X-ray absorption spectroscopy proves the trigonal-planar sulfur-only coordination of copper(I) with high-affinity tripodal pseudopeptides

Article abstract of DOI:10.1021/ic401206u

A series of tripodal ligands L derived from nitrilotriacetic acid (NTA) and extended by three converging metal-binding cysteine chains were previously found to bind selectively copper(I) both in vitro and in vivo. The ligands L¹ (ester) and L

Full text of DOI:10.1021/ic401206u

Article
pubs.acs.org/IC
X‑ray Absorption Spectroscopy Proves the Trigonal-Planar Sulfur-
Only Coordination of Copper(I) with High-Affinity Tripodal
Pseudopeptides
Anne-Solene Jullien,† Christelle Gateau,† Isabelle Kieffer,‡,§ Denis Testemale,‡,⊥ and Pascale Delangle*,†
̀
†Service de Chimie Inorganique et Biologique (UMR_E 3 CEA UJF), Commissariat a
Alternatives, INAC, 17 rue des martyrs, 38054 Grenoble Cedex 9, France
̀
l′Energie Atomique et aux Energies
‡BM30B/FAME beamline, European Synchotron Radiation Facility (ESRF), F-38043 Grenoble Cedex 9, France
§Observatoire des Sciences de l′Universite
́ ́
de Grenoble, UMS 832 CNRS Universite Joseph Fourier, F-38041 Grenoble Cedex 9,
France
⊥Institut Nee
́
l, CNRS et Universite
́
Joseph Fourier, BP 166, F-38042 Grenoble Cedex 9, France
S
* Supporting Information
ABSTRACT: A series of tripodal ligands L derived from
nitrilotriacetic acid (NTA) and extended by three converging
metal-binding cysteine chains were previously found to bind
selectively copper(I) both in vitro and in vivo. The ligands L1
(ester) and L2 (amide) were demonstrated to form copper(I)
species with very high affinities, close to that reported for the
metal-sequestering metallothioneins (MTs; log KCu‑MT ≈ 19).
Here, an in-depth study by Cu K-edge X-ray absorption
spectroscopy (XAS) was performed to completely characterize
the copper(I) coordination sphere in the complexes, previously
evidenced by other physicochemical analyses. The X-ray
absorption near-edge structure (XANES) spectra shed light
on the equilibrium between a mononuclear complex and a
cluster for both L1 (ester) and L2 (amide). The exclusive symmetric CuS3 geometry adopted in the mononuclear complexes
(Cu−S ≈ 2.23 Å) was clearly demonstrated by extended X-ray absorption fine structure (EXAFS) analyses. The EXAFS analyses
also proved that the clusters are organized on a symmetric CuS3 core (Cu−S ≈ 2.26 Å) and interact with three nearby copper
atoms (Cu---Cu ≈ 2.7 Å), consistent with the Cu6S9-type clusters previously characterized by pulsed gradient spin echo NMR
spectroscopy. XAS data obtained for other architectures based on the NTA template (L3 acid, L4 without a functionalized
carbonyl group, etc.) demonstrated the formation of polymetallic species only, which evidence the necessity of the proximal ester
or amide group to stabilize the CuS3 mononuclear species. Finally, XAS was demonstrated to be a powerful method to quantify
the equilibrium between the two copper(I) environments evidenced with L1 and L2 at different copper concentrations and to
determine the equilibrium constants between these two complexes.
