1
−1
T and T were observed with an increase of the ethylene glycol
316 nm (ε = 2.14 × 10 L mol cm ) and a broad peak at 477
3
1
2
−1
−1
chain length. For the free ligands, the T increased from 1.06 s
nm (ε = 7.01 × 10 L mol cm ) (Figure 5A). Titrating
1
for 1 to 1.49 s for 4, while T increased from 0.90 s for 1 to 1.31
concentrated aqueous Na S O into this solution rendered a
2
2
2
4
s for 4. This enhancement was attributed to the improved
flexibility of the fluorinated group as the length of the ethylene
glycol chain linking it to the ATSM backbone increases. Cu1−4
followed a similar trend for both T (17−43 ms) and T (7.7−
red-shift of the sharp band at 311 nm, a dramatic quench of the
absorbance band at 477 nm, and a slight increase of a broad
band at ∼600 nm. Full disappearance of the peak at 477 nm
was observed after addition of 40 equiv of Na S O . These
1
2
2
2
4
3
4 ms). In addition to the enhanced flexibility of the fluorine-
absorbance changes are consistent with a metal-centered
2
+
71
containing tail, the attenuated Cu paramagnetic relaxation
enhancement (PRE) effect likely contributes to the increasing
relaxation times for the Cu complexes as the linker is extended.
We calculated the ratio between the relaxation times of the
reduction as has been observed with similar complexes.
During titration, a small amount of water was added upon
addition of Na S O . Thus, the observed product is likely a
2
2
4
−
mixture of the anionic [Cu(I)3] complex and the free ligand
3, with the former being the major species. Back oxidation by
air diffusion into the cuvette for 30 min recovered the broad
peak at 477 nm, with the restored spectrum overlapping well
with the initial.
2
+
ligands and corresponding Cu complexes (T (ligand)/T (Cu
n
n
complex), n = 1, 2). The results, described as longitudinal (Γ )
1
and transverse (Γ ) PRE rates, respectively, are shown in Figure
2
2
+
4C. Considering that Cu only has one unpaired electron and
a long electronic relaxation time, we assumed a major
The reduction was further monitored by 19F NMR, which
was performed in a mixture of 70% DMSO and 30% aqueous
buffer (Figure 5B). Prior to reduction, Cu3 showed a broad
peak with a peak width at half-height of 38 Hz. This larger peak
width relative to that observed in pure DMSO (21 Hz) is likely
due to the increased water content in the solvent. After
component of the PRE effect is through direct dipole−dipole
6
8
interactions. In this case, both longitudinal and transverse
PRE rates are conventionally described by the Solomon−
Bloembergen equations to give a proportional relationship
6
between Γ1 and 1/r (r is the distance between the metal
,2
69,70
19
center and the interacting nuclei).
of Γ and Γ versus 1/r (r values estimated from DFT-
Thus, by fitting the plot
incubation of Cu3 with Na S O for 5 min, the F NMR
2
2
4
6
spectrum of the crude reaction revealed an intense singlet at δ =
−69.5 ppm, which matched up well with the spectrum of ligand
3. At this water concentration, the ligand has likely dissociated
from the Cu(I), giving 3 as the major product. The transverse
1
2
optimized structures), we observed an approximate linear
relationship (Figure 4C). We note that computational
optimization cannot precisely predict the structure in solution
phase in which fast conformational motions are expected, but
can provide a means of estimating the maximum predicted
nuclear relaxation time T was further measured. After the
2
reduction, the system displayed a T of 0.76 s, a 129-fold
2
2
+
distance between the Cu center and the fluorinated spins.
increase as compared to the T of Cu3 in the same solvent
2
The above results suggest a regular pattern of PRE rates tuned
system. The corresponding peak intensity increased by 22-fold.
2.8. Stability, Cytotoxicity, and Cell Uptake. Prior to
initiating cell experiments, we examined the stability of our
CuATSM derivatives in cell-culture media using UV−vis
spectroscopy. After leaving 30 μM solutions of complexes
Cu1−4 for 24 h, only small to negligible changes in absorbance
were recorded (Figure S3) for Cu2−4, indicating their
considerable stability in biological media. The absorbance
peaks for Cu1, however, disappeared after 24 h incubation; we
2
+
by the distance between Cu and F atoms, paving the way for
19
future design of fluorinated CuATSM derivatives for F PRE-
based MR detection of hypoxia.
