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\DOI{10.5802/crchim.255}
\datereceived{2023-04-05}
\datererevised{2023-07-12}
\dateaccepted{2023-08-22}
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\begin{DefTralics}
\newcommand{\upbeta}{\beta}
\end{DefTralics}
\begin{DefSom}
\newcommand{\upbeta}{\beta}
\end{DefSom}

%\begin{noXML}

%\makeatletter
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%\def\cdr@specialtitle@english{Chemical Biology}
%\def\cdr@specialtitle@french{Ch\'emobiologie}
%\makeatother

\title{Reduced Schiff-base derivatives to stop reactive oxygen species
production by the Cu(A$\upbeta$) species: a~structure--activity
relationship}

\alttitle{Ligands de type base de Schiff r\'eduite pour arr\^eter la
production d'esp\`eces r\'eactives de l'oxyg\`ene g\'en\'er\'ee par le
complexe Cu(A$\upbeta$)~:~une relation structure--activit\'e}

\author{\firstname{Margot} \lastname{Lef\`{e}vre}\CDRorcid{0009-0006-1924-7937}}
\address{LCC-CNRS, Universit\'{e} de Toulouse, CNRS, Toulouse, France}
\email[M. Lef\`{e}vre]{margot.lefevre@lcc-toulouse.fr}

\thanks{C. H. acknowledges ERC StG 638712, C. E. and M. L. the ANR-20-CE07-0009 for
financial support.}

\author{\firstname{Lielou} \lastname{Lantigner}}
\addressSameAs{1}{LCC-CNRS, Universit\'{e} de Toulouse, CNRS, Toulouse, France}
\email[L. Lantigner]{lielou.lantigner@lcc-toulouse.fr}

\author{\firstname{Laura} \lastname{Andolfo}}
\addressSameAs{1}{LCC-CNRS, Universit\'{e} de Toulouse, CNRS, Toulouse, France}
\address{LSPCMIB, CNRS UMR 5068, Universit\'{e} Toulouse III-Paul
Sabatier, 118 route de Narbonne, 31062 Toulouse cedex 9, France}
\email[L. Andolfo]{laura.andolfo21@gmail.com}

\author{\firstname{Corinne} \lastname{Vanucci-Bacqu\'{e}}\CDRorcid{0000-0002-7232-6267}}
\addressSameAs{2}{LSPCMIB, CNRS UMR 5068, Universit\'{e} Toulouse III-Paul
Sabatier, 118 route de Narbonne, 31062 Toulouse cedex 9, France}
\email[C. Vanucci-Bacqu\'{e}]{corinne.bacque@univ-tlse3.fr}

\author{\firstname{Eric} \lastname{Benoist}\CDRorcid{0000-0003-0764-9024}}
\addressSameAs{2}{LSPCMIB, CNRS UMR 5068, Universit\'{e} Toulouse III-Paul
Sabatier, 118 route de Narbonne, 31062 Toulouse cedex 9, France}
\email[E. Benoist]{eric.benoist@univ-tlse3.fr}

\author{\firstname{Charl\`{e}ne} \lastname{Esmieu}\CDRorcid{0000-0003-1412-033X}}
\addressSameAs{1}{LCC-CNRS, Universit\'{e} de Toulouse, CNRS, Toulouse, France}
\email[C. Esmieu]{charlene.esmieu@lcc-toulouse.fr}

\author{\firstname{Florence} \lastname{Bedos-Belval}\CDRorcid{0000-0002-6792-6815}\IsCorresp}
\addressSameAs{2}{LSPCMIB, CNRS UMR 5068, Universit\'{e} Toulouse III-Paul
Sabatier, 118 route de Narbonne, 31062 Toulouse cedex 9, France}
\email[F. Bedos-Belval]{florence.bedos@univ-tlse3.fr}

\author{\firstname{Christelle} \lastname{Hureau}\CDRorcid{0000-0003-3339-0239}\IsCorresp}
\addressSameAs{1}{LCC-CNRS, Universit\'{e} de Toulouse, CNRS, Toulouse, France}
\email[C. Hureau]{christelle.hureau@lcc-toulouse.fr}

\shortrunauthors
\CDRGrant[ERC]{StG 638712}
\CDRGrant[ANR]{ANR-20-CE07-0009}
\begin{abstract}
A${\upbeta}$ is the peptide involved in Alzheimer's disease. Its
binding to the redox copper ions and the subsequent production of
reactive oxygen species (ROS) contributing to the overall oxidative
stress observed in the disease is among the deleterious effects
A${\upbeta}$ may have. Here seven reduced Schiff-base ligands were
studied for their ability to stop Cu(A${\upbeta}$)-induced ROS
production. The spectroscopic UV--vis and EPR characterizations of the
Cu(II) complexes are reported as well. While all the ligands except one
are able to stop Cu(A${\upbeta}$)-induced ROS, only two maintain this
ability in the presence of the endogenous Zn ions, due to kinetic
competitiveness. 
{\vspace*{-3pt}}
\end{abstract}