Indeed, because the cytoplasm is a reducing environment, the
predominant oxidation state of copper in cells is CuI.8 The
copper(I) chelators are inspired from proteins involved in
copper homeostasis such as metallochaperones2,9 and metal-
lothioneins (MTs),10−13 and therefore the copper(I) binding
site is based on cysteines.14,15 Some of these copper(I)
chelators have been targeted to the hepatocytes to localize
their activity because the main organ of copper accumulation in
Wilson’s disease is the liver. Promising results have been
obtained because the targeted molecules have been demon-
strated to be able to effectively chelate excess intracellular
copper.16,17
INTRODUCTION
■
Metal overload is involved in several diseases and intoxica-
tions.1 Among the metals that can accumulate in the body,
copper is an essential element used as a cofactor by many redox
proteins. However, free copper can also promote Fenton-like
reactions and thus can be toxic. That is why the intracellular
concentration of copper is rigorously tuned in the body.2,3
Actually, one of the major genetic disorders of copper
metabolism is Wilson’s disease, which results in copper
overload.4,5 Chelation therapy has been found to be quite
efficient in many cases to treat this disease. Nevertheless, new
and more efficient treatments are necessary because many
current drugs induce side effects, mainly due to a lack of
specificity.4−7 We have been involved for a few years in the
development of intracellular copper(I) chelators that could
provide innovative and specific treatments of Wilson’s disease.5
Received: May 14, 2013
Published: August 12, 2013
© 2013 American Chemical Society
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We recently developed a series of tripodal derivatives based
on a nitrilotriacetic acid (NTA) scaffold extended with three
converging cysteine groups (Figure 1).18,19 We established that
be responsible for the apparent symmetry of the copper(I)
complexes observed at the NMR time scale at 298 K. So, to
ensure the C3 symmetry in the copper(I) complexes, Cu K-
edge XAS at cryogenic temperature was used.
In this paper, we aim at presenting a complete XAS study of
the copper(I) species formed with the tripodal ligands L1 and
L2, which are promising intracellular copper(I) chelating agents.
Ligand L3 and the new compound L4, derived from cysteamine
(Figure 1), which form only polymetallic species, were also
investigated to evaluate the general impact of the NTA
template on the copper(I) cluster species formed in solution.
For both L1 and L2, X-ray absorption near-edge structure
(XANES) spectra provide evidence of trithiolato trigonal
environments around the copper(I) ion in the mononuclear
complexes, whereas extended X-ray absorption fine structure
(EXAFS) analyses give direct proof of the symmetric trigonal-
planar coordination of copper(I) with three sulfur atoms at
average distance Cu−S ≈ 2.23 Å, characteristic of symmetric
CuS3 complexes.24−26 The polymetallic species are also
organized on a CuS3 core, with typical distances Cu−S ≈
2.26 Å, which interacts with three nearby copper atoms at an
average distance Cu---Cu ≈ 2.7 Å, as in many copper(I)
transport proteins such as Atox1,26,27 Cox17,28,29 Mac1, Ace1,30
Amt1,31 Ctr1,32 MTs,33−35 and many inorganic model
clusters.36 Moreover, analyses of edge spectra allowed us to
quantify the amount of mononuclear and polymetallic species
formed in solution with both L1 and L2 and confirmed the
partial formation constants K63 of the polynuclear species
Figure 1. Copper(I) complexes with the tripodal ligands L1−L4.
the two ligands L1 (ester) and L2 (amide) promote the
formation of very stable copper(I) complexes, with a large
selectivity with respect to zinc(II), another cation highly
present in cells. A variety of spectroscopic methodsUV
spectroscopy, circular dichroism (CD), mass spectrometry
(MS), and nuclear magnetic resonance (1H/13C NMR)were
used to characterize the copper(I) complexes formed with
these two ligands, which demonstrate very similar binding
properties: a very stable mononuclear complex, CuL, is formed
and transforms into a unique cluster, (Cu2L)3, in excess of
metal ion.18,19 Moreover, the diffusion coefficients measured by
NMR using a pulsed gradient spin echo (PGSE) sequence
provided the molecularity of these complexes. It was evidenced
that the chelators L form (Cu2L)3 clusters based on Cu6S9
cores, as found in the β domains of some MTs,12,20 which are
small cysteine-rich proteins, forming metal clusters and
biosynthesized in the presence of toxic metal ions or essential
metals such as copper and zinc in excess of physiological
requirements. These structural similarities with MTs were
found to be related to the high affinities of both L1 and L2 for
L1
L2
(Cu2L1−2)3 (log K63 = 20.8 and log K63 = 21.5). These
results are in very good agreement with data previously
obtained by competition experiments with bathocuproine
disulfonate (BCS).18,19 Therefore, both EXAFS and XANES
analyses provided very powerful tools to fully characterize the
copper(I) complexes formed with the tripodal pseudopeptides
in terms of both structures and affinities.
