Octadentate Zirconium(IV)-Loaded Macrocycles with Varied Stoichiometry Assembled From Hydroxamic Acid Monomers using Metal-Templated Synthesis

Article abstract of DOI:10.1021/acs.inorgchem.7b00362

The reaction between Zr(IV) and the forward endo-hydroxamic acid monomer 4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoic acid (for-PBH) in a 1:4 stoichiometry in the presence of diphenylphosphoryl azide and triethylamine gave the octadentate Zr(IV)-loaded tetrameric hydroxamic acid macrocycle for-[Zr(DFOT₁)] ([M + H]⁺ calc 887.3, obs 887.2). In this metal-templated synthesis (MTS) approach, the coordination preferences of Zr(IV) directed the preorganization of four oxygen-rich bidentate for-PBH ligands about the metal ion prior to ring closure under peptide coupling conditions. The replacement of for-PBH with 5-[(5-aminopentyl) (hydroxy)amino]-5-oxopentanoic acid (for-PPH), which contained an additional methylene group in the dicarboxylic acid region of the monomer, gave the analogous Zr(IV)-loaded macrocycle for-[Zr(PPDFOT₁)] ([M + H]⁺ calc 943.4, obs 943.1). A second, well-resolved peak in the liquid chromatogram from the for-PPH MTS system also characterized as a species with [M + H]⁺ 943.3, and was identified as the octadentate complex between Zr(IV) and two dimeric tetradentate hydroxamic acid macrocycles for-[Zr(PPDFOT_1D)₂]. Treatment of for-[Zr(PPDFOT₁)] or for-[Zr(PPDFOT_1D)₂] with EDTA at pH 4.0 gave the respective hydroxamic acid macrocycles as free ligands: octadentate PPDFOT₁ or two equivalents of tetradentate PPDFOT_1D (homobisucaberin, HBC). At pH values closer to physiological, EDTA treatment of for-[Zr(DFOT₁)], for-[Zr(PPDFOT₁)], or Zr(IV) complexes with related linear tri- or tetrameric hydroxamic acid ligands showed the macrocycles were more resistant to the release of Zr(IV), which has implications for the design of ligands optimized for the use of Zr(IV)-89 in positron emission tomography (PET) imaging of cancer.

Full text of DOI:10.1021/acs.inorgchem.7b00362

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Article
pubs.acs.org/IC
Octadentate Zirconium(IV)-Loaded Macrocycles with Varied
Stoichiometry Assembled From Hydroxamic Acid Monomers using
Metal-Templated Synthesis
William Tieu,† Tulip Lifa,† Andrew Katsifis,‡ and Rachel Codd*,†
†School of Medical Sciences (Pharmacology) and Bosch Institute, The University of Sydney, New South Wales 2006, Australia
‡Department of Molecular Imaging, Royal Prince Alfred Hospital, Camperdown, New South Wales 2050, Australia
S
* Supporting Information
ABSTRACT: The reaction between Zr(IV) and the forward
endo-hydroxamic acid monomer 4-[(5-aminopentyl)-
(hydroxy)amino]-4-oxobutanoic acid (for-PBH) in a 1:4
stoichiometry in the presence of diphenylphosphoryl azide
and triethylamine gave the octadentate Zr(IV)-loaded
tetrameric hydroxamic acid macrocycle for-[Zr(DFOT1)]
([M + H]+ calc 887.3, obs 887.2). In this metal-templated
synthesis (MTS) approach, the coordination preferences of
Zr(IV) directed the preorganization of four oxygen-rich
bidentate for-PBH ligands about the metal ion prior to ring
closure under peptide coupling conditions. The replacement of
for-PBH with 5-[(5-aminopentyl) (hydroxy)amino]-5-oxopen-
tanoic acid (for-PPH), which contained an additional
methylene group in the dicarboxylic acid region of the monomer, gave the analogous Zr(IV)-loaded macrocycle for-
[Zr(PPDFOT1)] ([M + H]+ calc 943.4, obs 943.1). A second, well-resolved peak in the liquid chromatogram from the for-PPH
MTS system also characterized as a species with [M + H]+ 943.3, and was identified as the octadentate complex between Zr(IV)
and two dimeric tetradentate hydroxamic acid macrocycles for-[Zr(PPDFOT1D)2]. Treatment of for-[Zr(PPDFOT1)] or for-
[Zr(PPDFOT1D)2] with EDTA at pH 4.0 gave the respective hydroxamic acid macrocycles as free ligands: octadentate
PPDFOT1 or two equivalents of tetradentate PPDFOT1D (homobisucaberin, HBC). At pH values closer to physiological, EDTA
treatment of for-[Zr(DFOT1)], for-[Zr(PPDFOT1)], or Zr(IV) complexes with related linear tri- or tetrameric hydroxamic acid
ligands showed the macrocycles were more resistant to the release of Zr(IV), which has implications for the design of ligands
optimized for the use of Zr(IV)-89 in positron emission tomography (PET) imaging of cancer.
which is a metabolite of the siderophore class produced for
Fe(III) acquisition by many Streptomyces species,17−19 has been
used to coordinate Zr-89 in a number of preclinical and clinical
studies,20−27 although as a hexadentate ligand, it is unable to
saturate the preferred octadentate Zr(IV) coordination
sphere.28 The complex [Zr(DFOB)(OH2)2]+ (Chart 1,
2)15,29 has only modest in vivo stability, which can result in
the release and deposition of some of the radionuclide in
nontargeted organs, and a reduction in the image quality.29−31
In recent work,32,33 the DFOB scaffold was improved as a
Zr(IV) ligand by undertaking a chain-extension reaction
between DFOB and the endo-hydroxamic acid monomer 4-
[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoic acid (for-
PBH), which is a biosynthetic fragment of desferrioxamine-
type siderophores.34−37 This produced a linear octadentate
tetrameric hydroxamic acid ligand DFOB-for-PBH that formed
[Zr(DFOB-for-PBH)] (Chart 1, 3), which was more stable to
INTRODUCTION
■
Good clinical decision making in the diagnosis and manage-
ment of patients with cancer is supported by access to high-
definition images of the tumor with positron emission
tomography (PET) imaging in wide use for this purpose.1−4
Zirconium-89 has been under recent scrutiny as a radionuclide
with properties that show promise for its use in antibody-
coupled PET imaging.5−9 The half-life of Zr-89 (3.27 d, β+ =
22.7%, maximum β+ energy = 0.897 MeV) is well-matched to
the circulation half-life of antibodies, which makes Zr-89
attractive for immunological PET applications designed to
concentrate the radionuclide at the site of the antigen-
expressing tumor to improve the tumor-to-nontumor signal
and the image resolution.9−12 Progressing preclinical and
clinical evaluation of Zr-89 as a PET radionuclide would
benefit from a ligand suitably tailored to bind Zr(IV) with high
affinity and selectivity. As a hard acid, Zr(IV) favors
coordination with ligands rich in oxygen donor atoms,13
which include hydroxamic acids.14−16 The linear trimeric
hydroxamic acid desferrioxamine B (Chart 1, 1, DFOB),
Received: February 9, 2017
© XXXX American Chemical Society
A
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89 PET imaging is reported. The importance of macrocycles in
drug design,48−51 warrants the development of new methods
for the streamlined production of structurally diverse macro-
cycles.52,53
Chart 1. Desferrioxamine B (DFOB) (1) and Its Complex
with Zr(IV) (2)
a
RESULTS AND DISCUSSION
■
Synthesis of endo-Hydroxamic Acid Ligands. A series
of six endo-hydroxamic acid amino carboxylic acid monomers
were prepared using reported methods: for-PBH, for-PPH, for-
HBH, ret-PBH and ret-HBH.47 The monomer 4-(6-amino-N-
hydroxyhexanamido)butanoic acid (ret-PPH) was prepared in a
fashion similar to ret-PBH, with the substitution of tert-butyl-3-
[(benzyloxy)amino]propanoate with ethyl-4-[benzyloxy)-
amino]butanoate (Scheme S1 and Figures S1−S6). The first
letter in the nomenclature system for these ligands denotes the
component derived from either pentane-1,5-diamine (P) or
hexane-1,6-diamine (H), with the second letter referring to the
component derived from butandioic acid (B) or pentandioic
acid (P). The terminal ‘H’ is reference to the acidic proton of
the hydroxamic acid. Native siderophores are assembled from
forward (for-) endo-hydroxamic acids, which feature the
internal −N(OH)C(O)− motif inserted in the direction as
written, between the amine terminus and the carboxylic acid
terminus. The reverse or retro (ret-) analogues feature the
hydroxamic acid insert in the reverse orientation: −C(O)N-
(OH)−. This work sought to determine whether Zr(IV)
demonstrated a preference in the MTS-directed assembly of
macrocycles from ligands that differed in length and thereby
cavity size, as defined by the number of internal main-chain
atoms (PBH, 10; PPH and HBH, 11); the position of the
additional methylene group, as inserted in the diamine region
(HBH) or the dicarboxylic acid region (PPH); and the
orientation of the hydroxamic acid group (for-, ret-), which
could affect the electronic structure and the physicochemical
properties of the macrocycle (Scheme 1).