\begin{altabstract}
A$\upbeta$ est le peptide impliqu\'e dans la maladie d'Alzheimer. Son
complexe de cuivre Cu(A$\upbeta$) catalyze la production d'esp\`eces
r\'eactives de l'oxyg\`ene (ROS) qui contribuent au stress oxydatif global
observ\'e dans la maladie. Ici, sept ligands de type base de Schiff
r\'eduits ont \'et\'e \'etudi\'es pour leur capacit\'e \`a arr\^eter la production de
ROS induite par Cu(A$\upbeta$). Les caract\'erisations spectroscopiques
UV--vis et EPR des complexes de Cu(II) obtenues avec ces sept ligands
sont \'egalement rapport\'ees. Alors que tous les ligands sauf un sont
capables d'arr\^eter les ROS induits par le Cu(A$\upbeta$), seuls deux
conservent cette capacit\'e en pr\'esence d'ions Zn endog\`enes, en raison
d'une \guillemotleft{}~comp\'etitivit\'e cin\'etique~\guillemotright{}.
\end{altabstract}

\keywords{\kwd{Copper}\kwd{Alzheimer}\kwd{Peptide}\kwd{2N2O ligand}\kwd{Oxidative stress}}

\altkeywords{\kwd{Cuivre}\kwd{Alzheimer}\kwd{Peptide}\kwd{Ligands 2N2O}\kwd{Stress oxydant}}


\dateposted{2023-10-31}
\begin{document}
\maketitle

{\vspace*{-3pt}}

\twocolumngrid

%\end{noXML}

\section{Introduction}\label{sec1}

Alzheimer's disease (AD) is an incapacitating disease that represents
the major proportion of dementia cases in the elderly~\cite{1, 2}. AD
is a multifactorial pathology with genetic predispositions and multiple
probable causes~\cite{3}. Among them, a first hypothesis is known as ``the amyloid
cascade''~\cite{4,5,6}. It is based on the detection of senile plaques
consisting of aggregated amyloid-${\upbeta}$ (A${\upbeta}$) peptides,
where A${\upbeta}$ are peptides of approximately 40 amino acid residues
in length. Therefore, the process from the monomeric peptides to the
amyloid fibrils, so-called self-assembly or aggregation, is regarded as
a key element in the development of the disease. Another hypothesis is
the ``metal hypothesis''~\cite{7,8,9}. It relies on a deregulation of
metal ions levels, mainly copper (Cu) and zinc (Zn) ions. It is based
on the following facts: (1) metal ions are exchanged with the synaptic
cleft where A${\upbeta}$ peptides aggregate, (2) their respective
affinities for the A${\upbeta}$ peptides match within their biological
concentration, making possible their interaction with A${\upbeta}$, (3)
aberrant metal levels (up to mM to be compared to ${\upmu}$M in
the cerebrospinal fluid) are found in the senile plaques. In addition
to their likely participation in the modulation of A${\upbeta}$
aggregation~\cite{10, 11}, and because they are redox-active, Cu ions
can produce highly deleterious Reactive Oxygen Species (ROS) when bound
to the A${\upbeta}$ peptide and thus can participate in the oxidative
stress observed in AD~\cite{12}. Zn ions are present in much higher
quantities than Cu ions in the synaptic cleft and, in contrast to Cu
ions, they are redox silent. 

Due to its redox ability and participation in oxidative stress,
A${\upbeta}$-bound Cu are thus considered as a therapeutic target of
interest among others. We and many other groups have developed many
copper-targeting strategies to overrule the deleterious effects of Cu
(for recent reviews on that topic, see
refs.~\cite{13,14,15,16,17,18,19,20,21} and references therein). Among
the various properties, if Cu ions are considered as the target of
choice of the intended ligands, they should possess a high Cu over
Zn(II) thermodynamic selectivity, much higher than that of A${\upbeta}$
since there are about 10--100 fold more Zn(II) than Cu ions in the
synaptic cleft~\cite{22, 23}.  

