Thermodynamic and Structural Investigation of Synthetic Actinide-Peptide Scaffolds

Article abstract of DOI:10.1021/acs.inorgchem.5b02379

The complexation of uranium and europium, in oxidation states +VI and +III, respectively, was investigated with pertinent bio-inorganic systems. Three aspartate-rich pentapeptides with different structural properties were selected for study to rationalize the structure-affinity relationships. Thermodynamic results, crosschecked by both isothermal titration calorimetry and time-resolved laser fluorescence spectroscopy, showed different affinity depending on the peptide for both Eu(III) and U(VI). The thermodynamic aspects were correlated to structural predictions, which were acquired by density functional theory quantum chemical calculations and from IR and extended X-ray absorption fine structure experiments. The combination of these microscopic properties revealed that carbonyl-metal interactions affected the entropy in the case of europium, while the larger uranyl cation was mostly affected by preorganization and steric effects, so that the affinity was enhanced through enthalpy. The approach described here revealed various microscopic aspects governing peptide actinide affinity. Highlighting these mechanisms should certainly contribute to the rational synthesis of higher affinity biomimetic aspartic ligands.

Full text of DOI:10.1021/acs.inorgchem.5b02379

Article
pubs.acs.org/IC
Thermodynamic and Structural Investigation of Synthetic Actinide−
Peptide Scaffolds
Samir Safi,† Aurelie Jeanson,† Jerome Roques,† Pier Lorenzo Solari,‡ Florence Charnay-Pouget,§
́
́
Christophe Den Auwer,∥ Gaelle Creff,∥ David J. Aitken,§ and Eric Simoni*,†
̈
†Institut de Physique Nuclea
́
ire d’Orsay and ICMMO, CNRS, Univ. Paris-Sud, Universite
́
Paris-Saclay, 91405 Orsay cedex, France
§
‡MARS Beamline, Synchrotron SOLEIL, L’Orme des Merisiers, 91192 Gif-sur-Yvette, France
∥Institut de Chimie de Nice, Universite
́
de Nice Sophia Antipolis, 28 Avenue Valrose, 06108 Nice, France
S
* Supporting Information
ABSTRACT: The complexation of uranium and europium, in
oxidation states +VI and +III, respectively, was investigated
with pertinent bio-inorganic systems. Three aspartate-rich
pentapeptides with different structural properties were selected
for study to rationalize the structure−affinity relationships.
Thermodynamic results, crosschecked by both isothermal
titration calorimetry and time-resolved laser fluorescence
spectroscopy, showed different affinity depending on the
peptide for both Eu(III) and U(VI). The thermodynamic
aspects were correlated to structural predictions, which were acquired by density functional theory quantum chemical calculations
and from IR and extended X-ray absorption fine structure experiments. The combination of these microscopic properties
revealed that carbonyl−metal interactions affected the entropy in the case of europium, while the larger uranyl cation was mostly
affected by preorganization and steric effects, so that the affinity was enhanced through enthalpy. The approach described here
revealed various microscopic aspects governing peptide actinide affinity. Highlighting these mechanisms should certainly
contribute to the rational synthesis of higher affinity biomimetic aspartic ligands.
In this context, we have undertaken an investigation of
aspartate-rich pentapeptides bestowed with different geo-
metrical constraints. These peptides are relevant partners
when studying the factors, which govern An complexation since
they are model fragments of protein primary structures. In this
work we focus on trivalent and hexavalent An; An(IV)
coordination has been covered elsewhere.6 The use of various
aspartate scaffolds to relate their microscopic structural and
thermodynamic properties may provide valuable leads for a
better understanding of the biomolecular interactions as well as
for the development of new complexes implicating these ions,
for use in various applications (anticancer drugs, decorporating
reagents, and so forth). Furthermore, we examine the influence
of the intrinsic cation geometry by comparing the complexation
of the linear uranyl(VI) oxo-cation with that of the “spherical”
europium(III), which we adopt as a non-radioactive surrogate
of trivalent actinides.
INTRODUCTION
■
In the wake of the Fukushima accident, many radioactive
elements were released into the environment. Plutonium and
non-natural uranium were indeed found as far as 120 km from
the explosion site.1 Although lighter elements are the major
source of radioactive contamination,2 from a public health and
societal standpoint, the lack of understanding of actinide (An)
migration pathways in the biosphere is of particular concern.
Earlier studies emphasized that exogenic actinides, and more
specifically uranium, can contaminate aspartate side chains and,
to a lesser extent, amide oxygens in native proteins.3 This
observation is significant in the light of the Pearson hard acid/
hard base relationship. The description of the structural and
thermodynamic aspects of such complexes is therefore
imperative for an understanding of the different contamination
pathways, which are governed by both structure (steric
hindrance, affinity) and stability.4 Most bio-inorganic studies
have concentrated on amino acid transition metal complexes
and have been reviewed extensively by Sigel and Martin;5 in
contrast, descriptions of biologically relevant actinide and
lanthanide complexes remain scarce. This is partly due to both
the highly soluble nature of the complexes involved, which
makes their crystallization difficult, and the complex speciation
of actinides, which may undergo hydrolysis at biologically
relevant pH.