EXPERIMENTAL SECTION
■
Sample Preparation. The syntheses of the ligands L1 (ester), L2
(amide), and L3 (acid) were described elsewhere.18,19 The synthesis of
L4 (without functionalized carbonyl group R; Figure 1) is reported in
the Supporting Information (SI). The whole set of samples L was
prepared in a phosphate buffer (100 mM, pH 7.4)/glycerol/
acetonitrile (70:20:10, v/v/v) mixture, and the final concentration of
the ligand L solution was determined by measuring the free thiol
concentration following Ellman’s procedure.37 This procedure uses
5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) as an indicator. Each free
thiol group present in the peptide yields 1 equiv of TNB2− [λ = 412
nm, ε(TNB2−) = 14150 M−1 cm−1]. The copper(I) solutions were
prepared by dissolving the appropriate amount of Cu(CH3CN)4·PF6
in deoxygenated acetonitrile. The final concentration was determined
by adding an excess of sodium bathocuproine disulfonate (Na2BCS)
1
2
copper(I) in the mononuclear complexes (log KCuL ,CuL = 19.2
and 18.8)18,19 close to those reported for MTs in the literature
(log KCu‑MT ≈ 19).21,22
On the contrary, L3 (Figure 1), which is functionalized with
three carboxylate units, was found to only form polymetallic
species. The mononuclear complex with L3 was not observed,
which may be assigned to charge repulsions between the three
negatively charged groups at physiological pH in the tripodal
ligand.18
The soft sulfur ligands L1−L3 were also found to be efficient
mercury chelating agents, and we demonstrated previously by
low-temperature Hg L-edge X-ray absorption spectroscopy
(XAS) that the apparent average C3 symmetry of the HgS3
environment evidenced by NMR at 298 K was, in fact, a highly
distorted geometry with three Hg−S bonds with very different
lengths.23 These data suggested that the NTA-based tripods are
too constrained to accommodate a trigonal-planar geometry
around the large mercury(II) cation. In fact, the apparent
symmetrical species detected by NMR at 298 K mostly results
from an average of highly dissymmetric complexes (distorted T
shape) that rapidly equilibrate on the NMR time scale at 298 K.
That is why we wondered whether such a fast equilibrium could
3−
and measuring the absorbance of Cu(BCS)2 (λmax = 483 nm, ε =
13300 M−1 cm−1).32 Then, aliquots corresponding to x equiv of
copper(I) (0.5 < [CuI] < 5 mM, i.e., 0.15−2 equiv) were added to the
ligand solution. Because the samples were sensitive to oxygen, the
preparations were performed in a glovebox before each XAS
experiment. Then, the samples were transferred to the XAS beamline
under an argon atmosphere to avoid any oxidation. Drops (50 μL) of
the solutions were then deposed on the sample holders, frozen in
liquid nitrogen, and placed in a helium cryostat to be analyzed by XAS
at Cu K-edge (8979 eV). The compositions of the samples are
reported in Table 1.
XAS. XAS measurements were carried out at the European
Synchrotron Radiation Facility (ESRF, Grenoble, France), which
was operating with a ring current of 200 mA. Cu K-edge XAS spectra
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set to 0.9 because this value was reported for CuS3 model compounds
also studied at the BM30B (Fame) and here chosen as references.26
Then the distances Cu−S and Cu−Cu (Å), the Debye−Waller factor
Table 1. List of Samples Analyzed by XAS in a Phosphate
Buffer (100 mM, pH 7.4)/Glycerol/Acetonitrile (70:20:10,
v/v/v) Mixture
2
(σ2 in Å2), and the goodness-of-fit (χn ) values were compared to data
ligand
sample
[L] (mM)
[CuI] (mM)
Cu/L
reported for model compounds to assert the validities of the EXAFS
fitting results.26−36
L1
1A
1B
1C
1D
1E
1F
2A
2B
2C
2D
2E
2F
2G
2H
3A
4A
4B
2.75
2.68
2.61
2.46
2.39
2.43
3.08
2.90
2.81
2.97
2.81
2.75
2.65
2.47
2.76
2.25
2.04
0.50
1.25
2.00
3.50
4.25
4.83
0.50
0.74
1.41
1.50
2.50
3.50
4.50
5.00
2.50
1.12
3.05
0.18
0.47
0.77
1.41
1.78
1.98
0.16
0.25
0.50
0.51
0.89
1.27
1.70
2.00
0.92
0.50
1.50
RESULTS
■
Most of the results are presented for the ester ligand L1. Similar
data were obtained with the amide ligand L2 and are therefore
mainly presented in the SI.