{In the present article, we aim to complete seminal works on Schiff
base derivatives, used as Cu(II) ligands able to retrieve Cu(II) from
A${\upbeta}$, redox-silence it and be selective enough for Cu(II)
versus Zn(II) to maintain such ability in the co-presence of
stoichiometric amount of Zn(II). In a seminal paper, Storr and coworkers
described the synthesis of glucose derivatives of reduced Schiff base
ligands, such as} $^{\mathrm{G}}${L}$_{\mathrm{Me}}$ {(Scheme~\ref{sch1}),
and the thorough characterization of the corresponding Cu(II)
complexes}~\cite{24}. {Later on, with the objective of helping \textit{in vitro}
characterizations by increasing the solubility of the ligand, the}
$^{\mathrm{s}}${L}$_{\mathrm{Me}}$ {analogue was reported (Scheme~\ref{sch1}). 
It showed the ability to remove Cu(II) from A${\upbeta}$, to stop
Cu(A${\upbeta}$)-induced ROS formation, and to restore apo-like
aggregation of Cu(A${\upbeta}$)}~\cite{25}. {Then the}
$^{\mathrm{s}}${L}$_{\mathrm{Me}}$ {ligand was shown to have an
appropriate selectivity to maintain its ability to stop
Cu(A${\upbeta}$)-induced ROS production in the presence of one
equivalent of Zn(II)}~\cite{23}. {These ligands have higher affinity and
Zn(II) over Cu(II) selectivity than A${\upbeta}$ and hence they
maintain the ability to extract Cu from A${\upbeta}$ in the presence of
one equiv. of Zn(II). The affinity for a ligand} $L$ {and a metal ion}
$M$ {is defined as} ${K}_{M}^{L}=\sfrac{(LM)}{((L)(M))}$ {while the Cu(II)
over Zn(II) selectivity for a ligand} $L$ {as}
$S^{L}=\sfrac{{K}_{\mathrm{Cu}}^{L}}{{K}_{\mathrm{Zn}}^{L}}${.\break The affinity and
selectivity values for} $^{\mathrm{G}/\mathrm{s}}${L}$_{\mathrm{Me}}$ {are
reported along those for A${\upbeta}$ in Table~\ref{tab1}.}

\begin{table}[t!]
\caption{\label{tab1}Apparent affinity values (for Cu(II) and Zn(II))
at pH 7.1 for the A${\upbeta}$ peptide and the two reference ligands}
\begin{tabular}{ccccc}
\thead
$L$ & $\log (K_{\mathrm{Cu}}^{L})$ & $\log({K}_{\mathrm{Zn}}^{L})$ &
$\log (S^{L})$ & Ref.  \\
\endthead
A${\upbeta}$ & \09.2 & 5.0 & 4.2 & \cite{26, 27} \\
$^{\mathrm{G}}$L$_{\mathrm{Me}}$ & 12.1 & 4.6 & 7.5 & \cite{24} \\
$^{\mathrm{s}}$L$_{\mathrm{Me}}$ & 13.8 & 6.1 & 7.7 & \cite{25, 27}
\botline
\end{tabular}
\end{table}

\begin{scheme*}
\includegraphics{sc01}
\vspace*{7pt}
\caption{\label{sch1}Scheme of the various ligands {${\bmathcal{L}}$}
under study. Main variations are methylation of the amine (indicated by
the \textit{Me} subscript), sulfonation of the phenol arms (indicated
by the \textit{s} superscript), and introduction of a cyclohexyl on the
ethylene bridge ({${\bmathcal{L}}'$} series).
{${\bmathcal{L}}$} corresponds to the fully deprotonated form of any of
the ligands. Charges are omitted for clarity.} 
\vspace*{-5pt}
\end{scheme*}

Here, we report on the synthesis and characterizations of a series of
ligands based on the two $^{\mathrm{G}/\mathrm{s}}$L$_{\mathrm{Me}}$
previously described (Scheme~\ref{sch1}). They will be noted
{${\bmathcal{L}}$} as a generic term. We aimed to improve the
ligand design and several lines were followed: effect of the presence
of (i) sulfonato groups on the phenol rings
($^{\mathrm{s}}{\bmathcal{L}}$ versus
{${\bmathcal{L}}$}), (ii) methyl substituents on the amine
functions (${\bmathcal{L}}_{\mathrm{Me}}$ versus
{${\bmathcal{L}}$}) and (iii) a (${\pm}$)-trans-1,2-cyclohexyl as
bridge (${\bmathcal{L}}'$ versus ${\bmathcal{L}}$) inspired by previous
works on similar scaffolds~\cite{28,29,30}. Rationales for such ligand
alterations were (i) to demonstrate that
$^{\mathrm{s}}{\bmathcal{L}}$ can be studied \textit{in vitro} while
{${\bmathcal{L}}$} could be used for further \textit{in vivo}
applications, since the $^{\mathrm{s}}{\bmathcal{L}}$ would
not have a correct drug profile, especially to cross the blood--brain
barrier, a key step when AD is targeted, (ii) to draw a 
structure--activity relationship, linking Cu(II) first coordination
sites and the arrest of Cu(A${\upbeta}$)-induced ROS production
including in the presence of Zn(II), and (iii) to question kinetic
issues beyond the thermodynamic approach mostly described until now. 