Indeed spherical hard metals such as lanthanides or actinides
may coordinate with peptides in a similar manner to amino
acids. The carbonyl group originating from the amide function
interacts with such cations,7 which could not be the case with
UO22+oxo-cation due to the constrained pentagonal ion
structure. We selected here Eu(III) as a representative of all
Received: October 15, 2015
© XXXX American Chemical Society
A
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series of TRLFS spectra were measured at pH 3.5 for U(VI) and pH 6
for Eu(III) since, under these conditions, the formation of hydrolytic
species is minimal and therefore, according to the meal speciation
diagram, only the free aquo metals must be taken into account in
solution.11,12 Solutions were prepared at peptide concentrations
ranging from 0 to 3 × 10−3 M.
Experimental Setup. TRLFS experiments were performed using a
Continuum R Nd:YAG laser (frequency 10 Hz, pulse duration of ∼7
ns) as the excitation source coupled with a Panther OPO. Detection
was achieved using a Spectra-Pro-300 monochromator (Acton
Research corporation) coupled with a CCD camera (Princeton
Instruments). The excitation wavelength was chosen as 430 nm for
uranium and 466 nm for europium, and laser power was ∼2 mJ.
Emission spectra were recorded using WINSPEC software (Princeton
Instruments). The fluorescence spectra had better than 0.2 nm
resolution. The error in measured fluorescence intensity was less than
5%. All fluorescence intensity measurements were performed on
nonaerated freshly prepared solutions in a quartz cuvette at
the +III elements of the f families (lanthanides, heavy actinides)
and U(VI) ({UO22+}) as the ubiquitous form of uranium in
natural conditions but also as the representative of the actinyl
species (uranyl, neptunyl, plutonyl).
To investigate these characteristics, three N- and C-terminal
protected pentapeptide sequences were selected: DGDGD,
ADPDA, and DPDPD (A = alanine, G = glycine, D = aspartate,
P = proline). The latter two contain proline (P) residues that
induce an angular strain, while the first peptide contains two
flexible glycine (G) residues. The alanine (A) residues in the
second peptide introduce partial conformational restrictions. In
all cases, the expected complexing functions are the side-chain
carboxylates of the aspartate (D) residues. The number of
carboxylate side chains was also varied between 2 and 3, as were
the positions of the coordinating residues in the pentapeptide
sequences.
Among the structural investigation techniques for bio-
inorganic systems, X-ray absorption spectroscopy (XAS) was
found suitable since it is an element-specific local structural and
electronic probe that is increasingly used to study actinide
interaction in biological systems.8 This technique, coupled with
other spectroscopic methods such as infrared spectroscopy and
time-resolved laser fluorescence spectroscopy (TRLFS),
enabled us to achieve a full understanding of the metal
environment and the affinity for the coordination partners.
Additionally, density functional theory (DFT) was used to
model the actinide complex stability. This approach was already
applied successfully in our group to study the coordination of
uranyl with proteins.9
Affinity studies were used to investigate the thermodynamics
of peptide complexes with both Eu(III) and U(VI) by
isothermal titration calorimetry (ITC). Lately, this method
emerged as the key technique for quantifying the thermody-
namics of metal−biomolecule interactions. Furthermore, it is a
true solution-phase method, since it does not require labeling
or immobilization.10 Finally, the stoichiometry and the affinity
were independently cross checked by both ITC and TRLFS,
which gives further information about the species present in
solution, through fluorescence lifetime measurement.
temperature 25
1 °C. Every measurement was repeated at least
three times. The fluorescence spectra integration (from 450 to 650
nm) and fluorescence decay curves were calculated with the Origin
Pro 8.0 software program. The stability constants and spectral
deconvolutions were refined using the least-squares fitting program
HypSpec.
Isothermal Titration Calorimetry. The overall procedure for
ITC experiments was similar to that described by Lin et al.13 The VP-
ITC from Microcal (Northampton, MA) was used in all experiments.
