Structural and Thermodynamics Studies on Polyaminophosphonate Ligands for Uranyl Decorporation

Article abstract of DOI:10.1021/acs.inorgchem.0c02145

The development of actinide decorporation agents with high complexation affinity, high tissue specificity, and low biological toxicity is of vital importance for the sustained and healthy development of nuclear energy. After accidental actinide intake, sequestration by chelation therapy to reduce acute damage is considered as the most effective method. In this work, a series of bis- and tetra-phosphonated pyridine ligands have been designed, synthesized, and characterized for uranyl (UO22+) decorporation. Owing to the absorption of the ligand and the luminescence of the uranyl ion, UV-vis spectroscopy and time-resolved laser-induced fluorescence spectroscopy (TRLFS) were used to probe in situ complexation and structure variation of the complexes formed by the ligands with uranyl. Density functional theory (DFT) calculations and X-ray absorption fine structure (XAFS) spectroscopy on uranyl-ligand complexes revealed the coordination geometry around the uranyl center at pH 3 and 7.4. High affinity constants (log K ～17) toward the uranyl ion were determined by displacement titration. A preliminary in vitro chelation study proves that bis-phosphonated pyridine ligands can remove uranium from calmodulin (CaM) at a low dose and in the short term, which supports further uranyl decorporation applications of these ligands.

Full text of DOI:10.1021/acs.inorgchem.0c02145

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Structural and Thermodynamics Studies on Polyaminophosphonate
Ligands for Uranyl Decorporation
Gaoyang Ye,* Jérome Roques, Pier-Lorenzo Solari, Christophe Den Auwer, Aurélie Jeanson,
́
̀
Jérémy Brandel, Loïc J. Charbonniere, Wangsuo Wu, and Eric Simoni*
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ABSTRACT: The development of actinide decorporation agents with high
complexation affinity, high tissue specificity, and low biological toxicity is of vital
importance for the sustained and healthy development of nuclear energy. After
accidental actinide intake, sequestration by chelation therapy to reduce acute
damage is considered as the most effective method. In this work, a series of bis-
and tetra-phosphonated pyridine ligands have been designed, synthesized, and
characterized for uranyl (UO22+) decorporation. Owing to the absorption of the
ligand and the luminescence of the uranyl ion, UV−vis spectroscopy and time-
resolved laser-induced fluorescence spectroscopy (TRLFS) were used to probe in
situ complexation and structure variation of the complexes formed by the ligands
with uranyl. Density functional theory (DFT) calculations and X-ray absorption fine structure (XAFS) spectroscopy on uranyl−
ligand complexes revealed the coordination geometry around the uranyl center at pH 3 and 7.4. High affinity constants (log K ∼17)
toward the uranyl ion were determined by displacement titration. A preliminary in vitro chelation study proves that bis-
phosphonated pyridine ligands can remove uranium from calmodulin (CaM) at a low dose and in the short term, which supports
further uranyl decorporation applications of these ligands.
unfortunately, after in vivo tests, only a few of them could
reduce the retention. Furthermore, the toxicities of these
ligands are needed to be taken into account in order to pass
the clinical trial.8,9
INTRODUCTION
■
With the development of the nuclear energy industry, the
manipulation of the world stock of uranium leads us to
consider uranium decorporation after possible accidental
contamination. The hexavalent uranyl ion (UVIO22+) is the
most stable form in aqueous solutions and biological media.1
After an internal contamination, the solubilized uranium could
be distributed to the target organs via the bloodstream. The
uranyl ion preferentially transfers to the skeletal, liver, and
kidney tissues and is retained in these target organs.2 Due to its
chemotoxicity and radiotoxicity, the acute effect on health
could cause death and the long-term effects will result in
cancer, anemia, and necrosis.3 In order to limit sequence
damage to organs, chelating agents with high stability constants
and high selectivities toward the uranyl ion can be used to
promote uranyl excretion from the target organs. The resulting
uranyl complexes should also be soluble in biological
conditions in order to be excreted by urine or bile.4,5
We therefore considered a series of multidentate phospho-
nated pyridine ligands for the uranyl decorporation study.