L2
Copper K-Edge Spectral Analyses. Several copper
complexes have been extensively studied by XAS, and a large
amount of data are available in the literature.26−36,41 In
particular, it is well-known that a peak centered at 8982−
8985 eV in the XANES region accounts for the existence of
copper(I) species. This peak can be interpreted in terms of
ligand-field theory. The transitions from the 1s orbital to the 4p
orbitals of the copper(I) ion are responsible for the absorption
observed in the 8982−8985 eV area of the spectrum (Figure
2).42 Both the peak intensity and the peak energy position
L3
L4
were collected at the BM30B (FAME) beamline using a Si(220)
double-crystal monochromator with dynamic sagittal focusing. The
photon flux was on the order of 1012 photons s−1, and the spot size was
full-width at half-height (fwhm) 300 μm horizontal × 150 μm
vertical.38 The sample holder was loaded in a helium cryostat with the
temperature set to 10 K in order to avoid X-ray beam damage of the
sample during data collection. The spectra were collected in
fluorescence mode by measuring the Cu Kα fluorescence with a 30-
element solid-state germanium detector (Canberra). From 4 to 10
scans, depending on the copper concentration in the sample, 40 min
each were averaged. Data from each detector channel were inspected
for glitches or dropouts before inclusion in the final average. The
energy calibration was achieved by measuring a metallic copper foil for
the Cu edge and assigning the first inflection point of the copper foil
spectrum to 8979 eV.
Figure 2. Normalized Cu edge spectra of copper(I) references (linear
REF(CuS2) = [Et4N][Cu(SAd)2] and trigonal REF(CuS3) =
[Et4N]2[Cu(SC6H4-p-Cl)3]) and experimental spectra of L1-based
copper(I) species [CuL1 = sample 1A and (Cu2L1)3 = sample 1F].
Spectra are offset for clarity.
The data analyses were performed using the Horae package,39
including ATHENA for the data extraction and ARTEMIS for the shell
fitting. XANES spectra were background-corrected by a linear
regression through the preedge region and a low-order polynomial
curve through the postedge region and normalized to the edge jump.
The spectra for L-based copper(I) species were compared qualitatively
to the reference spectra for copper(I)-based thiolate complexes
available in the literature.26−36 For extraction of the EXAFS, the
threshold energy E0 was defined at the half-height of the absorption
edge step. A cubic spline was fitted through the EXAFS energy range
and subtracted to remove the background. The k3-weighted EXAFS
spectra were Fourier-transformed over the k range 2−13 Å−1 using a
Hanning window. The upper limit of the k range, 13 Å−1, was chosen
to avoid signals from minute traces of zinc (threshold 9660 eV, i.e., 13
Å−1) always present in liquid samples.36 The fits were performed on
the Fourier-transformed spectra over the R range 1−3 Å. Two
structural fit models were then applied. The first one (a coordinate-
based model) was derived from the atomic coordinates of the sulfur
and copper atoms in the [Cu4 (CH3S−)6]2−[(C3H7)4N+]2 cluster
described by Baumgartner et al. (see Table S1, SI).40 On the basis of
this structure, for the mononuclear complexes, the atomic coordinates
of Cu(1), S(1), S(2), and S(3) were used in Atoms (ARTEMIS
module). For the clusters, data were fitted using a model based on the
whole set of copper and sulfur coordinates because in this case one
copper atom is surrounded by three sulfur and three copper atoms, as
expected for the L-based copper(I) clusters. The second model
consisted of using structural tetrahedral models (see the SI for further
information). In each case, the coordination number was set to 3 for
sulfur atoms in the mononuclear complexes and 3 for both sulfur and
depend on the ligands that bind the copper(I) ion. Actually,
both the atom types and the coordination sphere around the
copper(I) ion determine the shape of the peak as seen on the
spectra of copper(I) reference compounds given in Figure 2. In
the complex [Et4N][Cu(SAd)2],26,41,43 the copper(I) ion is
linearly coordinated to two sulfur atoms, and the peak appears
sharp and intense [REF(CuS2) in Figure 2].26 By contrast, the
peak intensity related to the complex [Et4N]2[Cu(SC6H4-p-
Cl)3],25,26 where copper(I) is trigonally coordinated by three
sulfur atoms, is weaker [REF(CuS3) in Figure 2].
