A Responsive Magnetic Resonance Imaging Contrast Agent for Detection of Excess Copper(II) in the Liver in Vivo

Article abstract of DOI:10.1021/jacs.8b13493

The design, synthesis, and properties of a new gadolinium-based copper-responsive magnetic resonance imaging (MRI) contrast agent is presented. The sensor (GdL₁) has high selectivity for copper ions and exhibits a 43% increase in r₁ relaxivity (20 MHz) upon binding to 1 equiv of Cu²⁺ in aqueous buffer. Interestingly, in the presence of physiological levels of human serum albumin (HSA), the r₁ relaxivity is amplified further up to 270%. Additional spectroscopic and X-ray absorption spectroscopy (XAS) studies show that Cu²⁺ is coordinated by two carboxylic acid groups and the single amine group on an appended side chain of GdL₁ and forms a ternary complex with HSA (GdL₁-Cu²⁺-HSA). T₁-weighted in vivo imaging demonstrates that GdL₁ can detect basal, endogenous labile copper(II) ions in living mice. This offers a unique opportunity to explore the role of copper ions in the development and progression of neurological diseases such as Wilson's disease.

Full text of DOI:10.1021/jacs.8b13493

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Article
A responsive MRI contrast agent for detection
of excess copper(II) in the liver in vivo
Namini N. Paranawithana, Andre F Martins, Veronica Clavijo Jordan,
Piyu Zhao, Sara Chirayil, Gabriele Meloni, and A. Dean Sherry
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b13493 • Publication Date (Web): 20 Jun 2019
Downloaded from http://pubs.acs.org on June 21, 2019
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A responsive MRI contrast agent for detection of excess copper (II) in
the liver in vivo
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Namini N. Paranawithana,1 Andre F. Martins,1 Veronica Clavijo Jordan,2 Piyu Zhao,1 Sara Chirayil,2
Gabriele Meloni1 and A. Dean Sherry1,2*.
1Department of Chemistry and Biochemistry, University of Texas at Dallas, Richardson, Texas, USA.
2Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
KEYWORDS: Copper-responsive, Magnetic Resonance Imaging, Contrast agents, Human serum albumin, Wilson disease.
ABSTRACT: The design, synthesis, and properties of a new gadolinium-based copper-responsive MRI contrast agent is
presented. The sensor (GdL1) has high selectivity for copper ions and exhibits a 43% increase in r1 relaxivity (20 MHz) upon
binding to 1 equivalent of Cu2+ in aqueous buffer. Interestingly, in the presence of physiological levels of human serum
albumin (HSA), the r1 relaxivity is amplified further up to 270%. Additional spectroscopic and XAS studies show that Cu2+ is
coordinated by two carboxylic acid groups and the single amine group on an appended side-chain of GdL1 and forms a ternary
complex with HSA (GdL1-Cu2+-HSA). T1-weighted in vivo imaging demonstrates that GdL1 can detect basal, endogenous labile
copper(II) ions in living mice. This offers a unique opportunity to explore the role of copper ions in the development and
progression of neurological diseases such as Wilson’s disease.
1. INTRODUCTION.
differences in proton content, cell density, and water perfusion
and diffusion. Image contrast can be altered by the use of
paramagnetic inorganic complexes that shorten the T1,2
relaxation times of water molecules in various compartments.
These agents are commonly known as contrast agents (CAs).
Among all paramagnetic complexes designed for use as CAs,
the
Gd3+-based agents have proven to be the safest and most
versatile agents for clinical use over the past ~30 years. The
efficiency of an agent per unit concentration is commonly
reported as r1 (T1) or r2 (T2) relaxivity.26-28 Notably, the design
of contrast agents that alter the T1 of water protons in response
to a given analyte is of major importance. Many responsive
probes have been reported including sensors for metal ions,9
enzyme activity,29 pH,30 pO2,31 and temperature.32 One of the
first reports of a copper-activated MR sensor was based on a
Copper is the third most abundant transition metal in the body
and a required dietary nutrient. The average healthy human has
a total of ~110mg of copper in tissue.1-4 Copper is typically
bound to specific proteins and enzymes where it plays
fundamental catalytic and structural roles.5-7 Copper has also
been associated with signaling events in the brain.8-10 In
biological environment, copper is present in two oxidation
states, the cuprous (Cu+) and cupric (Cu2+) ions.11 Typically,
total extracellular Cu2+ can vary widely from nM to µM while
intracellular Cu+ can vary from µM to mM.12-14 Due to its redox
properties, copper homeostasis is tightly regulated in cells and
disruption of this is associated with
neurodegenerative diseases including
a
number of
Alzheimer’s,
Parkinson’s, prion diseases, familial amyotrophic lateral
sclerosis, Menkes and Wilson’s diseases.15-18 For instance,
genetic mutations of copper-transporting proteins ATP7A and
ATP7B results in afflictions of systemic brain copper
deficiency in Menkes disease and hyperaccumulation of hepatic
copper ions in Wilson’s disease, respectively.19-22
Accumulation of copper in the liver of Wilson’s disease patients
ranges from a few micromolar to several millimolar.23 Although
copper ion homeostasis and the impact of abnormal copper
levels on physiology have been widely studied, details about the
functional role of copper ions in various tissues in vivo remain
insufficiently understood due to lack of real-time copper
imaging techniques in live animals.24,25
GdDO3A
(1,4,7,10-tetraazacyclododecane-1,4,7,-triacetic
acid) derivative having a iminodiacetate pendant arm for Cu2+
recognition. This derivative displayed a 41% increase in r1
relaxivity upon binding Cu2+.33 A later paper reported a
GdDO3A derivative having a quinolone-based pendant arm34
and it displayed a 71% increase in r1 upon addition of Cu2+.
Neither of these agents were examined in vivo. Derivatives
have also been designed to show greater selectivity for Cu+ over
Cu2+.35 In addition to MRI agents, other imaging approaches for
imaging copper have included positron emission tomography
(PET), optical imaging and bimodal imaging techniques.36,37
The positron emitter, 64Cu, was successfully used to image
greater uptake and accumulation of copper in livers of Wilson’s
disease mouse model.24 Similarly, a near-infrared fluorescent
sensor for detection of Cu+ ions has been shown to be capable
of monitoring fluctuations in exchangeable copper stores in
Magnetic resonance imaging (MRI) is a powerful medical
diagnostic technique that allows noninvasive, three-
dimensional visualization of tissue with high spatial and
temporal resolution. MRI is largely based on detection of water
and fat protons so that image contrast among tissues reflects
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Figure 1. Gadolinium-DO3A based copper responsive (GdL) agents
living cells and mice under basal conditions, as well as in
situations of copper overload or deficiency.38
Water proton relaxivity measure,ments in the presence of
various metal ions. The longitudinal relaxivity (r1) of GdL1
(4.7 ± 0.1 mM-1s-1 at 20 MHz) was unchanged upon addition of
Ca2+, Mg2+, Cu+ or Fe3+ ions (Figure 2 and Figure S4). However,
addition of Zn2+ increased r1 to 5.3 mM-1s-1 (a 12% increase)
while addition of Cu2+ increased the r1 to 6.7 mM-1s-1 (a 47%
increase). The background relaxivity due to the weak
paramagnetism of Cu2+ and Fe3+ was subtracted from the r1
values shown in Figure 2 and reported in Table 1.50 This
suggests that GdL1 has some selectivity for Cu2+ over Zn2+ in
agreement with the Irving-William series and Pearson’s Hard-
Soft Acid Base (HSAB) theories.51 Even though the origin of
this r1 enhancement is unclear from these data alone, one
possibility is that the linker side-arm with the anionic carboxyl
groups on the bis-benzoic acid motif may form a hydrogen bond
with the single exchanging inner-sphere water molecule on the
Gd3+ ion and this interaction is reduced when Cu2+ or Zn2+ binds
to GdL1. This could in principle alter the water exchange rate in
this system and result in a small change in r1. A second
contributing factor might be that GdL1 experiences relatively
slower molecular rotation (R) upon binding to Cu2+ and this
could result in a slight increase in r1 relaxivity. These two
possibilities were examined in more detail below.