2
.7. In Vitro Reduction Studies. To demonstrate the
reduction of our fluorinated Cu(II)ATSM derivatives in
reducing environments, we monitored the reduction of Cu3
in vitro with UV−vis and NMR spectroscopies. By UV−vis, the
initial DMF solution of Cu3 exhibited an intense absorbance at
D
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attribute this to precipitation of the complex due to its low
aqueous solubility. Indeed, visible precipitate was observed
when aqueous solutions of this complex were left for 24 h.
Thus, Cu1 was deemed not suitable for further biological
studies, and further cell experiments were performed solely
with Cu2−4.
Cytotoxicity of Cu2−4 was tested in a human breast cancer
cell line (MCF-7) using a live/dead assay. Cells were treated
with the three synthesized complexes, respectively, at a
concentration of 30 μM for 4 h, and the cell viability was
evaluated using the ReadyProbes Cell Viability Imaging Kit
= 10 ms and TR (repetition time) = 100 ms, was used. In
comparison, the slowly relaxing 4 was best imaged with a long
echo pulse sequence employing TE = 100 ms and TR = 2040
ms. We determined the limit of detection (LOD) for 4 and
Cu4 at a total scan time of 2 h (Figure 7). Good linearity was
achieved when plotting the signal-to-noise ratio (SNR) versus
sample concentration for both 4 (r = 0.996) and Cu4 (r =
0.996) (Figure 7B,D). The LOD was further evaluated from the
fitted line by using a threshold of 3-fold of the SNR for the
blank sample and was determined to be 0.44 mM for 4 (4.1
2
2
1
9
19
mM F) and 0.099 mM for Cu4 (0.89 mM F). The LOD of
12,72
(
blue/green). The results are compiled in Figures S4 and S5. In
4 falls on the same scale as the previously reported values.
19
both normoxic (20% O ) and hypoxic (0.1% O ) environ-
The paramagnetically relaxed F MR signal (i.e., Cu4) showed
a 4.5-fold increase in SNR due to the increased scan rate
enabled by the short echo pulse sequence. This submillimolar
probe detection limit holds great promise for future use of this
class of probes in biological systems.
2
2
ments, little fluorescence was recorded in the green channel for
dead cell staining, revealing low cytotoxicity of the Cu2+
complexes (>98% viability for all Cu-treated cells, see Figure
2+
S6). Thus, at an incubation concentration of 30 μM Cu -
complex (270 μM fluorine concentration), the cell condition
was ideal for further studies to differentiate hypoxic and
The large difference in relaxation times between 4 and Cu4
19
allowed us to differentiate them through F MRI. Figure 8A
1
9
19
normoxic cells using F NMR/MRI-based detection.
Cellular uptake levels were investigated by treating MCF-7
cells with Cu2−4 for 4 h and measuring the intracellular Cu
shows F MR images of four tubes containing a solvent blank
(DMSO), a 1 mM DMSO solution of 4, a 1 mM DMSO
solution of Cu4, and a mixed DMSO solution of 1 mM 4 and 1
mM Cu4. We scanned these samples using both the long and
short echo sequences described above. As expected, the DMSO
content through ICP-OES, together with CuCl as a control.
2
The results are compiled in Figure 6. Compared to the CuCl2
19
blank solution had zero detectable F signal when either pulse
sequence was used. When using the long echo sequence, the
solution of 4 and the mixed solution of Cu4 and 4 both gave a
strong signal (SNR 16.0 and 16.4, respectively), while the Cu4-
only solution signal was not above the detection threshold
(
SNR 2.0) as its magnetization relaxes before image acquisition
begins. On the other hand, when using the short echo
sequence, the opposite results were obtained. Bright signals
were seen in the solutions containing Cu4 (SNR 11.5 and 13.4
for the Cu4-only solution and solution mixture of Cu4 and 4,
respectively). Only a negligible signal of 4 was detected (SNR
3.4), as very little diamagnetic species relaxes during the short
echo pulse sequence time frame.
