Link between Affinity and Cu(II) Binding Sites to Amyloid-β Peptides Evaluated by a New Water-Soluble UV-Visible Ratiometric Dye with a Moderate Cu(II) Affinity

Article abstract of DOI:10.1021/acs.analchem.6b04979

Being able to easily determine the Cu(II) affinity for biomolecules of moderate affinity is important. Such biomolecules include amyloidogenic peptides, such as the well-known amyloid-β peptide involved in Alzheimer's disease. Here, we report the synthesis of a new water-soluble ratiometric Cu(II) dye with a moderate affinity (10⁹ M^-1 at pH 7.1) and the characterizations of the Cu(II) corresponding complex by X-ray crystallography, EPR, and XAS spectroscopic methods. UV-vis competition was performed on the Aβ peptide as well as on a wide series of modified peptides, leading to an affinity value of 1.6 × 10⁹ M^-1 at pH 7.1 for the Aβ peptide and to a coordination model for the Cu(II) site within the Aβ peptide that agrees with the one mostly accepted currently.

Full text of DOI:10.1021/acs.analchem.6b04979

Subscriber access provided by UNIV OF CALIFORNIA SAN DIEGO LIBRARIES
Article
Link Between Affinity and Cu(II) Binding Sites to Amyloid-
Peptides Evaluated by a New Water-Soluble UV-
#
Visible Ratiometric Dye with a Moderate Cu(II) Affinity.
Amandine Conte-Daban, Valentina Borghesani, Stephanie Sayen, Emmanuel
Guillon, Yves Journaux, Geoffrey Gontard, Laurent Lisnard, and Christelle HUREAU
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04979 • Publication Date (Web): 12 Jan 2017
Downloaded from http://pubs.acs.org on January 16, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth
Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 9
Analytical Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Link Between Affinity and Cu(II) Binding Sites to Amyloidꢀβ Pepꢀ
tides Evaluated by a New WaterꢀSoluble UVꢀVisible Ratiometric Dye
with a Moderate Cu(II) Affinity
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a,b‡
a,b‡
c
c
Amandine ConteꢀDaban, Valentina Borghesani, Stéphanie Sayen, Emmanuel Guillon, Yves
d,e
d,e
d,e
a,b,*
Journaux, Geoffrey Gontard, Laurent Lisnard and Christelle Hureau
a,b
CNRS; LCC (Laboratoire de Chimie de Coordination) ; 205, route de Narbonne, Fꢀ31077 Toulouse, France. Université de
Toulouse; UPS, INPT ; LCC ; Fꢀ31077 Toulouse, France
c
Université de Reims Champagne Ardenne, Institut de Chimie Moléculaire de Reims (ICMR), UMR 7312 CNRSꢀURCA,
Moulin de la Housse, BP 1039, 51687 Reims Cedex 2, France
d
Sorbonne Universités, UPMC Univ. Paris 06, UMR 8232, IPCM, Fꢀ75005, Paris, France
e
CNRS, UMR 8232, Institut Parisien de Chimie Moléculaire, Fꢀ75005, Paris, France
ABSTRACT: Being able to easily determine the Cu(II) affinity for biomolecules of moderate affinity is important. Such biomoleꢀ
cules include amyloidogenic peptides, such as the wellꢀknown amyloidꢀβ peptide involved in Alzheimer’s disease. Here, we report
9
ꢀ1
the synthesis of a new water soluble ratiometric Cu(II) dye with a moderate affinity (10 M at pH 7.1) and the characterizations of
the Cu(II) corresponding complex by Xꢀray crystallography, EPR and XAS spectroscopic methods. UVꢀVis competition were
9
ꢀ1
performed on the Aβ peptide as well as on a wide series of modified peptides, leading to an affinity value of 1.6 10 M at pH 7.1
for the Aβ peptide and to a coordination model for the Cu(II) site within the Aβ peptide that agrees with the one mostly accepted
currently.
1
Copper ions play key biological roles. They are essential
metal ions that play an important role as catalytic centers in
several processes including very fundamental ones like energy
insights into the coordination sphere of the metal center when
a series of modified peptides is studied. As a direct conseꢀ
quence, the straightforward and accurate determination of Cu
affinity for peptides is an important objective. However, this is
an intricate task since it necessitates appropriate analytical
tools. The complexity of such studies is well illustrated by the
1
production. They have also been linked to several diseases.
Dyshomeostasis of Cu is very dangerous and is well docuꢀ
mented by two lethal genetic diseases, called Wilson's and
Menkes' diseases, linked to an overload of Cu and to a depleꢀ
tion of Cu, respectively. Cu imbalance has also been inꢀ
volved in the etiology of most of neurological disorders such
abundant and differing reports on the evaluation of Cu(II)
2ꢀ5
16ꢀ20
affinities for the Aβ peptide (see refs.
and refs. therein).
6
18,22ꢀ24
This is also true for the +I redox state of Cu,
which is
7ꢀ9
10,11
as Alzheimer's disease (AD), Parkinson's disease (PD),
however not under focus in the present study. While Cu(II)
1
0,12
6
ꢀ1
19
ꢀ1
Prion diseases.
affinity values ranging from 10 M to 10
M
have been
25
proposed, a relative consensus has recently been reached
At a molecular scale, two parameters are important regardꢀ
ing Cu and the peptides or proteins involved in the aboveꢀ
mentioned diseases: (i) the metal environment, i.e. the nature
of the atoms surrounding the metal center and (ii) the affinity
of the peptides for the metal center. In the context of AD
which is under focus in the present article, there are many
reports on the coordination sites of the Cu(II) center to the
1
0
ꢀ1
17,18
around a value of 10
M
at pH 7.4
(this value correꢀ
sponds to the conditional affinity value, i.e. the absolute affiniꢀ
ty value at a given pH ; the interaction with buffer is not conꢀ
sidered and will lead to a weaker soꢀcalled apparent affinity
25
value). Hence the objective of the present study is to define a
new tool appropriate for the rapid, easy and accurate evaluaꢀ
tion of Cu(II) binding affinities to peptides.
8,13ꢀ15
amyloidꢀβ peptide (Aβ) (for recent reviews, see refs.
) and
and refs. therein).
Some studies also aim at relating the Cu(II) affinity of modiꢀ
1
6ꢀ18
16
on the Cu(II) affinity for Aβ (see refs.
There are mainly two ways of determining affinity values.
The first one relies on potentiometric titrations that lead to
absolute constants, from which an apparent affinity value can
1
9ꢀ21
fied Aβ peptides with the coordination sites.
16,26
be calculated at any pH values.