LC-MS Data From Zr(IV)-Based MTS Reaction Sol-
utions. Standard MTS experiments involved the reaction
between Zr(IV) and a given ligand (e.g., for-PBH) in a 1:4 ratio
in a solution of 1:1 methanol:water to form the preassembled
complex, with ring closure effected after a 5-d reaction time
with diphenylphosphoryl azide (DPPA) and triethylamine. A
series of three native tetrameric hydroxamic acid macrocycles
has been characterized from cultures of Erwinia amylovora, with
the macrocycle assembled from for-PBH named DFOT1.54 This
naming system has been used here. The LC trace from the
Zr(IV)-for-PBH MTS system showed a single peak (SIM 887)
at tR 29.7 min (Figure 1a), which in the MS gave an isotope
pattern (Figure 2a) consistent with the calculated patterns
(Figure 2d) of the [M + 2H]2+, [M + H]+, and [M + Na]+
adducts of for-[Zr(DFOT1)] (4) (Scheme 1). The presence of
Zr(IV) in the macrocycle was clear from the characteristic
isotope pattern of natural Zr (90Zr 51.45, 91Zr 11.22, 92Zr 17.15,
94Zr 17.38, 96Zr 2.80). Two other species were identified from
the Zr(IV)-for-PBH MTS system, including the Zr(IV)
complex formed with the linear tetrameric ligand ([M + H]+
calc 905.4, obs 905.1), as the precursor of for-[Zr(DFOT1)]
(4), and a trimeric Zr(IV)-loaded macrocycle ([M]+ calc 687.2,
obs 687.1). The latter species was confirmed as [Zr(DFOE)]+
(with labile ancillary ligands (eg, H2O) likely displaced during
the MS procedure) from its coelution with an authentic sample
of [Zr(DFOE)]+ (Figure S7). No macrocycles were detected
from a DPPA and triethylamine solution containing for-PBH in
a
Complexes between Zr(IV) and a linear (3) or macrocyclic (4)
chain-extended variant of DFOB.
Zr(IV) release than [Zr(DFOB)(OH2)2]+ (2). Complexes
between Zr(IV) and other linear octadentate hydroxypyridin-
onate (HOPO)-type hydroxamic acid ligands38−40 or a DFOB-
squaramide ligand41 have also been characterized. The current
work considered that a further gain in the stability of the Zr(IV)
complex could result if the octadentate DFOB-type tetrameric
hydroxamic acid ligand was configured into a macrocycle
(Chart 1, 4). This principle was supported by a study of
macrocycles designed to coordinate Zr(IV) that were cyclized
from hydrophobic abiological tetrameric linear constructs using
ring-closing metathesis,42 and from other studies focused upon
developing macrocycles for Zr-89 PET imaging.43
The current work has used a metal-templated synthesis
(MTS) approach to examine the ability of Zr(IV) to assemble
its own best macrocycle from a selection of hydroxamic acid
monomers reminiscent of the fragments used in siderophore
biosynthesis. The stability of these macrocycles in retaining
Zr(IV) could be reasonably compared to the linear tetrameric
hydroxamic acid ligand DFOB-for-PBH, and trimeric DFOB.
The use of a metal ion to template the assembly of its ideal
coordination complex from a pool of fragment ligands that
contain reactive termini is attractive in its simplicity, and offers
the potential to deliver macrocycles with high affinity and
selectivity.44,45 Metal-templated synthesis (MTS) with Fe(III)
or Ga(III) as the template has been used to assemble the
trimeric hydroxamic acid macrocycle desferrioxamine E
(DFOE) from for-PBH.46 Other metal-loaded ring-expanded
desferrioxamine-type macrocyclic siderophores have since been
assembled using Fe(III) or Ga(III)-based MTS from a range of
endo-hydroxamic acid monomers, including forward (for- =
H2N−(CH2)x−N(OH)C(O)−(CH2)y−CO2H) and reverse
(ret- = H2N−(CH2)x−C(O)N(OH)−(CH2)y−CO2H) li-
gands.47
In this work, the utility of MTS for assembling Zr(IV)
macrocycles as constructs with potential relevance for Zr(IV)-
B
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Scheme 1. Zr(IV)-Templated Synthesis of Tetrameric
Macrocycles 4-9 from the Cognate Monomers for-PBH, for-
PPH, for-HBH, ret-PBH, ret-PPH, or ret-HBH
Figure 1. Liquid chromatograms from a solution of Zr(IV) and (a) for-
PBH, (b) for-PPH, (c) for-HBH, (d) ret-PBH, (e) ret-PPH, or (f) ret-
HBH, after reaction with peptide coupling reagents. Selected ion
monitoring (SIM) was used as the detection mode, with values set to
correspond with the [M + H]+ adduct of the cognate macrocycle 4−9.
the absence of Zr(IV), which demonstrated the essential nature
of the Zr(IV) ion template in the reaction.
In the Zr(IV)-for-PPH MTS system (SIM 943), two well-
resolved peaks were present in the LC at tR = 37.4 min and tR =
42.7 min (Figure 1b). Each of these peaks gave experimental
MS signals (tR = 42.7 min, Figure 2b; tR = 37.4 min, Figure 2c)
that correlated with the presence of the [M + 2H]2+, [M + H]+,
and [M + Na]+ adducts of for-[Zr(PPDFOT1)] (5) (calculated
data Figure 2e−f). The presence of two peaks with identical MS
characteristics was unexpected and was examined further in
later experiments. The presence of four additional methylene
groups in for-[Zr(PPDFOT1)] (5) compared to for-[Zr-
(DFOT1)] (4), was consistent with the increase of 56 m/z
units in the [M + H]+ adduct signal, with other adducts also
showing consistent increases. Signals ascribable to the linear
tetramer precursor of for-[Zr(PPDFOT1)] (5) or the trimeric
DFOE-type macrocyclic analogue were not detected in this
system, or for any other monomer.
No LC peaks were discernible from the Zr(IV)-for-HBH
(Figure 1c) or the Zr(IV)-ret-HBH (Figure 1f) MTS systems
(SIM 943), which suggested that the formation of for-
[Zr(HBDFOT1)] (6) and ret-[Zr(HBDFOT1)] (9) was
unfavorable. The relative intensities of the LC peaks observed
for the Zr(IV)-ret-PBH (Figure 1d) or the Zr(IV)-ret-PPH
(Figure 1e) MTS systems to form macrocycles ret-[Zr-
(DFOT1)] (7) or ret-[Zr(PPDFOT1)] (8), respectively, was
less than the corresponding for-PBH or for-PPH ligand systems,
which indicated for-endo-hydroxamic acid monomers were
favored above the ret-monomers in the Zr(IV)-macrocycle
Figure 2. MS signals from the LC peaks detected using SIM values
corresponding with (a) for-[Zr(DFOT1)], and the two peaks
consistent with for-[Zr(PPDFOT1)] at (b) tR = 42.7 min and (c) tR
= 37.4 min. The calculated isotope patterns for the corresponding
species are aligned at right (d−f).