Figure 6. Uptake levels of Cu2−4 in MCF-7 cells under both
normoxic (20%, white bars) and hypoxic (0.1%, gray bars) conditions.
MCF-7 cells were incubated with 30 μM Cu2 complex for 4 h before
the intracellular Cu content was analyzed by ICP-OES. The control
group represents cells that were not incubated with a copper source.
+
The efficient 19F signal differentiation, combined with sub-
millimolar detection limit for 4 and Cu4, prompted us to track
CuCl (30 μM) was used as an additional control to demonstrate the
19
2
the reduction of Cu4 through F MRI as a further step toward
effect of transportation of the complex into cells with incubation under
the same conditions.
application (Figure 8B). Cu4 (1 mM) was incubated with 2
equiv of Na S O in a DMSO/HEPES (7/3, v/v) mixture for 5
2
2
4
min, where the reduction happened immediately, changing the
solution color from reddish brown to bright yellow. With
application of the long echo pulse sequence, an increased signal
was detected. Analysis of the SNR gave a signal enhancement of
6.1-fold upon reduction of the Cu2+ complex. However, when
using the short echo pulse sequence, only negligible signal was
recorded (SNR 1.8), indicating an almost complete reduction
control group, improved cell uptake was observed for Cu2−4 at
femtomole levels per cell, suggesting that the synthesized
2
+
ATSM derivatives are effective to transport Cu into cells.
Better cell uptake was seen with an increase of the ethylene
glycol chain length under both normoxic (20% O ) and hypoxic
2
(
0.1% O ) conditions. Interestingly, the only complex that
2
1
9
exhibited a significant increase in cell uptake under hypoxic
conditions compared to normoxic conditions was Cu4,
showing a 52% enhancement in cell uptake when grown
under hypoxic conditions for 4 h. We note that, in a
noncancerous cell type (HEK293 cells), no difference in
cellular copper content was observed following incubation with
Cu4 under normoxic and hypoxic conditions.
of Cu4. Taken together, the above results suggest that F MRI
can be used to track both the ligand and its Cu2+ complex and
monitor hypoxia-based reduction events.
We finally used 19F NMR to test the potential for Cu4 to
differentiate cells cultured in normoxic (20% O ) and hypoxic
2
(0.1% O ) conditions. MCF-7 cells were incubated with 30 μM
2
Cu4 for 4 h under hypoxic and normoxic conditions, and whole
cell suspensions were transferred to NMR tubes under the
same oxygen levels. We also mixed cell-culture media with 30
μM Cu4 and used it as a cell-free control. The results are
2
.9. MR Imaging and in Vitro NMR Studies. We used 4
and Cu4 as representative compounds to demonstrate the
application of fluorinated CuATSM derivatives for MR-based
studies. A series of phantom images were acquired on a 7 T
preclinical MRI system using a rapid acquisition with relaxation
enhancement (RARE) sequence. Due to the short relaxation
time of Cu4, a short echo pulse sequence, with TE (echo time)
1
9
compiled in Figure 8C. While there was almost negligible
F
NMR signal (SNR 4.7 compared to the calculated spectral
1
9
noise level) observed in the cell-free control group, a F signal
increase at δ = −70.5 ppm in the hypoxic group (SNR 22.4)
E
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Figure 7. Determination of LOD on 4 and Cu4 through 19F MRI at a field of 7.0 T at 22 °C. (A, C) 19F MRI performed on phantoms containing 4
(
(
A) or Cu4 (C) diluted in DMSO at different concentrations. (B, D) Linearity of signal-to-noise ratio (SNR) versus concentration of 4 (A) or Cu4
C), together with a SNR threshold for the detection limit. A long echo pulse sequence was used for 4 (TE = 100 ms, TR = 2040 ms), and a short
echo pulse sequence was used for Cu4 (TE = 10 ms, TR = 100 ms).