\section{Results}\label{sec2}

\subsection{Ligands {${\bmathcal{L}}$}}\label{sec2.1}

The synthesis of the ligands has been adapted from literature
\cite{31,32,33,34,35,36} and is described in the Supporting
Information. 

\begin{table*}
\caption{\label{tab2} UV--vis and EPR parameters of the
Cu({${\bmathcal{L}}$}) complexes}
\begin{tabular}{ccccccccc}
\thead
\xmorerows{1}{${\bmathcal{L}}$} & \multicolumn{4}{c}{UV--vis} && 
\multicolumn{3}{c}{EPR$^{\mathrm{a}}$} \\\cline{2-5}\cline{7-9}
& \parbox[t]{1cm}{\centering $\lambda_{\max}^{\mathrm{d}\ndash\mathrm{d}}$ (nm)} & 
\parbox[t]{2cm}{\centering $\varepsilon^{\mathrm{d}\ndash\mathrm{d}}$\break (M$^{-1}{\cdot}$cm$^{-1}$)} & 
\parbox[t]{1.5cm}{\centering $\lambda_{\max}^{\mathrm{LMCT}}$\break (nm)} & 
\parbox[t]{2.3cm}{\centering $\varepsilon^{\mathrm{LMCT}}$\break (10$^3{\cdot}$M$^{-1}{\cdot}$cm$^{-1}$)} && 
$g_{//}$ & \parbox[t]{2cm}{\centering A$_{//}$\break (10$^{-4}{\cdot}$cm$^{-1}$)}\vspace*{2pt} & $g_{\bot}$ \\ 
\endthead
L & \textbf{595} & 232 & \textbf{382} & 1.24 && 2.23 ${\pm}$ 0.01 & 201 ${\pm}$ 2 & 2.05 ${\pm}$ 0.01 \\ 
L$_{\mathrm{Me}}$ & \textbf{603} & 264 & \textbf{394} & 1.17 && 2.24 ${\pm}$ 0.01 & 198 ${\pm}$ 2 & 2.06 ${\pm}$ 0.01 \\ 
L$'$ & \textbf{590} & 194 & \textbf{382} & 1.18 && 2.23 ${\pm}$ 0.01 & 204 ${\pm}$ 2 & 2.05 ${\pm}$ 0.01 \\ 
$\mathrm{L}'_{\mathrm{Me}}$ & \textbf{577} & 324 & \textbf{398} & 1.30 && 2.23 ${\pm}$ 0.01 & 206 ${\pm}$ 2 & 2.06 ${\pm}$ 0.01 \\ 
$^{\mathrm{S}}$L & \textbf{605} & 251 & \textbf{386} & 1.26 && 2.23 ${\pm}$ 0.01 & 201 ${\pm}$ 2 & 2.05 ${\pm}$ 0.01 \\ 
$^{\mathrm{S}}$L$_{\mathrm{Me}}$ & \textbf{620} & 254 & \textbf{392} & 1.23 && 2.24 ${\pm}$ 0.01 & 203 ${\pm}$ 2 & 2.06 ${\pm}$ 0.01 \\ 
$^{\mathrm{S}}$L$'$ & \textbf{600} & 264 & \textbf{382} & 1.35 && 2.23 ${\pm}$ 0.01 & 206 ${\pm}$ 2 & 2.06 ${\pm}$ 0.01
\botline
\end{tabular}
\tabnote{$^{\mathrm{a}}$The \textit{g} values were calculated using the
average position of the second and third hyperfine lines, while the
hyperfine coupling values correspond to the field differences between
the second and third lines to minimize second-order contributions to
hyperfine splittings. $^{\mathrm{65}}$Cu isotope was used.}
\vspace*{-3pt}
\end{table*}

\begin{figure*}
\includegraphics{fig01}
\caption{\label{fig1}UV--vis (Panel A) and EPR (panel B) spectra of the
Cu(II)({${\bmathcal{L}}$}) complexes. UV--vis (Panel C) and EPR (panel
D) spectra of Cu(II) removal by L from Cu(A${\upbeta}$) and
Cu,Zn(A${\upbeta}$) respectively. Experimental conditions: panels A and
C: {[}Cu$^{\mathrm{II}}${]} $=$ 500 ${\upmu}$M, {[}L{]} $=$
{[}A${\upbeta}${]} $=$ {[}Zn$^{\mathrm{II}}${]} $=$ 500~$\upmu$M,
{[}HEPES{]} $=$ 100 mM, pH 7.4, $T = 25$~\textdegree C; panel B:
{[}$^{\mathrm{65}}$Cu$^{\mathrm{II}}${]} $=$ 480 ${\upmu}$M, {[}L,
A${\upbeta}${]} $=$ 500 ${\upmu}$M; and panel D:
{[}$^{\mathrm{65}}$Cu$^{\mathrm{II}}${]} $=$ 480 ${\upmu}$M, {[}L,
A${\upbeta}${]} $=$ 500 ${\upmu}$M or
{[}$^{\mathrm{65}}$Cu$^{\mathrm{II}}${]} $=$ {[}Zn{]} $=$ 190
${\upmu}$M, {[}L, A${\upbeta}${]} $=$ 200 ${\upmu}$M;
panels B and D: {[}HEPES{]} $=$ 50 mM, pH~7.4, 10\% of glycerol as
cryoprotectant, $T = 120$ K, ${\upnu}$ ${\approx}$ 9.5 GHz, mod. ampl. $=$
5 G, microwave power: 5 mW. In panel D, EPR data corresponding to
Cu,Zn(A${\upbeta}$) $+$ L have been multiplied by 2 in order to account for
the 2.5-fold lower concentration used.}
\vspace*{3pt}
\end{figure*}

