A High-Denticity Chelator Based on Desferrioxamine for Enhanced Coordination of Zirconium-89

Article abstract of DOI:10.1021/acs.inorgchem.0c01629

Herein we report a new high-denticity chelator based on the iron siderophore desferrioxamine (DFO). Our new chelator-DFO2-is acyclic and was designed and synthesized with the purpose of improving the coordination chemistry and radiolabeling performance with radioactive zirconium-89. The radionuclide zirconium-89 ([89Zr]Zr4+) has found wide use for positron emission tomography (PET) imaging when it is coupled with proteins, antibodies, and nanoparticles. DFO2 has a potential coordination number of 12, which uniquely positions this chelator for binding large, high-valent, and oxophilic metal ions. Following synthesis of the DFO2 chelator and the [natZr]Zr-(DFO2) complex we performed density functional theory calculations to study its coordination sphere, followed by zirconium-89 radiolabeling experiments for comparisons with the "gold standard"chelator DFO. DFO (CN 6) can coordinate with zirconium in a hexadentate fashion, leaving two open coordination sites where water is thought to coordinate (total CN 8). DFO2 (potential CN 12, dodecadentate) can saturate the coordination sphere of zirconium with four hydroxamate groups (CN 8), with no room left for water to directly coordinate, and only binds a single atom of zirconium per chelate. Following quantitative radiolabeling with zirconium-89, the preformed [89Zr]Zr-(DFO) and [89Zr]Zr-(DFO2) radiometal-chelate complexes were subjected to a battery of in vitro stability challenges, including human blood serum, apo-transferrin, serum albumin, iron, hydroxyapatite, and EDTA. One objective of these stability challenges was to determine if the increased denticity of DFO2 over that of DFO imparted improved complex stability, and another was to determine which of these assays is most relevant to perform with future chelators. In all of the assays DFO2 showed superior stability with zirconium-89, except for the iron challenge, where both DFO2 and DFO were identical. Substantial differences in stability were observed for human blood serum using a precipitation method of analysis, apo-transferrin, hydroxyapatite, and EDTA challenges. These results suggest that DFO2 is a promising next-generation scaffold for zirconium-89 chelators and holds promise for radiochemistry with even larger radionuclides, which we anticipate will expand the utility of DFO2 into theranostic applications.

Full text of DOI:10.1021/acs.inorgchem.0c01629

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Article
A High-Denticity Chelator Based on Desferrioxamine for Enhanced
Coordination of Zirconium-89
Elaheh Khozeimeh Sarbisheh,∇ Akam K. Salih,∇ Shvan J. Raheem, Jason S. Lewis, and Eric W. Price*
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ABSTRACT: Herein we report a new high-denticity chelator
based on the iron siderophore desferrioxamine (DFO). Our new
chelatorDFO2is acyclic and was designed and synthesized
with the purpose of improving the coordination chemistry and
radiolabeling performance with radioactive zirconium-89. The
radionuclide zirconium-89 ([89Zr]Zr4+) has found wide use for
positron emission tomography (PET) imaging when it is coupled
with proteins, antibodies, and nanoparticles. DFO2 has a potential
coordination number of 12, which uniquely positions this chelator
for binding large, high-valent, and oxophilic metal ions. Following
synthesis of the DFO2 chelator and the [natZr]Zr-(DFO2)
complex we performed density functional theory calculations to
study its coordination sphere, followed by zirconium-89 radio-
labeling experiments for comparisons with the “gold standard” chelator DFO. DFO (CN 6) can coordinate with zirconium in a
hexadentate fashion, leaving two open coordination sites where water is thought to coordinate (total CN 8). DFO2 (potential CN
12, dodecadentate) can saturate the coordination sphere of zirconium with four hydroxamate groups (CN 8), with no room left for
water to directly coordinate, and only binds a single atom of zirconium per chelate. Following quantitative radiolabeling with
zirconium-89, the preformed [89Zr]Zr-(DFO) and [89Zr]Zr-(DFO2) radiometal−chelate complexes were subjected to a battery of in
vitro stability challenges, including human blood serum, apo-transferrin, serum albumin, iron, hydroxyapatite, and EDTA. One
objective of these stability challenges was to determine if the increased denticity of DFO2 over that of DFO imparted improved
complex stability, and another was to determine which of these assays is most relevant to perform with future chelators. In all of the
assays DFO2 showed superior stability with zirconium-89, except for the iron challenge, where both DFO2 and DFO were identical.
Substantial differences in stability were observed for human blood serum using a precipitation method of analysis, apo-transferrin,
hydroxyapatite, and EDTA challenges. These results suggest that DFO2 is a promising next-generation scaffold for zirconium-89
chelators and holds promise for radiochemistry with even larger radionuclides, which we anticipate will expand the utility of DFO2
into theranostic applications.
released from a chelate complex in vivo, and experiments in
mice have consistently shown a substantial amount of bone
uptake (∼5−10% ID/g after ∼3−7 days).2,9,24 Despite the
apparent instability of the [89Zr]Zr-(DFO) complex in mice,
human immuno-PET imaging studies using zirconium-89 do
not appear to suffer from the same degree of high bone
uptake.6,25−27 In recent years, a number of promising new
chelators have been reported which all aim to improve the
radiolabeling performance and in vivo stability with zirconium-
89 (Figure 1).3,18,28−44
INTRODUCTION
■
The radionuclide zirconium-89 has gained popularity over the
past decade due to its favorable decay characteristics
([89Zr]Zr4+, t1/2 = 78.4 h, β+ ratio = 22.7%, Eβ (mean) =
+
396 keV) for positron emission tomography (non-invasive
nuclear imaging modality, PET).1−3 The radiometal zirco-
nium-89 has a half-life that is well matched with large and
slowly distributing biological vectors such as antibodies and
nanoparticles.4−16 Previously, the only chelator capable of
effectively coordinating with zirconium-89 (radiolabeling) and
forming a complex with sufficient thermodynamic and kinetic
stability in vivo was the iron siderophore desferrioxamine
(DFO).17−21 DFO is typically non-site selectively conjugated
to lysine residues (primary amine) on antibodies to form
thiourea linkages, using the bifunctional chelator (BFC) p-
SCN-Bn-DFO (commercially available), although amide
linkages formed via DFO-activated esters are also used.22,23
Zirconium-89 is known to accumulate in bone when it is
Received: June 2, 2020
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Figure 1. Chemical structures of a selection of recently reported bifunctional chelators based on desferrioxamine (DFO) for zirconium-89,
including p-SCN-Bn-DFO, DFO*, oxoDFO*, DFO squarate ester (H3DFOSqOEt), DFO-cyclo*, and DFO2.
Over the past few years we have been working on the design,
synthesis, and evaluation of a new family of DFO-based
chelators. The first of these new chelators is presented herein
and is named DFO2, as it is comprised of two desferrioxamine
molecules tethered together using a bifunctional linker. The
resulting chelator is potentially 12-coordinate (six hydroxamic
acid groups, CN 12), with an octadentate (CN 8) coordination
sphere expected for Zr4+ with DFO2. One drawback of many
new chelators is their cumbersome synthesis and difficult
purificationtypically a result of the intrinsic need of chelators
to contain a large number of oxygen- and nitrogen-rich polar
functional groups with high hydrogen-bonding potential. We
have achieved simple synthesis and purification by using
commercial DFO as our major building block, although low
water solubility of the unconjugated DFO2 was encountered.