The binding stoichiometry between GdL1 and Cu2+ was
determined to be 1:1 as reported by the method of continuous
variations (see Job plot, Figure S9) and by an inflection point52
in the relaxivity data (Figure 3). This stoichiometry was
assumed in all calculations of dissociation constants (Kd). The
increases in r1 of GdL2 and GdL3 were considerably lower upon
addition of Cu2+ (Figure 3), suggesting that either these two
complexes have a weaker affinity for Cu2+ or the resulting
GdLx-Cu2+ complexes have quite different water exchange
properties.
Our group recently reported several MR zinc-sensors capable
of detecting the release of intracellular stores of zinc into
extracellular space in prostate39 and pancreas of live mice.40
Divalent zinc ions released by cells in these organs are
immediately chelated by a zinc-responsive MR agent, and the
resulting binary complex then forms a ternary complex with
serum albumin which results in reduced molecular motion and
an increase of r1.41,42 Copper is also known to bind to albumin
and other less abundant proteins in plasma.43-47 Hence, we
hypothesized that the key to an effective copper detection in
vivo by MRI might be to design a Cu-responsive agent that also
forms a ternary complex between Cu2+ ions, the sensor, and
albumin, similar to the Zn-sensor designs.
Herein, we report the synthesis of a novel copper-responsive
MRI contrast agent having a bis(benzoic acid)methylamine
recognition motif (GdL1) and the physicochemical properties of
the resulting GdL1-Cu2+ complex and the ternary complex
formed with HSA. X-ray absorption spectroscopy (XAS) of
GdL1 and some structural analogs were used to interrogate the
Cu2+ binding site in this system. Finally, GdL1 was injected into
mice to detect extracellular Cu2+ in the liver by MRI.
A
comparison of GdL1 (high Cu2+ affinity) with GdL2 and GdL3
(lower Cu2+ affinity) (Figure 1) indicated that only GdL1 detects
extracellular copper in mouse liver.
2. RESULTS and DISCUSSION
Design and synthesis. The structure of the Cu2+-responsive
agent reported here consists of a GdDO3A moiety with a
bis(benzoic acid)methylamine side-chain as a potential chelator
for Cu2+. This design was motivated by our previous MR-
responsive Zn2+ sensor scaffold where the ion of interest
initiates formation of a ternary complex between the agent and
serum albumin.41,42 Although Cu2+ has a preference for nitrogen
donor atoms, the coordination rigidity provided by the
bis(benzoic acid)methylamine could potentially favor
coordination by geometrical stabilization of tetragonal or
square pyramidal structures typical of Cu2+.48,49 This structural
feature precludes the possibility of binding with the more
abundant biological ions like Ca2+ and Mg2+. To evaluate the
impact of repositioning of the carboxylate groups on the
aromatic side-chain and lowering the charge on the carboxylate
groups, GdL2 and GdL3 were also studied for comparison. The
synthetic details of all three contrast agents are described in
supplementary materials.
Binding experiments in the absence of HSA (GdLx-Cu2+).
The equilibrium dissociation constants (Kd) between the three
GdLx complexes and Cu2+ were determined by fluorescence
spectroscopy by performing titrations in which Cu2+ was added
to a buffered solution containing GdLx. Addition of Cu2+ results
in quenching of the intrinsic fluorescence of the benzoic acid
moieties of GdLx (Figure S11).53,54 The resulting binding curves
were fit to a 1:1 binding model to give the Kd values reported in
Table 1. These data indicate that GdL1 has the highest affinity
for Cu2+ (84 + 10 µM), followed by GdL3 (352 + 9 µM) and
GdL2 (895 + 32 µM). This suggests that the position of the
carboxyl groups (meta versus para) and charge of
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6.0
GdL2
GdL3
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4.5
0.0
0.5
Cu2+ / mM
1.0
0
Fe3+
Ca2+
Zn2+
Cu+
Cu2+
Mg2+
Figure 2. The 20 MHz relaxivity (r1) of GdL1 in the
presence of various Mn+ ± HSA. The white bars reflect r1
after addition of 0.5 mM Mg2+, Ca2+, Fe3+, Zn2+, Cu+ or Cu2+
to 0.5mM solutions of GdL1. The black bars reflect r1 after
subsequent addition of 0.6mM HSA to the GdL1-Mn+
solutions. The data were collected in 0.1M MOPS buffer
(pH 7.4) at 37οC.
Figure 3. The 20 MHz r1 relaxivity of GdL1-3 as a function
of added Cu2+. The concentration of GdL1-3 was 0.5 mM in
0.1M MOPS buffer (pH 7.4). The data were collected at
37οC.
the complexes are both important for Cu2+ binding.
Binding experiments in the presence of HSA. Additional
proton relaxation enhancement (PRE) titrations were carried
out to examine the binding interactions and to quantitatively
evaluate the binding constants for each GdLx complex with
HSA in the presence of 1 molar equivalent of Cu2+. A fitting of
these data (Figure S13 and Table S2) to a 1:1 binding model
gave the Kd values reported in Table 1. GdL1 showed the highest
binding affinity to HSA-Cu2+ (Kd = 45 ± 3.1 μM) while the
binding affinities of GdL2 and GdL3 were surprisingly weaker
only by ~30%. This demonstrates a significance differences in
binding affinity between the different GdLx and Cu2+ are leveled
upon formation of the ternary GdLx-Cu2+-HSA complexes.
These data alone suggest that HSA plays a significant role in
stabilizing the binding interactions between GdLx and Cu2+
ions. To confirm the formation of the LnLx-Cu2+-HSA ternary
complex, a sample of LaL1 (a diamagnetic analog), Cu2+ and
HSA (from an EPR experiment, see below) were passed
through a size exclusion chromatography column, and the
eluent peaks were separately analyzed for Cu and La by ICP-
MS. Those results (Figure S9 and Table S1) showed that ~63%
of the total La eluted from the column in the form of a ternary
LaL1-Cu2+-HSA complex, confirming the formation of a stable
Cu mediated ternary complex.