\subsection{Characterizations of Cu({${\bmathcal{L}}$}) complexes}\label{sec2.2}

The Cu(II) complexes, formed in situ by the mixture of
quasi-stoichiometric ratio between {${\bmathcal{L}}$} and Cu(II) stock
solutions (see Figure S1 for the determination of concentration of
{${\bmathcal{L}}$}) were characterized by UV--vis and EPR
spectroscopies. Their signatures are shown in Figure~\ref{fig1}, 
panels A and B,
while the corresponding parameters are listed in Table~\ref{tab2}. All the
Cu({${\bmathcal{L}}$}) complexes show similar features (note that the
complexes are neutral with L, L$'$ and L$_{\mathrm{Me}}$,
$\mathrm{L}'_{\mathrm{Me}}$ and di-anionic for $^{\mathrm{s}}$L,
$^{\mathrm{s}}$L$'$ and $^{\mathrm{s}}$L$_{\mathrm{Me}}$, but that
charges will be omitted for clarity). In UV--vis spectra (Figure~\ref{fig1},
panel A), d--d bands and phenolato-to-Cu(II) CT (Charge Transfer)
transitions are observed near 600 nm and 380 nm, respectively. Some
weak differences are observed according to {${\bmathcal{L}}$}. In
contrast to the d--d band, the LMCT band is not affected by the presence
of sulfonato groups on the ligand phenol moiety, whereas the N-methyl
group affects it. It should also be noted that the presence of
cyclohexyl instead of an ethylene bridge did not affect the LMCT band
characteristics. In EPR spectroscopy (Figure~\ref{fig1}, panel B), typical
spectra of square-planar Cu(II) complexes are obtained, with hyperfine
and $g$-value parameters in line with a 2N2O equatorial site, according
to the Peisach an Blumberg correlation~\cite{37}. Again, some slight
differences in the EPR parameters are observed between the various
ligands, whereas the presence of two sulfonato groups on the ligands
has virtually no impact on the Cu({${\bmathcal{L}}$}) EPR signatures.
Again, as for UV--vis spectra, methylation of the secondary amine leads
to the most significant difference with a decrease in the hyperfine
coupling (A$_{//}$) values and an increase in the
$g_{//}$ values, while grafting of the cyclohexyl on the
ethylene diamine bridge has the opposite effect. 

In brief, the spectroscopic characterizations indicate that the first
coordination sphere of the complexes are identical in buffered
solution, but that some minor second sphere structural changes\break occur. 

\subsection{Cu(II) removal from Cu(A${\upbeta}$)}\label{sec2.3}

Next, we performed competition experiments, monitored by UV--vis and EPR
spectroscopies (Figure~\ref{fig1}, panels C and D in case of ligand L and 
Figures~S2 and S3 for the other ligands), to check that the ligands
{${\bmathcal{L}}$} were able to remove Cu(II) from Cu(A${\upbeta}$)
including in the presence of Zn. In other words, we check that
{${\bmathcal{L}}$} have higher Cu(II) affinity and Cu(II) over Zn(II)
selectivity than A${\upbeta}$. The spectroscopic signatures of
Cu({${\bmathcal{L}}$}) were recovered during the competition
experiments (Cu(A${\upbeta}$) $+$ {${\bmathcal{L}}$} or
Cu,Zn(A${\upbeta}$) $+$ {${\bmathcal{L}}$}). Thus all the ligands
{${\bmathcal{L}}$} are efficient in retrieving Cu from Cu(A${\upbeta}$)
regardless of the presence of one equiv. of Zn(II). Hence, the
thermodynamic parameters of {${\bmathcal{L}}$} are suitable for
performing Cu(II) removal from A${\upbeta}$ including in the presence
of one equiv. of Zn(II). However, during the course of the competition
experiment, we noticed that the thermodynamic equilibrium is reached
more slowly in the case of the ligand ${\mathrm{L}}'_{\mathrm{Me}}$ (about 30 min
versus less than 5 min for the others ligands, Figure S4). 

\subsection{Effect of ligands {${\bmathcal{L}}$} on
Cu(A${\upbeta}$)-induced ROS production}\label{sec2.4}

Finally, Cu(A${\upbeta}$)-induced ROS formation was evaluated using a
very suitable and straightforward experiment, namely the ascorbate
(Asc) consumption assay. This is an appropriate method to monitor ROS
formation. Briefly, it consists in measuring the absorption of Asc (at
265 nm, ${\varepsilon} = 14\text{,}500$ M$^{-1}{\cdot}$cm$^{-1}$)
that is the reductant fueling the incomplete reduction of
O$_{\mathrm{2}}$ to $\mathrm{O}_{{2}}^{\circ-}$,
H$_{\mathrm{2}}$O$_{\mathrm{2}}$ and HO$^\circ$~\cite{21}, and that
does not absorb once oxidized. It has been previously shown that Cu and
Cu(A${\upbeta}$)-induced Asc consumption mirrors the formation of
H$_{\mathrm{2}}$O$_{\mathrm{2}}$ and HO$^\circ$~\cite{38, 39}. A
ligand will be efficient in stopping Asc consumption if it can bind
Cu(II) or remove it from A${\upbeta}$, and then form a Cu(II) complex
resistant to reduction by Asc, eventually interrupting the redox cycle
of Cu and thus the production of ROS. 