The long-term goal of this new “DFO2” chelator family is a
rapid and modular synthesis platform to enable fast evaluation
of a variety of chelator derivatives, followed by rapid synthetic
iteration of the lead ligand scaffolds. The priorities of our
chelate system are thus simple and fast syntheses, with the
requirement to contain a modular design where we can easily
change the linker group, add different chelating moieties,
change the bifunctional conjugation chemistry, and tune
physical properties such as polarity/solubility. The first
example of this new chelator family, DFO2, has been
synthesized, characterized, radiolabeled with zirconium-89,
and evaluated via in vitro radiochemical stability assays. We
have additionally evaluated the coordination sphere using
density functional theory (DFT) calculations, as we did not
obtain diffractable crystals and therefore no X-ray crystallo-
graphic data. We have performed many different types of in
vitro stability assays with the purpose of determining which
assays are most relevant for probing the 89Zr-chelate stability.
We hope that the results of these assays will make this process
of screening chelators more efficient in the future by
identifying which in vitro assays best predict in vivo stability
differences.
RESULTS AND DISCUSSION
■
To expand the denticity of DFO from a potential coordination
number of 6 (three hydroxamic acid groups), we chemically
tethered two molecules of commercially available DFO
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Scheme 1. Synthesis of the Linker Molecule 3, Which Was Used to Tether Two DFO Molecules Together to Form DFO2
Scheme 2. Synthesis of DFO2 from Modified Desferrioxamine B (4) and a Linker Group (3), Followed by Nonradioactive Zr4+
Coordination Showing One Possible Conformational Isomer (6)
together. To achieve this, we required a linker that could both
tether two DFO molecules with suitable chemistry and also
provide orthogonal reactivity in the bifunctional component of
the chelator. The synthesis of DFO2 revolved around a
propylenediamine linker with a p-NO2-phenyl group extending
from the backbone. We synthesized this fragment in three
steps starting from p-NO2-benzyl bromide and diethyl
malonate (Scheme 1). An optimization was made to a
previously published synthetic method for this linker fragment,
where during step two of the synthesis to form compound 2 we
used ammonium hydroxide as the source of amine instead of
ammonia gas. This route does not require access to a
compressed cylinder of ammonia gas or the additional hazards
of ammonia gas.45 The functional linker moiety 3 was
synthesized in three chemical steps with a cumulative yield
of ∼8%.
modify DFO through a ring-opening reaction with succinic
anhydride. This step formed DFO-COOH (4), which
extended the length of the DFO chain by four atoms (from
31 to 35 atoms long) and also converted the terminal
functional group from a primary amine to a carboxylic acid.
The backbone linker 3 was then reacted with DFO-COOH (4)
to form DFO2 (5) using standard peptide coupling conditions.
As with the bifunctional chelators p-SCN-Ph-DFO and p-NO2-
Ph-DFO, the solubility of DFO2 is poor. During the synthetic
step to produce DFO2 (5), the solvent DMF was required to
achieve solubility and suitable reaction yields. The poor
solubility of the resulting DFO2 (5) chelator enabled us to
purify DFO2 by only precipitation and washing, where we
tested a large number of solvent combinations and found that
cold ethyl acetate was the most effective. Coordination of
DFO2 with the salt ZrCl4 rapidly formed the coordinated
Zr(DFO2) complex under mild ambient conditions. A small
amount of the strong polar solvent dimethylformamide (DMF)
To attach commercially available desferrioxamine B mesylate
(DFO, Scheme 2) to our linker fragment (3), we had to
C
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was required to solubilize DFO2 at the macro scale and
facilitate this nonradioactive coordination chemistry. At the
low concentrations of chelator required for radiochemistry
(μM to nM), sufficient solubility could be achieved by
transferring a small volume of a DFO2 stock solution dissolved
in DMF or DMSO into aqueous radiolabeling buffer. The non-
radioactive Zr(DFO2) complex was characterized by standard
techniques including 1H/13C NMR spectroscopy and low- and
high-resolution mass spectrometry. Despite the poor water
solubility, we have not been able to grow X-ray crystallography
quality crystals.
the Zr(DFO2) complex. The calculated average bond length of
2.244 Å for Zr−O bonds in the Zr(DFO2) complex is in
alignment with those of the single-crystal X-ray diffraction and
DFT calculation results for the Zr(Me-AHA)4 complex.46,47
The calculated structure of the Zr(DFO2) complex in Figure 2
shows the bottom hydroxamic acid of each DFO tail in the
DFO2 structure remaining unbound. It is possible that other
hydroxamates in DFO2 could bind instead of one or both of
the bottom hydroxamate moieties and form alternative
geometric isomers/conformers (over 500 possible isomers).
It is also possible that these hydroxamates could switch during
any reorganization of the coordination sphere, thereby forming
an equilibrium between geometric isomers. More detailed DFT
studies in combination with advanced spectroscopy techniques
will likely be required to better elucidate the precise details of
the coordination sphere; however, the huge number of possible
isomers will likely make any firm conclusions unlikely. At this
stage of research, the in vitro and in vivo stability data are the
most important, as we are seeking a practical and functional
improvement over existing chelators for use in molecular
imaging and targeted radionuclide therapy.
In lieu of crystallographic data, we performed density
functional theory (DFT) calculations to evaluate the
coordination sphere of the Zr(DFO2) complex (Figure 2).
It is possible that the two hydroxamate groups that are not
strictly needed in the octadentate Zr(DFO2) complex could
still improve the stability of the complex in comparison with
strictly octadentate chelators such as Zr(DFO*) through a
process that could be roughly analogous to avidity. Although
the resulting zirconium(IV) complexes for both DFO2 and
DFO* are octadentate and should have comparable complex
stabilities (both utilize identical hydroxamate groups), DFO2
could offer improved in vivo kinetic stability as a result of the
two additional hydroxamic acid groups being covalently
attached in close physical proximity to the coordination
sphere, which should displace the position of the equilibrium
toward Zr(DFO2) complex formation. This could hypotheti-
cally protect against in vivo competition from ligands such as
water, chloride, phosphate, and proteins, and effectively
increase the stability. We radiolabeled both our new chelator
DFO2 and the “gold standard” chelator DFO with zirconium-
89 and have performed many in vitro stability assays. In the
future it would be useful to obtain DFO* to serve as a
comparison and/or to synthesize an octadentate derivative of
DFO2. DFO* was not commercially available at the time of
performing these experiments. If the radiochemical stability of
[89Zr]Zr-DFO2 was compared with zirconium-89 complexes
of structurally similar chelators (e.g., DFO*) with a denticity of
only 8, and if [89Zr]Zr-DFO2 demonstrated improved stability,
it could hypothetically be attributed to the presence of the
extra two hydroxamate groups.
Figure 2. Energy-minimized and geometry-optimized molecular
structures calculating using DFT (Materials Studio, DMol3 /PBE,
solvent as water with COSMO) showing (a, left) the Zr(DFO2)
complex and (b, right) a closeup of the Zr(IV) ion geometry in
Zr(DFO2) complex. Hydrogen atoms are removed from the figure for
visual clarity but are included for calculations.
Calculations were performed using Materials Studio software
with DMol3 using the Perdew-Burke-Ernzerhof (PBE) func-
tional and employed double-numerical plus d-function (DND)
basis sets, with the solvent effects modeled by COSMO. All of
the calculations were performed in solution with water as the
solvent. Due to the large structure of the Zr(DFO2) complex,
it took substantial time to complete the calculations. The
DFO2 chelator with many polar hydroxamic acid and carbonyl
groups was prone to getting stuck in local minima, potentially
due to intramolecular hydrogen bonding between the two
DFO chains. This intramolecular hydrogen bonding could also
decrease the water solubility of DFO2 as it would minimize the
interactions of the polar hydroxamic acid groups with the
solvent. The calculated structure of the Zr(DFO2) complex at
the global minimum energy suggests an octadentate structure
with no bound water molecules (Figure 2).