The spectra of these reference compounds in the solid state26
are compared with those of copper(I) complexes of L1 in
solution in Figure 2. Clearly, the spectrum obtained for the
mononuclear complex CuL1, which is the species present in
excess of ligand, i.e., in sample 1A (Table 1), shows strong
similarities with the spectrum recorded for the trigonally
coordinated copper(I) in [Et4N]2[Cu(SC6H4-p-Cl)3] [REF-
(CuS3) in Figure 2]. These two spectra display very similar
intensities (∼0.65−0.68) of the peak centered at 8982−8985
eV and similar relative intensities and positions of the XANES
peaks in general. This suggests that the central copper(I) ion is
trigonally bound to the three sulfur atoms of the ligand scaffold
in the mononuclear complex CuL1. In excess of copper(I), the
spectrum is different and the peak intensity collapses
2
copper atoms in the clusters. The amplitude reduction factor S0 was
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completely when 2 equiv of copper(I) is added to L1 in sample
1F (Table 1 and Figure 2). This is consistent with formation of
the cluster (Cu2L1)3, in which the environment of the central
copper(I) ion differs from the one found in the mononuclear
complexes. For intermediate concentrations of copper(I), the
intensity of the peak centered at 8982 eV decreases gradually
before the final collapse for 2 equiv of copper(I) added. This
phenomenon is depicted in Figure 3 for L1.
molecularities (z) of the clusters (Cu2L3)z and (Cu2L4)z by
1
diffusion coefficient measurements by NMR because the H
NMR signals of the complexes with L3 or L4 were too large.
Anyway, the lack of an amide or ester function next to the
coordinating thiolate functions of L (Figure 1) is responsible
for the absence of well-defined mononuclear complexes in
solution with both L3 and L4.
To characterize the copper(I) species formed in solution and
refine the coordination spheres around the copper(I) ion in
each case, the EXAFS domains of the XAS spectra were then
looked into. The EXAFS data of the mononuclear complexes
CuL1 and CuL2 were analyzed in samples 1A and 2A with low
copper(I) concentrations. The clusters (Cu2L1)3 and (Cu2L2)3
were investigated in samples 1F and 2H, which contain 2 equiv
of copper(I). Both samples 1A (or 2A) and 1F (or 2H) were
shown previously by 1H NMR to contain only the mononuclear
adduct and the polymetallic one, respectively.
EXAFS Analyses: Mononuclear Complexes of L1 and
L2. The EXAFS fitting results obtained for the mononuclear
complex CuL1 are depicted in Figure 5a. Similar graphs were
obtained for the mononuclear complex CuL2 (Figure S2, SI).
Clearly, the model based on a central copper atom surrounded
by three sulfur atoms in a trigonal-planar geometry40 returns a
very good fit of the data collected. The quantitative results are
reported in Table 2. Importantly, models with only two sulfur
atoms in a digonal geometry give significantly poorer results,
and models with a T-shaped coordination return nonphysical
negative Debye−Waller values. The three S−Cu distances were
found to be almost equal, at ≈2.23 Å (Table 2). This distance
belongs to the characteristic distance range reported for
symmetric CuS3 compounds, ≈2.22−2.26 Å, in the liter-
ature.24−26 Consequently, these data confirm that both L1 and
L2 form mononuclear complexes with a typical symmetric CuS3
coordination. This demonstrates that the NMR spectra
recorded at 298 K17,18 are characteristic of C3-symmetric
mononuclear copper(I) complexes and are not the result of
dissymmetric species averaged at the NMR time scale at 298 K
like with mercury(II).23
EXAFS Analyses: Polymetallic Species (Cu2L)z. The
EXAFS fitting results obtained for the cluster (Cu2L1)3 are
depicted in Figure 5b. Analogous graphs were obtained for the
cluster (Cu2L2)3 (Figure S2, SI). The models based on a central
copper atom surrounded by three sulfur atoms in a trigonal-
planar geometry and three copper atoms40 give a good fit of the
data collected, either using the coordinate-based model or the
tetrahedral-based model. In particular, they reproduce the beat
at 8 Å−1, characteristic of the additional Cu---Cu interactions.