C
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formation. The LC trace from the Zr(IV)-ret-PBH MTS system
was also resolved into two peaks, representing ret-[Zr-
(DFOT1)] (7) and a second species of an uncertain nature.
Peak integration gave a for-PBH/ret-PBH (sum of both peaks)
ratio of 1.5, and a for-PPH/ret-PPH (sum of both peaks) ratio
of 52.9.
Overall, the results demonstrated a degree of ligand
selectivity was in operation in Zr(IV)-mediated macrocycle
assembly. The use of MTS for the preparation of trimeric
Fe(III)-loaded macrocycles,47 showed a similar selectivity ratio
with respect to the for-PBH/ret-PBH system. As distinct from
the Zr(IV)-based MTS system, Fe(III)-loaded macrocycles
were formed with for-HBH and ret-HBH, which demonstrated
metal ion selectivity was in operation in the MTS systems.
Characterization of the Two Species in the Zr(IV)-for-
PPH MTS System. The LC peaks for the higher-yielding
Zr(IV)-MTS systems using for-PBH or for-PPH were collected
for further characterization. The two peaks in the for-PPH
system (Figure 1b) were collected separately and the integrity
of the species confirmed by LC, with the species showing
elution times of tR = 35.5 min (Figure 3a) and 41.0 min (Figure
3g). The trace using TIC detection (black) was coincident with
the trace (gray) using SIM values representative of for-
[Zr(PPDFOT1)] (5). The absolute retention times were
different from the first acquired LC data, which was due to
instrumental variation. To better understand the nature of the
species, each fraction was incubated with EDTA at pH 4.0, with
samples analyzed by LC-MS at 24 and 60 h. After 24 h, the
earlier-eluting species gave a new signal using TIC detection at
tR = 28.1 min (Figure 3b), which gave an experimental MS
isotope pattern (Figure 3e) consistent with the calculated
pattern (Figure 3f) of [M + 2H]2+, [M + H]+ and [M + Na]+
adducts of a Zr(IV)-free dimeric hydroxamic acid macrocycle
assembled from two for-PPH units. After 60 h, the dimeric
hydroxamic acid macrocycle remained present (Figure 3c, gray)
with no remaining trace of the Zr(IV)-loaded parent species
(Figure 3c, black). This supported that the earlier-eluting
Zr(IV) species at tR = 35.5 min was a complex of Zr(IV), in
which the coordination sphere was saturated by two
tetradentate dimeric macrocycles: for-[Zr(PPDFOT1D)2]
(Scheme 2, 10). The complex for-[Zr(PPDFOT1D)2] (10)
was an isomer of for-[Zr(PPDFOT1)] (5), in which the
coordination sphere of the latter was saturated by one
octadentate tetrameric macrocycle.
The dimeric macrocycle 11 released from EDTA-treated 10
is an analogue of the small family of dimeric hydroxamic acid
macrocycles identified as natural bacterial siderophores,
including alcaligin,55 bisucaberin,56 putrebactin,57 and avar-
oferrin.58−60 Since for-PPH contains one extra methylene group
than for-PBH, which is the native monomer of the symmetric
dimer bisucaberin, macrocycle 11 has been given the common
name homobisucaberin (HBC).
After 24 h incubation with EDTA, the later-eluting Zr(IV)
species gave a signal using TIC detection at tR = 36.7 min
(Figure 3h), with an experimental MS isotope pattern (Figure
3k) consistent with the calculated pattern of [M + 2H]2+, [M +
H + Na]2+, [M + H + K]2+, [M + H]+, and [M + Na]+ adducts
(Figure 3l) of the apo-macrocycle PPDFOT1 (12). There was a
trace of for-[Zr(PPDFOT1D)2] (10) in this solution, which gave
rise to a low intensity MS signal at m/z 451.2. After 60 h
incubation, there remained traces of the parent for-[Zr-
(PPDFOT1)] (5) (Figure 3i, black) in addition to the apo-
macrocycle (gray). This demonstrated that the Zr(IV) ion was
Figure 3. LC-MS data from a solution of 10 (column at left) or 5
(column at right), detected as (a, g) TIC (black) or at SIM values
(gray) 472, 943, 965, corresponding to 5 and 10, and following
incubation with EDTA (pH 4), detected as (b, h) TIC, or (c, (i) SIM
values (black) 472, 943, 965, corresponding to 5 and 10, or SIM values
(gray) 429, 857, 879, corresponding to 11 and 12, or after incubation
of the peak at (d) 28.1 min (gray) or (j) 36.7 min (gray) with Zr(IV),
detected as (d, j) SIM values (black) 429, 451, 472, 857, 879, 943, 965,
corresponding with 5 and 10 and 11 and 12. The MS signal from the
peak at 28.1 min attributable to 11 is shown as experiment (e) or
calculated (f); or at 36.7 min attributable to 12 as experiment (k) or
calculated (l).
more readily removed from for-[Zr(PPDFOT1D)2] (10) than
for-[Zr(PPDFOT1)] (5). The extraction of Zr(IV) was pH
dependent, with negligible Zr(IV) removed from for-[Zr-
1
(PPDFOT1)] (5) at pH 7.0. An H NMR spectrum of for-
[Zr(PPDFOT1)] (5) (Figure S8) showed signals that were
clustered in regions similar to those observed for a Zr(IV)-
DFOB complex,39 although the signal complexity from
conformational flux prevented the unambiguous assignment.
D
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and C1 (Scheme 2). As similar to for-[Zr(PPDFOT1)] (5), the
formation of for-[Zr(PPDFOT1D)2] (10) would require
coupling between N1 and C2, but a second coupling between
C1 and N2. The C1−N2 coupling would require the pendent
amine group of monomer 2 to reorient from below the x-y
plane to above the x-y plane. The other dimeric macrocycle
comprising for-[Zr(PPDFOT1D)2] would be formed upon
coupling between N3 and C4, as common to for-[Zr-
(PPDFOT1)], and between C3 and N4, requiring the pendent
amine group of monomer 4 move below the x-y plane (Scheme
2). In the case of the for-PPH MTS system, the distribution of
both conformers as determined by probability, and the
proximity of the relevant amine and activated carboxylic acid
termini, were favorably poised to promote the formation of for-
[Zr(PPDFOT1)] (5) and for-[Zr(PPDFOT1D)2] (10). LC-MS
analysis of the bulk solution from the ret-PBH MTS system
following incubation with EDTA at pH 4.0 supported the
presence of ret-[Zr(DFOT1)] (7) and ret-[Zr(DFOT1D)2].
Molecular Mechanics Calculations. Structures of the
Zr(IV) complexes with for-PBH, for-PPH or for-HBH to form a
1:1 Zr(IV):tetrameric macrocycle or a 1:2 Zr(IV):dimeric
macrocycle were built using X-ray crystallographic data for
[Zr(Me-AHA)4] (Me-AHAH = N-methylacetohydroxamic
acid)15 as the core of the molecule, and fragments of for-
PBH from the structure of [Fe(DFOB)] (Figure 4).61
Molecular mechanics calculations showed that from the six
complexes, for-[Zr(DFOT1)] (4) gave the highest value for the
minimum energy (Table 1), which was attributed to ring strain
imposed by the suboptimal chain length of the for-PBH ligand.
The average of the torsion angle of the four amide bonds was
used as a surrogate measure of ring strain, with the value for for-
[Zr(DFOT1)] deviating the most from 180°. There was a
significant decrease in the energy minimum for for-[Zr-
(DFOT1D)2], which was attributed to the reduction in the
ring strain from for-[Zr(DFOT1)] to the isomer for-[Zr-
(DFOT1D)2]. In each isomer pair of the 1:1 Zr(IV):tetrameric
macrocycle and the 1:2 Zr(IV):dimeric macrocycle, the latter
isomer gave a lower energy minimum, consistent with the
notion that the organization of dimers reduced the ring strain in
the octadentate complex. Molecular mechanics calculations
using an alternative model of the Zr(IV) coordination sphere42
gave high-energy structures of the dimer complexes, with one
wing of the dimer partially inserted into the other, which
supported the veracity of using the [Zr(Me-AHA)4] core in the
modeling.