Figure 8. Differentiation of Cu4 and 4 through long and short echo 19F MR pulse sequences at a field of 7.0 T at 22 °C. (A) 19F MR images of
solvent blank (left column) and DMSO solutions containing 1 mM 4 (2nd-left column), 1 mM Cu4 (2nd-right column), and 1 mM Cu4 plus 1 mM
4
(right column). (B) 19F MR images of in vitro reduction of Cu4 by using 2 equiv of Na S O in a DMSO/HEPES (7/3, v/v) mixture. Top row for
2 2 4
parts A and B: long echo pulse sequence was used, TE = 100 ms, TR = 2040 ms, 588 scans (∼2 h). Bottom row for parts A and B: short echo pulse
19
sequence was used, TE = 10 ms, TR = 100 ms, 588 scans (∼6 min). (C) F NMR spectra at 11.7 T of live MCF-7 cells using Cu4 demonstrating
increased signal intensity under hypoxic conditions (0.1% O ), together with cell-culture media mixed with 30 μM Cu4 as a cell-free control.
2
was observed, suggesting an efficient reduction of the complex
by the cellular machinery. On the other hand, only a weak
signal was observed for the normoxic group (SNR 8.2), which
we ascribe mainly to a limited reduction of the complex inside
cells in the normoxic environment and, in part, from the signal
of Cu4 itself. With the above results suggesting a potential for
this class of compounds to act as reporters to track hypoxic cells
using F MRI, future work will focus on introducing groups to
improve the water solubility and fluorine content of the
molecule to enable in cell and in vivo MR imaging of hypoxia.
functionalities. The PRE effect of Cu2+ in fluorinated CuATSM
derivatives was quantitatively studied for the first time,
2
+
exhibiting a T -increase of 129-fold between one of the Cu
2
complexes and its reduced system. Applying the transverse PRE
rates of the complex to the theoretical model of PRE through a
dipole−dipole interaction demonstrated a regular pattern for
6
the PRE effect through space tuning, with a 1/r dependence.
19
We note that there is significant relaxation enhancement even
when the fluorine tag is positioned ∼18 Å away from the S =
1
2+
/
Cu center, opening up a number of possibilities for next
2
generation probes. Introducing an ethylene glycol linker
effectively modulates the lipophilicity and redox properties of
the complexes, leading to different cell uptake levels and
selectivity between cells grown under normoxic and hypoxic
conditions. MR phantom images were collected using 4 and
Cu4 as representatives and demonstrated efficient differ-
entiation of 19F nuclei from the complex versus the ligand
through two different RARE pulse sequences. The F spin
density detection limits were further determined to be 0.44 mM
for the ligand and 0.099 mM for the complex using their
3
. CONCLUSION
In conclusion, we have synthesized a series of CuATSM
derivatives that bear nine identical fluorine atoms and a
variable-length ethylene glycol linker between the ATSM
backbone and the fluorinated tag. Attaching ethylene-glycol-
linked fluorine groups to the CuATSM framework maintained
the coordination geometry of the Cu center and rendered the
redox behavior of the new complexes similar to that of
CuATSM despite the presence of electron-withdrawing
2
+
19
F
DOI: 10.1021/acs.inorgchem.7b00500
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
optimized imaging pulse sequences. Further, we tracked 19F
signal changes upon reduction of Cu4 both chemically and in
the hypoxic cellular environment through MRI and NMR.
Ongoing work in this system is focused on optimization of cell
uptake levels as well as further enhancement of both fluorine
spin density and water solubility. Further, the reported imaging
results in which short echo sequences enable detection of
fluorinated Cu2 species provide an intriguing avenue for the
development of new imaging agents.
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Nano 2015, 9, 8239−8248.
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3
1
MRI contrast agent for imaging tumor hypoxia in vivo. JBIC, J. Biol.
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+
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CEST Images in Vivo by T Relaxation: A New Approach in the
Design of Responsive PARACEST Agents. J. Am. Chem. Soc. 2013,
1
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.7b00500.
■
1
35, 14904−14907.