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Water proton relaxivity measurements in the presence of
various metal ions and HSA. HSA, the most abundant protein
in serum (~600 μM), plays a key role in the transport of metal
ions, fatty acids, and other hydrophobic molecules including
many drugs. HSA has two Cu2+ binding sites, the N-terminal
site (NTS) and multi-metal binding site (MBS).55,56 It was
reported that Cu2+ has a significantly higher affinity for the NTS
site (~1 pM)57 than the MBS site (~10 nM)58. Thus, the NTS site
is considered to be the only site in HSA to be occupied by Cu2+
since the concentration of HSA is much higher than the
biological concentration of free Cu2+ ions.55-59 We recently
demonstrated that analogous Gd-based MR contrast agents
responded to an increase in free Zn2+ ions from pancreatic β-
cells40 and epithelial prostate cells stimulated by an increase in
plasma glucose.39 This functional response was shown to reflect
formation of a ternary GdLx-Zn-albumin complex at the MBS
site A.60 This previous data suggested that perhaps Cu2+ could
also be detected in vivo in those situations where excess free
Cu2+ ions in extracellular spaces might be available for binding
to a contrast agent. This motivated further relaxometric studies
to determine the magnetic contributions of all the GdLx with
Cu2+ in the presence of physiological levels of HSA. As
summarized in Table 1, the r1 values for all three complexes
increase slightly in the presence of HSA alone (likely reflecting
a slight increase in viscosity) but increase substantially after the
addition of Cu2+ ions. This suggests that the GdLx complexes
experience slower molecular rotation by the formation of a
GdLx-Cu-HSA ternary complex. This is particularly true for
GdL1 where r1 increases from 6.1 + 0.1 to 22.6 + 0.2 mM-1s-1 (a
270% increase). As shown in Figure 2 (and Figure S5), addition
of Mg2+, Ca2+, Fe3+ do not result in an increase in r1 in the
presence of HSA while Zn2+ ions do to a lesser extent, about 2-
fold lower than the increase in r1 induced by Cu2+.
Kinetic inertness. The kinetic stability of a GdLx complex is
also an important factor to consider when developing MRI
probes. Previous studies reported that Cu2+ could displace Gd3+
from a complex by transmetallation.61,62 This possibility was
examined by challenging GdL1 with 3-molar equivalents of
Cu2+ in 0.03 M phosphate buffer (pH =7.2). Under these
conditions, if transmetallation occurred, any unchelated Gd3+
would then precipitate from the solution as an insoluble
phosphate, a process that can be monitored by relaxometry.
R1obs values measured over the time (Figure S15) show that
complexes are kinetically inert, even in the presence of 3-fold
excess Cu2+. In addition, LC-MS data showed that in the
presence of
a
1-molar equivalent of Cu2+ no metal
transmetallation was observed at room temperature after 7 days
in MOPS buffer.
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Table 1. 20 MHz relaxivity values for GdL1-3 ± Cu2+ and ± HSA and Kd values for binding of GdL1-3-Cu2+ with HSA.
All experiments were performed in 0.1M MOPS buffer (pH 7.4) at 37οC.
1
2
3
4
In the absence of 0.6 mM HSA
In the presence of 0.6mM HSA
5
6
7
8
r1 (mM-1s-1)
%
r1 (mM-1s-1)
Kd
Kd
%
GdLx
increase
increase
in r1
(GdL-Cu2+)
(µM)a
(GdL-Cu2+-
HSA) (µM)b
No
1 eq.
Cu2+*
No Cu2+
1 eq. of Cu2+
in r1
Cu2+
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GdL1
4.7+0.1 6.7+0.1
4.9+0.2 5.5+ 0.1
4.8+0.1 5.4+0.2
43%
12%
12%
84 ± 10
895 ± 32
352 ± 9
6.1+0.1
5.7±0.1c
6.5+0.2
22.6+0.2
15.4±0.2c
14.5+ 0.1
270%
123%
90%
45 ± 3.1
59 ± 5
GdL2
GdL3
6.3+0.2
12.0+0.2
60 ± 10
a Kd(GdL-Cu2+) was determined by fluorescence titrations.
b Kd(GdL-Cu2+-HSA) was determined by proton relaxation enhancement titrations.
C Values measured in the presence of 0.6 mM mouse albumin.
Experiments to identify the Cu2+ donor atoms in GdL1. The
X-band EPR spectrum of LaL1-Cu2+ exhibited an unusual axial
spectrum (devoid of well-defined hyperfine features and a g 
1.99) both in the absence and presence of HSA (Figure S18).
The g values shown by these spectra significantly deviated
from the typical values of an axial EPR spectrum for Type-2
Cu2+ complex.535 This suggests that the Cu2+ center in both
complexes are electron poor, likely due to the strong electron
withdrawing effect of the lanthanum ion in the complex. In
comparison, the X-band EPR spectrum of HSA-Cu2+ exhibited
a typical Type-2 square pyramidal geometry very similar to
previously reported EPR spectra in the literature.63 However,
the broadened hyperfine features of the Cu2+ EPR spectra after
addition of LaL1 precluded a detailed structural analysis of the
copper center.
XAS studies. Copper K-edge X-ray absorption spectroscopy
(XAS) studies were also performed to identify the Cu2+ donor
atoms in GdL1-Cu2+ and GdL1-Cu2+-HSA. The XANES
spectrum of GdL1-Cu2+ (Figure 4) is characterized by an intense
absorption feature at 8987–8988eV with a broad low energy tail
in the region below 8985eV (normalized absorption of approx.
0.5 at 8988 eV) arising from a 1s → 4p transition characteristic
of Cu2+ complexes. The presence of the first major inflection
point at 8986eV and the absence of lower energy features
(normalized absorption 0.15 at 8984 eV and first inflection
point 8984 eV) is typical of classic tetragonal Cu2+ complexes
with nitrogen and oxygen ligands. The complex also presents a
weak 8979eV peak (more visible in the first derivative spectra)
corresponding to the 1s → 3d transition possibly reflecting a
less centrosymmetric nature of the center and thus a significant
degree of distortion from planarity.64 The XANES spectrum of
GdL1-Cu2+-HSA is nearly identical to the spectrum of Cu2+-
HSA, suggesting a very similar coordination environment in the
two complexes. The spectra are characterized by an intense
absorption feature at 8987–8988eV arising from a 1s → 4p
transition. Additional features for the Cu2+ site include a lower-
energy feature with normalized absorption of approximately
0.25 at 8984 eV, and the first inflection point determined in the
first derivative spectrum at 8982 eV, about ~1 eV higher than
the one observed in Cu+ complexes. Possible photoreduction of
Cu2+ was prevented experimentally by collecting the spectra at
different locations in the frozen sample in each scan. Also, the
XANES spectra were quite similar to the one observed for the
Cu2+-DAHK peptide complex representing the N-terminal Cu2+
binding site in HSA, thus supporting the same coordination
environment in the full-length protein.55
Additional information of coordination environment and ligand
metal distances were obtained by copper K-edge extended X-
ray absorption fine structure (EXAFS). The experimental
copper EXAFS spectra are presented in Figure S19 together
with best fits, and the corresponding EXAFS Fourier
transforms. The spectrum of GdL1-Cu2+ could be fitted with 2
ligand shells indicative of a Cu complex coordinated by 3 N/O
ligands at 1.99 Å and a N/O ligand at 2.51 Å (Table 2). The
XANES and EXAFS results are consistent with the formation
of a distorted tetragonal complex in which 3 donor atoms from
the two carboxylates and the tertiary amine of the bis(benzoic
Figure 4. XANES spectra for GdL1 in the presence of Cu2+
and HSA.