Scheme 2. Zr(IV):for-PPH Complexes (1:4) Preorganized to
Form for-[Zr(PPDFOT1)] (5) or for-[Zr(PPDFOT1D)2]
(10)
a
a
Demetallation of 10 gave homo-bisucaberin 11, and demetallation of
5 gave 12 in analytical yields.
The apo-macrocycles 11 and 12 were isolated and incubated
with Zr(IV) to examine the reincorporation of Zr(IV), as a
surrogate of the radiolabeling procedure. Incubation for 1 h of
the dimeric hydroxamic acid macrocycle 11 with Zr(IV) did not
reassemble for-[Zr(PPDFOT1D)2] (10), with the traces for the
free ligand (Figure 3d, gray) and the solution following Zr(IV)
incubation (Figure 3d, black) coincident. Incubation of the
tetrameric hydroxamic apo-macrocycle PPDFOT1 (12) (Figure
3j, gray) with Zr(IV) showed a signal (Figure 3j, black) that
was coincident with for-[Zr(PPDFOT1)] (5), which supported
the viability of radiolabeling these types of macrocycles.
A repeat MTS reaction between Zr(IV) and for-PPH gave an
LC trace with a similar distribution of [Zr(PPDFOT1)] (5) and
for-[Zr(PPDFOT1D)2] (10) (Figure S9) as first observed,
supporting the reproducibility of the system. Each peak was
isolated and a subsample subject to EDTA-based Zr(IV)
removal (pH 4.0) and purification to give a sample set of the
Zr(IV)-loaded macrocycles ([Zr(PPDFOT1)] (5), for-[Zr-
(PPDFOT1D)2] (10)), and the corresponding apo-macrocycles
(PPDFOT1 (12), and HBC (11)) for analysis by high
resolution MS. Each species analyzed in the HRMS as the
[M + Na]+ adduct, with HBC (11) showing an additional
adduct [M]+ attributable to a complex formed with adventitious
Al3+ (Figure S10−S13).
As expected, the complexes modeled with for-PPH and for-
HBH, that contained one additional methylene group than for-
PBH, had greater volumes than for-[Zr(DFOT1)] and for-
[Zr(DFOT1D)2]. The energy minimum for for-[Zr-
(PPDFOT1)] (5) was significantly less than for-[Zr(DFOT1)]
(4), which indicated that the additional methylene group in for-
PPH was beneficial in the formation of a tetrameric macrocycle
with a cavity size better optimized for the accommodation of
the Zr(IV) ion. The reduced energy minimum for for-
[Zr(PPDFOT1)] (48 main-chain atoms) than for-[Zr-
(DFOT1)] (44 main-chain atoms) could be considered more
amplified than the absolute value, based on calibration to the
number of main-chain atoms in the macrocycle, giving 11.6 or
14 kcal/mol per main-chain atom, respectively.
The structures of for-[Zr(PPDFOT1)] and for-[Zr-
(PPDFOT1D)2] showed the amide oxygen atom was oriented
away from the Zr(IV)-O8 coordination sphere, with an average
distance (as measured from each amide oxygen atom to the
The formation of for-[Zr(PPDFOT1)] (5) would require
coupling between the amine group of monomer 1 (N1) and the
DPPA-activated carboxylic acid group of monomer 2 (C2); and
contiguous coupling between N2 and C3, N3 and C4, and N4
E
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Article
degree of steric and electronic repulsion resulting from the
proximity of the amide oxygen atoms to the Zr(IV)-O8
coordination sphere. These effects provide a possible rationale
for the preference of the for-PPH ligand in the macrocycle
assembly, and the lower concentration or no complex
formation in the for-PBH and for-HBH ligand systems,
respectively.
Release of Zr(IV) with EDTA Treatment. It was posited
that in the presence of EDTA at physiological pH values, the
Zr(IV) ion would be better retained by the Zr(IV)-loaded
macrocycles, compared to related linear ligands. In the presence
of a significant excess of EDTA, Zr(IV) was completely
removed at pH 7.0 from [Zr(DFOB)(OH2)2]+ (2) after 1 h
incubation time (Figure 5). About 60% of Zr(IV) was retained
Figure 5. Percentage (n = 3) of initial concentration (LC-MS, SIM
detection of the [M]+ or [M + H]+ adduct) of [Zr(DFOB)]+ (open
square), [Zr(DFOB-for-PBH)] (closed square; n = 1 for t = 4, 5 d),
for-[Zr(DFOT1)] (open circle) or for-[Zr(PPDFOT1)] (closed circle)
at t = 0, 1−6 days, in the presence of 1800 equiv of EDTA at pH 7.0.
in the linear, tetrameric chain-extended DFOB analogue
[Zr(DFOB-for-PBH)] (3) after 72 h (refer to the ESI for the
synthesis of DFOB-for-PBH, Figure S14). The trend in the
increased stability of [Zr(DFOB-for-PBH)] (3) above [Zr-
(DFOB) (OH2)2]+ (2) due to the increased ligand denticity
was in agreement with previous work.32 The current work
showed a greater loss of Zr(IV) from [Zr(DFOB-for-PBH)]
(3), which was ascribed to differences in reaction conditions
and/or the use of a significant excess of EDTA, as was
necessary for nonradioactive methods of detection. The loss of
Zr(IV) from for-[Zr(DFOT1)] (4) or for-[Zr(PPDFOT1)] (5)
was minimal under the same conditions, with 93% or 96% of
the respective Zr(IV)-loaded macrocycles detected after 72 h.
The minimal loss of Zr(IV) from for-[Zr(DFOT1)] (4) and for-
[Zr(PPDFOT1)] (5) at pH 7.0 indicates the value that these
types of macrocycles could have in Zr(IV)-89 PET imaging
applications, to attenuate the loss of the Zr(IV)-89 radiolabel in
vivo. The stability of the 44- or 48-membered macrocycles, for-
[Zr(DFOT1)] (4) or for-[Zr(PPDFOT1)] (5), respectively,
was in accord with previously reported Zr(IV) macrocycles.42,43
Figure 4. Structures of (a) Zr(IV)-(Me-AHA)4 from X-ray
crystallography15 or from molecular mechanics calculations of the
complex between Zr(IV) and a tetrameric macrocycle (Zr(IV):ligand
1:1) or the cognate dimeric macrocycle (Zr(IV):ligand 1:2) assembled
from (b, e) for-PBH, (c, f) for-PPH, or (d, g) for-HBH.
Table 1. Data from Molecular Mechanics Calculations of the
Zr(IV)-Loaded Macrocycles
distance COamide
−
CO/NOHX (Å)
energy
(kcal/mol)
vol.