The second one consists in
Determining the affinity of Cu(II) for peptides is thus of inꢀ
terest, first because this is intrinsically an important parameter
that mirrors the possibility to have the metal ionꢀpeptide interꢀ
action in biological conditions, and second because it give
measurements at a given pH either by (i) isothermal calorimeꢀ
try (ITC), fluorescence when a fluorophore is natively present
in the peptide sequence or added for purpose and when the
1
ACS Paragon Plus Environment

Analytical Chemistry
Page 2 of 9
fluorescence is quenched by the addition of the metal ion of DAEFGHDSGYEVRHQK; Aβ , DAEFRHDSGYEVHHQKꢀ
28
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
interest or (ii) by competition experiments with a ligand of
known affinity followed by the appropriate method. Potentiꢀ
ometry is likely the most powerful technique since it gives
access to the affinity value at any pH values but could be
extremely difficult to implement in case of biomolecules and
requires important quantity of the biological material. Results
from ITC are far from straightforward although additional data
apart from the affinity can be evaluated (enthalpy, entropy...)
LVFFAEDVGSNK;
LVFFAEDVGSNKGAILGLMVGGVV were bought from
GeneCust (Dudelange, Luxembourg) with purity grade > 95
%.
Aβ ,
DAEFRHDSGYEVHHQKꢀ
40
Stock solutions of the peptides were prepared by dissolving
powder in milliQ water (resulting pH ~ 2), except for the Aβ28
and Aβ40 peptides which were dissolved in NaOH 50 mM.
Peptide concentration was determined by UVꢀVis absorption
16
(reviewed in ref. ). Competition experiments appear to be a
of Tyr10 considered as free tyrosine (at pH ~ 2.0, ε ꢀ ε
=
276 296
ꢀ1 ꢀ1 31
ꢀ
1
ꢀ1
wellꢀsuited method. With respect to Cu(II), UVꢀVis and fluoꢀ
rescent spectroscopic tools are of interest. In a seminal paper,
Wedd and coworkers have designed four peptideꢀbased fluorꢀ
1
410 cm .M ; at pH ~ 12.5, ε – ε360 = 2400 cm .M ). For
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
293
the mutants without Tyr10, the absorption of the two Phe was
used (ε258 – ε280 = 390 cm .M ).
ꢀ
1
ꢀ1 32
8
ꢀ1
ophores of various Cu(II) affinities ranging from 10 M to
14
ꢀ1
As the Cu(II) coordination sites in the Aβ peptide are localꢀ
ized in the first 16 amino acids residues, the Aβ and its
counterparts were used as a model of the fullꢀlength peptides.
1
0
M . They were further used to probe the affinity of Cu(II)
8
16
for different peptides and proteins by competition monitored
27
by fluorescence. While Cu(II) quenches the fluorescence of
the competitor, the addition of the peptide of interest restores
it, removing the Cu(II) from the probe.
ABH and BAH where B corresponds to the βꢀalanine were
bought from Protéogenix (Strasbourg, France) and stock soluꢀ
tions were prepared by solubilizing the peptides in milliQ
water. Their concentrations were determined by UVꢀVis titraꢀ
tion with a titrated Cu(II) solution following the dꢀd band
In the present paper, we propose an alternative strategy that
relies on a UVꢀvisible competition experiment against an easy
9
ꢀ1
to synthesize Cu(II) dye with moderate affinity (3.2 10 M at
pH 7.1) that is anticipated to be perfectly appropriate for evalꢀ
uation of Cu(II) affinity to weakly structured peptide, such as
Aβ, at physiological pH. By UVꢀVis, the addition of the pepꢀ
tide of interest on the colored Cu(II) complex induces the
disappearance of the absorption bands.
33
34
absorption of the complex, according to ref.
.
Ligand. The 3,4ꢀbis(oxamato)benzoic acid ligand, L (see
Scheme S1), was synthetized as described below and used in
its sodium salt form. A 8.5 mM stock solution was prepared,
increasing the pH until solubilization, determined by UVꢀVis
titration with a titrated Cu(II) solution following the band
Using this new dye, we have investigated more than sevenꢀ
teen peptidic sequences derived from the human Aβ peptide,
including murine, Nꢀterminally modified peptides and biologiꢀ
cally relevant mutants. Their Cu(II) affinities were evaluated
and discuss with respect to the coordination models previously
absorption at 330 nm of the complex (see text below and Figꢀ
ure 3), according to ref.
34
.
Synthesis of H L.
5
H Et L: 3,4-(diethyloxamate)benzoic acid:
3
2
1
3,21,28ꢀ30
proposed in the literature.
The ester form of the L ligand was prepared following the
known procedure for oxamate ligands
ously reported synthesis. To 5 g of 3,4ꢀdiaminobenzoic acid
(32.8 mmol) in THF (200 mL) is added with strong stirring
35,36
instead of the previꢀ
37
EXPERIMENTAL SECTION
Chemicals. Reagents were commercially available and
were used as received. All the solutions were prepared in
milliQ water (resistance: 18.2 Mꢁ.cm).
8
.25 mL of ethyl oxalyl chloride (72 mmol, 2.2 eq). The mixꢀ
ture is refluxed for 2 hours, filtered while hot and let to cool
down to room temperature. Removal of the solvent leads to a
brown precipitate. The solid is abundantly washed with water,
filtered, washed with 50 % v. ethanol and then with a miniꢀ
mum of cold 96 % v. ethanol, and dried in air. Yield: 8.9 g (77
The Cu(II) ion source was CuSO .5H O, bought from Sigꢀ
4
2
maꢀAldrich. A stock solution was prepared at 25 mM.
HEPES buffer (sodium salt of
2ꢀ[4ꢀ(2ꢀ
%
). See Supporting Info for elemental analysis, NMR and IR
hydroxyethyl)piperazinꢀ1ꢀyl]ethanesulfonic acid) was bought
from SigmaꢀAldrich. A stock solution was prepared at 500
mM, pH = 7.3 in order to reach a resulting pH 7.1 and the
same buffer stock solution was used over the course of the
present study.
characterizations.
Na H L·2.2H O
1.5 3.5
2
Aqueous NaOH (2 M, 57 mL) was added dropwise to a susꢀ
pension of H Et L (8 g, 22.7 mmol) in water (400 mL). The
3
2
solution was stirred for 30 min and filtered. Addition of 4 M
HCl (28 mL) to the resulting solution leads to the rapid forꢀ
mation of a brown precipitate. After 10 min the precipitate is
collected on a sintered glass filter, washed with cold water,
then with 96 % v. EtOH, and finally with ether before being
dried for several hours in air and overnight at 45°C in an oven.
Yield: 5.4 g (64%). See Supporting Info for elemental analyꢀ
sis, NMR and IR characterizations.