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acid)methylamine moiety with possible additional coordination
by a solvent water molecule to the Cu2+ ion.
chelating sites in bis(benzoic acid)methylamine moiety of
GdL1. It should be noted that the bond distances for the distorted
1
2
3
4
Table 2 . Structural and coordination parameters obtained from fitting Cu K-edge EXAFS.
(N- Coordination Number, R -interatomic distance, σ2- Debye-walker factor)
5
6
7
8
9
Complex
N
3
bond
R (Å)
1.994(3)
2.51(1)
σ2(Å2)
0.0008
0.0001
F-factor
0.488
GdL1-Cu(II)
Cu-N/O
Cu-N/O
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Cu(II)-HSA
3
1
1
Cu-N/O
Cu-N/O
Cu-N/O
1.991(2)
2.278(7)
2.515(7)
0.0016
0.0013
0.0008
0.398
GdL1-Cu(II)-HSA
GdL1-Cu(II)-HSA
3
1
1
4
1
Cu-N/O
Cu-N/O
Cu-N/O
Cu-N/O
Cu-N/O
1.954(6)
2.33(1)
2.86(1)
1.965(6)
2.31(2)
0.0054
0.0037
0.0001
0.0054
0.0037
0.576
0.587
1
Cu-N/O
2.87(1)
0.0001
Coordination numbers are indicated by N, interatomic distances R are given in Å (the values in parentheses are the estimated
standard deviations), Debye–Waller factors σ2 (the mean-square deviations in interatomic distance) in Å2, and the fit-error
function F is defined by F = [∑ k6(χ (k)calcd – χ (k)exp)2 /∑ k6(χ (k)exp)2]1/2 where χ(k) are the EXAFS oscillations and k is the
photoelectron wavenumber.
In the HSA-Cu2+ complex, the EXAFS data were best fit with 3
coordinating shells around Cu2+ with 3 Cu-O/N bonds at 1.99 Å
and an additional (likely equatorial) N/O bond at 2.28 Å. In
addition, a third shell corresponding to a N/O ligand at 2.51 Å
was obtained in the fit suggesting the presence of an axial
ligand. This analysis was in agreement with a previously
reported square pyramidal Cu2+-HSA coordination at the N-
terminal site (also known as ATCUN) with high-affinity for
Cu2+.53 Copper is coordinated to four nitrogen donors in the
NTS site consisting of Asp, Ala, His amide nitrogen atoms and
the His side chain in an equatorial position and a water molecule
or N-terminal amine nitrogen in the axial position.55,59 For the
GdL1-Cu2+-HSA ternary complex, the EXAFS data could be
best fitted with 3 or 4 N/O ligands (resulting in similar F-values)
at 1.96 Å and 2 additional N/O ligands at 2.33 Å and 2.89 Å.
The analysis predicts the presence of a distorted square
pyramidal/ octahedral coordination around the Cu2+ in the
ternary complex with 4 equatorial ligands with short bond
distances and 1 or 2 axial ligands with longer bond distance
(Table 2). This coordination anticipates the replacement of a
loosely bound axial ligand in the Cu2+-HSA complex by the
square pyramidal Cu2+-HSA complex are distinctly different
from the bond distances of the distorted square pyramidal
ternary complex. Despite being difficult to unambiguously
distinguish the coordination 1 or 2 axial ligands by XAS, the
EXAFS analysis confirms small differences in the coordination
shells between GdL1-Cu2+-HSA and HSA- Cu2+. This distorted
octahedral or square pyramidal coordination of Cu2+ in the
ternary complex compared to the distorted square pyramidal
coordination of Cu2+-HSA supports the formation of a copper-
mediated complex between HSA and GdL1.
Molecular Modelling. Molecular models of the Cu centers in
GdL1, HSA, and the ternary complex were generated based
upon the coordination geometry and bond lengths for N and O
atoms obtained from the EXAFS experimental data (Table 2)
using standard MM+ methods. The energy-minimized models
are presented in Figure 5. The model of GdL1-Cu2+ reflects a
distorted tetragonal geometry around the Cu center as predicted
by XAS. The geometry of the NTS Cu2+ site in HSA reflects a
square pyramidal geometry similar to the previously reported
crystal structures,53 while Cu center in the ternary complex is a
distorted octahedral geometry. These models support the EPR
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and XAS data by predicting only small differences in
maximum intensity after ~6 minutes post-injection, reflective
of relatively fast excretion of GdL1 characteristic of most low
molecular weight Gd-based extracellular agents. In a separate
coordination geometry of GdL1-Cu2+-HSA compared to HSA-
Cu2+.
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Figure 5. MM+ minimized structures of (A) Domain structure of albumin (PDB ID code 1AO6): domain I and II are colored green
(residues 1–373), domain III in yellow (residues 380–571), long chain fatty acid sites (FA), Sudlow’s drug binding sites, Cu2+
binding NTS site and zinc binding site A (MBS/Site A) are also shown. (B)GdL1-Cu2+ complex (C) HSA-Cu2+ complex (CCDC-
809109)52 (D) HSA-Cu2+- GdL1 distorted square pyramidal complex (E) HSA-Cu2+- GdL1 distorted octahedral complex consistent
with all NMR, XAS and EXAFS data. Hydrogen atoms and other sites of HSA have been removed to simplify visualization. Only
residues at NTS site in HSA are included. These figures were generated using HyperchemTM7.5.
In vivo imaging of free copper pools in living mice. Most
dietary absorbed copper is transported to the liver via
enterohepatic circulation where serum albumin acts as
transporter protein to maintain total exchangeable forms of
copper in the µM range.10,65-70 Hence, the liver plays a key role
in copper homeostasis by facilitating copper storage and
incorporating copper into ceruloplasmin and other copper
binding proteins. Either elevated or reduced copper in the liver
has been associated with neurological disorders and acute liver
diseases. Therefore, a non-invasive method to image those
abnormalities in copper levels in vivo is of broad interest.
group, mice were pretreated with the copper chelator, ATN-224
(5 mg/kg), two hours prior to injection of GdL1.38,70 In those
animals, the average MR liver enhancement after injection of
0.1mmol/kg GdL1 was lower, ~11% (p-value = 8.6 X 10-3).
This suggests that pre-treatment with the copper chelator
removed some of the excess Cu2+ prior to injection of GdL1. The
tissue distribution of Cu and Gd in the same mice used in the
imaging experiments were determined by ICP-MS analysis
(Figure 6C). These results confirmed that the higher MR signal
intensities directly correlated with higher copper levels in the
liver of healthy mice while the Gd content was identical in both
ATN-224 treated and non-treated animals.