θamide
av
complex
(Å3)
(deg) (n = 16)
min
for-[Zr(DFOT1)]
for-[Zr(DFOT1D)2]
for-[Zr(PPDFOT1)]
563
544
555
543
1928
1961
2113
2173
176.7
177.7
178.2
178.2
4.971
5.141
6.478
6.346
3.033
3.572
4.756
4.859
for-
[Zr(PPDFOT1D)2]
for-[Zr(HBDFOT1)]
552
551
2102
2117
178.1
177.5
5.176
4.829
3.087
2.922
for-
[Zr(HBDFOT1D)2]
CONCLUSION
■
four oxygen donor atoms in the relevant quadrant) > 6.3 Å,
with the minimum distance of about 4.7 Å. In the case of for-
[Zr(DFOT1)] and for-[Zr(DFOT1D)2] or for-[Zr(HBDFOT1)]
and for-[Zr(HBDFOT1D)2], there appeared to be a greater
Reaction between Zr(IV) and a selection of endo-hydroxamic
acid amino carboxylic acid ligands in a 1:4 stoichiometry in the
presence of peptide coupling reagents was used to prepare
Zr(IV)-loaded macrocycles. Of the six hydroxamic acid
F
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Inorganic Chemistry
Article
Liquid Chromatography−Mass Spectrometry. Reverse-phase
LC-MS was performed using an Agilent Technologies system (Santa
Clara, CA), consisting of an injector (100 μL loop), Agilent 1260
Infinity degasser, a binary pump, a fraction collector, a diode array
detector and an Agilent 6120 Series Quadrupole electrospray
ionization (ESI)-mass spectrometer. An Agilent C18 column reverse-
phased prepacked column (particle size: 5 μm; 4.6 × 150 mm internal
diameter) was used at flow rate of 0.5 mL min−1. Agilent OpenLAB
Chromatography Data System (CDS) ChemStation Edition was used
to process mass chromatograms in both the scan mode and the
selected ion monitoring (SIM) mode. Two different gradients were
used, as follows (A, H2O/formic acid 99.9:0.1; B, ACN/formic acid
99.9:0.1). Method A: 0−42% B over 60 min (Figure 1, Figure 3,
Figure S1). Method B: 0−15% B for 10 min, then 15−30% B over 35
min (semipreparative isolation of for-[Zr(PPDFOT1)] and for-
[Zr(PPDFOT1D)]2). MS isotope patterns were calculated using
ChemCalc.65
monomers examined using this MTS approach, for-PPH gave
the highest concentration of Zr(IV)-loaded macrocycle, which
was produced as an octadentate tetramer: for-[Zr(PPDFOT1)]
(5). An unexpected product was an octadentate Zr(IV)-loaded
complex formed with two for-PPH derived dimeric macro-
cycles: for-[Zr(PPDFOT1D)2] (10). These two complexes are
isomers. The MTS-based synthesis of for-[Zr(PPDFOT1)] (5)
was facile, albeit with the product formed in a modest yield
(about 11%). MTS may have most value in identifying the ideal
monomer required for the assembly of the optimal macrocycle,
with more conventional synthetic methods,62,63 including
metal-ion-assisted ring closure,64 required to obtain more
material. MTS also has value in revealing new coordination
chemistry for lesser known metal ions. The apo-macrocycles
PPDFOT1 (12) or homobisucaberin (HBC) (11) were
obtained from the treatment of the respective compounds
for-[Zr(PPDFOT1)] (5) or for-[Zr(PPDFOT1D)2] (10) with
EDTA at pH 4.0. Incubation of PPDFOT1 with Zr(IV) for 1 h
produced for-[Zr(PPDFOT1)] (5). At pH values closer to
physiological values, for-[Zr(DFOT1)] (4) and for-[Zr-
(PPDFOT1)] (5) were stable toward the loss of Zr(IV), with
the metal ion leached more readily from compounds containing
linear hydroxamic acid−based ligands. The design of chelators
that have high affinity and selectivity toward a given
radionuclide is a field that is strongly coupled to the efficacy
and clinical value of PET imaging.1−4 The use of MTS to
interrogate the coordination preferences of a given metal ion in
the presence of a selection of fragment ligands, could have
untapped potential in this regard. We are currently using MTS
to inform optimal ligand design of a selection of frontier
radionuclides, including Lu-177 and others.
Synthesis of Ligands. for-PBH, for-PPH, for-HBH, ret-PBH, and
ret-HBH34,47 or DFOB-for-PBH32 were prepared as described
previously. The ligand ret-PPH was prepared from precursors P1−
P4 (Scheme S1 and Figures S2−S5), with the final step undertaken
from P4 as follows.
4-(6-Amino-N-hydroxyhexanamido)butanoic acid (ret-PPH).
A sample of P4 (100 mg, 0.23 mmol) was dissolved in dichloro-
methane (4.5 mL) and trifluoroacetic acid (0.5 mL) was added. The
reaction mixture was stirred for 3 h and concentrated in vacuo to give a
yellow gum. The residue was dissolved in tert-butanol and ethyl acetate
mixture (9:1, 5 mL) and 10% Pd/C was added under a nitrogen
atmosphere. The mixture was purged with nitrogen, degassed, purged
with hydrogen and then stirred under a hydrogen atmosphere for 8 h.
The reaction mixture was quenched with water, filtered through cotton
wool and concentrated in vacuo to give the title compound as a yellow
1
gum. Yield: 86 mg, 93%. H NMR (400 MHz, CDCl3): δ 3.66 (t, J =
6.8 Hz, 2H), 2.93 (t, J = 7.6 Hz, 2H), 2.51 (t, J = 7.2 Hz, 2H), 2.32 (t,
J = 7.2 Hz, 2H), 1.90 (apparent qn, J = 6.8 Hz, 2H), 1.62−1.69 (m,
4H), 1.39−1.47 (m, 2H) (Figure S6). 13C NMR (101 MHz, CDCl3):
δ 176.9, 175.8, 49.9, 48.3, 40.5, 32.8, 32.0, 28.2, 27.0, 25.1, 23.1 (Figure
S7).
EXPERIMENTAL SECTION
■
Reagents. The following chemicals were obtained from Sigma-
Aldrich: ethyl 4-bromobutyrate (95%), O-benzylhydroxylamine hydro-
chloride (99%), Dowex 1 × 8 (Cl form), dimethyl sulfoxide (99.9%),
diphenyl phosphoryl azide (97%), di-tert-butyl dicarbonate (Boc2O;
≥98%), tert-butyl acrylate (98%), 1,5-dibromopentane (99%),
potassium phthalimide (97%), triethylamine (TEA; 99%), N,N-
dimethylformamide (DMF; 99.8%), 10% palladium on carbon, N,N-
ethyldiisopropylamine (DIPEA; 99%), tert-butanol (≥99.0%), 1,4-
dioxane (99.8%), tetrahydrofuran (THF; 99.8%), benzylchloroformate
(95%), trifluoroacetic acid (TFA; ≥ 98%), sodium carbonate (99%),
sodium hydride (60% suspension in oil), pyridine (99%), succinic
anhydride (≥97%), glutaric anhydride (99%), 6-aminohexanoic acid
(95%), 7-aminoheptanoic acid (98%), and magnesium sulfate
(MgSO4; 97%). Sodium sulfate (NaSO4) and sodium bicarbonate
(99.5%) were obtained from Chemsupply. Hydrazine hydrate (N2H4·
H2O; 45−55%) was obtained from Ajax Chemicals. N-[3-(dimethyl-
amino)propyl]-N′-ethylcarbodiimide hydrochloride (EDC·HCl; ≥
98%), N-hydroxybenzotriazole (HOBt; ≥ 99%), and N-[(dimethyl-
amino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methyl-
methanaminium hexafluorophosphate N-oxide (HATU, 97%) were
obtained from ChemImpex. Hydrogen (H2) and nitrogen (N2) gases
were sourced from BOC. All chemicals were used as received. Milli-Q
water was used for all experiments.
Metal-Templated Synthesis. A 1:4 solution of Zr(IV) (acac)4
and the endo-hydroxamic acid (2 mg/mL concentration with respect to
the ligand) was prepared in 1:1 methanol/H2O and the solution was
stirred for 1 h. The mixture was concentrated to dryness in vacuo and
was redissolved in DMF to a concentration of 1 mg/mL with respect
to the ligand. Diphenylphosphoryl azide (6 equiv) and Et3N (6 equiv)
were added and the mixture was stirred at ambient temperature for 5
days. The reaction mixture was then quenched with cold H2O (a
volume equal to the volume of DMF). Aliquots (10 μL) were analyzed
by LC-MS following HPLC method A. The mixture containing for-
[Zr(DFOT1)] or the mixture containing for-[Zr(PPDFOT1)] and for-
[Zr(PPDFOT1D)2] was further purified as follows. To the reaction
mixture was added H2O (10 mL per 1 mL reaction mixture) and
Dowex 1 × 8 (0.1 g per 1 mL of reaction mixture) and was stirred for
24 h. The mixture was filtered and washed with 1:1 DMF/H2O (2 ×
50 mL), and the filtrate was concentrated in vacuo. The residue was
redissolved in 1:1 DMSO/H2O and purified by semipreparative HPLC
following method C. Sufficient quantity of for-[Zr(PPDFOT1)] was
1
obtained for an H NMR spectrum (400 MHz, CDCl3): δ 8.39−8.46
(m, 2.3 H, exchange with solvent), 3.87−3.98 (m, 4H), 3.70−3.83 (m,
4H, 3.51−3.50 (m, 4H), 3.01−3.12 (m, 4H), 2.63−2.72 (m, 2H),
1.81−2.26 (m, 26H), 1.48−1.76 (m, 14H), 1.16−1.38 (m, 12H)
(Figure S8).