The thermodynamic stability of metal−ligand complexes is
an interesting and useful parameter that is often discussed for
new chelators or new chelator−metal pairings. Although it
gives valuable information, to date it does not appear that
thermodynamic stability constants predict in vivo stability.18
Despite a lack of predictive power for in vivo stability,
formation constants can serve as a valuable tool for comparing
the overall thermodynamic stabilities of different ligands with a
metal, therefore providing information about which types of
metal-donor groups and which denticities are preferred to
achieve maximum stability. The high acidity (solvent activation
and hydrolysis, pKa ∼ 0.22) of Zr4+ gives it the propensity to
precipitate and form oxo-/hydroxo-bridged species in aqueous
solution (unless very acidic), making it challenging for physical
chemistry studies. Recent studies have overcome these issues
Hydroxamic acid groups are known as being exceptionally
well suited for binding Zr4+, more so than endogenous
biological ligands that would be available to compete (e.g.,
transferrin, albumin, chloride, phosphate). Brechbiel et al. in an
investigation of the complexation of Zr4+ with hydroxamates
showed that Zr4+ forms an eight-coordinated complex with
four bidentate hydroxamates.46,47 As ligands they used two
hydroxamate derivatives, acetohydroxamic acid (AHA) and N-
methylacetohydroxamic acid (Me-AHA). Single-crystal X-ray
diffraction studies of Zr(Me-AHA)4 revealed an average bond
length of 2.193 Å for the Zr−O bonds. Also, data obtained via
DFT calculations for the Zr(Me-AHA)4 complex suggested an
average bond length of 2.248 Å for eight Zr−O bonds. These
studies also suggested that a “cis-cis-trans-trans” geometry was
most stable. Table S1 shows our calculated bond lengths for
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and determined the formation constants for ligands with Zr4+
using potentiometric, UV/Vis spectroscopic, isothermal
titration calorimetric, and mass spectrometric methods.48−50
With a very high pM value of 32.250 for Zr(DFO) and a log
used for these experiments. Regardless, improved water
solubility is a priority for future iterations of this new DFO2
family of chelators.
One of the most common in vitro stability assays performed
for radiolabeled molecules is some form of a blood serum
incubation/competition. For zirconium-89, DFO is known to
form a stable complex that is not appreciably transchelated by
human or murine blood serum, even out beyond 7 days of
incubation time. For zirconium-89 stability assays in blood
serum, radio-iTLC is most commonly used to assess the degree
of transchelation to serum proteins due to its simple and fast
execution. Spotting the iTLC strips and eluting with mobile
phase takes ∼10−15 min, and many can be eluted
simultaneously. Then using a standard radio-TLC reader
(Bioscan/EZ AR2000) or even an automated γ counter
(cutting strips into pieces), these eluted radio-iTLC strips can
be measured at a rate of about 1−3 min each. When the
radiolabeling of large proteins such as antibodies is monitored,
size-exclusion HPLC chromatography provides more reliable
information, including the detection of antibody aggregates,
but it requires ∼20−50 min per HPLC run.
We radiolabeled both DFO and DFO2 with zirconium-89
quantitatively (>99% RCY) to form [89Zr]Zr-(DFO) and
[89Zr]Zr-(DFO2) complexes (no free radiometal) and then
without purification transferred aliquots of these complexes to
human blood serum and assessed their stability for 7 days
(Figure 3). We evaluated their serum stability using two
versions of the same assay: standard radio-iTLC with EDTA
mobile phase as eluent (50 mM, pH 5−5.5) (Figure 3A) and
precipitation of the serum proteins with cold acetonitrile,
centrifugation to pellet the proteins, and decanting of the
water/acetonitrile which contained the intact [89Zr]Zr-
(chelator), followed by rinsing the protein pellet with more
ice-cold acetonitrile/water mix (∼70/30) and measuring the
radioactivity in each decanted fraction (Figure 3B). The results
of both assays suggested the superior stability of the [89Zr]Zr-
(DFO2) radiometal complex by demonstrating less zirconium-
89 associated with serum proteins. The precipitation method
resulted in the largest difference in stability, with the percent
intact radiometal complexes after 7 days incubation being 81%
4% for the [89Zr]Zr-(DFO) complex and 87% 1% for the
[89Zr]Zr-(DFO2) complex.
The iron transport protein transferrin is the most obvious
culprit for zirconium-89 transchelation in vivo due to the
similarities in binding properties between Zr4+ and Fe3+.52−54
As such, we performed an apo-transferrin (apo, metal free; holo,
metal bound) competition challenge, which should be more
strict than blood serum for two reasons: (1) blood serum
contains transferrin that is partially bound by iron (holo), and
therefore not all binding sites are available, and (2) we used
super-physiological concentrations of ∼250 μM. For reference,
human blood serum contains ∼36 μM transferrin (∼80 kDa
protein), which is partially iron bound (holo).55 Another major
blood protein that can bind to a variety of metal ions is serum
albumin (∼66.5 kDa), which is typically found at around 70
μM in the blood. For this assay we used human serum albumin
(Aldrich, >96%) at a concentration of ∼1.125 mM for a super-
physiological quantity, but this was not the apo form (Figure
4B). The results of these radiometal−chelator stability assays
again suggest that the [89Zr]Zr-(DFO2) complex possesses
superior stability to transchelation by blood serum proteins in
comparison to the [89Zr]Zr-(DFO) complex (Figure 4). The
difference in stability (percent intact radiometal−chelator
K
ML value of 36.14,48 it is clear that any instability in vivo is not
a result of insufficient thermodynamic stability. Now that these
research groups have overcome the solution chemistry
challenges of determining stability constants with Zr4+, it
would be timely to obtain more values with new chelators. In
addition, recent work has proposed a fascinating DFT-based
method for predicting thermodynamic stability constants in
silico, which is particularly useful for metal ions such as Zr4+
that are challenging to handle in aqueous solutions.51
Although it is possible that DFO2 could simply chelate two
separate atoms of zirconium-89 at the same time with an
expected coordination environment for each zirconium atom
of three hydroxamate groups (CN 6, with two H2O; total CN
8), the huge molar excess of chelator over the radiometal used
for radiolabeling experiments makes this very unlikely to occur.
Even when coordination chemistry was performed with DFO2
and ∼0.9 mol equiv of a non-radioactive Zr4+ salt, there was no
Zr2-DFO2 observed via mass spectrometry. Radiolabeling
experiments with zirconium-89 suggest the same result, where
no appreciable difference in radiochemical yields (RCYs) or
specific activity were observed when 500, 50, 5, or 0.5 nmol of
DFO or DFO2 was used (Table 1). If each molar equivalent of
Table 1. Results from Radiolabeling DFO and DFO2 with
a
Zirconium-89
[89Zr]Zr-
Chelator
(nmol)
oxalate
RCY 60 SA (mCi μmol−1
min (%)
MBq μmol−1
/
(μCi/MBq)
)
log DPBS,pH7.4
−0.71 01
[89Zr]Zr-(DFO2)
500
50
5
459/17.0
282/10.4
290/10.7
283/10.5
99
99
7.4
1
0.9/33.6
5.6/207
4.3/159
0.5
[89Zr]Zr-(DFO)
500
50
5
273/10.1
276/10.2
269/10.0
282/10.4
99
99
2.3
1.4
0.5/20.0
5.5/202
−2.70 06
0.5
a
At chelator concentrations of 500−0.5 nmol, radiochemical yields
(RCYs) were determined via radio-iTLC (n = 3), and log D octanol−
buffer partition coefficient values were obtained by shake-flask
methods (phosphate buffered saline pH 7.4) (n = 5).