Briefly, peptide solutions (1.4 mL) were prepared at 0.02 to 0.05 mM
concentrations in buffer (25 mM HEPES, pH 3.5, and 100 mM NaCl)
and were placed in the titrating cell, while solutions at 0.4 mM
(Eu(III)) and 10 mM (U(VI), at this pH value (according to the metal
speciation diagrams, only the free aquo metals are present in solution)
prepared in the same buffer were placed in the syringe. The peptide
solutions were then titrated at 25 °C with 20 μL injections (spaced at
4 min intervals). The blank injections of the metal solution into the
corresponding buffer were used to account for the heat of mixing and
dilution. In each experiment 15 injections were made, so that the final
molar ratio of U(VI) and Eu(III) to peptide was 4 to 5. The heat of
the reaction, Q, was obtained by integrating the peak after each
injection of peptide ligand using ORIGIN software provided by the
manufacturer (Microcal, Northampton, MA). The heat of the reaction
at each injection is related to the calorimetric enthalpy of binding; in
our case the total cumulative heat was composed of contributions from
a single binding site. The model equations for fitting the binding
isotherms are included with the Microcal software and are detailed
elsewhere.14
ATR-FTIR. Peptide solutions (10 mM) were mixed at pH 4 and 6
for uranium and europium, respectively, with increasing metal-to-
peptide molar ratios (0.5 to 5). The freshly prepared solutions were
then deposited on a diamond crystal surface (Pike Technologies).
Absorption spectra were recorded in the range of 2400−600 cm−1
(resolution: 2 cm−1, 50 scans per spectrum) on a Bruker IFS 66 V
spectrometer with an ATR module and an MCT detector. Absorption
spectra of the peptides were obtained after subtracting the absorption
spectra of the buffers, recorded in the same conditions. Under these
conditions and using H2O and D2O as solvents the difference spectra
were found to be accurate in the 1650−900 cm−1 frequency domain.
Density Functional Theory Computational Details. All
structures were optimized using the DFT approach as implemented
in the Gaussian09 package.15 Calculations were performed using the
hybrid B3LYP functional.16 MWB60 Stuttgart relativistic effective core
potentials were used to describe uranium atoms, while the 6-31+G*
basis set was used for others. To take into account part of the solvent
effect, solvation was introduced using a dielectric continuum model of
permittivity ε = 80. The conductor-like polarizable continuum model
implemented in Gaussian09 was used. Vibrational harmonic
frequencies, corresponding to the optimized structures, were also
calculated and compared to the experimental ones.
EXPERIMENTAL PROCEDURES
■
Materials and Stock Solutions. A stock of UO2 solution (0.1 M
in 0.63 M HClO4) was obtained by dissolving UO2(NO3)2·6H2O
(99% Fluka puriss) in 12 M HClO4 (Merck Suprapur) and
evaporation. This operation (adding HClO4 and evaporation) was
repeated three times. An analogous protocol was followed to prepare a
stock solution of Eu(III) (0.1 M in 0.63 M HClO4). The pH values of
the test solutions were measured using a pH microelectrode (semi
micro, Mettler Toledo, France) and pH meter (seven multi, Mettler
Toledo, France). The electrode was calibrated using standard buffers.
The pH measurements were made at 20 °C with a precision of 0.05
pH units. The pH values of the test solutions were adjusted with
NaOH (p.a., CO2-free, Merck) and HClO4 (Suprapur, Merck).
Peptide Synthesis. N- and C-protected derivatives of the two
peptides ADPDA and DGDGD were synthesized in-house, using
solution-state peptide coupling procedures; full details are provided in
the Supporting Information document. A commercially available
sample of DPDPD-NH2 (Polypeptide, France) was found to be of
sufficient purity for the experiments conducted in this work.
Time-Resolved Laser Fluorescence Spectroscopy. Sample
Preparation. The U(VI) an Eu(III) concentrations were adjusted by
diluting the stock solution (the concentration of which is regularly
measured using liquid scintillation counting) to 0.01 mM. The ionic
strength of the experimental solutions was kept constant at 0.15 M,
using NaClO4 (p.a., Merck) under an argon atmosphere. Multiple
EXAFS. EXAFS Sample Preparation. Peptide-Eu(III) and peptide-
U(VI) complexes were prepared in a 10 to 1 molar ratio at a U(VI)/
B
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Figure 1. Peak deconvolution of a normalized fluorescence spectrum of a solution containing 1 × 10−5 M U(VI) or Eu(III) and 1 × 10−4 M peptides
at pH 3.5 for U(VI) and pH 6 for Eu(III) and I = 0.1 M.
Eu(III) HCl solution concentration of 0.5 mM. Higher stoichiometries
were tested without any significant effect on the EXAFS spectrum.
Final pH was adjusted either with NaOH or HCl (provided by
Sigma−Aldrich). The U(VI)/Eu(III) solutions were added slowly to
an acidic peptide solution to avoid hydrolysis, then the pH was raised
to the desired value. The solutions were found to be stable at pH
values as high as 6.
Extended X-ray Absorption Fine Structure Data Acquisition.