These ligands were originally designed for the formation of
magnetic resonance imaging (MRI) contrast agents and for
imaging by single-photon emission computed tomography
(SPECT).11,12 Experiments carried out on mice with 99mTc
and the bis-phosphonated ligand L1 and tetra-phosphonated
ligand L2 (Figure 1) have shown that the complexes formed
with L1 could rapidly be excreted though the kidneys and
bladder while those of the tetra-phosphonated ligand L2 were
fixed on the bone tissues.11 A further test with the Gd3+
complex of ligand L2 revealed a fast clearance in blood, while
the contrast agent was distributed to kidneys, liver, and muscle.
Along the experiment, the mouse could tolerate the
administration of the contrast agent.13,14 Meanwhile, the
complex of Eu3+ with ligand L2 showed that the ligand could
Up to now, several ligands have been proven to be efficient
for uranyl decorporation with acceptable side effects.6−9
2+
According to Pearson’s rules, the UO2 ion is defined as a
Received: July 18, 2020
Published: February 1, 2021
hard acid and thus is prone to react with hard bases like
phosphonate anions. 1-Hydroxyethane-1,1-diphosphonic acid
(HEDP) is used in clinical studies for uranyl decorporation
due to the high affinity of bisphosphonates toward the uranyl
ion.10 Many phosphonate ligands that are designed for uranyl
decorporation could complex the uranyl ion tightly, but,
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Figure 1. Bis-phosphonated ligands L1, L3, L4, and tetra-phosphonated ligand L2.
solid products using an AG 245 Mettler Toledo analytical balance
(precision 0.01 mg). Sodium hydroxide (NaOH) and hydrochloric
acid (HCl) were used to adjust pH during titrations. The ionic
strength of all the solutions was fixed to 0.1 M with sodium
perchlorate (Merck, p.a., NaClO4). All the experiments described
were repeated at least three times.
UV−Visible. UV−vis spectrophotometric analysis was performed
on an Agilent Technologies Cary 60 UV−vis apparatus equipped with
a deuterium lamp and a tungsten lamp. The measurements were
carried out in scanning mode from 200 to 800 nm (a scanning speed
of 3.3 nm/s).
Displacement Titration. 0.0125 M HEPES pH 7.4 buffers were
prepared by dissolving the acid form of HEPES buffer (12.5 mmol)
and 112.5 mmol of n-Bu4Cl in 1 L of Millipore water. The pH was
adjusted using a pH meter by addition of nBu4N+OH− in H2O. For
the 100 μM Sulfochlorophenol S (SCP) solution, 6.25 mL of 4 mM
SCP stock solution was mixed in 244 mL of appropriate buffer. For
the SCP-UO22+ solution at 100 μM, 6.25 mL of stock solution of SCP
at 4 mM was mixed in 243 mL of water, and 1.2 mL of uranyl stock
solution (20 mM) were added. Both stock solutions were stored for 1
h before use. The ligand solutions were prepared by diluting the stock
solutions of 2 mM ligands with water to obtain a final concentration
of 400 μM.
In each 1 mL centrifugation tube, 100 μL of SCP-UO2 solution (40
μM) in HEPES buffer was added, and different volumes of each ligand
solution (5 mM) were added to reach 0 to 10 equiv. of ligand
concentration. Samples of SCP solution, SCP-UO2 solution, and
ligand solution at the same concentration were also prepared as
reference. The tubes were stored in the dark at room temperature for
36 h for equilibration. The samples were read using a Cary 60 UV−vis
apparatus. Measurements were made at 690 nm for pH = 7.4.
Using the Hyss speciation program, the complexation conditional
constants were calculated assuming that the complex formed L
(tested)−uranyl has a stoichiometry of 1:1 in our experimental
conditions.18
offer four five-membered chelate rings at the equatorial
plane,12 which is well suited for uranyl ion coordination.
Considering the fast excretion, low toxicity, and high affinity,
phosphonated pyridine ligands designed for uranyl ion
coordination could represent a promising alternative for uranyl
decorporation studies.
In this study, two pentadentate bis-phosphonated pyridine
ligands, L3 and L4 (Figure 1), bearing five donor atoms for an
equatorial planar coordination, were designed and synthesized
to improve the lipophilic property and oral efficiency.4,5,15
Structural and thermodynamic studies were performed for the
two bis-phosphonated ligands, L3 and L4 (Figure 1). In
addition, the stability of the uranyl−ligand complex obtained
with L4 was tested by competition with cations, and first
chelation results with the calmodulin protein (CaM) supports
potential use in decorporation. Three additional tetra-
phosphonated ligands L1, L2, and L5 were also prepared but
are not discussed in this report.