DFO2 was binding 2 molar equiv of zirconium-89, one would
expect precisely 2 times higher specific activity. The difference
in polarity between [89Zr]Zr-(DFO) and [89Zr]Zr-(DFO2)
was assessed by log D partition coefficients, determined using
standard shake-flask methods with octanol and phosphate-
buffered saline (Table 1; pH 7.4). These results suggest that
the [89Zr]Zr-(DFO) complex is more water soluble with a log
D value of −2.70 0.06; however, it is important to note that
this DFO is not the bifunctional derivative containing a phenyl
ring but rather non-functionalized desferrioxamine B mesylate,
which is far more water soluble, as it contains a protonated
primary amine at physiological pH. The log D value obtained
for the [89Zr]Zr-(DFO2) complex was −0.71 0.01; however,
this version contains a p-NO2-Ph moiety, which will
substantially decrease the water solubility relative to the
protonated primary amine found in the non-bifunctional DFO
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Figure 3. Results from in vitro stability assays (n = 3) comparing the stability of zirconium-89 complexes of the new chelator DFO2 to that of the
gold standard chelator DFO, with (A) competition against human blood serum over a 7 day duration and evaluated via radio-iTLC and (B) the
same blood serum stability assay evaluated by precipitating proteins (cold acetonitrile) and decanting the supernatant via centrifugation.
Figure 4. Results from in vitro stability assays comparing [89Zr]Zr-(DFO) and [89Zr]Zr-(DFO2) complexes, both evaluated via radio-iTLC, with
(A) competition against the human iron transport protein apo-transferrin (5-fold molar excess) over a 9-day duration and (B) human serum
albumin (45-fold molar excess) over a 7-day duration.
Figure 5. Results from in vitro stability assays comparing the [89Zr]Zr-(DFO) complex and the [89Zr]Zr-(DFO2) complex, with (A) competition
against free iron(III) chloride over an 11-day duration, evaluated via radio-iTLC, and (B) competition against hydroxyapatite (HTP, BioRad Bio-
Gel) over a 24 h duration, evaluated via centrifugation and decanting of the supernatant from the HTP pellet.
complex) was very small between both chelators (∼1−3%),
with the exception of the 9 day time point for the apo-
transferrin challenge (Figure 4A). Only at this long time point
was a substantial difference in stability manifested, with the
percent intact radiometal complex after 9 days of incubation
being 65% 11% for the [89Zr]Zr-(DFO) complex and 80%
1% for the [89Zr]Zr-(DFO2) complex. This curious and rapid
drop in stability at 9 days of incubation is perhaps explained by
an increasing baseline noise during the radio-iTLC measure-
ment as the remaining zirconium-89 had decayed to low levels.
As such, the reliability of this 9 day data point is dubious.
The chelator desferrioxamine is an iron siderophore
produced by bacteria, and since our new chelator DFO2 is
based on this siderophore, it is reasonable to assume that they
will both have high selectivity and stable binding for Fe3+.56,57
Following the methods of Deri et al.,32 we mixed the [89Zr]Zr-
(DFO) and [89Zr]Zr-(DFO2) complexes with a 10-fold molar
excess of ferric chloride (FeCl3) and monitored the stability via
radio-iTLC for 11 days (Figure 5A). Within error, there
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a
Table 2. Results from In Vitro EDTA Transchelation Stability Assays
a
Pre-radiolabeled [89Zr]Zr-(DFO) and [89Zr]Zr-(DFO2) complexes were mixed with a 100-fold molar excess of EDTA chelator at various pH
values and monitored by radio-iTLC for 7 days.
appeared to be no difference in the ability of these two
zirconium-89 chelate complexes to resist transchelation with
ferric iron. Because iron homeostasis is very tightly regulated in
the body, there is essentially no “free” iron in the body and so
we do not believe the lack of stability against transchelation by
“free” iron is a practical issue for these hydroxamate-based
zirconium-89 chelators.
After 7 days of incubation and at pH 7.5, the [89Zr]Zr-(DFO)
complex remained 23.7% 9.6% intact where the [89Zr]Zr-
(DFO2) complex was 95.0% 0.5% intact.
Altogether, these results suggest that our new chelator
DFO2offers a superior chelation environment for zirco-
nium-89 in comparison with DFO by offering a higher
coordination number and improved stability to a wide variety
of in vitro challenges. Although DFO2 having an abnormally
high potential denticity of 12 might not improve its ability to
complex zirconium relative to other octadentate chelators such
as DFO*, we believe it uniquely positions DFO2 to bind high-
valent metal ions such as lanthanides and actinides. We are
working on next-generation DFO2 derivatives with further
simplified synthesis, improved solubility, and different bifunc-
tional linker groups with these goals in mind. For example, to
obtain the p-NO2-phenyl group in the backbone of DFO2, we
performed three synthetic steps, which has proved to be a
challenge due to low yields and difficult purification. In the
future we plan to optimize the design of DFO2 by exploring
linker options alternative to the p-NO2-phenyl propylenedi-
amine linker that can help us achieve our goals of enhanced
solubility and facile bioconjugation. New DFO2 derivatives
will undergo bioconjugation with a model antibody, radio-
labeling, and in vivo stability assessment in mice. Long term, we
hope to link in vivo results back to this study and determine
which of these in vitro stability assays were ultimately the most
predictive of the chelators in vivo behavior.
A high uptake of zirconium-89 is routinely observed in the
bones of mice, and therefore the stability of these complexes in
the presence of hydroxyapatite is potentially very relevant. We
mixed hydroxyapatite resin (BioRad biogel HTP) in TRIS/
HCl buffer (50 mM, pH 7.4) with precomplexed [89Zr]Zr-
(DFO) and [89Zr]Zr-(DFO2) to determine the degree of
transchelation and/or radiometal−chelate adsorption to the
hydroxyapatite. After 24 h of incubation, competition mixtures
were centrifuged to pellet the hydroxyapatite and the
remaining solution was decanted via pipet. The hydroxyapatite
pellet was resuspended and then repelleted and the solution
decanted. The amount of radioactivity present in the pellet vs
that of the supernatant (plus rinse) was used to determine the
percent stability, which was expressed as the percent of
radiometal−chelate complex remaining in solution and not
associated with the hydroxyapatite pellet. These results showed
a substantial difference in stability, where the most stringent
competition containing ∼40 mg of hydroxyapatite revealed a
stability of 71% 1% for [89Zr]Zr-(DFO) and 90% 1% for
[89Zr]Zr-(DFO2) (Figure 5B). A control of [89Zr]Zr-oxalate
(most zirconium-89 is delivered in 1 M oxalic acid and then
neutralized to pH 7−7.4 for radiolabeling) was also added to
∼40 mg of hydroxyapatite, where only 1.4% 0.4% remained
in solution and nearly all zirconium-89 was tightly associated
with the hydroxyapatite.
As a final in vitro stability experiment we again followed a
method by Deri et al.,32 where preformed radiometal−chelate
complexes were mixed with a 100-fold molar excess of the
chelator ethylenediaminetetraacetic acid (EDTA). Competi-
tion mixtures ranging from pH 5.0 to 8.0 (pH increments of
0.5) were incubated with shaking (as with all stability assays
discussed) and monitored via radio-iTLC for 7 days (Table 2).
This stability assay revealed a large difference in stability
between [89Zr]Zr-(DFO) and the [89Zr]Zr-(DFO2) complex,
particularly at physiologically relevant pH values of 6.5−7.5.
CONCLUSIONS
■
The new chelator DFO2 presents a promising new modular
and high-denticity chelator scaffold, which we will build upon
using its modular synthesis scheme to produce highly stable
and customizable bifunctional chelators for zirconium-89 and
other interesting radiometals. With double the potential
coordination number of the “gold standard” chelator for
zirconium-89DFO (CN 12 vs 6)DFO2 appears to
saturate the octadentate coordination sphere of zirconium
and prevent the coordination of water. This conclusion is
supported by high-resolution mass spectrometry of the non-
radioactive Zr(DFO2) complex, density functional theory
calculations, and radiolabeling results (specific activity).