EXAFS experiments, at the Eu and U LIII edge, were performed on the
MARS beamline at the SOLEIL synchrotron facility (a ring operated at
2.75 GeV with 400 mA), which is the French bending magnet
beamline dedicated to the study of radioactive materials.17 The optics
of the beamline essentially consist of a water-cooled double-crystal
monochromator, which is used to select the incident energy of the X-
ray beam and for horizontal focalization, as well as two large water-
cooled reflecting mirrors that are used for high-energy rejection
(harmonic part) and vertical collimation and focalization.18 In this case
of uranium samples, the monochromator was set with the Si(220)
crystals and the mirrors with the Pt strips at 3.1 mrad, whereas, in the
case of europium the monochromator was set with the Si(111) crystals
and the mirrors with the Si strips at the same angle. The spectra were
acquired in fluorescence mode using a multielement Ge detector from
ORTEC. No metal reduction was observed in the X-ray absorption
near-edge structure region, and thus no beam attenuation was
necessary. All measurements were recorded in double-layered 200
μL solution cells specifically designed for radioactive samples, at room
temperature. Data were processed using the ATHENA code.19
Background removal was performed using a pre-edge linear function.
Atomic absorption was simulated with a square spline function. Phases
and amplitudes were calculated with Feff82 code20 with the model
clusters obtained by the above computational methods. The EXAFS
signal extracted was fitted in R space without any additional filtering,
using the ARTEMIS code.19 In all the fits, only one global amplitude
factor and one energy threshold factor were considered for all the
contributions. Hanning windows with k2 [3 ; 11.5] Å−1 for uranium
and [1.5 ; 9.5] Å−1 for europium and a fitting range with R [1 ; 4.0] Å
χ 2
for both were used. The R factor (r) and quality factor
provided as an indication of fit quality in the R space.
are both
i /n
C
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one found for the neptunyl ion, which is an oxo-cation as well,
but with only one positive charge (corresponding to a
pentavalent neptunium).24 Considering the chemical similar-
ities and therefore the analogies between trivalent lanthanides
and actinides, the obtained formation constant values for
europium could be used to get an order of magnitude for the
values that could be expected for trivalent americium and
curium. This is corroborated by the values associated with the
interactions between these to trivalent actinides and amino
acids such as aspartate or glutamate.24
Isothermal Titration Calorimetry Analysis of the
Peptide-U(VI) Complex. ITC was used to investigate the
thermodynamics of U(VI) and Eu(III) with all three peptides.
To increase the reliability of the thermodynamics model, the
constants determined with TRLFS were used as constraints in
the fitting process, considering that the metal speciation
conditions are the same (only free aquo ions are present in
solution) for both techniques. The stoichiometry, which is
determined from the inflection point of the titration curve, was
fitted between 0.85 and 0.95, increasing the confidence interval
of our values. These obtained values, under unity, are common
in literature due to molecular diffusion between the syringe and
the calorimeter cell. Moreover, as the K values were held
constant in the fitting procedure, the stoichiometry numbers
obtained are quite reliable and thus allow us to conclude on the
1:1 complex formation. Consequently, this result (and the
unique lifetime value found for each system) rules out the
possibility of the 2:1 complex formation. The error in the
determined thermodynamic values was negligible, of the order
of 20 cal. Figure 2 shows an example of raw data and the fit
obtained from the U(VI)/ADPDA titration.
RESULTS AND DISCUSSION
■
Time-Resolved Laser Fluorescence Spectroscopy
Analysis. The stoichiometry and pseudo formation constant
were determined by varying the concentration of the peptide
and using the Hypspec deconvolution program to determine
each of the species concentrations. Stoichiometries larger than
unity were rejected by Hypspec for all the studied systems.
These data were used to calculate the corresponding
concentrations of the complex and of the non-complexed
ligand. This well-established method for analyzing fluorescence
spectra is detailed in the literature.21,22 All spectral
deconvolutions are presented in Figure 1. The observed
spectral edge at 570 nm for U (VI) is due to spectrometer
cutoff.
In the case of uranyl complexes small spectral shifts from 2 to
5 nm toward the higher wavelengths and a decrease of
fluorescence intensity were observed upon addition of the
peptides to the solution (Figure 1). This decrease is also related
to the increased contribution from a species with a lower
lifetime, of ∼1 μs, that we assign to the complex. The DGDGD
uranium complex presents the lowest lifetime value (0.5 μs),
which is consistent with its relative flexibility compared to the
relative rigidity induced to the proline group in the two others
systems. In the case of europium an increase of the fluorescence
5
7
yield of the D0 → F2 (at 615 nm) transition marks the
coordination process in solution, as this band is hypersensitive
to the coordination environment. Higher lifetimes are also
detected to mark the replacement of the water molecules by
coordinating amino acids in the peptide’s sequences.
Noninteger values of determined q numbers (q = number of
water molecules in the coordination sphere) are relatively
common. This phenomenon was investigated by Supkowski et
al.23 and was attributed to the mixed nature of europium
coordination (eight to nine oxygens) or, as in the present case,
to the contributions from the N−H oscillators (which affect the
lifetime) in which the amide carbonyl oxygen is in the first
coordination sphere. Both the formation constants and the
determined fluorescence lifetimes are presented in Table 1.