{With Asc consumption assays, two distinct experiments can be
performed, either by incubating the various chemical partners at play
and then triggering the reaction by addition of Asc (named
``experiments A'', Figure S5 and Figure~\ref{fig2}, panel D), or by adding
ligand} {{${\bmathcal{L}}$}} {during Cu(A${\upbeta}$)-induced Asc
consumption (named ``experiments B'', Figure~\ref{fig2}, panels A to C). In
experiments A, if the incubation time is long enough, the results
indicate the ability of} {{${\bmathcal{L}}$}} {to remove Cu from
Cu(A${\upbeta}$) including in the presence of increasing stoichiometry
of Zn(II), thus documenting the thermodynamics of the reaction of
Cu(II) extraction out of A${\upbeta}$. This is a very straightforward
way to evaluate whether the selectivity of a ligand} $L$ {is
appropriate}~\cite{22, 23}. {More specifically, such a method is more
suited to screen between various ligands than the individual
determination of Cu(}$L${) and Zn(}$L${) formation that would also
release the selectivity of the} $L$ {(see introduction). In experiments
B, additional kinetic parameters are involved 
(Scheme~\ref{sch2}A)}~\cite{40,41,42,43}. 
{Indeed, during Asc consumption, Cu oscillates between
the +I and +II redox states. To be efficient, the tested ligand thus
has to be faster in removing Cu(II) from A${\upbeta}$ than the
reduction of Cu(II)(A${\upbeta}$) to Cu(I)(A${\upbeta}$) by Asc. In
the} {{${\bmathcal{L}}$}} {series, we wanted to decipher the importance
of Zn stoichiometry in both the thermodynamics (experiments ``A'') and
kinetics (experiments ``B'') of Cu(II) removal. In the following, all
the experiments were performed with 10 ${\upmu}$M of Cu(II) ions, 1.2
equiv. of A${\upbeta}$ and/or} {{${\bmathcal{L}}$}}{, and 0, 1 or 10
equiv. of Zn(II). Note that for practical reasons, the experiments are
run with a slight excess of A${\upbeta}$ and/or} {{${\bmathcal{L}}$}}
{to avoid any possibility of unbound Cu(II) ions and/or
A${\upbeta}$-bound Cu(II) complexes, respectively. Indeed, this would
dramatically change the rate of Asc. consumption.} 

\begin{figure*}
\includegraphics{fig02}
\caption{\label{fig2}Ascorbate consumption is followed at 265 nm (with
a baseline subtraction at 800 nm) induced by Cu(A${\upbeta}$). Panels A
to C: starting from a Cu(I)/Cu(II) mixture and in the presence of
increasing Zn(II) stoichiometry.~{[}{{${\bmathcal{L}}$}}{{]} $=$
{[}A}${\upbeta}${{]} $=$ 12 ${\upmu}$M, {[}Zn}$^{\mathrm{II}}${{]} $=$ 0,
12 and 120 ${\upmu}$M, {[}Cu}$^{\mathrm{II}}${{]} $=$ 10 ${\upmu}$M,
{[}Asc{]} $=$ 100 ${\upmu}$M, {[}HEPES{]} $=$ 100 mM, 
pH 7.4, $T = 25$~\textdegree C. 
Panel D: starting from Cu(II) and after increasing
incubation times from 1 to 48 hours. {[}L}$'_{\mathrm{Me}}${{]} $=$
{[}A}${\upbeta}${{]} $=$ 12 ${\upmu}$M, {[}Zn{]} $=$ 120 ${\upmu}$M,
{[}Cu}$^{\mathrm{II}}${]} $=$ 10 ${\upmu}$M, {[}Asc{]} $=$ 100 ${\upmu}$M,
{[}HEPES{]} $=$ 100 mM, pH 7.4, $T = 25$~\textdegree C. The arrows indicate
the time at which the different components are added into the cuvette.}
\end{figure*}

\begin{scheme*}
\includegraphics{sc02}
\vspace*{10pt}
\caption{\label{sch2}Mechanisms of arrest of Cu(A${\upbeta}$)-induced
ROS production by a Cu(II)-targeting ligand {${\bmathcal{L}}$}, in the
absence (Panel A) and presence of Zn (panel B). In the absence of
Zn(II), the reaction of Cu(II) extraction from A${\upbeta}$ is in
competition with Cu(II)(A${\upbeta}$) reduction by Asc. In the presence
of Zn(II), an additional competition reaction occurs due to the
possible formation of Zn({${\bmathcal{L}}$}) that will retard the
formation of Cu({${\bmathcal{L}}$}).}
\vspace*{-12pt}
\end{scheme*}