To examine whether GdL1 can detect and respond to changes in
extracellular copper in vivo, T1-weighted MR images of
C57BL/6 mice were collected at 4.7T (Figure 6). This imaging
field was chosen from the equipment available to us because the
r1 differences between GdL1 versus GdL1-Cu-HSA, although
smaller at 4.7T versus 0.47T (20 MHz), remain significantly
different. After i.v. injection of a bolus of 0.1 mmol/kg GdL1,
the average gain in signal intensity throughout the liver of a
healthy mouse was ~25% when compared to the pre-contrast
images. This enhancement returned to baseline after reaching
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Figure 6. (A) In vivo MRI images of wild type mouse (n=3) pre- and post-injection of GdL1 (0.1mmol/kg) without (top) or with
(bottom) pretreatment with ATN-224 (5mg/kg in 50ul). All images were obtained at 4.7 T. (B) The average MRI signal intensity
of mouse liver 6 min after injection of either GdL1 or Gadavist™ in control mice (black bars) versus mice pretreated with ATN-224
(white bars). The columns on the right show average liver signal intensities at 13 min after injection with Multihance™. The data
were compared using a two-tailed student t-test. *p < 0.05 (n=3); error bars reflect ± SD. (C) Total Cu and Gd (µg/g tissue) in
various tissues collected from control mice (black bars) and from mice pretreated with ATN-224 (white bars) 6 min after the
injection of GdL1. Tissue copper levels relative to tissue wet weight were determined by ICP-MS. The data were compared using a
two-tailed student t-test. *p < 0.05 (n=3); error bars reflect ± SD.
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Mice were also imaged using two different Gd-based agents as
controls, Gadavist (an extracellular agent) and Multihance (a
hepatobiliary agent). After injection of an equivalent amount
of Gadavist, the signal intensity of liver gained intensity as
expected for a typical extracellular agent but only by ~8%. Like
GdL1, the signal gain in liver reached a maximum at ~6 min
then returned to pre-injection baseline values at about the same
clearance rate as GdL1. In mice pretreated with ATN-224, the
liver enhancement was unchanged, consistent with a lack of
affinity of Gadavist for Cu2+. Given that the possibility a small
amount of GdL1 may clear via hepatobiliary excretion (a
complete biodistribution study has not been done), it was
important to perform a similar set of control experiments using
a known hepatobiliary agent, Multihance, to determine whether
ATN-224 treatment might alter liver function. Those imaging
results are also presented in Figure 6B. Since the clearance of
Multihance in liver was significantly slower than either GdL1 or
Gadavist, the signal intensity data shown here reflect the
maximum values at 13 min rather than at 6 min. As shown,
liver image enhancement resulting from the passage of
Multihance through the liver was identical in untreated mice
versus mice pretreated with ATN-224, showing that liver
function is unaltered by ATN-224. Therefore, the decreased
intensity we observed when using GdL1 must reflect a decrease
in freely available Cu2+ in liver.
3. CONCLUSIONS
In this study, we investigated whether a new macrocyclic
gadolinium complex, GdL1, could act as a Cu2+-responsive MRI
contrast agent. We also examined the physical-chemical
properties of the ternary GdL1-Cu2+-HSA complex that resulted
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in a magnified longitudinal r1 relaxivity (20 MHz) of 22.6 mM-
For example, Wilsons’ disease patients reported the
1s-1. Our results showed that the observed r1 enhancement due
to the slow tumbling of the ternary complex was sufficient to
allow detection of M levels of freely available Cu2+ in the liver
by T1-weighted MR imaging, even at 4.7T. After injection of
GdL1 into healthy untreated mice, the liver was nicely enhanced
at 6 min, and image contrast returned to background levels after
~ 20 min. However, when mice were treated with ATN-224, the
MR signal gain in liver images was ~50% less compared to
control animals. The lower contrast enhancement observed in
the liver of mice pretreated with ATN-224 paralleled the
reduction in total liver copper as detected by ICP-MS. Based
on the ICP results, one can estimate the concentration of GdL1
in liver at 6 min and ask the question of whether the decreased
levels of Cu as detected by ICP-MS are consistent with the
imaging results. The total amount of Gd in liver at 6 min was
40 µg/g wet tissue. If one assumes that GdL1 remains largely
extracellular and that the extracellular fraction of wet liver is
~24-26%,71,72 then the GdL1 concentration can be estimated as
~1 mM (40 µg/g divided by 157 g/mol = 0.25 µmol/g = 0.25
µmol/0.26 mL = 1 mM). This same calculation for Cu gives a
total [Cu] of 0.36 mM in liver of control mice (6 µg/g) and 0.18
mM in livers after treatment with ATN-224 (3 g/g). Although
not all of the Cu2+ is extracellular, a 50% change in available
Cu would easily be detected using GdL1 even if r1 is no higher
than 9 mM-1s-1 at 4.7T. One would expect to see even more
dramatic changes in MRI signal intensity in liver if these
experiments had been performed at typical clinical imaging
fields, 1.5T or 3T.
significantly higher concentrations of serum non-ceruloplasmin
copper (>4.0µM) in the blood74 and hepatic copper content of
>250µg per gram of dry liver weight.75,76 Similarly, deficiency
of copper has been reported in a variety of genetic, neurological,
cardiovascular and metabolic diseases.9,77 Furthermore, it has
recently been shown that elevated serum and tumor copper
levels are linked to the progression of cancer malignancy78 and
also plays an important role in the regulation of sleep-related
and arousal behaviors.79 Therefore, we believe that the
observations reported here using GdL1 will catalyze discoveries
of Cu2+-responsive MRI agents for imaging acute liver
conditions such as that found in Wilsons diseases or elevated
copper levels in other disease conditions.
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ASSOCIATED CONTENT
Supporting Information.
The SI contains the synthesis details for preparing the Gd
complexes, the experimental details of the relaxometric
experiments, titrations, 17O NMR experiments, EPR spectra,
EXAFS spectra, cyclic voltammetry, computational model details,
and MR images.
AUTHOR INFORMATION
Corresponding Author
* E-mail: dean.sherry@utsouthwestern.edu; sherry@utdallas.
edu.
The second goal of this study was to identify the Cu2+ donor
atoms on GdL1, the donor atoms in the ternary GdL1-Cu2+-HSA
complex, and the location of the GdL1-Cu2+ binding site in
HSA. The fact that Cu2+ has only one high-affinity site in HSA,
the N-terminal site,55,57 it is reasonable to assume that GdL1 also
binds at this site by contributing donor atoms to Cu2+. This
model is consistent with the changes in the Cu2+ coordination
sphere as reported by EPR and X-ray absorption spectroscopy
(XAS) data. The combined results indicate that the Cu2+ binds
to GdL1 via a single tertiary N atom and two carboxylate O
atoms on GdL1 and a single water molecule to form a distorted
tetragonal complex. The Cu2+ center in GdL1-Cu2+-HSA
ternary complex is most consistent with coordination by four
equatorial nitrogen donor atoms from the protein and one or two
axial O/N donor from GdL1 resulting in distorted
octahedral/square pyramidal geometry. The slight coordination
changes in the Cu2+ center with GdL1 in the presence of HSA
results in a stable ternary complex that results in a surprisingly
high r1 relaxivity at 20 MHz.
Author Contributions
All authors read and approved the manuscript.
ACKNOWLEDGMENTS
Financial support from the National Institutes of Health (DK-
095416 and EB-015908), the American Diabetes Association
(ADA 7-12-IN-42) and the Robert A. Welch Foundation (AT-584)
is gratefully acknowledged. We thank the staff at Beamline 9-3,
Stanford Synchrotron Radiation Light source (SSRL) for support
in XAS data collection. We also thank Dr. Alexendar Funk for help
with the mouse imaging experiments at UTSW, Drs. Hien Q.