EXPERIMENTAL SECTION
■
General Methods. Solvents and starting materials were purchased
from Aldrich, Acros, and Alfa Aesar and used without further
purification. Elemental analyses and mass spectrometry analysis were
carried out by the Service Commun d’Analyses of the University of
1
Strasbourg. H, 13C, and 31P NMR spectra were recorded on Bruker
Avance 300 and Avance 400 spectrometers operating at 300 and 400
MHz, respectively. Chemical shifts are reported in ppm, relative to
residual protonated solvent as internal reference.16 Elemental analysis
and monoisotopic masses were calculated with the Chemcalc
software.17
Physicochemical Studies. Materials. Distilled water was purified
by passing through a mixed bed of ion exchange (Bioblock Scientific
R3-83002, M3-83006) and activated carbon (Bioblock Scientific
ORC-83005). All the stock solutions were prepared by weighing the
TRLFS. The experiments were conducted using a laser excitation
source Nd:YAG (7 ns pulse at a frequency of 10 Hz with an energy of
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2 mJ) of the Continuum Company coupled to an Optical Parametric
Oscillator (OPO, Panther, Continuum). Detection was performed
using a Spectra-Pro-300 monochromator (Acton Research Corpo-
ration) coupled to a CCD camera (Princeton Instruments). The
beam passes through a quartz cuvette containing the solution to be
analyzed before reaching the detector. A lens and mirror system
focuses the fluorescence beam onto the input slot of the
spectrophotometer that selects the wavelength range to be observed.
The CCD camera makes it possible to adjust the various
measurement parameters for the resolution in time: the delay, the
gate width, the integration time, and the number of spectra. Spectra
were recorded using WINSPEC software (Princeton Instruments).
The excitation wavelength chosen for the measurement of uranium
was 430 nm. The error on the intensity of the fluorescence was less
than 5%, and the resolution was greater than 0.2 nm.
1,3,5-Tribenzylhexahydrotriazine (2). To a stirred solution of
benzylamine (20.7 g, 193 mmol) at 0 °C, 37% aqueous formaldehyde
solution (19.4 mL, 60.7 mmol) was slowly added keeping the
temperature below 5 °C. 1 M aqueous sodium hydroxide solution (5
mL) was added to the resulting precipitated gum, and the mixture was
submitted to an ultrasonic treatment for 1 h at 0 °C. Diethyl ether
(190 mL) was added, and the aqueous phase was further extracted
with diethyl ether (3 × 50 mL). The combined organic layers were
dried with Na2SO4, and the solvent was removed under reduced
pressure to yield a colorless oil. The crude product was purified by
flash chromatography (cyclohexane/ethyl acetate 100:0 to 85:15).
Compound 2 was obtained as a colorless oil (21.1 g, 91%). 1H NMR
(300 MHz, CDCl3): δ 7.33−7.05 (m, 15H), 3.59 (s, 6H), 3.34 (s
broad, 5H).
Diethyl ((Benzylamino)methyl)phosphonate (3). Compound 2 (4
g, 11.2 mmol) was added to diethyl phosphite (5.1 g, 36.9 mmol)
under nitrogen, and the mixture was heated at 100 °C for 16 h. The
resulting product was purified by flash chromatography (ethyl
DFT Computational Details. All the structures were optimized by
the DFT approach as implemented in the Gaussian09 package.19
Calculations were performed using the hybrid B3LYP functional.20
MWB60 Stuttgart relativistic effective core potentials21 were used to
describe the uranium atoms, MWB2 was used for the oxygen,
nitrogen, and carbon atoms, and the 6-31G basis set was used for
hydrogen. Solvation was introduced using a dielectric continuum
model of permittivity ε0 = 80. The conductor-like polarizable
continuum model (CPCM) implemented in Gaussian09 was used
as well.