In the absence of Zn and after short incubation (300~s) (Figure S5,
panel A), all the ligands except $\mathrm{L}'_{\mathrm{Me}}$ are able to stop
Cu(A${\upbeta}$) induced Asc consumption. After a longer incubation
time (${>}$18 h), $\mathrm{L}'_{\mathrm{Me}}$ becomes efficient
(Figure S6). This indicates that Cu(II) removal from A${\upbeta}$ by
$\mathrm{L}'_{\mathrm{Me}}$ is slower than that with the other
{${\bmathcal{L}}$} ligands, but thermodynamically possible, in line
with the competition experiments previously described. In addition to
showing that all the ligands are able to extract Cu(II) from
A${\upbeta}$, these experiments confirm that all the
Cu({${\bmathcal{L}}$}) complexes formed are resistant to reduction by
ascorbate. In line with these first results, all the ligands, except
$\mathrm{L}'_{\mathrm{Me}}$ and to a lesser extent L$'$ and L$_{\mathrm{Me}}$,
are able to stop Cu(A${\upbeta}$)-induced Asc consumption when added
during the course of the experiments (Figure~\ref{fig2}, panel A). 


\subsection{Effect of ligands {${\bmathcal{L}}$} on the
Cu(A${\upbeta}$)-induced ROS production in the presence of
Zn(II)}\label{sec2.5}

In the presence of Zn, the differences between the various ligands
appear more clearly. This may be due to the additional competition
reaction between Cu(II) or Zn(II) removal from Cu,Zn(A${\upbeta}$)
(Scheme~\ref{sch2}, panel B). With a short incubation (300~s) (Figure S5, panel
B) or when the ligand is added during Cu(A${\upbeta}$)-induced Asc
consumption (Figure~\ref{fig2}, panel B), the trends are the same. The
``efficiency trend'' (i.e. the ability of the ligand to stop
Cu(A${\upbeta}$)-induced Asc consumption in the presence of Zn) is as
follows: L~${\sim}~{}^{\mathrm{s}}$L ${>}$ L$_{\mathrm{Me}} \sim {}^{\mathrm{s}}$L$_{\mathrm{Me}}$~${\gtrsim}$~$^{\mathrm{s}}$L$'$
${\gtrsim}$ L$'$~$\gg$~$\mathrm{L}'_{\mathrm{Me}}$. With higher stoichiometry
of Zn(II) (10 equiv.), the differences are more obvious and only the L
and $^{\mathrm{s}}$L ligands maintain their ability to stop
Cu(A${\upbeta}$)-induced Asc consumption (Figure~\ref{fig2}, panel C). 

Hence, it appears here that Zn(II) induces a change in the ability of
the ligands {${\bmathcal{L}}$} to stop Cu(A${\upbeta}$)-induced ROS
production and that this effect is dependent on the ligands
{${\bmathcal{L}}$}, with a dependence that is more clearly revealed in
the presence of 10~equiv. of Zn(II). Two explanations can be proposed
(i): when increasing the ratio of Zn(II), the thermodynamic equilibria
at play change and the ligand becomes thermodynamically unable to
remove Cu(II) from A${\upbeta}$, or (ii) Zn(II) has a kinetic impact on
Cu extraction from A${\upbeta}$. This kinetic effect would be due to
the faster formation of the Zn({${\bmathcal{L}}$}) complexes that have
further to swap their metallic center with Cu(A${\upbeta}$) (Scheme~\ref{sch2},
panel B). To discriminate between these two hypotheses, we also
performed experiments A with a longer pre-incubation time of
Cu(A${\upbeta}$) $+$ {${\bmathcal{L}}$} in the presence of 10 of equiv.
Zn(II). Several incubation times were tested: 1, 3, 18, 24, and 48 h
(Figure~\ref{fig2}, panel D and Figure S7). The results obtained show that with
a sufficient incubation time, all the ligands prevent
Cu(A${\upbeta}$)-induced ROS production and thus that the overall
effect of Zn(II) is kinetic. For all ligands, except $\mathrm{L}'_{\mathrm{Me}}$
which requires at least 24 h of incubation, (Figure~\ref{fig2}D), 1 h of
incubation is sufficient to prevent Asc consumption (Figure S7). 