The complexation of the peptides under study with U(VI)
and Eu(III) ions is driven predominantly by the favorable
entropic change (TΔS = 7003−13112 cal/mol) since enthalpy
changes were positive (ΔH = 193−8279 cal/mol). A control
run with TRIS instead of HEPES gave the same results,
indicating that no heat contribution from buffer ionization is
detected.
One possible explanation for the interaction’s large entropy
drive is that both dissociated metal ions and free peptides are
heavily solvated by both ion−dipole and hydrogen-bonding
interactions. Therefore, the cation binding mechanisms
demands the extensive desolvation of both host and guest,
thus affording the highly positive entropy change upon
complexation. This phenomenon was also reported in the
case of transition metals by Trapaidze et al.25
Enthalpy changes in the case of biomolecules, and more
specifically peptides, are often related to the formation or
rupture of noncovalent interactions (complexation, hydrogen
bonds, van der Waals, and London interactions). Lopez et al.26
used metal binding to determine the α-helix formation
enthalpy, which is in part due to hydrogen/van der Waals
bonds formation. Furthermore, they reported the dependence
of this enthalpy contribution on the peptide backbone and
specifically in the case of glycine and proline as compared to
alanine. This is not surprising, considering that glycine provides
more flexibility to the backbone, while proline limits the
degrees for freedom. Considering our results, the smaller global
endothermic enthalpies reported for DGDGD for both uranyl
and europium can thus be explained as follows: DGDGD
contains probably less hydrogen intramolecular bonds than the
two peptides with proline residue (due to the cis-amide bonds
next to the proline, which gives an intramolecular structura-
Table 1. Formation Constants and Fluorescence Lifetime
Attributions
uranium
europium
log kU
0.2
lifetime
(μs) 0.1
log kEu
lifetime
(μs) 0.1
sample
0.2
q
free metal
1.7
1.0
112
212
8.9
4.5
ADPDA-
metal
3.2
3.8
5.0
3.1
3.5
3.8
DPDPD-
metal
0.9
0.5
196
224
4.8
4.2
DGDGD-
metal
We observed that in both cases the formation constants
present the highest value with the peptide containing no
proline residue. The thermodynamics of this affinity enhance-
ment are explored by ITC in the next paragraph. Concerning
the uranyl ion, the obtained formation constants values are the
same order of magnitude than the ones found with different
kind of amino acids such as, for instance, aspartate, glutamate,
or alanine, which correspond to the typical values associated
with uranyl−carboxylate interactions.3 In contrast, these
formation constants values are about twice more than the
D
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Figure 2. (left) Calorimetric titrations with 15 injections of U(VI) (black) and blank buffer (gray) into ADPDA peptide in a 1.4 mL reaction cell
(see Experimental Section). An exothermic reaction is shown by a negative heat effect, whereas an endothermic reaction is shown by a positive heat
effect. (right) The blank dilution heat values are subtracted from the sample integrated heat values from the left panel and plotted as a function of the
◆
molar ratio of U(VI) to ADPDA in the reaction cell ( ), and the corresponding fit is represented by the straight line.
tion). Therefore, to break these hydrogen bonds, the enthalpy
values needed are higher for the ADPDA and DPDPD than for
the DGDGD peptide.
This calorimetric study also reveals that both the entropy and
enthalpy changes show, for both metals, a higher affinity for the
peptide without proline residue (Table 2). However, in the case
of uranium it is the enthalpy contribution that affects the Gibbs
free energy (Table 2). To understand this phenomenon we
characterized the structure of the formed complexes by
theoretical calculation coupled to FTIR and EXAFS.
Fourier Transform Infrared Analysis. To observe
structural changes induced by metal addition, FTIR difference
spectra were recorded. The structural changes revealed
themselves via the appearance of negative and positive peaks
in the spectra. To eliminate artificial negative peaks due to
solvent subtraction the experiment was performed in both D2O
and H2O for all peptides (Figure 3). In each of the spectra we
noted the presence of the negative peaks at 1575−1581 and
1463−1486 cm−1, which were attributed, respectively, to the
deprotonated ionic carboxylate’s asymmetric (νas) and
symmetric (νsym) stretch modes. This depicts the carboxylate’s
contribution to the coordination of Eu(III) and U(VI).