1
acetate) to give compound 3 as an oily product (3.65 g, 79%). H
NMR (300 MHz, CDCl3): δ 7.33−7.09 (m, 5H), 4.07 (m, 4H), 3.80
(s, 2H), 2.88 (d, JPH = 12.6 Hz, 2H), 1.61 (s broad, 1H), 1.26 (t, J =
7.1 Hz, 6H). 31P NMR (162 MHz, CDCl3): δ 25.7.
Tetraethyl (((Pyridine-2,6-diylbis(methylene))bis-
(benzylazanediyl))bis(methylene))bis(phosphonate) (4). Under a
nitrogen atmosphere, amine 3 (1.1 g, 4.3 mmol) and freshly flame-
dried K2CO3 (1.2 g, 9.1 mmol) were added to a solution of 2,6-
bis(bromomethyl)pyridine 1 (600 mg, 2.26 mmol) in MeCN (20
mL). The mixture was heated for 16 h at 70 °C. After filtration, the
solvent was removed in vacuo, and the crude oil was purified by flash
chromatography (ethyl acetate/MeOH 100:0 to 90:10). Compound 4
EXAFS. EXAFS Sample Preparation. The uranyl/L2 1/1.2 solutions
(∼2 mM) were prepared by increasing the pH to 10 with sodium
hydroxide (1 M) to solubilize the precipitate formed in acidic
conditions after mixing the uranyl stock solution and L2 stock
solution, and then hydrochloric acid (0.1 M) and sodium hydroxide
(0.1 M) were used to reach pH 3 and 7.4, respectively. The uranyl/L4
1:1.2 solutions were prepared similarly.
1
was obtained as an oily light-yellow product (1.1 g, 79%). H NMR
(400 MHz, CDCl3): δ 7.59 (t, J = 7.7 Hz, 1H), 7.38 (d, J = 7.7 Hz,
2H), 7.34−7.10 (m, 10H), 4.05−3.93 (qd, J = 7.2 Hz, J = 7.2 Hz,
8H), 3.85 (s, 4H), 3.73 (s, 4H), 2.88 (d, J = 10.6 Hz, 4H), 1.21 (t, J =
7.1 Hz, 12H). 13C NMR (100 MHz, CDCl3): δ 158.6, 138.6, 137.0,
129.2, 128.4, 127.3, 121.5, 61.9 (d, J = 6.7 Hz), 61.2 (d, J = 8.3 Hz),
59.8 (d, J = 9.0 Hz), 49.1 (d, J = 161.5 Hz), 16.6 (d, J = 6.0 Hz). 31P
NMR (162 MHz, CDCl3): δ 25.4 (Figure S1a).
((((6-((Benzyl((diethoxyphosphoryl)methyl)amino)methyl)-
pyridin-2-yl)methyl)amino)methyl)diphosphonic Acid L3. The L3
ligand was isolated as a by-product of the synthesis of L4 (0.15 g,
13%). 1H NMR (400 MHz, D2O): δ 7.64 (t, J = 7.8 Hz, 1H), 7.33 (d,
J = 7.7 Hz, 1H), 7.25−7.04 (m, 6H), 3.78 (d, J = 21.9 Hz, 4H), 3.63
(s, 2H), 2.58 (d, J = 11.6 Hz, 2H), 2.51 (d, J = 12.7 Hz, 2H). 13C
NMR (101 MHz, D2O) δ 163.10 (s), 158.72 (s), 138.56 (s), 138.56
(s), 129.86 (s), 128.18 (s), 127.08 (s), 122.89 (s), 120.84 (s), 60.28
(d), 59.24 (d, J = 8.2 Hz), 55.19 (d, J = 12.8 Hz), 54.34 (d, J = 140.4
Hz), 47.70 (d, J = 137.0 Hz). 31P NMR (162 MHz, D2O) δ 16.39,
15.91 (Figure S1b). ESI+/MS (D2O): m/z = 416.1 ([M + H]+,
100%); m/z = 831.2 ([2M + H]+, 15%) (Figure S2a). Calc. for
C16H23N3O6P2·TFA: C, 41.6; H, 5.77; N, 8.66; Found: C, 40.9; H,
4.57; N, 7.94.
(((Pyridine-2,6-diylbis(methylene))bis(benzylazanediyl))bis-
(methylene))diphosphonic Acid L4. Compound 4 (1.7 g, 2.79 mmol)
was dissolved in 6 M HCl (36.5 mL), and the mixture was heated in
reflux for 16 h. The solvent was removed under reduced pressure.