1
1H NMR and 13C NMR Spectroscopy. H NMR and 13C NMR
Stability of Zr(IV) Complexes. Stock solutions (1 mg/mL) of for-
[Zr(DFOT1)] (4), for-[Zr(PPDFOT1)] (5), for-[Zr(PPDFOT1D)2]
(10), DFOB-for-PBH or DFOB were prepared in methanol. An
aliquot (10 μL) of the stock solution of the relevant compound was
added to an aliquot (190 μL) of EDTA (100 mM) at pH 4.0 (Figure
3) or pH 7.0 (Figure 5). Aliquots (10 μL) were removed at specified
time points and the solutions were analyzed by LC-MS.
spectroscopy was carried out using a Varian 400-MR NMR
spectrometer (Lexington, MA) at a frequency of 399.73 MHz at 24
°C operated with VnmrJ 3.1 software (Agilent Technologies, Santa
Clara, CA). The spectral data are reported in ppm (δ) relative to their
residual solvent peaks for CDCl3 (7.27 ppm (1H), 77.23 ppm (13C))
or CD3OD (3.31 ppm, 4.87 ppm (1H), 49.00 ppm (13C)) or DMSO-
d6 (2.50 ppm (1H), 39.52 ppm (13C)). Coupling constants (J) are
reported in Hz and splitting are reported as singlet (s), doublet (d), t
(triplet), q (quartet), and qn (quintet).
Reassembly of Zr(IV) Complexes. Solutions containing EDTA
(171 μL, 100 mM) and for-[Zr(PPDFOT1)] (5) (9 μL of 1 mg/mL)
G
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Inorganic Chemistry
Article
or for-[Zr(PPDFOT1D)2] (10) (9 μL of 1 mg/mL) were pH adjusted
to 4.0 and after 60 h were purified by HPLC (Method A). PPDFOT1
(12) was collected from 34.5−39.5 min and homobisucaberin (11)
was collected from 26.5−29.5 min. The fractions were pooled,
lyophilized and redissolved in Milli-Q water (200 μL). A sample of
PPDFOT1 (12) (10 μL) or homobisucaberin (11) was mixed with an
aliquot (10 μL) of Zr(acac)4 (0.25 mg/mL in 1:100 methanol:H2O)
and after incubation for 1 h was analyzed by LC-MS.
(4) Boros, E.; Marquez, B. V.; Ikotun, O. F.; Lapi, S. E.; Ferreira, C.
L. In Ligand Design in Medicinal Inorganic Chemistry; Storr, T., Ed.;
John Wiley & Sons, Ltd: Chichester, UK, 2014.
(5) Fischer, G.; Seibold, U.; Schirrmacher, R.; Wangler, B.; Wangler,
̈
̈
C. 89Zr, a radiometal nuclide with high potential for molecular imaging
with PET: chemistry, applications and remaining challenges. Molecules
2013, 18, 6469−6490.
(6) Blower, P. J. A nuclear chocolate box: the periodic table of
nuclear medicine. Dalton Trans. 2015, 44, 4819−4844.
(7) Brasse, D.; Nonat, A. Radiometals: towards a new success story in
nuclear imaging. Dalton Trans. 2015, 44, 4845−4858.
(8) van Dongen, G. A. M. S.; Huisman, M. C.; Boellaard, R.;
Hendrikse, N. H.; Windhorst, A. D.; Visser, G. W. M.; Molthoff, C. F.
M.; Vugts, D. J. 89Zr-immuno-PET for imaging of long circulating
drugs and disease targets: why, how and when to be applied? Q. J.
Nucl. Med. Mol. Imaging 2015, 59, 18−38.
(9) Scharli, R. K.; Price, R. I.; Chan, S.; Cryer, D.; Jeffery, C. M.;
Asad, A. H.; Morandeau, L.; Eu, P.; Cullinane, C.; Kasbollah, A.;
Katsifis, A. Establishing reliable production of the PET isotope 89Zr
for research use: From target fabrication to preclinical imaging. AIP
Conf. Proc. 2012, 1509, 101−107.
(10) Verel, I.; Visser, G. W. M.; Boellaard, R.; Stiger-van Walsum, M.;
Snow, G. B.; van Dongen, G. A. M. S. 89Zr immuno-PET:
Comprehensive procedures for the production of 89Zr-labeled
monoclonal antibodies. J. Nucl. Med. 2003, 44, 1271−1281.
(11) Rice, S. L.; Roney, C. A.; Daumar, P.; Lewis, J. A. The next
generation of positron emission tomography radiopharmaceuticals in
oncology. Semin. Nucl. Med. 2011, 41, 265−282.
(12) Deri, M. A.; Zeglis, B. M.; Francesconi, L. C.; Lewis, J. S. PET
imaging with 89Zr: From radiochemistry to the clinic. Nucl. Med. Biol.
2013, 40, 3−14.
Molecular Mechanics Calculations. Structures of for-[Zr-
(DFOT1)] and for-[Zr(DFOT1D)2] were built using HyperChem
15
(version 7.5) from the X-ray coordinates of Zr(Me-AHA)4 and
fragments of for-PBH from Fe(III)-DFOB.61 Additional methylene
groups were inserted for for-[Zr(PPDFOT1)], for-[Zr(PPDFOT1D)2],
for-[Zr(HBDFOT1)], and for-[Zr(HBDFOT1D)2]. Structures beyond
atoms that included Zr(IV) and those in the first (O8) and second
(C4N4) coordination spheres were subject to energy minimization,
using the AMBER2 force field. Further details and coordinates for all
structures are provided (Table S1−S6).
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.7b00362.
Data is provided for the synthesis of ret-PPH (Scheme 1,
Figures S1−S6), the Zr(IV)-based for-PBH MTS system
showing for-[Zr(DFOT1)], the linear precursor of for-
1
[Zr(DFOT1)] and [Zr(DFOE)]+ (Figure S7), the H
NMR spectrum of for-[Zr(PPDFOT1)] (Figure S8); the
repeat MTS-based synthesis with for-PPH (Figure S9);
HRMS data for for-[Zr(PPDFOT1)], for-[Zr-
(PPDFOT1D)2], PPDFOT1 or PPDFOT1D (HBC)
(13) Baggio, R.; Garland, M. T.; Perec, M. Preparation and X-ray
crystal structure of the polymeric zirconium(IV) oxalate complex
[K2{Zr(C2O4)3}·H2C2O4·H2O]n. Inorg. Chem. 1997, 36, 737−739.
(14) Baroncelli, F.; Grossi, G. The complexing power of hydroxamic
acids and its effect on the behaviour of organic extractants in the
reprocessing of irradiated fuels. I. The complexes between
benzohydroxamic acid and zirconium, iron(III) and uranium(IV). J.
Inorg. Nucl. Chem. 1965, 27, 1085−1092.
1
(Figures S10−13, respectively); the H NMR spectrum
of DFOB-for-PBH (Figure S14); and the coordinates of
the structures of for-[Zr(DFOT1)], for-[Zr(DFOT1D)2],
for-[Zr(PPDFOT1)], for-[Zr(PPDFOT1D)2], for-[Zr-
(HBDFOT1)], and for-[Zr(HBDFOT1D)2] (Tables
S1−S6) (PDF)
́
(15) Guerard, F.; Lee, Y.-S.; Tripier, R.; Szajek, L. P.; Deschamps, J.