The quantitative results are reported in Table 2. The distances
Cu−S ≈ 2.26 Å and Cu---Cu ≈ 2.7 Å were found to be in
accordance with values given in the literature for several
copper(I) proteins,26−32 in particular MTs,33−35 and some
inorganic model clusters36 organized on a CuS3 core, which
interact with one to three nearby copper atoms in either Cu4S6
or Cu6S9 clusters. The Cu---Cu distances obtained with the
coordinate-based model are not equal (≈2.65−2.82 Å; see
Table 2), but the resolution expected for these second-shell
atoms is too low (∼0.1 Å)44 to allow a discrimination between
so close distances. Therefore, the tetrahedral-based model,
which sets the three copper atoms at the same distance (2.73
Å) from the central copper atom, is preferred.
Figure 3. Normalized Cu edge spectra obtained for ligand L1 with
increasing amounts of copper(I).
The analogous graph for L2 is reported in Figure S1 in the SI.
This regular decrease clearly indicates that the cluster is
forming at the expense of the mononuclear complex when the
copper concentration increases in solution. Besides, isosbestic
points in the edge area of the spectra (Figure 3) are detected at
8981, 8984, and 8988 eV, which demonstrates the presence of
only two copper(I) complexes in equilibrium. These observa-
tions corroborate previous physicochemical studies (UV, CD,
1
MS, H/13C NMR, and PGSE-NMR), which have shown that
both L1 and L2 form mononuclear complexes and the clusters
(Cu2L1)3 and (Cu2L2)3 in solution.18,19
By contrast, L3 (Figure 1) was shown to be unable to form
mononuclear complexes in solution, even at low concentrations
of copper and would rather complex copper in (Cu2L3)z
polymetallic species.18 Similar observations were obtained
with the analogue L4. As expected, the peak centered at 8982
eV in the XAS spectra of these complexes was very flat and the
shapes of the curves were similar to the one observed for the
clusters (Cu2L1)3 and (Cu2L2)3 in samples 1F and 2H (Figure
4), even in excess or stoichiometric amounts of the ligand.
Unfortunately, it was not possible to determine the
Figure 4. Normalized Cu edge spectra of L1 complexesCuL1
(sample 1A) and (Cu2L1)3 (sample 1F)compared to the spectra
measured for CuL3 (sample 3A; 1 equiv of copper) and CuL4 (samples
4A and 4B; 0.5 and 1.5 equiv of copper, respectively) species. Spectra
are offset for clarity.
The EXAFS analyses performed for the copper(I) complexes
formed with the ligands L3 and L4 gave very similar results
(Table 2). The corresponding graphs are depicted in Figure S3
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Figure 5. XAS data for (a) the mononuclear complex CuL1 (sample 1A) and (b) the cluster (Cu2L1)3 (sample 1F). Left: Spectra of the k3-weighted
EXAFS experimental data and corresponding fit. Right: Fourier transforms of the k3-weighted EXAFS experimental data and corresponding fit. FT
and IM FT are the real and imaginary parts for Fourier transformation, respectively. Solid lines: experimental data. Dotted lines: fit.
a
Table 2. EXAFS Fitting Results
complex
sample
3 × Cu−S
σ(S)2
3 × Cu−Cu
σ(Cu)2
ΔE
χn
R
2
CuL1
1A
2A
2.23
2.23
2.26
2.26
2.26
2.26
2.26
2.26
2.26
2.26
5(1)
5(1)
5(1)
5(1)
5(1)
5(1)
5(1)
4(1)
4(1)
4(1)
4(2)
3(2)
5(2)
5(2)
4(1)
4(2)
5(1)
5(1)
5(1)
5(1)
15
4.0
7.1
4.5
7.0
2.0
2.4
2.0
2.5
1.3
2.0
CuL2
33
92
118
37
51
23
34
9
b
(Cu2L1)3
1F
2.65, 2.71, 2.80
2.73
10(3)
15(3)
9(2)
c
1F
b
(Cu2L2)z
(Cu2L3)z
(Cu2L4)z
2H
2.66, 2.72, 2.82
2.74
c
2H
14(4)
5(1)
b
3A
2.66, 2.71, 2.81
2.73
c
3A
10(1)
5(1)
b
4A
2.66, 2.73, 2.82
2.73
c
4A
10(1)
16
a
Distances are in angstroms with 0.01 or 0.02 errors for the Cu−S and Cu−Cu distances, respectively. Other experimental errors on the last
character are indicated in parentheses. Debye−Waller factors (σ2) are in Å2 × 103. The threshold energy shifts ΔE are in electronvolts. χn2 and R (%)
are the reduced χ2 and the R factor of the fit, respectively. Samples 1A and 2A (mononuclear complexes) were analyzed with atomic coordinates of
b
Cu(1), S(1), S(2), and S(3) only. Samples 1F, 2H, 3A, and 4A (polynuclear complexes) were analyzed with the whole set of copper and sulfur
atomic coordinates of sulfur and copper atoms in the [Cu4(CH3S−)6]2−[(C3H7)4N+]2 cluster described by Baumgartner et al.40 Samples 1F, 2H, 3A,
c
and 4A (polynuclear complexes) were analyzed with the tetrahedral-based model of coordinates (see the Experimental Section).