After completion of the reaction, the mixture was evaporated to
dryness, and the crude product was purified by flash chromatography
with a C18 reverse phase column (eluent system H2O/ACN/TFA
100:0:0.1% to 70:30:0.1%) to give ligand L4 (0.74 g, 52%). 1H NMR
(400 MHz, D2O): δ 7.47 (t, J = 7.8 Hz, 1H), 7.14 (d, J = 6.7 Hz, 4H),
7.07−6.72 (m, 8H), 4.73 (s, 4H), 4.30 (s, 4H), 3.17 (d, J = 12.4 Hz,
4H). 13C NMR (126 MHz, D2O): δ 158.40 (s), 138.78 (s), 137.90
(s), 129.96 (s), 128.30 (s), 127.17 (s), 122.78 (s), 60.62 (d, J = 5.5
Hz), 59.42 (d, J = 8.7 Hz), 53.52 (t, J = 190.4 Hz). 31P NMR (162
MHz, D2O): δ 7.64 (Figure S1c). ESI+/MS (D2O): m/z = 506.2 ([M
+ H]+, 100%), m/z = 1011.3 ([2M + H]+, 23%) (Figure S2b). Elem.
Anal. Calc. for C23H29N3O6P2·TFA: C, 50.5; H, 5.64; N, 7.43; Found:
C, 48.5; H, 4.88; N, 6.78.
Data Acquisition Processing and Analysis. EXAFS experiments at
the U LIII edge (E0 = 17172.4 eV) were carried out on the MARS
beamline at the SOLEIL synchrotron facility (ring operated at 2.75
GeV with 430 mA), which is the French bending magnet beamline
dedicated to the study of radioactive materials.22 The optics of the
beamline essentially consists of a water-cooled double crystal
monochromator, which is used to select the incident energy of the
X-ray beam and for horizontal focalization, and two large water-
cooled reflecting mirrors that are used for high-energy rejection
(harmonic part) and vertical collimation and focalization.23 Detection
was performed using a Ge multielement fluorescence detector
(Ortech). In this case, the monochromator was set with the Si
(220) crystals and the mirrors with the Pt strips at 3.1 mrad. All the
measurements were recorded in double-layered solution cells (200
μL) specifically designed for radioactive samples at room temperature.
The data were processed using the ATHENA code.24 Background
removal was performed by means of a pre-edge linear function.
Atomic absorption was simulated with a square-spline function. The
EXAFS signal extracted was fitted in R space without any additional
filtering using the ARTEMIS code.24 The DFT calculation of L2 at pH
3 was used to calculate the XAS phases and amplitudes with the Feff7
code.25 In all fits, a single global amplitude factor (S0 ) and a single
2
energy threshold factor (ΔE0) were considered for all contributions. A
Hanning adjustment window in k2 χ(k) (3.5−13.3 Å−1) was used, and
the adjustment of spectra was performed in R (1−4 Å).
Synthesis of the Ligands. Ligands L1 and L2 were synthesized
according to a previous publication from Abada et al.11 The syntheses
of ligands L3, L4, and L5 (L5 as a by-product) are described below.
2,6-Bis(bromomethyl)pyridine (1). To a solution of 2,6-pyridine-
dimethanol (3.36 g, 24.1 mmol) in DMF (20 mL) at 0 °C, PBr3 (5.22
mL, 55.6 mmol) was added dropwise. The mixture was stirred at
room temperature for 6 h, and then water was added (300 mL) to
solubilize the formed solid. After extraction with CH2Cl2 (3 × 100
mL), the organic layer was dried with Na2SO4 and the solvent was
removed in vacuo to yield a yellow solid. The crude product was
purified by flash chromatography (CH2Cl2) to give compound 1 as a
pure white solid (5.26 g, 82%). 1H NMR (400 MHz, CDCl3): δ 7.64
(t, J = 7.7 Hz, 1H), 7.31 (d, J = 7.7 Hz, 2H), 4.47 (s, 4H).
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Figure 2. Synthesis of the ligands (fully protonated species).