Peptides. Aβ16 (DAEFRHDSGYEVHHQK) and the muꢀ
tants:
AcꢀAβ ,
AcꢀDAEFRHDSGYEVHHQK;
D1NꢀAβ16,
E3QꢀAβ ,
1
6
NAEFꢀRHDSGYEVHHQK;
DAQFRHDSGYEVHHQK;
DAEFRHNSGYEVHHQK;
16
D7NꢀAβ16,
E11QꢀAβ16,
DAEFRꢀ
HDSGYQVHHQK; H6RꢀAβ , DAEFRRDSGYEVHHQK;
1
6
H6AꢀAβ , DAEFRADSGYEVHHQK; H13AꢀAβ , DAEFRꢀ
1
6
16
Synthesis of Li [Cu(L)]
HDSGYEVAHQK; H14AꢀAβ , DAEFRHDSGYEVHAQK;
3
1
6
mAβ , DAEFGHDSGFEVRHQK; R5GꢀAβ , DAEFGHDꢀ
Suitable crystals of the copper dye, Cu(L), were obtained as
follow: A suspension of H Et L (0.100 g, 0.28 mmol) in water
(10 mL) was treated with 0.71 mL of LiOH 2 M (1.42 mmol)
16
16
SGYEVHHQK; Y10FꢀAβ , DAEFRHDSGFEVHHQK;
1
6
3
2
H13RꢀAβ , DAEFRHDSGYEVRHQK; R5GꢀH13RꢀAβ16,
16
2
ACS Paragon Plus Environment

Page 3 of 9
Analytical Chemistry
and stirred at room temperature until complete dissolution of
the ligand. An aqueous solution of Cu(NO ) ·3H O (0.068 g,
pean Synchrotron Radiation Facility (ESRF, Grenoble,
43
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
France). The storage ring was operated in 7/8+1 mode at 6
GeV with a 200 mA current. The beam energy was selected
3
2
2
0.28 mmol, 5 mL) was then added dropwise to the oxamate
solution resulting in a deep blue solution of the copper comꢀ
plex that was stirred for 30 min at room temperature and filꢀ
tered. Slow diffusion of acetone yields crystals of
Li [Cu(L)]·5.5H O in 15 days. Yield: 0.054 g (41 % based on
using a Si(220) N cryoꢀcooled doubleꢀcrystal monochromator
2
with an experimental resolution close to that theoretically
predicted (namely ~ 0.5 eV FWHM at the Cu energy). The
2
beam spot on the sample was approximately 300 x 100 µm (H
3
2
Cu). See Supporting Info for elemental analysis, NMR and IR
characterizations.
x V, FWHM). Because of the low Cu(II) concentration, specꢀ
tra were recorded in fluorescence mode with a 30ꢀelement
solid state Ge detector (Canberra) in frozen liquid cells in a He
cryostat. The temperature was kept at 20 K during data collecꢀ
tion. The energy was calibrated with Cu metallic foil, such that
the maximum of the first derivative was set at 8979 eV.
EXAFS Cu(II) data were collected from 8840 to 8960 eV
using 5 eV step of 2 s, from 8960 to 9020 eV using 0.5 eV
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
METHODS
XꢀRay structure. Crystallographic data were collected on
a Bruker KappaꢀAPEX II CCD diffractometer (CuKα, λ =
1
2
1
,54178). Crystal data: purple plates, monoclinic C2/c, a =
6.3214(15), b = 6.7217(4), c = 20.4183(11) Å, β =
ꢀ1
step of 3 s, and from 9020 to 9300 eV with a kꢀstep of 0.05 Å
3
and 3 s per step. At least six scans recorded on different spots
were averaged. XAS samples were prepared from stock soluꢀ
tions of ligand and Cu(II) diluted down to approx. 1.0 mM in
buffered solution. Samples were frozen in the sample holder
after addition of 10% glycerol as a cryoprotectant and stored
in liquid nitrogen until used. Cu(II) photoreduction was conꢀ
trolled by recording successive scans at the same spot. It was
considered that during the first 20 minutes of recording the
photoreduction is insignificant.
12.590(3)°, V = 3335.3(3) Å , Z = 8, T = 200(1) K, ρ = 1.567
ꢀ3
ꢀ1
g.cm , F(000) = 1560, ꢂCuKα = 2.293 mm . Crystals were
mounted on a Hamilton cryoloop using ParatoneꢀN oil and
placed in the cold flow produced with an Oxford Cryocooling
device. Partial hemispheres of data –preselected with the
38
APEX II software were collected using ϕ and ω scans. Inteꢀ
grated intensities were obtained with SAINT+ and were corꢀ
3
8,39
rected for absorption with SADABS;
the structure was
4
0
41
solved with SIR92 and refined with SHELXLꢀ2014/7
4
2
(
WinGX software package). Data refinement gives (using
71 parameters and 6 restraints) wR2 = 0.2432 (2923 unique
reflections), R = 0.0720 (2514 reflections with I>2σ(I)), GOF
The data analysis was performed using the ‘‘Multiꢀ
Plateform Applications for Xꢀray Absorption’’ package, inꢀ
2
4
4
cluding Cherokee and Roundmidnight programs, according
to the standard and previously reported data analysis proceꢀ
1
=
1.128. Crystallographic details are available in CIF format,
free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html
or from the Cambridge Crystallographic Data Centre, 12,
Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223ꢀ336ꢀ
33; or deposit@ccdc.cam.ac.uk). CCDC number 1516020.
4
5,46
dures.
regression through the preꢀedge region and a polynomial
through the postꢀedge region. The backscattering phase, Φ (k,
), and amplitude, A (k, R ), functions were obtained using
Spectra were backgroundꢀcorrected by a linear
(
i
0
R
i
i
i
47
1
13
the ab initio FEFF7 code. Since theoretical phase shifts were
NMR, IR and Elemental Analysis. H and C NMR
used, it is necessary to fit the energy threshold E by adding an
0
spectra were collected on a 400 MHz Bruker Avance specꢀ
trometer at 298 K.
extra fitting parameter, ꢃE . Moreover, the FEFF7 code was
0
used to check if the multiple scattering of our reference comꢀ
pounds of known crystallographic structure is negligible in the
0–3 Å range. The estimated errors for distances and coordinaꢀ
tion numbers are ± 0.02 Å and ± 20%, respectively.
ATR/FTꢀIR spectra were collected on a Bruker TENSOR 27
equipped with a simple reflexion ATR diamond plate of the
Harrick MPV2 series.
Elemental analysis was performed at the ICSN, CNRS UPR
UVꢀVisible spectrophotometry. UVꢀvis spectra were
recorded on a spectrophotometer SPECORD S600 Analytik
Jena at 25°C in 1 cm path length quartz cuvette.
2
301, in GifꢀsurꢀYvette, France.
Electron Paramagnetic Resonance. Electron Paraꢀ
magnetic Resonance (EPR) data were recorded using an
Elexsys E 500 Bruker spectrometer, operating at a microwave
frequency of approximately 9.5 GHz. Spectra were recorded
using a microwave power of 20 mW across a sweep width of
Competition experiments between the ligand and the pep-
tides
The experiments have been monitored by UVꢀVis in
HEPES buffer 0.1 M (at a resulting pH = 7.1). The ligand (50
µM in theory) and Cu(II) (45 µM in theory) were mixed and
successive additions of the different peptides (0 to 500 µM in
total) were added. Each addition of the peptide is made once
the thermodynamic equilibrium of the previous reaction is
reached (this takes about 20 min). To be reproducible with
respect to pH condition, the same stock buffer solution (pH =
7.3 to reach a resulting pH of 7.1) was used for all the experiꢀ
ments. The competition experiments were performed at 25°C
in triplicate.