Nguyen and Alexios Papadimitrators for their support and training
in the ICP-MS, EPR experiments at UT Dallas, and Dr. Limei
Zhang at University of Nebraska-Lincoln for discussions on XAS
data collection and analysis.
ABBREVIATIONS
HSA, Human Serum Albumin, XAS, X-ray absorption
spectroscopy, MRI, Magnetic resonance imaging.
In summary, our study shows that GdL1 can be used as a sensor
of excess freely available Cu2+ ions in tissue. In the presence of
HSA, the freely available Cu2+ forms a stable ternary complex
GdL1-Cu2+-HSA that magnifies the r1 relaxivity to such an
extent that in vivo detection of exchangeable Cu2+ MR imaging
was possible. To our knowledge, this is the first time that
extracellular copper levels in the liver could be detected and
with a remarkable statistical difference. Although this work has
not included a mouse disease model to validate the results
obtained here, there is enough evidence to suggest that this
sensor can be used in mouse models with known abnormal
levels of copper. The total serum copper levels can be markedly
elevated in acute liver failure due to its release of excess copper
ions from liver tissue stores. This results in elevated total serum
Cu2+ not bound to ceruloplasmin referred to as “free-copper”.73
REFERENCES
1. Puig, S.; Thiele, D.J. Molecular mechanisms of copper
uptake and distribution. Curr. Opin. Chem. Biol. 2002, 6,
171-180.
2. Maryon, E.B.; Molloy, S.A.; Zimnicka, A.m.; Kaplan, J.H.
Copper entry into human cells: progress and unanswered
questions. Biometals. 2007, 20, 355- 364.
3. Linder MC, Wooten L, Cerveza P, Cotton S, Shulze S,
Lomeli N. Copper Transport. Am
1998,67,965S–971S.
J
Clin Nutr.
4. Prohaska, J.R.; Gybina, A.A. Intracellular coppert transport
in mammals. J. Nutr. 2004, 134, 1003-1006.
5. Lutsenko, S.; Barnes, N.L.; Bartee, M.Y.; Dmitriev. O.Y.
Function and Regulation of Human Copper-Transporting
ATPases. Physiol Rev.2007. 87,1011-1046.
ACS Paragon Plus Environment

Page 9 of 11
Journal of the American Chemical Society
6. Balamurugan K.; Schaffner W. Copper homeostasis in
Structure, Dynamics, and Applications. Chem. Rev., 1999,
99, 2293-2352.
eukaryotes: teetering on a tightrope. Biochim Biophys Acta.
2006,1763,737–746.
7. Macreadie I.G. Copper transport and Alzheimer's disease.
Eur. Biophys. J. 2008, 37, 295–300.
8. Waggoner, D.J.; Bartnikas, T.B.; Gitlin, J.D. The role of
copper in neurodegenerative disease. Neurobiol. Dis. 1999,
6, 221-230.
9. Que, E.L.; Domaille, D.W.; Chang, C.J. Metals in
neurobiology: probing their chemistry and biology with
molecular imaging. Chem. Rev. 2008, 108, 1517–1549.
10. Zatta, P.; Frank A. Copper deficiency and neurological
disorders in man and animals. Brain Res Rev. 2007, 54,19–
33.
1
2
3
4
5
6
7
8
29. Moats, R.A.; Fraser, S.E.; Meade, T.J. A ‘smart’ magnetic
resonance imaging agent that reports on specific enzymatic
activity. Angew. Chem. Int. End. Eng., 1997, 36, 726-732.
30. Raghunand, N.; Howison, C.; Sherry, A.D.; Zhang, S.;
Gillies, R.J. Renal and Systemic pH Imaging by Contrast-
Enhanced MRI. Magn. Reson. Med. 2003, 49, 249-257.
31. Aime, S.; Botta, M.; Gianolia, E.; Terreno, E. A p(O(2))-
Responsive MRI Contrast Agent Based on the Redox Switch
of Manganese(II / III) - Porphyrin Complexes. Angew.
Chem. Int. Ed. 2000, 39, 747-754.
9
32. Berkowitz, B.A.; Wilson, C.A.; Hatchell, D.L.; London,
R.E. Quantitative-Determination of the Partial Oxygen-
Pressure in the Vitrectomized Rabbit Eye In vivo Using F-
19 NMR. Magn Reson Med. 1991, 21, 233–241.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
11. Bush, A.I. Metals and neuroscience. Curr. Opin. Chem. Biol.
2000, 4, 184-191.
12. Brown, D.R.; Qin, K.; Herms, J.W.; Madulaung,
A.;Manson, J.; Strome, R.; Fraser, P.E.; Kuck, T.; von
Bohlen A.; Schulz- Schaeffer, W.; Giese, A.; Westway, D.;
Kretzscmar, H. The cellular prion protein binds copper in
vivo. Nature, 1997, 390, 684-687.
13. Morgan, M. T.; Bagchi, P.; Fahrni, C. J. Fluorescent probes
for monovalent copper. Encyclopedia of Inorganic and
Bioinorganic Chemistry.2013, 1–19.
33. Que, E.L.; Chang, C.J. A smart magnetic resonance contrast
agent for selective copper sensing. J. Am. Chem. Soc. 2006,
128, 15942.
34. Que, E. L.; Gianolio, E.; Baker, S. L.; Wong, A. P.; Aime,
S.; Chang, C. J. Copper-responsive magnetic resonance
imaging contrast agents. J. Am. Chem. Soc. 2009, 131, 8527.
35. Li, W.S.; Luo, J.; Chen, Z.N. A gadolinium(III) complex
with 8-amidequinoline based ligand as copper(II) ion
responsive contrast agent. Dalton Trans. 2011, 40, 484-492.
36. Jang, J.H.; Bhuniya, S.; Kang, J.; Yeom, A.; Hong, K.S.;
Tim, J.S., Cu2+-Responsive Bimodal (Optical/MRI) contrast
agent for cellular imaging, Organic Letters, 2013,15, 4702-
4705.
37. Zhang, X.; Jing, X.; Liu, T.; Han, G.; Li, H.; Dun, C.; Dual-
functional Gadolinium Based Copper (II) probe for selective
magnetic resonance Imaging and florescence sensing, Inor.
Chem. 2012, 51, 2325-2331.
38. Hirayama, T.; Van de Bittner, G.C.; Gray, L.W.; Lutsenko,
S.; Chang, C.J. Near-infrared fluorescent sensor for in vivo
copper imaging in a murine Wilson disease model. PNAS,
2012, 190, 2228-2233.
39. Jordan, M.V.C.; Lo, S.; Chen, S.; Preihs, C.; Chirayil, S.;
Zhang, S.; Kapur, P.; Li, W.; Leon-Rodriguez, L.M.D.;
Lubag, A.J.M.; Rofsky, N.M.; Sherry, A.D. Zinc-sensitive
MRI contrast agent detects differential release of Zn(II) ions
from the healthy vs. malignant mouse prostate. PNAS, 2016,
113, E 5464-E5471.