Following quantitative radiolabeling with zirconium-89, the
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[89Zr]Zr-(DFO) and [89Zr]Zr-(DFO2) complexes were
compared using a large number of in vitro stability challenges,
including human blood serum, apo-transferrin, serum albumin,
iron, hydroxyapatite, and EDTA. The results of these
numerous stability assays demonstrated that our new chelator
DFO2 formed a more stable zirconium-89 complex in
comparison to DFO, except for the iron challenge, where
both chelators were identical. Substantial differences in
stability were not observed for human blood serum using
radio-iTLC, but when a protein precipitation method of
analysis was used, the stability values after 7 days deviated in
favor of DFO2. To simulate transchelation/adsorption with
bone, we developed a hydroxyapatite resin challenge, and
finally we performed a competition against a 100-fold molar
excess of EDTAwhere both assays revealed the superior
stability of DFO2 with zirconium-89. All told, these results
suggest that our new DFO2 chelator is a promising next-
generation scaffold for the elaboration of zirconium-89
chelators. We plan to use the modular synthesis of the
DFO2 chelator to tune its polarity, conjugation chemistry, and
coordination chemistry.
GCv 4G instrument using field desorption ionization (FDI). For the
isotopic pattern only the mass peak of the isotope with the highest
natural abundance is given.
Density Functional Theory (DFT) Calculations. DFT geometry
optimizations were carried out using DMol3 and Biovia Materials
Studio, version 2017 R2,58,59 using the generalized gradient
approximation (GGA) employing the Perdew−Burke−Ernzerhof
(PBE)60 exchange-correlation functional both for the potential during
the self-consistent field (SCF) procedure and for the energy.61 All
calculations employed double-numerical plus d-function (DND) basis
sets, using the 4.4 basis file, as implemented within the DMol3
software, including polarization functions for all atoms with all-
electron core treatments. Quantum simulations of solvated molecules
were modeled using the conductor-like screening model (COSMO)
solvation model62,63 in DMol3, with a dielectric value representing
water (ε = 78.54). As part of these calculations, DMol3 confirmed that
the calculated structures were local minima. All graphical depictions of
the structures were generated using CYLview software64 using
coordinates from the optimized log file.
Synthesis of Diethyl 2-[(4-Nitrophenyl)methyl]-
propanedioate (1). This synthesis was adapted from Pandya et
al.65 Diethyl malonate (8.6 mL, 56.378 mmol) and potassium
carbonate anhydrous (2.808 g, 20.318 mmol) were dissolved in
acetone (8.0 mL). The mixture was stirred for 1 h at room
temperature. Then 4-nitrobenzyl bromide (2.213 g, 10.244 mmol)
was added and the mixture stirred for 2 h at 45 °C. After it was cooled
to room temperature, the yellow reaction mixture was filtered to
remove the remaining potassium carbonate and was washed with
acetone (20 mL). The filtrate was collected, the solvent was reduced
by rotary evaporation in vacuo, ethanol was added (∼10 mL), and
this mixture was then kept at −20 °C in the freezer overnight. Crystals
formed and were separated by filtration, and the filtrate was placed
back in the freezer at −20 °C overnight to produce more crystals.
This process was completed a total of three times. White crystals of 1
(1.393 g, 46.13%) were obtained from three rounds of precipitation
and filtration. 1H NMR (CDCl3, 500 MHz): δ 1.22 [t, 6H, J = 7.1 Hz,
CH2CH3], 3.32 [d, 2H, J = 7.8 Hz, CCH2CH], 3.66 [t, 1H, J = 7.8
Hz, CCH2CH], 4.14−4.21 [m, 4H, OCH2CH3], 7.39 [d, 2H, J =
8.7 Hz, CHCCH2Ar], 8.15 [d, 2H, J = 8.7 Hz, CHCNO2Ar].
Synthesis of 2-(4-Nitrobenzyl)malonamide (2). This synthesis
was adapted from Price et al.45 Compound 1 (295 mg, 0.999 mmol)
was dissolved in methanol (5 mL), followed by addition of
ammonium hydroxide (5 mL). The reaction mixture was stirred for
45 h at room temperature. A yellow precipitate formed and was
isolated by filtration and then washed with methanol and boiling
acetonitrile. The solvent was removed by rotary evaporation in vacuo.
Yellow crystals of compound 2 (180.78 mg, 61.7%) were obtained. 1H
NMR [(CD3)2SO, 500 MHz]: δ 3.10 [d, 2H, J = 7.6 Hz, 
CCH2CH], 3.38 [t, 1H, J = 7.7 Hz, CCH2CH], 7.07 [s, 2H,
CONH2], 7.27 [s, 2H, CONH2], 7.48 [d, 2H, J = 8.8 Hz, CH
CCH2Ar], 8.14 [d, 2H, J = 8.8 Hz, CHCNO2Ar].
EXPERIMENTAL SECTION
■
Reagents. All solvents and reagents were purchased from
commercial suppliers (Sigma-Aldrich, St. Louis, MO; TCI America,
Portland, OR; Fisher Scientific, Waltham, MA) and were used as
received unless otherwise indicated. DMSO used for chelator stock
solutions was of molecular biology grade (>99.9%; Sigma-Aldrich).
DFO·mesylate was purchased from Abcam. 1-[Bis(dimethylamino)-
methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluoro-
phosphate (HATU) was purchased from AK Scientific. N,N-
Diisopropylethylamine (DIEA), diethyl malonate, 4-nitrobenzyl
bromide, and borane−tetrahydrofuran solution (BH3·THF) were
purchased from Sigma-Aldrich. ZrCl4, potassium carbonate anhy-
drous, and ammonium hydroxide were purchased from Fisher
Scientific. All of the chemicals were used directly without purification.
Radiochemistry Instrumentation. [89Zr]Zr4+ was produced at
Memorial Sloan Kettering Cancer Center using an EBCO TR19/9
variable-beam energy cyclotron (Ebco Industries Inc., British
Columbia, Canada) via the 89Y(p,n)89Zr reaction. [89Zr]Zr4+ was
purified in accordance with previously reported methods to create
[89Zr]Zr4+r with a specific activity of 5.3−13.4 mCi/μg (195−497
MBq/μg).2 Radiolabeling reactions were monitored using silica gel
impregnated glass microfiber instant thin-layer chromatography paper
(iTLC-SG, iTLC-SA, Varian, Lake Forest, CA) and analyzed on a
Bioscan AR-2000 radio-TLC plate reader using Winscan Radio-TLC
software (Bioscan Inc., Washington, DC). All radiolabeling chemistry
was performed with ultrapure water (>18.2 MΩ cm−1 at 25 °C, Milli-
Q, Millipore, Billerica, MA), and treatment of water or buffers with
Chelex-100 resin was performed as indicated (1.2 g/L, 24 h) (BioRad
Laboratories, Hercules, CA). Radioactivity in samples was measured
using a Capintec CRC-15R dose calibrator (Capintec, Ramsey, NJ).
For [89Zr]Zr4+ log D experiments, a PerkinElmer (Waltham, MA)
Automated Wizard Gamma Counter was used for counting
radioactivity.
Synthesis of (2-(4-Nitrobenzyl)-1,3-propylenediamine (3).