1
50 mT (centred at 310 mT) with modulation amplitude of 0.5
mT. Experiments were carried out at 110 K using a liquid
nitrogen cryostat.
EPR samples were prepared from stock solution of ligand
6
5
diluted down to 0.2 mM in H O. 0.9 eq. of Cu(II) was added
2
6
5
from 25 mM Cu(NO ) stock solution homeꢀmade from a
Cu foil. Samples were frozen in quartz tube after addition of
0% glycerol as a cryoprotectant and stored in liquid nitrogen
3
2
6
5
1
until used.
Xꢀray Absorption Spectroscopy (XAS). Cu(II) Kꢀ
edge EXAFS (Extended X-Ray Absorption Fine Structure) and
XANES (Xꢀray Absorption Near–Edge Structure) spectra
were recorded at the BM30B (FAME) beamline at the Euroꢀ
Analysis of the data
The data analysis was performed with a 2ꢀstep procedure for
each experiment (corresponding to a given peptide).
3
ACS Paragon Plus Environment

Analytical Chemistry
Page 4 of 9
Then, the ꢔ ꢞꢍ ꢎꢑ value was adjusted to obtain the best reproꢀ
duction of the experimental data.
Step 1. The theoretical concentrations of the ligand L and
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the Cu(II) were adjusted using the absorbance of ligand at 246
ꢀ
1
ꢀ1
nm (ε246 ꢀ ε450 = 18000 cm .M ) and the absorbance of the
NB: Although ꢔ / ꢔ are dimensionless values, they are
ꢀ
1
ꢀ1
ꢞ
ꢝ
Cu(L) complex at 330 nm (ε330 ꢀ ε450 = 18000 cm .M ) respecꢀ
tively.
ꢀ1
given here in M or M , respectively, for convenience. Indeed
for 1:1 system, the ꢔ value in M gives a direct idea at which
ꢞ
Step 2. The absorbance of the competition experiments
measured at 330 nm after completion of the reaction was plotꢀ
ted as a function of the number of peptides equivalents recalꢀ
culated with respect to the real ligand concentration (see step
). Then these data were fitted with an inꢀhouse procedure
using the adjusted concentrations as starting parameters.
concentration the complex is predominantly formed.
NB: in a preliminary experiment, ꢔ ꢞꢍ ꢎꢉ was evaluated by
ꢍꢎ(ꢦꢧꢨ)
ꢞ
competition with ABH and BAH knowing ꢔ
and
ꢍꢎ(ꢧꢦꢨ)33
1
ꢔꢞ
with the similar procedure.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Absorbance was calculated according to:
RESULTS
ꢍꢎꢉ
ꢀ
ꢁꢂ =ꢃꢄꢉ
. ꢊꢋꢌ +ꢃꢄ
. ꢊꢏꢐꢋꢌ
The competitor used in the present work to evaluate the
Cu(II) affinity of a wide series of Aβ derived peptides by UVꢀ
ꢅꢅꢆꢃꢇꢈ
ꢅꢅꢆꢃꢇꢈ
ꢑ
ꢍꢎꢑ
+
ꢃꢄ
. ꢊꢒꢌ +ꢃꢄ
. ꢊꢏꢐꢒꢌ
ꢅꢅꢆꢃꢇꢈ
ꢅꢅꢆꢃꢇꢈ
Vis competition experiments, is an oxamate ligand (H L,
5
5ꢀ
Scheme S1, for the sake of clarity L will be written L. Simiꢀ
where P stands for peptide, L for the ligand, and CuP and
CuL for the complex between Cu(II) and peptide and the ligꢀ
and, respectively.
3ꢀ
larly, the [Cu(L)] will be noted Cu(L)). Oxamate ligands
have largely proved their appeal for the design of metalloligꢀ
ands and the subsequent preparation of multidimensional
The same equation can be expressed as:
48
compounds with controlled architectures and properties. In
ꢀꢁꢂ =ꢃꢄꢉ
. (ꢊꢋꢌ − ꢊꢏꢐꢌ + ꢊꢓꢌ)
ꢆ ꢆ
particular, the use of oxamate ligands and the multiꢀstep asꢀ
sembly of oxamateꢀbased complexes have successfully led to
relevant objects in molecular magnetism: switchable moleꢀ
ꢅꢅꢆꢃꢇꢈ
ꢍꢎꢉ
+
+
+
ꢃꢄ
ꢃꢄ
ꢃꢄ
. (ꢊꢏꢐꢌ − ꢊꢓꢌ)
ꢅꢅꢆꢃꢇꢈ
ꢑ
ꢆ
. (ꢊꢒꢌ − ꢊꢓꢌ)
ꢅꢅꢆꢃꢇꢈ
ꢍꢎꢑ
ꢆ
cules, high nuclearity coordination complexes, singleꢀchain
49,50
. ꢊꢓꢌ
magnets, porous magnets.
Recently oxamateꢀbased sysꢀ
ꢅꢅꢆꢃꢇꢈ
tems have also shown attractiveness in broader coordination
51,52
where α stands for the progression of the following reaction
peptide + Cu(II) →ꢃꢃpeptideꢀCu(II)
chemistry fields such as catalysis.
Here, we will take adꢀ
:
vantage of the mild Cu(II) affinity and of the presence of an
intense UVꢀVisible band for the complex with Cu(II) (λ = 330
and the [ ] correspond to the starting values in which the
0
ꢀ
1
ꢀ1
nm, ε = 18000 cm .M ).
dilution due to addition of the peptide has been taken into
account when required.
The Xꢀray structure of Cu(L) is shown in Figure 1 and the
crystallographic parameters are given in Table 1, top, and in
the Supporting Info. The complex adopts a squareꢀplanar
geometry where the L ligand coordinates the Cu(II) ion via its
two oxamate groups each binding the metal center through one
nitrogen and one oxygen atom. The squareꢀplanar geometry is
fairly regular with a 2.4° dihedral angle between the oxamate
groups and a 3.1° dihedral angle between the Cu(II) coordinaꢀ
tion plane and the phenyl plane of the ligand. The carboxylate
group of the ligand remains uncoordinated. Supramolecular
arrangements of the complexes are described in the Supporting
Information (Figure S1).
Cu(II) is supposed to be chelated by the peptide or by L,
there is no free Cu(II).