R.; Brechbiel, M. W. Investigation of Zr(IV) and 89Zr(IV) complex-
ation with hydroxamates: progress towards designing a better chelator
than desferrioxamine B for immuno-PET imaging. Chem. Commun.
2013, 49, 1002−1004.
AUTHOR INFORMATION
Corresponding Author
*E-mail: rachel.codd@sydney.edu.au.
■
(16) Codd, R. Traversing the coordination chemistry and chemical
biology of hydroxamic acids. Coord. Chem. Rev. 2008, 252, 1387−1408.
(17) Hider, R. C.; Kong, X. Chemistry and biology of siderophores.
Nat. Prod. Rep. 2010, 27, 637−657.
(18) Braich, N.; Codd, R. Immobilized metal affinity chromatography
for the capture of hydroxamate-containing siderophores and other
Fe(III)-binding metabolites from bacterial culture supernatants.
Analyst 2008, 133, 877−880.
(19) Ejje, N.; Soe, C. Z.; Gu, J.; Codd, R. The variable hydroxamic
acid siderophore metabolome of the marine actinomycete Salinispora
tropica CNB-440. Metallomics 2013, 5, 1519−1528.
ORCID
Rachel Codd: 0000-0002-2703-883X
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The Australian Research Council (DP140100092) is acknowl-
edged for research support to R.C. and A.K. and for salary
support to W.T. The University of Sydney is acknowledged for
the provision of a postgraduate research award (cofunded) to
T.L.
(20) Meijs, W. E.; Herscheid, J. D. M.; Haisma, H. J.; Pinedo, H. M.
Evaluation of desferal as a bifunctional chelating agent for labeling
antibodies with Zr-89. Appl. Radiat. Isot. 1992, 43, 1443−1447.
(21) Meijs, W. E.; Haisma, H. J.; Klok, R. P.; van Gog, F. B.; Kievit,
E.; Pinedo, H. M.; Herscheid, J. D. Zirconium-labeled monocloncal
antibodies and their distribution in tumor-bearing nude mice. J. Nucl.
Med. 1997, 38, 112−118.
REFERENCES
■
(1) Price, E. W.; Orvig, C. Matching chelators to radiometals for
radiopharmaceuticals. Chem. Soc. Rev. 2014, 43, 260−290.
(22) Borjesson, P. K. E.; Jauw, Y. W. S.; Boellaard, R.; de Bree, R.;
̈
Comans, E. F. I.; Roos, J. C.; Castelijns, J. A.; Vosjan, M. J. W. D.;
Kummer, J. A.; Leemans, C. R.; Lammertsma, A. A.; van Dongen, G.
A. M. S. Performance of immuno-positron emission tomography with
zirconium-89-labeled chimeric monoclonal antibody U36 in the
detection of lymph node metastases in head and neck cancer patients.
Clin. Cancer Res. 2006, 12, 2133−2140.
(2) Zeglis, B. M.; Houghton, J. L.; Evans, M. J.; Viola-Villegas, N.;
Lewis, J. S. Underscoring the influence of inorganic chemistry on
nuclear imaging with radiometals. Inorg. Chem. 2014, 53, 1880−1899.
(3) Price, T. W.; Greenman, J.; Stasiuk, G. J. Current advances in
ligand design for inorganic positron emission tomography tracers
68Ga, 64Cu, 89Zr and 44Sc. Dalton Trans. 2016, 45, 15702−15724.
H
DOI: 10.1021/acs.inorgchem.7b00362
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
(23) van Rij, C. M.; Sharkey, R. M.; Goldenberg, D. M.; Frielink, C.;
Molkenboer, J. D.; Franssen, G. M.; van Weerden, W. M.; Oyen, W. J.;
Boerman, O. C. Imaging of prostate cancer with immuno-PET and
immuno-SPECT using a radiolabeled anti-EPG-1 monoclonal anti-
body. J. Nucl. Med. 2011, 52, 1601−1607.
(24) Dijkers, E. C. F.; Kosterink, J. G. W.; Rademaker, A. P.; Perk, L.
R.; van Dongen, G. A. M. S.; Bart, J.; de Jong, J. R.; de Vries, E. G. E.;
Lub-de Hooge, M. N. Development and characterisation of clinical-
grade 89Zr-trastuzumab for HER2/neu imminoPET imaging. J. Nucl.
Med. 2009, 50, 974−981.
with 89Zr4+: Comparison with desferrioxamine B. Dalton Trans. 2015,
44, 4884−4900.
(40) Deri, M. A.; Ponnala, S.; Kozlowski, P.; Burton-Pye, B. P.; Cicek,
H. T.; Hu, C.; Lewis, J. S.; Francesconi, L. C. p-SCN-Bn-HOPO: a
superior bifunctional chelator for 89Zr immunoPET. Bioconjugate
Chem. 2015, 26, 2579−2591.
(41) Rudd, S. E.; Roselt, P.; Cullinane, C.; Hicks, R. J.; Donnelly, P.
S. A desferrioxmaine B squaramide ester for the incorporation of
zirconium-89 into antibodies. Chem. Commun. 2016, 52, 11889−
11892.
(25) Holland, J. P.; Caldas-Lopes, E.; Divilov, V.; Longo, V. A.;
Taldone, T.; Zatorska, D.; Chiosis, G.; Lewis, J. S. Measuring the
pharmacodynamic effects of a novel Hsp90 inhibitor on HER2/neu
expression in mice using 89Zr-DFO-trastuzumab. PLoS One 2010, 5,
e8859.
(26) Petrik, M.; Zhai, C.; Novy, Z.; Urbanek, L.; Haas, H.;
Decristoforo, C. In vitro and in vivo comparison of selected Ga-68
adn Zr-89 labelled siderophores. Mol. Imaging Biol. 2016, 18, 344−
352.
(27) Jauw, Y. W. S.; Menke-van der Houven van Oordt, C. W.;
Hoekstra, O. S.; Hendrikse, N. H.; Vugts, D. J.; Zijlstra, J. M.;
Huisman, M. C.; van Dongen, G. A. M. S. Immuno-positron emission
tomography with zirconium-89-labeled monoclonal antibodies in
oncology: waht can we learn from initial clinical trials? Front.
Pharmacol. 2016, 7 (131), 15.
(28) Holland, J. P.; Vasdev, N. Charting the mechanism and
reactivity of zirconium oxalate with hydroxamate ligands using density
functional theory: Implications in new chelate design. Dalton Trans.
2014, 43, 9872−9884.
́
(42) Guerard, F.; Lee, Y.-S.; Brechbiel, M. W. Rational design,
synthesis, and evaluation of tetrahydroxamic acid chelators for stable
complexation of zirconium(IV). Chem. - Eur. J. 2014, 20, 5584−5591.
(43) Boros, E.; Holland, J. P.; Kenton, N.; Rotile, N. J.; Caravan, P.
Macrocycle-based hydroxamate ligands for complexation and
immunoconjugation of 89zirconium for positron emission tomography
(PET) imaging. ChemPlusChem 2016, 81, 274−281.
(44) McMurry, T. J.; Raymond, K. N.; Smith, P. H. Molecular
recognition and metal ion template synthesis. Science 1989, 244, 938−
943.
(45) McMurry, T. J.; Rodgers, S. J.; Raymond, K. N. Template and
stepwise synthesis of a macrobicyclic catechoylamide ferric ion
sequestering agent. J. Am. Chem. Soc. 1987, 109, 3451−3453.
́
(46) Kachadourian, R.; Chuilon, S.; Merienne, C.; Kunesch, G.;
Deroussent, A. A New Total Synthesis of Ferrioxamine E through
Metal-templated Cyclic Trimerization. Supramol. Chem. 1997, 8, 301−
308.
(47) Lifa, T.; Tieu, W.; Hocking, R. K.; Codd, R. Forward and
reverse (retro) iron(III)- or gallium(III)-desferrioxamine E and ring-
expanded analogs prepared using metal-templated synthesis from endo-
hydroxamic acid monomers. Inorg. Chem. 2015, 54, 3573−3583.