This synthesis was adapted from Price et al.45 A mixture of compound
2 (1.005 g, 4.237 mmol) in dry THF (15 mL) was cooled in an ice
bath, followed by dropwise addition of a diborane (BH3·THF)
solution (8.14 mL, 1.0 M in THF) under nitrogen gas. After it was
stirred for 1/2 h, the solution was warmed to room temperature and
then it was heated to reflux under nitrogen gas overnight. Before HCl
(12 M; 15 mL) was added, the reaction mixture was cooled to room
temperature. The reaction mixture was then warmed to reflux with no
inert gas for 1 h. Then the solvent was evaporated by a rotary
evaporator and NaOH (6 M; 30 mL) was added to the residue, and
this mixture was then extracted with dichloromethane (5 × 15 mL).
The organic fractions were collected and dried with anhydrous
Na2SO4. The solvent was then removed by rotary evaporation in
vacuo, and ethanol (20 mL) was added followed by HCl (12 M; 4
mL). The solution was placed in the freezer at −20 °C overnight. A
white solid formed, was filtered, and was dried. Compound 3 (250
mg, 28.20%) was obtained. 1H NMR (D2O, 500 MHz): δ 2.57 [sept,
1H, J = 6.9 Hz, CH2CHCH2], 2.99 [d, 2H, J = 7.6 Hz, CCH2CH],
Chemical Characterization Methods. 1H and 13C NMR spectra
were recorded at the University of Saskatchewan on 500 MHz Bruker
Avance and 500 MHz Bruker Avance III HD NMR spectrometers at
25 °C in (CD3)2SO and CD3OD. 1H chemical shifts were referenced
to the residual protons of the deuterated solvents (δ 2.50 ppm for
(CD3)2SO; δ 3.31 ppm for CD3OD); 13C chemical shifts were
referenced to the (CD3)2SO signal at δ 39.52 ppm and the CD3OD
signal at δ 49.00 ppm. Coupling constants are reported to the nearest
0.5 Hz (1H NMR spectroscopy) or rounded to integer values in Hz
(13C NMR spectroscopy). Assignments were supported by additional
NMR experiments, DEPT, HMQC, and COSY, for compounds 3−6.
High-resolution mass spectra were measured on a JEOL AccuTOF
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3.07 [dd, 2H, J = 6.7 Hz, J = 13.7 Hz, CH2NH2], 3.19 [dd, 2H, J = 6.7
Hz, J = 13.7 Hz, CH2NH2], 7.55 [d, 2H, J = 8.8, NO2CCH], 8.28 [d,
2H, J = 8.8, CCHCH]. 13C NMR (D2O, 500 MHz): δ 34.7, 36.5,
39.9, 124.0, 130.1, 145.2, 146.7. HRMS (ESI; m/z): [M + H]+ calcd
for C10H15N3O2, 210.1242; found, 210.1241.
145.9, 146.1, 148.0, 149.1 (C6H4), 160.9, 161.0, 162.7, 162.8, 170.1,
170.4, 170.7, 170.8, 171.0, 171.17, 171.23, 171.3, 171.4, 171.8, 171.9,
173.6, 173.8, 173.9, 177.8 (CO) ppm. HRMS (TOF): m/z calcd for
C68H111N15O22Zr + H+, 1580.7153 [M + H]+; found, 1580.7109;
calcd for [C68H111N15O22Zr + 2H]2+/2, 790.8610 [M + 2H]2+/2;
found, 790.8643.
Synthesis of DFO-COOH (4). Compound 4 (DFO-COOH) was
prepared according to a published procedure.66 To a solution of
succinic anhydride (0.857 g, 8.56 mmol) in pyridine (4.0 mL) was
added desferrioxamine (DFO) mesylate salt (0.250 g, 0.380 mmol).
This formed a white suspension. The reaction mixture was stirred at
room temperature overnight. To this mixture was added an aqueous
solution of sodium hydroxide (0.16 M; 45 mL), and this mixture was
stirred for 5 h and formed a clear solution. Hydrochloric acid (12 M;
6.0 mL) was added dropwise to this solution until the pH of the
solution reached 2.0 (pH strip). The solution turned cloudy upon
addition of hydrochloric acid. The mixture was left at 5 °C in the
refrigerator for further precipitation. After vacuum filtration the
precipitate was washed several times with a 0.01 M solution of
hydrochloric acid, followed by lyophilization. Compound 4 was
[89Zr]Zr4+ Radiolabeling Studies. Zirconium-89 was received
after target dissolution and purification as the [89Zr]Zr-(oxalate)
complex in 1.0 M oxalic acid. Phosphate-buffered saline (PBS, pH 7.4,
pretreated with Chelex resin) was used as a radiolabeling buffer,
sodium carbonate (1.0 M) was used to neutralize aliquots of
zirconium-89 in oxalic acid to pH 6.8−7.2 (checked by a pH strip),
and an EDTA solution (50 mM, pH 5−5.5) was used as a mobile
phase for elution of radio-iTLC (Varian iTLC-SG) to check RCY and
purity. For radio-iTLC analysis, intact [89Zr]Zr-(chelate) (chelate =
DFO or DFO2) remained at the baseline, while the [89Zr]Zr4+ that
remained unbound or was transchelated to serum proteins eluted with
the solvent front. Radio-iTLC measurements were analyzed using a
BioScan AR2000 instrument, and the integration of activity at the
baseline and solvent front was used to calculate yields and degree of
transchelation during stability assays. The commercially available
chelator DFO·mesylate (Macrocyclics) was used as a control
alongside our new chelator DFO2. Stock solutions of both chelators
were prepared in DMSO at a concentration of 17.0 mg/mL. Aliquots
of these stock solution were transferred to radiolabeling buffer
(Chelex treated PBS, pH 7.4) to prepare separate solutions of DFO
and DFO2 chelators at final volumes of 2.5 mL (100 μM). For each
chelate solution, aliquots of 4−5 mCi (148−185 MBq) of [89Zr]Zr-
(oxalate) were neutralized with sodium carbonate as described above
and diluted to a total volume of 2.5 mL in PBS. This solution was
combined with the chelate solution to produce the final radiolabeling
solution containing 5 mL total volume with 50 μM chelate and 4−5
mCi of zirconium-89. The radiolabeling reaction mixture was mixed
on an Eppendorf thermomixer (800 rpm, 60 min, 37 °C).
Radiochemical yields via radio-iTLC for both DFO and DFO2
chelators were >99% after 60 min reaction time. These preformed
[89Zr]Zr-(chelate) solutions were used without purification for
stability assays. Radiolabeled complexes of DFO and DFO2 chelators
with zirconium-89 were also prepared at 100 μM final concentration
in the same manner as described above and used for some of the
stability assays described below.
1
obtained as a white solid (0.232 g, 93%). H NMR [(CD3)2SO, 500
MHz]: δ 1.20 (m, 7H, CH), 1.37 (m, 7H, CH), 1.49 (m, 6H, CH),
2.27 (m, 6H, CH), 2.40 (t, 2H, CH), 2.57 (t, 4H, CH), 2.99 (q, 6H,
CH), 3.45 (t, 6H, CH), 7.80 (m, 3H, NH), 9.62 (s, 2H, N−OH), 9.66
(s, 1H, N−OH), 12.07 (m, 1H, COOH) ppm. 13C{1H} NMR
[(CD3)2SO, 126 MHz]: δ 20.4, 23.5, 26.1, 27.6, 28.8, 29.2, 29.9, 30.0,
38.4, 46.8, 47.1 (CH), 170.1, 170.7, 171.3, 172.0, 173.9 (CO) ppm.