ꢏꢐꢒ
ꢔ
ꢊ
ꢒ
ꢌ
ꢃ.ꢊꢏꢐꢌ ꢊꢏꢐꢋꢌ
ꢃ.ꢊꢋ
.ꢊꢏꢐꢌ
ꢕ
=ꢃ
ꢏꢐꢋ
ꢊꢓꢌ
ꢌ
ꢔ
ꢕ
With the starting concentrations:
ꢏꢐꢒ
ꢔ
ꢔ
ꢊꢒꢌ − ꢊꢓꢌꢃ
ꢊꢏꢐꢌ − ꢊꢓꢌ
0
ꢕ
0
=
ꢃ
ꢃ.
ꢏꢐꢋ
ꢕ
ꢊꢓꢌ
ꢊꢋꢌ −ꢃ
ꢊ
ꢏꢐ + ꢊꢓꢌ
ꢌ
0
0
This is a quadratic equation having the form
ꢗ
ꢖꢓ + ꢁꢓ + ꢘ = 0
, with
ꢙꢚꢃꢛꢃ√ꢜ
ꢓ =ꢃ
, with
ꢗꢝ
ꢍꢎꢑ
ꢔ
ꢞ
ꢁ
=ꢃ
ꢃ. (ꢊꢋꢌ − ꢊꢏꢐꢌ ) + ꢊꢒꢌ + ꢊꢏꢐꢌ
ꢆ ꢆ ꢆ ꢆ
ꢍꢎꢉ
ꢔ
ꢞ
ꢗ
ꢟ =ꢃꢁ − 4. ꢖ. ꢘ
ꢢꢣꢤ
ꢠ
ꢡ
ꢖ =ꢃ
− 1
ꢢꢣꢥ
ꢠ
ꢡ
ꢆ
ꢘ =ꢃ−ꢃꢊꢒꢌ ꢃ. ꢊꢏꢐꢌꢆ
4
ACS Paragon Plus Environment

Page 5 of 9
Analytical Chemistry
spectrum of Cu(L) is given in Figure 2B. It is in line with
1
2
3
4
5
6
7
8
9
Cu(II) complexes where the Cu(II) center lies in a square
planar environment.
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Panel A. EPR signature of Cu(L). [L] = 200 µM,
65
[
Cu(II)] = 180 µM, [HEPES] = 50 mM, pH = 7.1, T = 120 K.
Inset: superhyperfine structure on the first parallel line. Panel B.
Normalized Cu Kꢀedge Xꢀray Absorption Near –Edge Structure
(XANES) spectrum of Cu(L). [L] = 1 mM, [Cu(II)] = 0.9 mM,
[HEPES] = 0.1 M, pH = 7.0, T = 20K.
Figure 1. Ellipsoid plot (H and disorders atoms are omitted for
clarity) of Cu(L).
The persistence of the structure in aqueous solution has
been probed by several complementary techniques. The most
appropriate one is EXAFS by which the CuꢀN and CuꢀO disꢀ
tances have been determined in solution and are in perfect
agreement with those obtained for the crystal structure (for
crystallography, see Table 1, bottom and Figure S1, for
EXAFS fitting, see Figure S2).
The other important parameter of this ligand is its affinity
constant for Cu(II). This affinity was evaluated by the same
type of competition experiments than those detailed below,
performed first between L and two short peptides, namely
33
ABH and BAH (B stands for βꢀalanine). These peptides were
studied by potentiometry to determine the impact of the 5ꢀ
membered versus 6ꢀmembered metallacycles with regard to
the Cu(II) affinity in an ATCUNꢀlike (Amino Terminal CU
Table 1. Crystallography distances between Cu(II) and
their neighbours (top) and EXAFS parameters (bottom): N
54
and Nickel) sites. Their conditional Cu(II) affinity values
calculated at pH 7.1 from the potentiometric data equal: 3.0
=
number of neighbours, R = absorberꢀneighbour distance,
8
9
ꢀ1
1
0 and 3.7 10 M , respectively. Competition experiments
σ = DebyeꢀWaller factor.
with the ABH (resp. BAH) lead to a Cu(II) affinity value of
9
ꢀ1
9
ꢀ1
ꢉ
N
Distance (Å)
1.9061(55)
1.9127(37)
1.9588(34)
1.9642(51)
2.2 10 M (resp. 4.1 10 M ) for L. The average value of ꢔ
ꢝ
9 ꢀ1
= 3.2 ± 1.0 10 M was used in the rest of the study.
CuꢀN1
CuꢀN2
The competition experiment between L and the Aβ peptides
for Cu(II) binding were performed by using the peptides to
remove the Cu(II) from the colored Cu(L) complex leading to
a disappearance of the signature at 330 nm characteristic of the
Cu(L) complex. Figure 3A shows the typical series of spectra
CuꢀO1
CuꢀO4
obtained for a given peptide (here Aβ ). L was first added
16
N
Distance (Å)
1.92
(dotted line), then Cu(II) (solid line) and then successive addiꢀ
2
2
CuꢀN
CuꢀO
tions of the peptide were performed (grey lines, See Experiꢀ
mental Section for more details). Note that the increase at 276
nm is due to Tyr10 absorbance and perfectly matches the
amount of peptide added.
1.97
ꢀ
2
σ2 (Å )
.00471
R factor
0.18 %
0
The parameters estimated from the EPR signature (Figure
A) are also in line with the Cu(L) solidꢀstate structure. Inꢀ
2
6
5
4
ꢀ1
deed, the g and A ( Cu) values equal 2.22 and 212 10 cm ,
/
/
//
respectively and are thus in line with a 2N2O equatorial bind5ꢀ3
ing mode according to the Peisach and Blumberg correlation.
A weak rhombicity linked to the presence of four non equivaꢀ
lent equatorial ligands is at the origin of the complex features
observed in the g region (coupling between I = 3/2 with SCu
⊥
Cu
=
1/2 for the two g and g transitions). The presence of two
x y
Figure 3. Panel A. UVꢀVis spectra of a solution of L (dotted line),
in the presence of 0.9 eq. of Cu(II) (solid line) and after addition
of increasing amounts (approx.. 1, 3, 7 and 10 eq.) of Aβ16 peptide
equivalent nitrogen atoms is consistent with the five superꢀ
hyperfine lines observed on the first parallel transition, arising
from the coupling between I = 1 with S = 1/2. The XANES
N
Cu
5
ACS Paragon Plus Environment

Analytical Chemistry
Page 6 of 9
a
b
(
grey lines). The arrows indicate the variation of the UVꢀVis
The error bars are evaluated in the S.I. (see Figure S4). The
K value for AcꢀAβ is too weak to be thoroughly determined by
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
spectra upon addition of the peptide. The increase at 276 nm is
a
16
due to Tyr10 absorption. Panel B. Experimental absorbance at
this method. Only a higher value can be given.