in the SI. Like the analogous architectures L1 and L2, both L3
and L4 form polymetallic species based on a symmetric CuS3
core (Cu−S ≈ 2.26 Å), which interacts with three copper
atoms at the distance Cu---Cu ≈ 2.7 Å. These results provide
the evidence that, as a main rule, ligands based on the NTA
template tend to form the same type of first-shell clusters in
solution.
The two binding constants were evaluated in previous studies
with competition experiments starting from the mononuclear
complex or the cluster and adding BCS as a competitor for
copper(I).18,19 The two ligands L1 and L2 were found to tightly
bind the copper(I) ion in the mononuclear complexes with
affinities log β11 = 19.2 and 18.8, respectively, close to those
determined for MTs (log KCu‑MT ≈ 19).21,22 The equilibrium
constant between the mononuclear complexes and the
polymetallic ones, K63, was also determined: log K63 = 20.719
and 21.545 for L1 and L2, respectively. The expected speciation
in the conditions used for the XAS experiments could thus be
calculated from these constants with the program Hyss 200946
and is represented by the dotted red lines in Figure 6.
Speciation of the Copper(I) Complexes with L1 and L2
from the XANES Spectra. The thermodynamic equilibria for
the formation of copper(I) complexes with L1 and L2 were
shown to be the following:19
CuI + L = CuL (β11)
(1)
On the other hand, because the XANES spectra are sensitive
to both the nuclearity and geometry of the copper(I) species
formed in solution,36,42 it was possible to calculate the
proportions of each complex in the samples. Linear
1
CuL + CuI = (Cu2L)3 (K63)
(2)
3
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(amide) exhibit the most interesting copper(I) chelating
properties with very high affinities and an equilibrium between
a mononuclear complex and a unique polymetallic species
(Cu2L1−2)3.
Cu K-edge XAS was used here to gain insights into the
structures of the copper(I) complexes and to completely
characterize the coordination sphere of the copper(I) ion.
Analyses of the XANES spectra of L1 and L2 confirm both the
existence of the mononuclear complex at low copper
concentration and the equilibrium between only two different
copper environments, the mononuclear complex and a unique
polymetallic species, as evidenced by several isosbestic points.
The exclusive CuS3 geometry adopted in the mononuclear
complexes was clearly supported by EXAFS fitting, which
provide three equal distances Cu−S ≈ 2.23 Å, characteristic of
symmetric CuS3 coordination. It is quite interesting to notice
that these symmetric copper(I) mononuclear complexes
dramatically differ from the dissymmetric analogous mercury-
(II) mononuclear complexes recently investigated by Hg L-
edge XAS spectroscopy.23 Whereas other spectroscopic
methods used at ambient temperature (1H and 199Hg NMR
and UV spectroscopy) indicated a trigonal-planar trithiolato
coordination of mercury(II), the EXAFS data revealed a
dissymmetrical HgS3 binding environment with three sulfur
atoms at 2.38, 2.51, and 2.67 Å (Figure 7, left). Even though
Figure 6. Comparison between the proportions of mononuclear
complexes (a) CuL1 and (b) CuL2, obtained after linear combinations
of the XANES spectra in ATHENA (black circles) and the proportions
calculated from thermodynamic constants obtained from BCS
competitions (dotted red lines). (a) L1: log β11 = 19.2, log K63
=
20.7, [L] = 2.5 mM. (b) L2: log β11 = 18.8, log K63 = 21.5, [L] = 2.75
mM.