Synthesis of p-NO2-Bn-DFO2 (5). Compound 4 (0.348 g, 0.527
mmol) is only sparingly soluble in DMF (10.5 mL) at room
temperature and was dissolved at 80 °C. N,N-Diisopropylethylamine
(DIEA; 0.072 g, 0.557 mmol) was added to a solution of 1-
[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxide hexafluorophosphate (HATU; 0.211 g, 0.555 mmol) in DMF
(11.0 mL) in a separate vial. This solution then was added to the
solution of compound 4 in DMF followed by addition of a solution of
compound 3 (0.052 g, 0.248 mmol) in DMF (5.3 mL). The reaction
mixture was stirred at room temperature for 24 h. Volatiles were
evaporated to reduce the volume to approximately one-sixth. The
crude product was then precipitated by slow addition of ice-cold
ultrapure water (70.0 mL) to this residue. The product was separated
by centrifugation, followed by two rounds of resuspension in ice-cold
ultrapure water and centrifugation. Finally, the product was dissolved
in a minimum volume of DMF, followed by addition of ice-cold ethyl
acetate to precipitate the product and then centrifugation and
decanting. The final powder was mixed with ultrapure water and
lyophilized. The p-NO2-Bn-DFO2 (5) product was obtained as a
bone white powder (0.2216 g, 60%). 1H NMR [(CD3)2SO, 500
MHz]: δ 1.21 (m, 17H, CH), 1.38 (m, 17H, CH), 1.49 (m, 15H,
CH), 2.26 (m, 11H, CH), 2.57 (m, 8H, CH), 2.99 (m, 17H, CH),
3.45 (m, 12H, CH), 7.51 (d, 2H, J = 8.5 Hz, C6H4), 7.76 (m, 6H,
NH), 7.82 (m, 2H, NH), 8.14 (d, 2H, J = 8.1 Hz, C6H4), 9.59 (s, 4H,
N−OH), 9.63 (s, 2H, N−OH) ppm. 13C{1H} NMR[(CD3)2SO, 126
MHz]: δ 20.3, 23.5, 26.0, 27.5, 28.0, 28.7, 28.8, 29.8, 29.9, 30.8, 30.9,
38.4, 46.8, 47.1 (CH), 123.2, 130.3, 145.9, 149.0 (C6H4), 170.0,
170.1, 171.1, 171.3, 171.7, 171.9, 177.7 (CO) ppm. HRMS (TOF):
m/z calcd for C68H115N15O22 + H+, 1494.8413 [M + H]+; found,
1494.8565; calcd for [C68H115N15O22 + 2H]2+/2, 747.9243 [M +
2H]2+/2; found, 747.9241.
Serum Stability Assay. [89Zr]Zr-(DFO) and [89Zr]Zr-(DFO2)
complexes were prepared as described above, and 400 μL aliquots
(100 μM) were transferred to microcentrifuge tubes (Eppendorf Low-
Bind, 1.5 mL) containing 600 μL of previously frozen human blood
serum (Sigma-Aldrich). Samples were placed on Eppendorf
Thermomixers at 37 °C and 800 rpm agitation. The assay progress
was monitored by radio-iTLC at times of 1, 5, and 7 days. As with
other assays performed for this study, zirconium-89 transchelated by
blood serum components elutes to the solvent front in an EDTA
mobile phase (50 mM, pH 5.0−5.5), where [89Zr]Zr-(chelate)
remains at the baseline.
An alternative analysis method was performed, where aliquots (300
μL) of the [89Zr]Zr-(chelate)/human blood serum competition
mixture were removed, mixed with ice-cold acetonitrile (700 μL) to
precipitate proteins, and then centrifuged at 10000 rpm for 10 min (4
°C). The supernatant was decanted via pipet and measured by a dose
calibrator, and the process was repeated with an ice-cold water/
acetonitrile mixture (30/70). The amount of zirconium-89 remaining
stuck to the serum proteins in the original microcentrifuge tube was
assumed to be transchelated, and the amount of radioactivity in the
supernatant was assumed to be chelate-bound. This ratio was used to
calculate the percent stability of the [89Zr]Zr-(chelate) complexes. As
a further control, 300 μL of only [89Zr]Zr-(chelate) complex with no
blood serum was put through the same process (ice-cold acetonitrile,
centrifugation) and it was found that for both [89Zr]Zr-(chelate)
complexes ∼10% of the activity in solution remained stuck in the
microcentrifuge tubes after centrifugation and decanting twice. The
stability values reported were not corrected for this sticking factor.
Synthesis of the Complex Zr(DFO2) (6). Compound 5 (50.00
mg, 33.45 μmol) was dissolved in a solvent mixture of DMF and H2O
(1/5; 1.3 mL) at 80 °C. A solution of ZrCl4 (7.64 mg, 32.78 μmol) in
H2O (0.7 mL) was slowly added to the resulting mixture. The
reaction was stirred at room temperature for 24 h. After all volatiles
were removed under high vacuum, the product was obtained as a thin
1
yellow glassy film. H NMR [(CD3)2SO, 500 MHz]: δ 1.23, 1.27,
1.39, 1.49, 1.63, 1.96, 2.12, 2.26, 2.31, 2.58, 2.61, 2.99, 3.09, 3.45, 3.61
(m, CH), 7.53 (m, C6H4), 7.80, 7.88, 7.95 (m, NH), 8.14 (d, 2H, J =
8.3 Hz, C6H4), 9.73 (br, N−OH) ppm; 13C{1H} NMR [(CD3)2SO,
126 MHz]: δ 16.8, 20.4, 22.3, 22.5, 23.2, 23.5, 24.9, 25.7, 26.0, 26.6,
26.9, 27.7, 28.0, 28.8, 28.9, 29.2, 29.3, 30.0, 34.1, 38.4, 46.8, 47.1,
49.7, 50.0, 50.3, 50.6, 63.8 (CH), 119.1, 123.2, 123.4, 127.5, 130.4,
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apo-Transferrin Assay. [89Zr]Zr-(chelate) complexes were
prepared as described above. For this assay, a 5-fold molar excess of
apo-transferrin (Sigma-Aldrich) was used relative to [89Zr]Zr-
(chelate) complex, from a stock solution of apo-transferrin in PBS
(Chelex treated, pH 7.4, 500 μM). The final competition solutions (n
= 3) for each of [89Zr]Zr-(DFO) complex and [89Zr]Zr-(DFO2)
complex contained 200 μL total volume, 50 μM of [89Zr]Zr-(chelate)
complex (PBS, 100 μL of 100 μM), and 250 μM of apo-transferrin
(PBS, 100 μL at 500 μM). Samples were placed on Eppendorf
Thermomixers at 37 °C and 800 rpm agitation. Assay progress was
monitored by radio-iTLC at times of 0, 1, 3, 5, 7, and 9 days.
Serum Albumin Assay. [89Zr]Zr-(chelate) complexes were
prepared as described above. Human serum albumin (Sigma-Aldrich,
>96%) was dissolved in PBS (chelex treated, pH 7.4) at a
concentration of 100 mg/mL (∼1.5 mM), and 150 μL aliquots
were transferred to microcentrifuge tubes (n = 3). The [89Zr]Zr-
(chelate) complex was then added (50 μL, 100 μM, ∼50 μCi) to each
tube for a final concentration of 25 μM [89Zr]Zr-(chelate) complex
and ∼1.125 mM albumin (∼45-fold molar excess). Samples were
placed on Eppendorf Thermomixers at 37 °C and 800 rpm agitation.
The assay progress was monitored by radio-iTLC at times of 0, 1, 3, 5,
and 7 days.