3
30 nm (dots) and the best fits (dashed lines) of the Cu(L) system
upon addition of peptides (Aβ16 (a), H6AꢀAβ16 (b) and AcꢀAβ16
(
[
c)). [L] = 50 µM, [Cu(II)] = 45 µM, [peptides] = 0 to 500 µM,
HEPES] = 0.1 M, pH = 7.1, T = 25°C.
DISCUSSION
The Aβ peptides used in this study were modified with reꢀ
spect to the human sequence on any potential binding aminoꢀ
acids, namely the carboxylate containing aminoꢀacid residues,
the His residues and the Nꢀterminal amine. The mutation inꢀ
volved between the human and murine sequences were also
studied.
The absorbance at λ = 330 nm of the mixture was plotted as
a function of the equivalent numbers of peptide added (dots in
Figure 3B). Then, these data were fitted with an inꢀhouse
procedure (dashed lines, for more details see Experimental
section).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Experimental absorbance values and their fits are shown in
10
Figure 3B as a matter of illustration for Aβ , H6AꢀAβ and
16
16
AcꢀAβ , (curves a, b, c, respectively). Qualitative analysis
16
indicates that the Cu(II) affinity of the three peptides follow
1
the trend: Aβ16 > H6AꢀAβ16 > AcꢀAβ . All the Cu(II) affinity
16
values obtained in the present study are gathered in Table 2
while the corresponding reproductions of the experimental
data are given in the Supporting Info (Figure S3). The accuraꢀ
cy of the fitting procedure is shown in the Figure S4. All the
calculated Cu(II) affinity values were normalized with respect
to the value of Aβ16 for an easier comparison, as shown in
Table 2 and in Figure 4.
0.1
0.01
Figure 4. Bar graph of the normalized affinity constants in log
units of the Aβ peptides and some of their mutants for Cu(II). The
Table 2. Cu(II) affinity values of the Aβ peptides studied
here. The reference for the normalization is the Aβ . A
1
6
reference for the normalization is the hAβ . The error bars indiꢀ
16
9
ꢀ1
Cu(II) affinity value of 3.2 ± 1.0 10 M for the competing
ligand L was taken.
cate the standard deviation. [L] = 50 µM, [Cu(II)] = 45 µM, [pepꢀ
tides] = 0 to 500 µM, [HEPES] = 0.1 M, pH = 7.1, T = 25°C. The
9
ꢀ1
affinity constant of the ligand is K = 3.2 ± 1.0 10 M .
a
Ka
K (peptide)
a
Peptide
Humanꢀderived peptides. The first modification is the
acetylation of the Nꢀterminal amine (Asp1) precluding the
possibility of Cu(II) binding. The affinity constant of Cu(II)
for this modified peptide is at least 10 times weaker than that
of the Aβ16 peptide, in line with an important contribution of
the Nꢀterminal amine in Cu(II) binding to Aβ , as discussed in
9
ꢀ1 a
/ K (hAβ )
(
10 M )
a
16
Aβ16
Aβ28
Aβ40
1.6
1.0
1.1
1.4
1.8
2.2
1
6
28,55,56
the literature.
In the second group of modified peptides,
b
AcꢀAβ16
< 0.1
< 0.1
the impact of the carboxylate function is evaluated. The carꢀ
boxylate group of each amino acid residues Asp1, Glu3, Asp7
and Glu11 is mutated with an amide function. Only a very
weak impact is observed with a decrease in the affinity upon
D1NꢀAβ16
1.1
1.0
1.1
1.3
0.7
0.6
0.7
0.8
E3QꢀAβ16
modification that doesn’t exceed a factor of 2. In the Aβ16
D7NꢀAβ16
there are 4 carboxylate groups available for the Cu(II) coordiꢀ
nation. Therefore, if one carboxylate is mutated, 3 other ones
remain to complete the coordination sphere. This is in perfect
agreement with the possibility of having an equilibrium beꢀ
E11QꢀAβ16
57
H6RꢀAβ16
H6AꢀAβ16
H13AꢀAβ16
0.3
0.3
0.9
0.8
0.2
0.2
0.6
0.5
tween the four carboxylate groups for one binding position.
The high similarity of affinity values for the four carboxylateꢀ
modified peptides also suggest that there is no preference for
one given carboxylate group. The last group of modified pepꢀ
tides consist in the His mutants (His6, His13 and His14).
While His6 mutation (H6A or H6R) has a significant impact (a
5ꢀfold decrease of the affinity value), His13 or His14 mutation
has a lesser effect (with a maximal 2ꢀfold decrease of the
affinity value). This indicates that (i) none of the His is essenꢀ
tial but (ii) the His13 and His14 can more easily exchange for
the binding position on the peptide than His6 and His13 or
than His6 and His14 do. This is in line with the proposition
that in the main coordination site His13 and His14 exchange
H14AꢀAβ16
mAβ16
R5GꢀAβ16
3.0
3.1
1.2
0.7
2.7
1.9
1.9
0.8
0.4
1.7
Y10FꢀAβ16
H13RꢀAβ16
R5GꢀH13RꢀAβ16
1
3,30,58
for one binding position while His6 is constantly bound.
6
ACS Paragon Plus Environment

Page 7 of 9
Analytical Chemistry
range. However, outside this range, competition with L will
It is worth mentioning that similar differences have been preꢀ
1
9,20
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
viously determined by ITC.
well with the coordination site of Cu(II) proposed based on
All together these data fit very
not give reliable values. In addition, the molar extinction coefꢀ
ficient is relatively low implying the use of quite large bioꢀ
molecule concentration. As a consequence, to generalize this
approach to the evaluation of Cu(II) affinity for any other
(bio)molecules relies on the ability to modulate the Cu(II)
affinities and to increase the molar extinction coefficient of
oxaloaminoꢀbenzoic acid derivatives by playing with the scafꢀ
fold.
1
3,30
spectroscopic data (reviewed in refs. ) and reminded in
Scheme 1. In such proposition, the Cu(II) ion is equatorially
bound to the Nꢀterminal amine, the adjacent carbonyl group
from the Asp1ꢀAla2 bond, the imidazole group form His 6 and
8
from His13 or 14 in equilibrium (see Scheme S2, top). A
carboxylateꢀcontaining residue has been proposed to occupy
the apical position. Last, the Cu(II) affinity seems to weakly
5
7
increase with the length of the peptide, in line with previous
reports,
the increase in the error bar when working with the Aβ40 pepꢀ
tide. This confirms that the Aβ moiety is a good model with
respect to Cu(II) binding site within the fullꢀlength peptide.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
17,59
but this result has to be taken with cautious due to
ASSOCIATED CONTENT
Supporting Information
1
6
The Supporting Information is available free of charge on the
ACS Publications website. Characterization of the ligand and of
the Cu(L) complex, EXAFS parameters, Competition data, Error
bar, Cu coordination model.
AUTHOR INFORMATION
Corresponding Author:
*
Eꢀmail: christelle.hureau@lccꢀtoulouse.fr.
Author Contributions
A.C.D. and V.B. contributed equally to this work.