Furthermore, the positive peaks obtained by metal addition
reveal complex formation through carboxylate symmetric and
asymmetric stretch modes,27 recapitulated in Table 3.
a
Table 2. Thermodynamic Parameters Determined with a
1:1 Binding Isotherm Model
The type of coordination of carboxylate groups to metals
ions can be derived from vibrational spectra by studying the
splitting (Δν) between the νas and νs (COO) modes. It has
been shown that Δν monodentate > Δν ionic = Δν bridging >
Δν bidentate.28 In our case the Δν values of the deprotonated
peptides serve as a reference and are equal to 100 15 cm−1,
a
The ΔG was calculated from the affinity constants determined by
TRLFS. All values in the table are reported in calories per mole.
Figure 3. FTIR difference (peptide-(U(VI) or Eu(III)) minus peptide). The difference spectra were obtained as detailed in the Methods Section.
E
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Table 3. Experimental and Theoretical Infrared Carboxylate Energy Gap Calculated by DFT Band Position for “Free” Peptide
and Complexes
peptides
bands
free peptide υ (cm−1
υas COO− υsym COO−
)
Eu(III) peptide υ (cm−1
)
U(VI) peptide υ (cm−1
)
υgap
υamide(I) υas COO− υsym COO−
Δυ
DFT-Δυ υassUO2 υas COO− υsym COO−
Δυ
DFT-Δυ
a
b
a
b
a
b
a
b
a
b
a
b
a
b
a
b
a
b
a
b
a
b
a
b
ADPDA
1583
1587
1578
1477
1484
1465
106
1626
1620
1643
1536
1560
1521
1556
1522
1541
1449
1426
1439
1398
1451
1422
87
134
82
89
151
55
937
936
937
1434
1532
1438
1528
1459
1521
1379
1352
1376
1351
1389
1349
55
180
62
42
180
56
DPDPD
DGDGD
103
113
158
71
161
68
177
70
189
51
120
142
172
128
a
b
Bidentate carboxylate coordination. Monodentate carboxylate coordination.
Figure 4. Theoretical models calculated using DFT for the three Eu(III) (blue) and the three U(VI) (yellow) interaction sites. Hydrogen atoms are
omitted for clarity.
depending on the peptide (cf. Table 3). In general, bidentate
coordination of the carboxylate groups to the Eu(III) or U(VI)
metal ion is expected to show a significantly lower spectral
splitting than monodentate binding. For europium, Δν values
of 71−87 cm−1 and 120−134 cm−1 reveal, respectively, the
presence of both monodentate and bidentate complexes. These
values are in agreement with those we determined from
vibrational DFT calculations, using the structural models
described in the latter section. The same conclusions can be
drawn for uranium. However, the band splits for U(VI)
complexes were more pronounced compared to the free
peptide (Δνbi 55−62 and Δνmono 172−180 cm−1), probably
due to a stronger covalent character of the interaction.
In the spectra of Eu(III) complexes we noticed an positive
band increasing in the 1620−1643 cm−1 region, blue-shifting
from the negative band at 1585 cm−1. This corresponds to the
trans-amide I (primarily CO stretch) mode that appears for
many peptide−metal cation complexes29 and is consistent with
strong coordination of carbonyl oxygen atoms to trivalent metal
cations, resulting in greater partial charge transfer.30,31 Finally,
F
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Table 4. Description of EXAFS Parameters Used for the Fit to Both Europium and Uranium Complexes
europium complex
uranium complex
a
b
c
d
a
b
c
d
path
Ndegen
ΔR
σ
path
U−Oyle
U−O1mono
U−O2bi
U−Cbi
U−Cmono
Ndegen
ΔR
ΔR1
ΔR2
ΔR3
ΔR4
ΔR5
σ
2
2
2
2
Eu−Ofirst‑sphere
Eu−Cbi
Eu−Cmono
9
1
1
ΔR1
ΔR2
ΔR3
σ1
σ2
σ3
2
1
4
1
1
2
2
2
σ1
σ2
σ3
σ4
σ5
2
2
2
2
2
2
2
Eu→ C→Cbi
2
2
ΔR2
ΔR4
σ4
σ5
U→Oyle1→Oyle2
2 × ΔR1
2 × ΔR1
2 × ΔR1
2 × σ1
2 × σ1
4 × σ1
2
2
Eu→ O →Ccarb
U→Oyle1→U→Oyle2
U→Oyle1→U→Oyle1
a
b
c
d
Atom types in the scattering path. Degeneracy from the theoretical models. Deviation from the theoretical bond length. Debye−Waller Factor
(bi: bidentate; mono: monodentate caboxylate; carb: amide carbonyl carbon).