EDTA Transchelation Assay. These transchelation challenges
were set up following the methods of Deri et al.,32 with a 100-fold
molar excess of EDTA to preformed [89Zr]Zr-(DFO) complex or
[89Zr]Zr-(DFO2) complex. The solutions prepared for this assay were
(1) 2.5 mL of [89Zr]Zr-(DFO) complex and [89Zr]Zr-(DFO2)
complex (50 μM each) as described above, (2) EDTA stock solution
(5 mM) split and then each substock solution adjusted by a pH meter
to pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0, and (3) ammonium acetate
buffer (500 mM) split and then each substock solution adjusted by a
pH meter to pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0. These three stock
solutions were combined in microcentrifuge tubes (1.5 mL tubes, n =
3) for each chelator and pH range in ratios of [89Zr]Zr-(chelate)
complex (200 μL, 50 μM), EDTA (200 μL, 5 mM), and ammonium
acetate (100 μL, 500 mM), with the EDTA and ammonium acetate
solutions matching the pH (e.g., pH 7.0 each for the pH 7.0 challenge,
n = 3). The final EDTA transchelation assay solutions contained a
total volume of 500 μL with [89Zr]Zr-(chelate) complex (20 μM,
∼100 μCi, ∼ 3.7 MBq) and EDTA solution (2 mM) for a 100-fold
molar excess of EDTA challenge. Transchelation was monitored by
radio-iTLC as described above (EDTA mobile phase, 50 mM, pH
5.0−5.5), where zirconium-89 associated with DFO or DFO2
chelators remained at the baseline and zirconium-89 transchelated
to EDTA eluted with the solvent front. Samples were placed on
Eppendorf Thermomixers at 37 °C and 800 rpm agitation. The assay
progress was monitored at times of 0, 1, amd 3 h and 1, 3, 5, and 7
days.
(chelate) complex in each competition was 10 μM. HTP competition
mixtures were placed on Eppendorf Thermomixers at 37 °C and 800
rpm agitation. After 24 h, these samples were centrifuged (10000 rpm,
10 min) to pelletize the HTP, the supernatant was decanted via pipet,
and the pellet was washed with TRIS/HCl buffer (1.0 mL) twice,
followed by centrifugation and decanting. The amount of zirconium-
89 adsorbed/bound by the HTP and the amount remaining chelate-
bound in the supernatant and washes was measured via a dose
calibrator. The percent stability was determined from a ratio of the
percent radioactivity in the supernatant/washes and the HTP pellet.
log D Octanol/Buffer Partition Coefficient. Radiolabeled
[89Zr]Zr-(DFO) and [89Zr]Zr-(DFO2) complexes were prepared as
described above (100 μM). After radiolabeling, log D values were
determined by transferring an aliquot of each radiolabeled chelator
(∼5 μCi, ∼5−10 μL) to 3 mL of PBS (pH 7.4) in 15 mL Falcon
centrifuge tubes (n = 5). An equal volume of 1-octanol (3 mL) was
then added, the tubes were capped, and the contents were then vortex
mixed for 60 s each. The tubes were then centrifuged for 10 min
(3000 rpm) to aid in phase separation. Samples were removed from
the centrifuge, and allowed to sit for 10 min, and 1.0 mL aliquots of
each phase were removed carefully via pipet and transferred to small
microcentrifuge tubes (1.5 mL) and the radioactivity measured on a γ
counter. The ratio of radioactivity (counts) in each phase was used to
calculate the log D values (eq 1). The amount of radioactivity in the
octanol phase was used in place of solute concentrations (ionized and
un-ionized), and the amount in the PBS (pH 7.4) phase was used in
place of solute concentration (ionized and un-ionized).
ionized
un‐ionized
i
j
j
y
z
z
[solute]
+ [solute]
octanol
octanol
j
z
z
z
log Doctanol,PBSpH7.4 = logj
j
ionized
un‐ionized
j
j
z
z
[solute]PBSpH7.4 + [solute]PBSpH7.4
k
{
(1)
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.inorgchem.0c01629.
NMR spectra and DFT coordinates (PDF)
AUTHOR INFORMATION
■
Corresponding Author
Eric W. Price − Department of Chemistry, University of
Saskatchewan, Saskatoon, Saskatchewan S7N 5C9, Canada;
orcid.org/0000-0002-8619-1199; Phone: +1-306-966-
4788; Email: eric.price@usask.ca; Fax: +1-306-966-4666
Iron(III) Competition Study. The radiolabeled [89Zr]Zr-(DFO)
complex and [89Zr]Zr-(DFO2) complex were prepared as described
above. The metal salt iron(III) chloride was prepared as a stock
solution (1.0 mM, Chelex-treated Millipure water). Aliquots of the
[89Zr]Zr-(chelate) complex (200 μL, 100 μM) were combined with
an iron(III) solution (200 μL, 1.0 mM) to yield final completion
mixtures (n = 3) containing 50 μM of [89Zr]Zr-(chelate) complex and
500 μM of iron(III) solution (10-fold molar excess). Samples of
[89Zr]Zr-(chelate) complexes in competing iron solutions were placed
on Eppendorf Thermomixers at 37 °C and 800 rpm agitation. The
assay progress was monitored by radio-iTLC as described above at
times of 0, 1, 3, 5, 7, and 9 days.
Hydroxyapatite (HTP) Stability Challenge. Hydroxyapatite
resin (HTP, BioRad Bio-Gel) was weighed into microcentrifuge tubes
(20 and 40 mg sets, n = 3 per weight and per chelator/control). In
each tube containing HTP was placed TRIS/HCl buffer (50 mM, 900
μL, pH 7.4). [89Zr]Zr-(DFO) and [89Zr]Zr-(DFO2) complexes were
radiolabeled as described above, with the exception that TRIS/HCl
buffer (50 mM, pH 7.4) was used, and aliquots were transferred to
each tube (100 μM, 100 μL, ∼100 μCi, ∼3.7 MBq), as well as a set of
unchelated but neutralized [89Zr]Zr-(oxalate) complex as controls
(∼100 μCi, ∼3.7 MBq, n = 3). The final concentration of [89Zr]Zr-
Authors
Elaheh Khozeimeh Sarbisheh − Department of Chemistry,
University of Saskatchewan, Saskatoon, Saskatchewan S7N
5C9, Canada
Akam K. Salih − Department of Chemistry, University of
Saskatchewan, Saskatoon, Saskatchewan S7N 5C9, Canada
Shvan J. Raheem − Department of Chemistry, University of
Saskatchewan, Saskatoon, Saskatchewan S7N 5C9, Canada
Jason S. Lewis − Department of Radiology, Radiochemistry and
Molecular Imaging Probes Core, and Molecular Pharmacology
Program, Memorial Sloan Kettering Cancer Center, New York,
New York 10065, United States; Department of Pharmacology
and Department of Radiology, Weill Cornell Medical College,
New York, New York 10065, United States; orcid.org/
0000-0001-7065-4534
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.inorgchem.0c01629
J
https://dx.doi.org/10.1021/acs.inorgchem.0c01629
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
pubs.acs.org/IC
Article
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Author Contributions
∇E.K.S. and A.K.S. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
E.W.P. was supported by the Natural Sciences and Engineering
Research Council (NSERC) (Discovery Grant, RGPIN-2017-
03952 2017-2022), the Canada Research Chairs program
(CRC in Radiochemistry, 2016-2021, #231072), the Canadian
Institutes of Health Research (CIHR Project Grant #388243),
the Canadian Foundation for Innovation John Evans Leader-
ship Fund (2016 #35162), the Sylvia Fedoruk Canadian
Centre for Nuclear Innovation, the Saskatchewan Health
Research Foundation (SHRF Establishment grant), and
startup funds from the University of Saskatchewan Department
of Chemistry and College of Arts and Sciences. E.K.S. was
supported by a Saskatchewan Health Research Foundation
(SHRF) postdoctoral fellowship (2017−2019). J.S.L. was
supported by R01 CA204167 and R35CA232130 as well as
the Mr. William H. Goodwin and Mrs. Alice Goodwin and the
Commonwealth Foundation for Cancer Research and The
Center for Experimental Therapeutics at Memorial Sloan
Kettering Cancer Center.
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ABBREVIATIONS
PET, positron emission tomography; 89Zr, zirconium-89; RCY,
radiochemical yield
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