‡
ACKNOWLEDGEMENTS
Scheme 1. Predominant forms at pH 7.1 of the Cu(II) coꢀ
ordination with human (left) and murine (right) Aβ pepꢀ
tides.
CH thanks the ERC aLzINK ꢀ Contract n° 638712 for financial
support. The authors thank the European Synchrotron Radiation
Facility for provision of beamtime (experiment 30ꢀ02ꢀ1060), the
FAME staff for their support, F. Collin, and C. Cheignon for their
help in recording the XANES and EXAFS data. Prof. P. Faller is
acknowledged for interesting discussions.
Human versus murine peptides. The murine sequence
differs from the human one by 3 mutations: R5G, Y10F and
H13R. With respect to Cu(II) affinity values, the murine pepꢀ
tide is twice stronger than the human one, in line with previous
REFERENCES
(1) Solomon, E. I.; Heppner, D. E.; Johnston, E. M.; Ginsbach, J. W.;
Cirera, J.; Qayyum, M.; KieberꢀEmmons, M. T.; Kjaergaard, C. H.; Hadt,
R. G.; Tian, L. Chem. Rev. 2014, 114, 3659ꢀ3653.
1
7,29
19
measurements by direct competition,
ITC, and potentiꢀ
55
ometry. The main mutation responsible for such a difference
is the R5G mutation. This has been previously related to the
formation of a 6ꢀmembered metallacycle between the side
chain of His6 and the deprotonated Gly5ꢀHis6 peptide funcꢀ
tion for the murine peptide that is not observed in the human
(
2) Sarkar, B. Chem. Rev. 1999, 99, 2535ꢀ2544.
(3) Delangle, P.; Mintz, E. Dalton Trans. 2012, 41.
4) Lalioti, V.; Muruais, G.; Tsuchiya, Y.; Pulido, D.; Sandoval, I. V.
Frontiers Biosciences 2009, 14, 4878ꢀ4903.
5) Scheiber, I. F.; Mercer, J. F. B.; Dringen, R. Prog. Neurobiol. 2014,
16, 33ꢀ57.
(
29,30
case (see Schemes 1 and S2 bottom).
(
1
(6) Kozlowski, H.; Luczkowski, M.; Remelli, M.; Valensin, D. Coord.
Chem. Rev. 2012, 256, 2129ꢀ2141.
CONCLUSIONS
(
(
(
7) Ayton, S.; Lei, P.; Bush, A. I. Free Radic. Biol. Med. 2013, 62, 76ꢀ89.
8) Hureau, C. Coord. Chem. Rev. 2012, 256, 2164ꢀ2174.
9) Barnham, K. J.; Bush, A. I. Chem. Soc. Rev. 2014, 43, 6727ꢀ6749.
In the present study, a new ratiometric dye was synthetized
and fully characterized. It was further used to determine the
affinity constant of Cu(II) for several Aβ peptides by UVꢀVis
competition experiments. This was made possible by the inꢀ
tense (18000 cm .M ) LMCT band displayed at λ = 330 nm
by the Cu(L) complex. The affinity values obtained in this
study for the various modified peptides agree perfectly with
published coordination sites, thus validating the proposed
strategy that relies on the determination of affinity constants of
a wide series of modified peptides to extrapolate a coordinaꢀ
tion model for a given peptide. In addition, the designed dye
could be further used to straightforwardly determine Cu(II)
affinity for other flexible peptides such as alphaꢀsynuclein
(10) D'Ambrosi, N.; Rossi, L. Neurochem. Int. 2015, 90, 36ꢀ45.
(11) Binolfi, A.; Quintanar, L.; Bertoncini, C. W.; Griesinger, C.;
Fernández, C. O. Coord. Chem. Rev. 2012, 256, 2188ꢀ2201.
ꢀ
1
ꢀ1
(12) Arena, G.; La Mendola, D.; Pappalardo, G.; Sóvágó, I.; Rizzarelli, E.
Coord. Chem. Rev. 2012, 256, 2202ꢀ2218.
(13) Drew, S. C.; Barnham, K. J. Acc. Chem. Res. 2011, 44, 1146ꢀ1155.
(14) Faller, P.; Hureau, C.; Berthoumieu, O. Inorg. Chem. 2013, 52,
12193ꢀ12206.
(
15) Telpoukhovskaia, M. A.; Orvig, C. Chem. Soc. Rev. 2013, 42, 1836ꢀ
1846.
16) Zawisza, I.; Rozga, M.; Bal, W. Coord. Chem. Rev. 2012, 256, 2297ꢀ
307.
17) Alies, B.; Renaglia, E.; Rozga, M.; Bal, W.; Faller, P.; Hureau, C.
Anal. Chem. 2013, 85, 1501ꢀ1508.
(
2
(
1
1
12
involved in Parkinson’s disease or Prion protein, for which
the Cu(II) affinities are proposed to lie in the 10 ꢀ10
8
10
ꢀ1
M
7
ACS Paragon Plus Environment

Analytical Chemistry
Page 8 of 9
(
6
(
18) Young, T. R.; Kirchner, A.; Wedd, A. G.; Xiao, Z. Metallomics 2014,
, 505ꢀ517.
19) Hong, L.; Carducci, T. M.; Bush, W. D.; Dudzik, C. G.; Millhauser,
G. L.; Simon, J. D. J. Phys. Chem. B. 2010, 114, 11261ꢀ11271.
20) Hong, L.; Simon, J. D. Metallomics 2011, 3, 262ꢀ266.
(43) Proux, O.; Biquard, X.; Lahera, E.; Menthonnex, J. J.; Prat, A.;
Ulrich, O.; Soldo, Y.; Trévisson, P.; Kapoujvan, G.; Perroux, G.; Taunier,
P.; Grand, D.; Jeantet, P.; Deleglise, M.; Roux, J.ꢀP.; Hazemann, J.ꢀL.
Phys. Scr. 2005, 115, 970ꢀ973.
(44) Michalowicz, A.; Moscovici, J.; MullerꢀBouvet, D.; Provost, K. J.
Phys.: Conf. Ser. 2009, 190, 012034ꢀ012035.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
(21) Alies, B.; Bijani, C.; Sayen, S.; Guillon, E.; Faller, P.; Hureau, C.
Inorg. Chem. 2012, 51, 12988ꢀ13000.
(45) Lengeler, B.; Eisenberg, P. Phys. Rev. B: Condens. Matter Mater.
Phys. 1980, 4507ꢀ4520.
(46) Guillon, E.; Merdy, P.; Aplincourt, M. Chemistry - A European
Journal 2003, 9, 4479ꢀ4484.
(
22) Alies, B.; Badei, B.; Faller, P.; Hureau, C. Chem. Eur. J. 2012, 18,
161ꢀ1167.
23) Feaga, H. A.; Maduka, R. C.; Foster, M. N.; Szalai, V. A. Inorg.