40. Lubag, A.J.M.; Leon-Rodriguez, L.M.D.; Burgess, S.C.;
Sherry, A.D. Noninvasive MRI of β-cell function using a
Zn2+-responsive contrast agent. PNAS, 2011, 108, 18400-
18405.
41. Yu, J.; Martins, A.F.; Preihs, C.; Clavijo-Jordan,V.;
Chirayil, S.; Zhao, P.; Wu, Y.; Nasr, K.; Kiefer, G.E.;
Sherry, A.D. Amplifying the sensitivity of Zinc(II)
responsive MRI contrast agents by altering water exchange
rates. J. Am. Chem. Soc., 2015, 137, 14173–14179.
42. Esqueda, A. C.; López, J. A.; Andreu-de-Riquer, G.;
Alvarado-Monzón, J. C.; Ratnakar, J.;Lubag, A. J. M.;
14. Rae, T.D.; Schmidt, P.J.; Pufahl,R.A.; Culotta,V.C.;
O’Halloran, T.V. Undetectable intracellular free copper: the
requirement of
a copper chaperone for superoxide
dismutase. Science, 1999, 284, 805.
15. Gaggelli, E.; Kozlowski, H.; Valensin, D.; Valensin, G.
Copper homeostasis and neurodegenerative disorders
(Alzheimer's, prion, and Parkinson's diseases and
amyotrophic lateral sclerosis). Chem. Rev. 2006, 106, 1995-
1998.
16. Waggoner, D.J.; Bartnikas, T.B.; Gitlin, J.D. The role of
copper in neurodegenerative disease. Neurobiol. Dis. 1999,
6, 221-230.
17. Desai, V.; Kaler, S.G. Role of copper in human neurological
disorders. Am J Clin Nutr. 2008, 88, 855S-858S.
18. Multhaup, G.; Schlicksupp, A.; Hesse, L.; Beher,
D.; Ruppert, T.; Masters, C.L.; Beyreuther, K. The amyloid
precursor protein of Alzheimer's disease in the reduction of
copper(II) to copper(I). Science, 1996, 271, 1406-1409.
19. Bertini, I.; Rosato, A. Menkes disease. Cell Mol. Life Sci.
2008, 65, 89-91.
20. Kaler, S.G. ATP7A-related copper transport diseases-
emerging concepts and future trends. Nat. Rev. Neurol.
2011, 7, 15-29.
21. Lutsenko, S.; Gupta, A.; Burkhead, J.L.; Zuzel, V. Cellular
multitasking: the dual role of human Cu-ATPases in cofactor
delivery and intracellular copper balance. Arch. Biochem.
Biophys. 2008, 476, 22-32.
22. Cox, D.; Moore, S.D. Copper transporting P-type ATPases
and human disease. J Bioenerg. Biomembr. 2002, 34, 333-
338.
23. Valentine, J.S.; Doucette, P.A.; Potter, S.Z. Copper-zinc
superoxide dismutase and amyotrophic lateral sclerosis.
Annu. Rev. Biochem. 2005, 74, 563- 572.
24. Peng, F.; Lutsenko, S.; Sun, X.; Musik, O. Positron emission
tomography of copper metabolism in the Atp7b-/- knock-out
mouse model of Wilson's disease. Mol. Imaging Biol. 2012,
14, 70-78.
25. Peng, F. Positron emission tomography for measurement of
copper fluxes in live organisms. Ann. N.Y. Acad. Sci.
2014,1314, 24–31.
26. Aime, S.; Botta, M.; Terreno, E. Gd (III)-based contrast
agents for MRI. Adv. Inorg. Chem., 2005, 57,173-232.
27. Aime, S.; Botta, M.; Fasano, M.; Terreno, E. Lanthanide
(III) chelates for NMR biomedical applications. Chem. Soc.
Rev.,1998, 27, 19-29.
Sherry, A. D.; De León-Rodríguez, L. M. A new
gadolinium-based MRI zinc sensor. J. Am. Chem. Soc. 2009,
131,11387- 11391.
43. Gaur, A.; Klysubun, W.; Nair, N.N.; Shrivastava, B.D.;
Prasad. J.; Srivatava, K. XAFS study of copper(II)
complexes with square planar and square pyramidal
coordination geometries. Journal of Molecular Structure,
2016,1118, 212-218.
44. Laussac, J.P; Sarkar, B. Characterization of the copper(II)-
and nickel(II)-transport site of human serum albumin.
Studies of copper(II) and nickel(II) binding to peptide 1-24
of human serum albumin by 13C and 1H NMR
spectroscopy. Biochemistry, 1984, 23,2832-2838
45. Zgirski, A.; Frieden, E. Binding of Cu(II) to non-prosthetic
sites in ceruloplasmin and bovine serum albumin. J. Inorg.
Biochem. 1990, 39, 137-148.
28. Caravan, P.; Ellison, J. J.; McMurry, T.J; Lauffer, R.B.
Gadolinium(III) Chelates as MRI Contrast Agents:
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 10 of 11
46. Bal, W. Christodoulou, J.; Sadler, P.J.; Tucar, A. Multi-
64. Sano, M.; Komorita, S.; Yamatera, H. XANES spectra of
metal binding site of serum albumin. J. Inorg. Biochem.
1998, 70, 33-39.
47. Lu, J.; Stewart, A.J.; Sadler, P.J.; Pinheiro, T.J.T.; Blindauer,
C.A. Albumin as a zinc carrier: properties of its high-affinity
zinc-binding site. Biochem. Soc. Trans. 2008, 36, 1317-
1321.
48. Shakdofa, M.; Shtaiwi, M.; Morsy, N.; Abdel-raseel, T.M.A.
Metal complexes of hydrazones and their biological,
analytical and catalytic applications: A review. Main Group
Chemistry, 2014, 13, 187-218.
copper(II) complexes: correlation of the intensity of the 1s
.fwdarw. 3d transition and the shape of the complex. Inorg.
Chem 1992, 31, 459-463.
1
2
3
4
5
6
7
8
65. Askwith, C.; Eide, D.; Van Ho, A.; Bernard, P.S.; Li L.;
Davis-Kaplan, S.; Sipe, D.M.; Kaplan J. The FET3 gene of
S. cerevisiae encodes a multicopper oxidase required for
ferrous iron uptake. Cell, 1994, 76,403–10.
66. Linder, M.C.; Massaro, E.J., editor. Handbook of copper
pharmacology and toxicology. Humana Press: New Jersey;
2002. 3–32.
9
49. Zhang, G.; Zhong, W.; Wei, Z.; Liu, Z.; Liu, X. Roles of
phenol groups and auxiliary ligand of copper(ii) complexes
with tetradentate ligands in the aerobic oxidation of benzyl
alcohol. Dalton Transactions, 2017, 46, 8286-8297.
50. Vymazal, J.; Bulte, J. W. M.; Frank, J. A.; Chiro, G. D.;
Brooks, R. A. Frequency dependence of MR relaxation
times I. Paramagnetic ions. J. Magn. Reson. Imaging, 1993,
3, 637–640.
51. Casella, L.; Gullotti, M.; Pallanza, G.; Pinter, A.;
Marchesini, A. Spectroscopic and binding studies of azide to
type-2-copper-depleted ascorbate oxidase from zucchini.