Table 5. Fitted Values (R, σ2, AMP, ΔE0) Determined for Each Peptide−Actinide Complex
uranium
bond
fitted length Å
(σ2 in Å2)
europium
bond
fitted length Å
(σ2 in Å2)
structure
ADPDA
N
2
DFT bond Å
1.80
N
9
DFT bond Å
AMP 1.0 ΔE0 2.1 ev R
factor 0.002 ChiR(88)
UOyl
1.77 (0.003) AMP 1.0 ΔE0 6.7 ev R
EuOfirst sphere
2.39−2.60
2.44 (0.008)
factor 0.02 ChiR(63)
UO1
1
4
1
1
2
2.20
2.48
2.92
3.53
1.80
2.25 (0.002)
2.43 (0.007)
2.92 (0.001)
3.38 (0.004)
EuCbi
1
2
1
1
9
2.94
2.87 (0.01)
4.37 (0.004)
3.45 (0.004)
3.63 (0.001)
2.44 (0.009)
UO2
EuCbi-C
4.44
UCbi
UCmono
UOyl
EuCmono
3.36
EuCcarb (C‑o)
EuOfirst sphere
3.70
DPDPD
AMP 1.0 ΔE0 1.8 ev R
factor 0.003 ChiR(90)
1.78(0.003)
AMP 1.0 ΔE0 6.88 ev R
factor 0.01 ChiR(55)
2.39−2.89
UO1
1
4
1
1
2
2.20
2.49
2.92
3.52
1.80
2.29 (0.004)
2.43 (0.009)
2.93 (0.001)
3.50 (0.005)
EuCbi
1
2
1
1
9
2.89
2.84 (0.01)
4.35 (0.001)
3.53 (0.004)
4.04 (0.005)
2.44 (0.008)
UO2
EuCbi-C
EuCmono
EuCcarb (C‑o)
EuO
4.39
UCbi
UCmono
UOyl
3.37
4.02
DGDGD AMP 1.0 ΔE0 3.6 ev R
1.78 (0.003) AMP 1.0 ΔE0 6.6 ev R
2.30−2.58
factor 0.002 ChiR(78)
factor 0.02 ChiR(63)
UO1
1
4
1
1
2.33
2.48
2.92
3.46
2.29 (0.003)
2.45 (0.009)
2.88 (0.001)
3.46 (0.004)
EuCbi
1
2
1
1
2.90
4.39
3.36
3.59
2.80 (0.01)
4.29 (0.002)
3.40 (0.006)
3.51 (0.003)
UO2
EuCbi-C
EuCmono
EuCcarb (C‑o)
UCbi
UCmono
a
All parameters were already defined in Table 4
in the case of uranyl, the υas UO2 stretch mode is sensitive to
the nature and number of uranyl equatorial ligands.32 Since all
three uranyl complex bands are shifted from 954 (free uranyl)
to 937 cm−1, we can presume a similar environment for all
three complexes.
carboxylate, one bidentate carboxylate, and two water
molecules, whatever the peptide. The DFT bond distances
are also presented in Table 5.
Extended X-ray Absorption Fine Structure Results.
The experimental europium and uranium peptide EXAFS data
were fitted using the independent structural parameters
summarized in Table 4. Only one first layer coordination
sphere (Ofirst‑sphere) was necessary to fit the europium data,
while three O layers were necessary in the case of uranyl: dioxo
uranyl atoms (Oyl), monodentate carboxylate (O1), bidentate
carboxylate, and water oxygen atoms (O2). Extra shells of
second sphere carbon atoms were added to the fits for both
coordinating carboxylates (monodentate Cmono and bidentate
Cbi) and an extra C shell in the europium case for the amide
peptidic bond carbonyl coordination.
Multiple scattering contributions, based on amplitude Feff
calculation, were also added to the fitting process. The second
sphere carbon MS paths in the case of europium (carbonyl,
carboxylate bidentate) were thus selected, and the dioxo MS
paths were selected in the case of uranium. We note that for the
latter cation second sphere carbon MS was minimal due to
distorted angles. The fitted values and the experimental and
fitted spectra are summarized in Table 5 and in Figure 5,
respectively. The fitted amplitude error was found to be of the
order of 0.05, which emphasizes fit quality. Further relevance of
Density Functional Theory Peptide-U(VI) and Peptide-
Eu(III) Model Complex Calculations. In the absence of
crystallographic data for the peptide complexes, indications
regarding the coordination scheme come from the knowledge
of the peptide amino acid sequence and the preliminary IR and
TRLFS data. We therefore tested in silico various geometrical
combinations around the U(VI) and Eu(III) that were in
agreement with IR and TRLFS, following a similar procedure
described elsewhere.9 This consisted of defining plausible
geometries for a first coarse-grain refinement, using initial
interatomic distances obtained from previously reported
work.33 The next step consisted of refining all the interatomic
distances in the presence of the peptide sequence strain and
steric effects using DFT calculations. The structures were then
cross-checked by comparing theoretical and experimental
vibrational spectra (Table 3). The structures that were found
to be most stable and that fitted the criteria are presented in
Figure 4. For the three peptides, the coordination site consists
in one bidentate carboxylate, one monodentate carboxylate, one
carbonyl, and four water molecules for europium. For uranium,
the coordination site is composed of one monodentate
G
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Inorganic Chemistry
Article
Figure 5. K3-weighted U and Eu LIII-edges EXAFS spectra of peptide complexes and the corresponding Fourier transforms. Black lines are the
experimental data, and dotted lines are corresponding fits.
structural values was analyzed separately for uranium and
europium.