9) Rojas-Quijano, F. A.; Tircso,
Tran Hoang, H.; Kalman, F. K.; Gulaka, P. K.; Kodibagkar, V. D.;
Aime, S.; Kovacs, Z.; Sherry, A. D. Synthesis and Characterization of a
*
S
(
́ ́ ́ ́
G.; Tircsone Benyo, E.; Baranyai, Z.;
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Komatsu, H.; Ito, T.; Yamada, H.; Chujo, Y.; Matsuda, T.; Hiraoka,
M.; Nishimoto, S.-i. Monitoring of Biological One-Electron Reduction
Details about syntheses and other methods, and
supplementary data files (PDF)
Accession Codes
19
19
by F NMR Using Hypoxia Selective Activation of an F-Labeled
CCDC 1549916 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_
request@ccdc.cam.ac.uk, or by contacting The Cambridge
Crystallographic Data Centre, 12 Union Road, Cambridge CB2
Indolequinone Derivative. J. Am. Chem. Soc. 2009, 131, 15982−15983.
(
11) Liu, S.; Shah, S. J.; Wilmes, L. J.; Feiner, J.; Kodibagkar, V. D.;
Wendland, M. F.; Mason, R. P.; Hylton, N.; Hopf, H. W.; Rollins, M.
D. Quantitative tissue oxygen measurement in multiple organs using
1
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1EZ, UK; fax: +44 1223 336033.
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AUTHOR INFORMATION
Corresponding Author
E-mail: emilyque@cm.utexas.edu.
■
(
13) Lu, M.; Zhu, X.-H.; Zhang, Y.; Chen, W. Intracellular redox state
31
+
revealed by in vivo P MRS measurement of NAD and NADH
contents in brains. Magn. Reson. Med. 2014, 71, 1959−1972.
(14) Keshari, K. R.; Kurhanewicz, J.; Bok, R.; Larson, P. E. Z.;
Vigneron, D. B.; Wilson, D. M. Hyperpolarized 13C dehydroascorbate
as an endogenous redox sensor for in vivo metabolic imaging. Proc.
Natl. Acad. Sci. U. S. A. 2011, 108, 18606−18611.
*
ORCID
Emily L. Que: 0000-0001-6604-3052
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
(15) Bohndiek, S. E.; Kettunen, M. I.; Hu, D.-e.; Kennedy, B. W. C.;
13
Boren, J.; Gallagher, F. A.; Brindle, K. M. Hyperpolarized [1- C]-
Ascorbic and Dehydroascorbic Acid: Vitamin C as a Probe for Imaging
Redox Status in Vivo. J. Am. Chem. Soc. 2011, 133, 11795−11801.
Funding
(16) Barskiy, D. A.; Shchepin, R. V.; Coffey, A. M.; Theis, T.;
This work was supported by start-up funds from the University
of Texas at Austin, the Welch Foundation (F-1883), a
University of Texas at Austin Undergraduate Research grant
V.K.), and a University of Texas at Austin Graduate School
Summer Fellowship (D.X.).
1
5
Warren, W. S.; Goodson, B. M.; Chekmenev, E. Y. Over 20%
Hyperpolarization in Under One Minute for Metronidazole, an
N
Antibiotic and Hypoxia Probe. J. Am. Chem. Soc. 2016, 138, 8080−
(
8
083.
(
́
17) Do, Q. N.; Ratnakar, J. S.; Kovacs, Z.; Sherry, A. D. Redox- and
Notes
Hypoxia-Responsive MRI Contrast Agents. ChemMedChem 2014, 9,
1116−1129.
The authors declare no competing financial interest.
(18) Iwaki, S.; Hanaoka, K.; Piao, W.; Komatsu, T.; Ueno, T.; Terai,
3+
ACKNOWLEDGMENTS
T.; Nagano, T. Development of hypoxia-sensitive Gd -based MRI
■
contrast agents. Bioorg. Med. Chem. Lett. 2012, 22, 2798−2802.
We thank Dr. Vincent Lynch for X-ray crystallography support
and Dr. Bradley Holliday for providing access to Gaussian. We
also acknowledge the Imaging Research Center at UTAustin
for access to their facilities.
(19) Takahashi, S.; Piao, W.; Matsumura, Y.; Komatsu, T.; Ueno, T.;
Terai, T.; Kamachi, T.; Kohno, M.; Nagano, T.; Hanaoka, K.
Reversible Off−On Fluorescence Probe for Hypoxia and Imaging of
Hypoxia−Normoxia Cycles in Live Cells. J. Am. Chem. Soc. 2012, 134,
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