(((Pyridine-2,6-diylbis(methylene))bis(azanediyl))bis-
determination of four protonation constants for each ligand
(Table 1). The other noncharacterized protonation constants
(methylene))bisphosphonic Acid L5. The L5 ligand was isolated as a
1
by-product of the synthesis of L4 (0.08 g, 9%). H NMR (400 MHz,
D2O) δ 7.74 (t, J = 7.8 Hz, 1H), 7.27 (d, J = 7.8 Hz, 2H), 3.82 (s,
4H), 2.53 (d, J = 12.5 Hz, 4H). 13C NMR (101 MHz, D2O) δ 158.13,
138.76, 121.34, 48.31, 46.95. 31P NMR (162 MHz, D2O) δ 16.50
(Figure S1d).
Table 1. Successive Protonation Constants of Ligands L3
a
and L4
pKn
L3
L4
H
H
H
H
H
pK1
pK2
pK3
pK4
Nali
Nali
RPO3
RPO3
9.5(1)
9.15(1)
6.17(6)
4.6(2)
9.28(5)
8.91(6)
6.36(8)
4.45(5)
RESULTS AND DISCUSSION
■
2−
2−
Design and Synthesis of the Ligands. The synthesis of
bis-phosphonated ligands L3 and L4 (Figure 2) was adapted
from known preparations of a phosphonated pyridine ligand.11
L3 and L4 were obtained by bromination of the commercially
available 2,6-dihydroxymethylpyridine 1 with PBr3 at 0 °C,
affording 2,6-dibromomethylpyridine 2 in 95% yield. Diethyl
((benzylamino)methyl)phosphonate 3 was obtained from
benzylamine in two steps (Figure 2). To a stirred solution of
benzylamine, formaldehyde was added to get 1,3,5-tribenzyl-
hexahydrotriazine 2 in 91% yield.26 The intermediate
compound 3 was synthesized by reaction of 2 with
diethylphosphite in 42% yield.27 N-alkylation reaction of 1
with 3 resulted in the formation of the bis-phosphonated ester
4 (79%), which led to ligand L4 (52%) after hydrolysis of
phosphonate groups using 6 M HCl. Interestingly, the
hydrolysis also resulted in the partial deprotection of the
benzyl groups, with formation of the monobenzylated ligand
L3 (13%), together with minute amounts (9%) of the bis-
deprotected ligand analogue L5.
a
Solvent: H2O; I = 0.1 M NaClO4; T = 25.0 (2) °C. The values in
parentheses correspond to the standard deviations expressed as the
last significant digit.
are associated with much lower values (log KLHx ≪ 2), which
could not be detected under our experimental conditions. The
two most basic protonation constants KLH and KLH2 were
attributed to the aliphatic amines, in agreement with the data
existing in the literature for other polyaminomethanephosph-
onate analogues.11,30−34 It can be noted that the preplacement
of a secondary amine in L3 by a benzylated tertiary one in L4
had little influence on the corresponding pK values. The third
and fourth protonation constants determined for the ligands
are associated with the first protonation constants of each of
the methanephosphonate groups.11
The distribution diagrams of the protonated species for
ligands L3 (Figure S4) and L4 (Figure 3) as a function of the
pH of the solution are very similar. They show that, under
acidic conditions (pH < 4), L3 and L4 mainly exist as their
neutral zwitterionic [LH4] species while under physiological
conditions (pH 7.4), the deprotonated form ([LH2]2−) with
two protonated aliphatic amines is predominant (∼80%). As a
comparison, it can be noted that, for ligand L2, the [LH3]5−
species was predominant under the same conditions.11
Protonation of Ligands L3 and L4. L3 and L4 have a
homogeneous series of chelating units with two terminal
2−
−PO3 phosphonate functions, two amines, and a pyridyl
ring. The difference between the two ligands is the number of
benzylene moieties, which might result in differences of
complexation affinity toward uranyl ions.
Investigation of the complexation of uranyl ions with the
ligands requires the complete characterization of ligand
protonation. Therefore, the acido-basic properties of L3 and
L4 were investigated in the pH range of 2.0−12 by
potentiometry. Potentiometric titrations of ∼1 mM solutions
of L3 or L4 followed by statistical processing of the data with
the HYPERQUAD software (Figure S3)28,29 resulted in the
In order to establish the ligand spectrophotometric proper-
ties as a function of the pH, spectrophotometric titration was
performed for ligand L4. The electronic spectra of the different
protonated L4 species were obtained by fitting the
spectrophotometric titration data with the protonation
constants deduced by potentiometric titrations (Figure 3).
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