combinations of the extreme XANES spectra obtained for the
lowest copper concentration (Table 1, samples 1A or 2A,
[Cu]/[L] = 0.16−0.18) and the highest copper concentration
(Table 1, samples 1F or 2H, [Cu]/[L] = 2) were performed
with the linear combination module available in ATHENA.47
Indeed, samples 1A (or 2A) and 1F (or 2H) were shown by 1H
NMR to contain only the mononuclear adduct and the
polymetallic one, respectively. The EXAFS threshold energy E0
was set at 8980 eV for the whole set of curves. The energy
range around the threshold energy was typically restrained to
10 eV around E0. An analysis close to E0 was preferred
because the long-range disorder and noise peculiar to each
EXAFS spectrum do not affect the edge area. The results are
reported in Tables S4 and S5 in the SI for L1 and L2,
respectively. Figure 6 shows the superimposition of the
experimental points derived from the edge analysis and the
speciation calculated with the constant values previously
obtained from competitions with BCS. The agreement is very
satisfying for the two ligands. The constants K63 were calculated
from the edge data and for samples containing a reasonable
Figure 7. Structures of the coordination spheres of mercury(II) and
copper(I) in their mononuclear complexes with L1 and L2.
coordination changes upon freezing cannot be totally excluded,
this result suggests that the tripodal ligands L1 and L2 are too
constrained to accommodate a trigonal-planar coordination
mode for the large cation mercury(II). On the contrary, the
EXAFS data obtained with copper(I) in this work demonstrate
that this metal ion is bound by three sulfur atoms in a very
symmetric environment, with three identical Cu−S bonds with
an average distance Cu−S = 2.23 Å (Figure 7, right). The
difference in the metal coordination spheres between mercury-
(II) and copper(I) complexes may be assigned to their ionic
radii: the mercury(II) ionic radius is approximately 50% greater
than that of copper(I).48
As for the polymetallic species, the EXAFS fitting
implemented with the coordinates of a Cu4S6 model cluster
was found to return good fits of the cluster data, thus
demonstrating that the central copper of the clusters is
surrounded by three sulfur atoms at equal distances (Cu−S
≈ 2.26 Å) and three copper atoms (Cu---Cu ≈ 2.7 Å). Of
course, the EXAFS data do not allow us to distinguish between
(Cu2L)z clusters of different molecularities (z > 1) because the
copper environments are very similar in clusters of different
sizes. However, the results are consistent with the structure of
the clusters (Cu2L1,2)3, which were evidenced by diffusion
NMR spectroscopy.18,19 More generally, because the two
L1
amount of the two complexes: log K63 = 20.8 0.1 (for L1,
L2
samples 1B, 1C, and 1D; Table S4, SI) and log K63 = 21.5
0.4 (for L2, samples 2B−2F; Table S5, SI).
DISCUSSION
■
The tripodal ligands L1−L4 derived from NTA and extended by
three converging metal-binding cysteine chains revealed to be
very efficient copper(I) chelating agents both in vitro18,19 and
in vivo.16 Besides, previous physicochemical analyses have
shown that two types of copper(I) complexes are formed in a
water solution at physiological pH.17,18 L1 (ester) and L2
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Inorganic Chemistry
Article
ligands L3 (acid) and L4 (no substituent R) were also found to
form clusters with similar geometries, it was concluded that, as
a main rule, the NTA-based chelator L tends to form
(Cu2L1)z−(Cu2L4)z clusters as found in copper(I) pro-
teins,26−32 in particular MTs,33−35 and some inorganic model
clusters.36 This result provides insight into an efficient design of
copper(I) chelators that mimic the coordination sphere found
in proteins in either Cu4S6 or Cu6S9 clusters.26−36
FAME CRG beamline for provision of the synchrotron
radiation beamline and facilities.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Syntheses of ligand L4 and supplementary figures and tables for
the XAS analyses. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: pascale.delangle@cea.fr.
Notes
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the “Agence Nationale pour la
Recherche” (COPDETOX; Grant ANR-11-EMMA-025), the
“Fondation pour la Recherche Medicale” (Grant
́
DCM20111223043), and the Labex ARCANE (Grant ANR-
11-LABX-0003-01). The authors acknowledge the ESRF and
4677.
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