Biol. Metals., 1991, 4, 81-87.
52. Job, P. Formation and Stability of Inorganic Complexes in
Solution. Ann.Chim(Paris), 1928, 113-115.
53. Guo, M.; Zou, J.; Li, P.; Shang, Z.; Hu, G.; Yu, Q.; Binding
interaction of gatifloxacin with bovine serum albumin. Anal.
Sci. 2004, 20, 465-470.
54. Cabaniss, S.E.; Shuman, M.S. Combined ion selective
electrode and fluorescence quenching detection for copper-
dissolved organic matter titrations. Anal. Chem. 1986, 58,
398-401.
55. Hureau, C.; Eury, H.; Guillot, R.; Bijani, C.; Sagen, S.;
Solari, P.; Guillon, E.; Faller, P.; Dorlet, P. X-ray and
Solution Structures of CuIIGHK and CuIIDAHK Complexes:
Influence on Their Redox Properties. Chem. Eur. J. 2011,
17, 10151-10160.
56. Bal, W.; Christodoulou, J.; Sadler, P.J.; Tucker, A. Multi-
metal binding site of serum albumin. Journal of Inorganic
Biochemistry, 1998, 70, 33-39.
57. Rozga, M.; Sokolowska, M.; Protas, A.M.; Bal, W., Human
serum albumin coordinates Cu(II) at its N-terminating
binding site with 1pM affinity. J. Biol. Inorg .Chem.
2007,12,913-918.
67. Vural, H.; Uzun, K.; Uz, E.; Kocgigil, A.; Cigli, A.; Akyol,
O. Concentration of copper, zinc and various elements in
serum of patients with bronchial asthma. J,Trace Elements
Med.Biol., 2000, 14, 88-91.
68. Alebic-Juretic. A.; Frkovic, A. Plasma copper
concentrations in pathological pregnancies. J. of Trace
Elements in Medicine and Biology, 2005, 19,191-194.
69. Linder, M.C.; Wooten, L.; Cerveza, P.; Cotton, S.; Shulze,
S.; Lomeli, N. Copper transport. Am J Clin Nutr., 1998,
67,965S–971S.
70. Lowndes, S.A.; Sheldon, H.V.; Cai, S.; Taylor, J.M.; Harris,
A.L. Copper chelator ATN-224 inhibits endothelial function
by multiple mechanisms. Microvascular Research, 2009,
77, 314-326.
71. Barratt, T.M.; Walser, M. Extracellular fluid in individual
tissues and in whole animals: the distribution of radiosulfate
and radiobromide. J.Clin.Invest. 1969,48,56-66.
72. Guo, S.L.; Su, L.N.; Zhai, Y.N.; Chirume, W.M.; Lei, J.Q.;
Zhang, H.; Yang, L.; Shen, X.P.; Wen, X.X.; Guo, Y.M. The
clinical value of hepatic extracellular volume fraction using
routine multi phasic contrast-enhanced liver Ct for staging
liver fibrosis. Clin. Radiol. 2017, 72, 242-246.
73. Ferenci, P.; Czlonkowska, A.; Merle, U.; Ferenc, S.;
Gromadzka, G.; Yurdaydin, C.; Vogel, W.; Bruha, R.;
Schmidt, H.T.; Strmmel, W. Late-onset Wilson's disease.
Roenterology, 2007, 132, 1294-1298.
74. Frommer, D.J. Direct measurement of serum non-
caeruloplasmin copper in liver disease. Clinica Chimica
Acta, 1976, 68, 3030-307.
75. Yang, X.; Tang X.P.; Zhang, Y.H.; Luo, K.Z.; Jiang Y.F.;
Luo H.Y.; Lei J.H.; Wang, W.L.; Li, M.M.; Chen, H.C.;
Deng, S.L.; Lai, L.Y.; Liang, J., Zhang, M.; Tian, Y.; Xu, Y.
Prospective evaluation of the diagnostic accuracy of hepatic
copper content, as determined using the entire core of a liver
biopsy sample. Hepatology. 2015, 62, 1731-41.
76. Poujois, A; Trocello, J-M; Djebrani-Oussedik, N; Poupon, J;
Collet, C; Girardot-Tinant, N; Sobesky, R; Habès, D;
Debray, D; Vanlemmens, C; Fluchère, F; Ory-Magne, F;
Labreuche, J; Preda, C; Woimant, F. Exchangeable copper:
a reflection of the neurological severity in Wilson's disease.
European Journal of Neurology. 2017, 24,154-160.
77. Heffern, M. C.; Park, H. M.; Au-Yeung, H. Y.; Van de
Bittner, G. C.; Ackerman, C. M.; Stahl, A. Chang, C. J. In
vivo bioluminescence imaging reveals copper deficiency in
a murine model of nonalcoholic fatty liver disease. Proc.
Natl. Acad. Sci. USA 2016, 113, 14219-14224.
78. Mumper, R.J.; Gupte, A. Elevated copper and oxidative
stress in cancer cells as a target for cancer treatment. Cancer
treatment Reviews, 2009,35,32-46.
79. Xiao, T; Ackerman, C.M.; Carroll, E.C.; Jia, S.; Hogland,
A.; Chan, J.; Thai, B.; Liu, C.S.; Iscacoff, E.Y.; Chang, C.J.
Copper regulates rest-activity cycles through the locus
coeruleus-norepinephrine system. Nat. Chem. Biol.
2018, 14,655–663.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
58. Bal, W.; Sokołowska, M.; Kurowska, E.; Faller, P. Binding
of transition metal ions to albumin: Sites, affinities and rates.
Biochim. Biophys. Acta, 2013, 1830, 5444-5455.
59. Neumann, P.Z.; Sass- Kortsark, A. The state of copper in
human serum: evidence for an amino acid-bound fraction.
Journal of Clinical Investigation, 1967, 46, 646-658.
60. Tweedle, M. F.; Hagan, J. J.; Kumar, K.; Mantha, S.; Chang,
C. A. Reaction of gadolinium chelates with endogenously
available ions. Magn. Reson. Imaging 1991, 9, 409–415
61. Xio, Y.; Zhao, G.; Fang, X.; Zhao, Y.; Wang, G.; Yang,
W.;Xu, J.
A
smart copper(II)-responsive binuclear
gadolinium(III) complex-based magnetic resonance imaging
contrast agent. RSC Adv, 2014, 4, 34421- 34427.
62. Sadler, P.J.; Viles, J.H., 1H and (113)Cd NMR
Investigations of Cd(2+) and Zn(2+) Binding Sites on Serum
Albumin: Competition with Ca(2+), Ni(2+), Cu(2+), and
Zn(2+).Inorg. Chem. 1996, 35, 4490-4496.
63. Martins, D.A.; gourea, L.R.; Muniz, G.S.; Louro, S.; Gama,
D.; Soeiro, M.; Teixeira, L.R., Norfloxacin and N-Donor
Mixed-Ligand Copper(II) Complexes: Synthesis, Albumin
Interaction,
and
Anti-
Trypanosoma
cruzi
Activity.Bioinorganic chemistry and application, 2016, 1-
11.
80.
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