(48) Giordanetto, F.; Kihlberg, J. Macrocyclic drugs and clinical
candidates: What can medicinal chemists learn from their properties? J.
Med. Chem. 2014, 57, 278−294.
(49) Driggers, E. M.; Hale, S. P.; Lee, J.; Terrett, N. K. The
exploration of macrocycles for drug discovery - an underexploited
structural class. Nat. Rev. Drug Discovery 2008, 7, 608−624.
(50) Marsault, E.; Peterson, M. L. Macrocycles are great cycles:
Applications, opportunities, and challenges of synthethic macrocycles
in drug discovery. J. Med. Chem. 2011, 54, 1961−2004.
(51) Heinis, C. Drug discovery. Tools and rules for macrocycles. Nat.
Chem. Biol. 2014, 10, 696−698.
(52) Marti-Centelles, V.; Pandey, M. D.; Burguete, M. I.; Luis, S. V.
Macrocyclization reactions: the importance of conformational,
configurational, and template-induced preorganisation. Chem. Rev.
2015, 115, 8736−8834.
(53) Marti-Centelles, V.; Burguete, M. I.; Luis, S. V. Macrocycle
synthesis by chloride-templated amide bond formation. J. Org. Chem.
2016, 81, 2143−2147.
(54) Feistner, G. J.; Stahl, D. C.; Gabrik, A. H. Proferrioxamine
siderophores of Erwinia amylovora. A capillary liquid chromato-
graphic/electrospray tandem mass spectrometric study. Org. Mass
Spectrom. 1993, 28, 163−175.
(55) Nishio, T.; Tanaka, N.; Hiratake, J.; Katsube, Y.; Ishida, Y.; Oda,
J. Isolation and structure of the novel dihydroxamate siderophore
alcaligin. J. Am. Chem. Soc. 1988, 110, 8733−8734.
(56) Takahashi, A.; Nakamura, H.; Kameyama, T.; Kurasawa, S.;
Naganawa, H.; Okami, Y.; Takeuchi, T.; Umezawa, H.; Iitaka, Y.
Bisucaberin, a new siderophore, sensitizing tumor cells to macrophage-
mediated cytolysis. II. Physico-chemical properties and structure
determination. J. Antibiot. 1987, 40, 1671−1676.
(57) Ledyard, K. M.; Butler, A. Structure of putrebactin, a new
dihydroxamate siderophore produced by Shewanella putrefaciens. JBIC,
J. Biol. Inorg. Chem. 1997, 2, 93−97.
(29) Holland, J. P.; Divilov, V.; Bander, N. H.; Smith-Jones, P. M.;
Larson, S. M.; Lewis, J. S. 89Zr-DFO-J591 for immunoPET of prostate-
specific membrane antigen expression in vivo. J. Nucl. Med. 2010, 51,
1293−1300.
(30) Abou, D. S.; Ku, T.; Smith-Jones, P. M. In vivo biodistribution
and accumulation of 89Zr in mice. Nucl. Med. Biol. 2011, 38, 675−681.
(31) Nayak, T. K.; Garmestani, K.; Milenic, D. E.; Brechbiel, M. W.
PET and MRI of metastatic peritoneal and pulmonary colorectal
cancer in mice with human epidermal growth factor receptor 1-
targeted 89Zr-labeled panitumumab. J. Nucl. Med. 2012, 53, 113−120.
(32) Patra, M.; Bauman, A.; Mari, C.; Fischer, C. A.; Blacque, O.;
Haussinger, D.; Gasser, G.; Mindt, T. L. An octadentate bifunctional
chelating agent for the development of stable zirconium-89 based
molecular imaging probes. Chem. Commun. 2014, 50, 11523−11525.
(33) Vugts, D. J.; Klaver, C.; Sewing, C.; Poot, A. J.; Adamzek, K.;
Huegli, S.; Mari, C.; Visser, G. W. M.; Valverde, I. E.; Gasser, G.;
Mindt, T. L.; van Dongen, G. A. M. S. Comparison of the octadentate
bifunctional chelator DFO*-pPhe-NCS and the clinically used
hexadentate bifunctional chelator DFO-pPhe-NCS for 89-Zr-immu-
no-PET. Eur. J. Nucl. Med. Mol. Imaging 2017, 44, 286−295.
(34) Kadi, N.; Oves-Costales, D.; Barona-Gomez, F.; Challis, G. L. A
new family of ATP-dependent oligomerization-macrocyclization
biocatalysts. Nat. Chem. Biol. 2007, 3, 652−656.
(35) Soe, C. Z.; Codd, R. Unsaturated macrocyclic dihydroxamic acid
siderophores produced by Shewanella putrefaciens using precursor-
directed biosynthesis. ACS Chem. Biol. 2014, 9, 945−956.
(36) Telfer, T. J.; Gotsbacher, M. P.; Soe, C. Z.; Codd, R. Mixing up
the pieces of the desferrioxamine B jigsaw defines the biosynthetic
sequence catalyzed by DesD. ACS Chem. Biol. 2016, 11, 1452−1462.
(37) Richardson-Sanchez, T.; Tieu, W.; Codd, R. Reverse biosyn-
thesis: Generating combinatorial pools of drug leads from enzyme-
mediated fragmentation of natural products. ChemBioChem 2017, 18,
368−373.
(38) Deri, M. A.; Ponnala, S.; Zeglis, B. M.; Pohl, G.; Dannenberg, J.
J.; Lewis, J. S.; Francesconi, L. C. Alternative chelator for 89Zr
radiopharmaceuticals: radiolabeling and evaluation of 3,4,3,-(LI-1,2-
HOPO). J. Med. Chem. 2014, 57, 4849−4860.
(39) Ma, M. T.; Meszaros, L. K.; Paterson, B. M.; Berry, D. J.;
Cooper, M. S.; Ma, Y. M.; Hider, R. C.; Blower, P. J. Tripodal
tris(hydroxypyridinone) ligands for immunoconjugate PET imaging
(58) Soe, C. Z.; Pakchung, A. A. H.; Codd, R. Directing the
biosynthesis of putrebactin or desferrioxamine B in Shewanella
putrefaciens through the upstream inhibition of ornithine decarbox-
ylase. Chem. Biodiversity 2012, 9, 1880−1890.
I
DOI: 10.1021/acs.inorgchem.7b00362
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
(59) Boettcher, T.; Clardy, J. A chimeric siderophore halts swarming
Vibrio. Angew. Chem., Int. Ed. 2014, 53, 3510−3513.
(60) Soe, C. Z.; Telfer, T. J.; Levina, A.; Lay, P. A.; Codd, R.
Simultaneous biosynthesis of putrebactin, avaroferrin and bisucaberin
by Shewanella putrefaciens and characterisation of complexes with
iron(III), molybdenum(VI) or chromium(V). J. Inorg. Biochem. 2016,
162, 207−215.
(61) Dhungana, S.; White, P. S.; Crumbliss, A. L. Crystal structure of
ferrioxamine B: A comparative analysis and implications for molecular
recognition. JBIC, J. Biol. Inorg. Chem. 2001, 6, 810−818.
(62) Bergeron, R. J.; McManis, J. S. The total synthesis of
desferrioxamines E and G. Tetrahedron 1990, 46, 5881−5888.
(63) Bergeron, R. J.; McManis, J. S.; Phanstiel, O. I.; Vinson, J. R. T.
A versatile synthesis of deferrioxamine B. J. Org. Chem. 1995, 60, 109−
114.
(64) Wencewicz, T. A.; Oliver, A. G.; Miller, M. J. Iron(III)-
templated macrolactonization of trihydroxamate siderophores. Org.
Lett. 2012, 14, 4390−4393.
(65) Patiny, L.; Borel, A. ChemCalc: a building block for tomorrow’s
chemical infrastructure. J. Chem. Inf. Model. 2013, 53, 1223−1228.
J
DOI: 10.1021/acs.inorgchem.7b00362
Inorg. Chem. XXXX, XXX, XXX−XXX