Uranium Peptide Complexes. The contribution of axial
oxygens was fitted from 1.77 to 1.78 Å with a coherent Debye
H
DOI: 10.1021/acs.inorgchem.5b02379
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Weller factor of 0.003 Å2. This confirms the U(VI) speciation
and is in complete agreement with literature.34,35 The absence
of a short distance contribution at 2.20 Å in the equatorial
range confirmed the absence of hydrolysis. The first equatorial
oxygen shell was fitted at 2.25−2.29 Å in accordance with a
monodentate carboxylate. The bidentate carboxylate was
confirmed by U−O distances at 2.48−2.49 Å and U···C
distances at 2.92 Å for all the peptide uranyl complexes, similar
to literature.36,37 These results are in line with the spectral IR
analysis. We note that no structural contribution for amide
peptidic bond carbonyl function in the uranyl coordination
sphere was needed to fit the data. This absence of the carbonyl
interaction, also suggested by IR, can be explained by the steric
hindrance due to the pentagonal bipyramidal coordination
geometry of uranyl. Since all three peptides have the same
coordination site we can conclude that the enhanced enthalpy
through the series relates mostly to the bond strength. The
more flexible DGDGD structure generates thus a more stable
complex. However, it is interesting to note that is has been
found, using EXAFS and NMR experiments, a structure
radically different with a same kind of linear pentapeptide
(Asp-Asp-Pro-Asp-Asp-NH2) interacting with Th(IV), Np(IV),
and Pu(IV) ions. Indeed, the results show the presence of an μ-
hydroxo oxygen atoms and a metal−metal contribution, which
could be due to a complex nuclearity higher than two.38
Consequently, in that case, the absence of the oxo-cation
structure could probably play the most important role in the
interactions between these tetravalent actinides and this
pentapeptide.
Europium Peptide Complexes. The inner coordination
sphere of europium was fitted with nine oxygens to determine a
mean value, for all three peptides, at 2.44 Å in agreement with
literature.39,40 As for carboxylate monodentate (3.36 Å) and
bidentate (2.89−2.94 Å) carbons, distances were fitted in
accordance with reported data.41 Moreover the amide carbonyl
multiple scattering contributes to the EXAFS signal. The fitted
amide carbonyl bond lengths were in agreement with our
calculated DFT models. Furthermore, we notice that the
carbonyl bond length follows the same trend as the entropy and
enthalpy determined by ITC. We can thus suggest that the
carbonyl bond makes an exothermic contribution and generates
a rigid complex, which is illustrated by decreased entropy.
These two factors contribute to a higher affinity.
the bond formation and a rigidified, ordered complex structure.
The directional coordination due to uranyl is mostly governed
by the enthalpy and thus by the bond stability. The high affinity
of DGDGD compared to the other two peptides is due to its
high degrees of freedom (no intramolecular structuration)
enabling it to easily reorganize itself to fit the coordination
sphere. Finally, for the three peptides, the strain caused by the
short backbone of the peptide sequence confined the
carboxylate to an exclusively monodentate coordination next
to the neighbor amide carbonyl function. This observation was
verified by DFT theoretical calculations that demonstrated the
instability of a bidentate carboxylate next to a neighboring
carbonyl. Finally, the rather strong interactions between these
metals and the studied peptides show that, in case of an internal
contamination, these elements could take the place of the
natural elements in larger biological system such as proteins.
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.5b02379.
Discussion of experimental procedures and spectroscopic
characterization of Boc-DGDGD-OMe and Moc-
ADPDA-NH2. Schematic descriptions of syntheses.
Numbered structures of organic synthesized products.
(PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: simoni@ipno.in2p3.fr.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors wish to thank B. Sitaud, S. Schultig, and I. Llorens
for beamline assistance on the MARS beamline of the SOLEIL
synchrotron. We would also like to thank M. Desmadril from
IBBMC, Paris Sud Univ. and L. Assairi from the Curie Institute
in Orsay for providing access to the ITC apparatus. We thank
C. Diarra for his precious help in managing the IPNO cluster:
GRIF (http://www.grif.fr). Finally we would like to acknowl-
edge P. Roy and J.-B. Brubach from the AILES beamline of the
SOLEIL synchrotron and R. Barillon from the Univ. of
Strasbourg for providing access to the IR apparatus.
The discussion of the EXAFS data in combination with
thermodynamic figures determined by ITC have revealed that
DGDGD flexibility enables structural rearrangement with a
thermodynamic positive effect in both europium and uranyl
cases. However, the origin of the affinity increase is cation-
dependent:
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