Chem. 2011, 50, 1614ꢀ1618.
24) Xiao, Z.; Gottschlich, L.; van der Meulen, R.; Udagedara, S. R.;
Wedd, A. G. Metallomics 2013, 5, 501ꢀ513.
25) Faller, P.; Hureau, C. Dalton Trans. 2009, 1080ꢀ1094.
1
(
(47) Zabinsky, S. I.; Rehr, J. J.; Ankudinov, A. L.; Albers, R. C.; Eller, M.
J. Phys. Rev. B: Condens. Matter Mater. Phys. 1995, 52, 2995ꢀ3009.
(48) Pardo, E.; RuizꢀGarcía, R.; Cano, J.; Ottenwaelder, X.; Lescouëzec,
R.; Journaux, Y.; Lloret, F.; Julve, M. Dalton Trans. 2008, 21, 2780.
(49) Dul, M.ꢀC.; Pardo, E.; Lescouëzec, R.; Journaux, Y.; FerrandoꢀSoria,
J.; RuizꢀGarcia, R.; Cano, J.; Julve, M.; Lloret, F.; Cangussu, D.; Pereira,
C. L. M.; Stumpf, H. O.; Pasan, J.; RuizꢀPerez, C. Coord. Chem. Rev.
2010, 254, 2281ꢀ2296.
(50) Grancha, T.; FerrandoꢀSoria, J.; Castellano, M.; Julve, M.; Pasán, J.;
Armentano, D.; Pardo, E. Chem. Commun. 2014, 50, 7569ꢀ7585.
(51) ForteaꢀPérez, F. R.; Schlegel, I.; Julve, M.; Armentano, D.; De
Munno, G.; Stiriba, S.ꢀE. J. Organomet. Chem. 2013, 743, 102ꢀ108.
(52) Pim, W. D. d.; Oliveira, W. X. C.; Ribeiro, M. A.; Faria, É. N. d.;
Teixeira, I. F.; Stumpf, H. O.; Lago, R. M.; Pereira, C. L. M.; Pinheiro, C.
B.; FigueiredoꢀJúnior, J. C. D.; Nunes, W. C.; Souza, P. P. d.; Pedroso, E.
F.; Castellano, M.; Cano, J.; Julve, M. Chem. Commun. 2013, 49, 10778ꢀ
10780.
(53) Peisach, J.; Blumberg, W. E. Arch. Biochem. Biophys. 1974, 165,
691ꢀ708.
(54) Bal, W.; Sokołowska, M.; Kurowska, E.; Faller, P. Biochim. Biophys.
Acta 2013, 1830, 5444ꢀ5455.
(55) KowalikꢀJankowska, T.; Ruta, M.; Wisniewska, K.; Lankiewicz, L. J.
Inorg. Biochem. 2003, 95, 270ꢀ282.
(56) Syme, C. D.; Nadal, R. C.; Rigby, S. E.; Viles, J. H. J. Biol. Chem.
2004, 279, 18169ꢀ18177.
(
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
(26) Arena, G.; Papppalardo, G.; Sovago, I.; Rizzarelli, E. Coord. Chem.
Rev. 2012, 256, 3ꢀ12.
(
27) Young, T. R.; Wijekoon, C. J. K.; Spyrou, B.; Donnelly, P. S.; Wedd,
A. G.; Xiao, Z. Metallomics 2015, 7, 567ꢀ578.
28) Alies, B.; Eury, H.; Bijani, C.; Rechignat, L.; Faller, P.; Hureau, C.
Inorg. Chem. 2011, 50, 11192ꢀ11201.
29) Eury, H.; Bijani, C.; Faller, P.; Hureau, C. Angew. Chem., Int. Ed.
Engl. 2011, 50, 901ꢀ905.
(
(
(30) Hureau, C.; Dorlet, P. Coord. Chem. Rev. 2012, 256, 2175ꢀ2187.
(31) Faller, P.; Hureau, C.; Dorlet, P.; Hellwig, P.; Coppel, Y.; Collin, F.;
Alies, B. Coord. Chem. Rev. 2012, 256, 2381ꢀ2396.
32) Fasman, G. D. H. o. B. a. M. B., Proteins, I, 3rd ed., CRC, 1976, p.
83.
33) Nagaj, J.; StokowaꢀSołtys, K.; Zawisza, I.; JeżowskaꢀBojczuk, M.;
(
1
(
Bonna, A.; Bal, W. J. Inorg. Biochem. 2013, 119, 85ꢀ89.
(34) Hureau, C.; Eury, H.; Guillot, R.; Bijani, C.; Sayen, S.; Solari, P. L.;
Guillon, E.; Faller, P.; Dorlet, P. Chem. Eur. J. 2011, 17, 10151ꢀ10160.
(35) Stumpf, H. O.; Pei, Y.; Kahn, O.; Sletten, J.; Renard, J. P. J. Am.
Chem. Soc. 1993, 115, 6738ꢀ6745.
(36) Cervera, B.; Sanz, J. L.; Ibanez, M. J.; Vila, G.; Lloret, F.; Julve, M.;
Ruiz, R.; Ottenwaelder, X.; Aukauloo, A.; Poussereau, S.; Journaux, Y.;
Munoz, M. C. J. Chem. Soc., Dalton Trans. 1998, 5, 781ꢀ790.
(57) Hureau, C.; Coppel, Y.; Dorlet, P.; Solari, P. L.; Sayen, S.; Guillon,
E.; Sabater, L.; Faller, P. Angew. Chem., Int. Ed. Engl. 2009, 48, 9522ꢀ
9525.
(58) Dorlet, P.; Gambarelli, S.; Faller, P.; Hureau, C. Angew. Chem., Int.
Ed. Engl. 2009, 48, 9273ꢀ9276.
(
(
37) PaulꢀRoth, C. O. C. R. Chimie 2005, 8, 1232ꢀ1236.
38) Bruker AXS Inc, B. Madison, Wisconsin, USA, 1998.
(39) Blessing, R. H. Acta Crystallogr., Sect. A: Found. Crystallogr. 1995,
1, 33.
40) Altomare, A.; Cascarano, G.; Giacovazzo, C.; Guagliardi, A.; Burla,
M. C.; Polidori, G.; Camalli, M. J. Appl. Crystallogr. 1994, 27, 435.
41) Sheldrick, G. M. Acta Crystallogr., Sect. C: Cryst. Struct. Commun.
2015, 71, 3ꢀ8.
42) Farrugia, L. J. J. Appl. Crystallogr. 2012, 45, 849ꢀ854.
5
(
(59) KowalikꢀJankowska, T.; RutaꢀDolejsz, M.; Wisniewska, K.;
Lankiewicz, L. J. Inorg. Biochem. 2001, 86, 535ꢀ545.
(
(
8
ACS Paragon Plus Environment

Page 9 of 9
For TOC only:
Analytical Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
ACS Paragon Plus Environment