\section{Discussion}

The various results obtained with the ligands {${\bmathcal{L}}$}, are
compiled in Figure~\ref{fig3}, where the rates of ascorbate consumption are
reported. 

\begin{figure}
\includegraphics{fig03}
\caption{\label{fig3}Rates of Cu(A${\upbeta}$)-induced ascorbate
consumption as a function of the ligand and equivalent of Zn(II),
starting from Cu(I)/Cu(II) (corresponding to data in Figure~\ref{fig2}, panels
A--C) and from Cu(II) after 24 h of incubation (corresponding to data in
Figure~\ref{fig2}, panel D and Figure S7). L in pink, L$_{\mathrm{Me}}$ in
blue, L$'$ in green, and $\mathrm{L}'_{\mathrm{Me}}$ in yellow, corresponding
sulfonated ligands are displayed in light colors. The rate of Asc
consumption was determined by measuring the slope of the Asc
consumption curves between the time ($t$) of the addition of
{${\bmathcal{L}}$} and $t + 300$~s, approximating the curves as straight
lines, and with {[}Asc{]} $=$ 100~${\upmu}$M and {[}Cu{]} $=$ 
10~${\upmu}$M. The dotted line indicates the level of Asc consumption
below which Asc consumption is considered to be similar to that of Asc
alone in the buffer (due to Asc auto-oxidation).}
\end{figure}

\subsection{Effect of p-sulfonation on phenol rings}\label{sec3.1}

The sulfonated version of three ligands were tested. Sulfonation has no
significant effect on the properties of the parent ligand regarding
Cu(A${\upbeta}$) and Cu,Zn(A${\upbeta}$)-induced ROS production. This
makes possible the use of such water-soluble counterparts for
\textit{in vitro} investigations, while the parent ligands could be
engaged in \textit{in vivo} experiments. 

\subsection{Structure--activity relationship}\label{sec3.2}

\subsubsection{Ligands other than $\text{L}'_{\mathrm{Me}}$}\label{sec3.2.1}

Ligands other than $\mathrm{L}'_{\mathrm{Me}}$ are all able to stop
Cu(A${\upbeta}$)-induced ROS production in the absence of Zn(II) when
added in the course of Cu(A${\upbeta}$)-induced Asc. consumption
(Experiments ``B'', Figure~\ref{fig3}, first set of columns). Besides, in the
presence of increasing ratios of Zn(II) and with long enough incubation
times, they all maintain this ability (Figure~\ref{fig3}, fourth set of columns,
Figures S5 and S7). Finally, when added during the course of Asc
consumption, some of them become less and less efficient as the
stoichiometry of Zn(II) is increased (Figure~\ref{fig3}, second and third
sets of columns).


To explain such observations, the chemical reactions shown in Scheme~\ref{sch3}
are proposed. According to Scheme~\ref{sch3}, the overall Zn(II)-induced
slowdown of Cu(II) removal out of A${\upbeta}$ (and consequent effect
on Cu(A${\upbeta}$)-induced Asc consumption) could be due to both
thermodynamic and kinetic factors for each\break individual reaction at play,
the determination of which is beyond the scope of the present study. 

\begin{scheme*}
\includegraphics{sc03}
\vspace*{10pt}
\caption{\label{sch3}Various possible reactions at play in Cu(II)
removal out of A${\upbeta}$ by {${\bmathcal{L}}$}, including in the
presence of one equiv. of Zn(II).}
\vspace*{-7pt}
\end{scheme*}

Reaction (1) corresponds to Cu(II) removal from A${\upbeta}$ by
{${\bmathcal{L}}$} in the absence of Zn(II). Then, in the presence of
Zn(II), the addition of {${\bmathcal{L}}$} to Cu,Zn(A${\upbeta}$) leads
to the two possible reactions (2) and (3) and the formation of
Cu({${\bmathcal{L}}$}) or Zn({${\bmathcal{L}}$}), respectively. From a
thermodynamic point of view, reaction (2) is much more favored than
reaction (3) as probed by the experiments with long incubation times
(Experiments ``A''). However, reactions (2) and (3) are in kinetic
competition, with reaction (3) being faster than reaction (2) because
otherwise the presence of Zn(II) would have no strong effect on
experiments ``B''. The progressive loss of the ability to stop
Cu(A${\upbeta}$)-induced Asc consumption with increasing levels of
Zn(II)\break (Figure~\ref{fig3}) thus mirrors the impact of Zn(II) on the overall rate
of Cu(II) removal from A${\upbeta}$. Such an effect may be due (i) to
the kinetic competition of Zn(II) with Cu(II) for binding to
{${\bmathcal{L}}$} (reaction (2) versus reaction (3)) and/or (ii) to a
modification in the rate of reaction (6) (Scheme~\ref{sch3}).

With the series of ligands {${\bmathcal{L}}$}, Zn(II) induces an
overall slowdown of Cu(II) removal. This contrasts with a previous
study on peptide-based ligands~\cite{44}, where an acceleration effect
due to Zn-induced Cu expelling from Cu,Zn(A${\upbeta}$) was reported
(corresponding to the path shown by reactions (4) and (4$'$)). This
indicates that reaction (4) is not predominant in the case of
{${\bmathcal{L}}$}. Hence, it will not be considered in the rest of the
discussion. 

The best ligands able to resist up to 10 equiv. of Zn are L and
$^{\mathrm{s}}$L, whereas $^{\mathrm{s}}$L$'$, L$_{\mathrm{Me}}$, and
$^{\mathrm{s}}$L$_{\mathrm{Me}}$ work correctly only in the presence of
one equiv. of Zn(II). Hence, the cyclohexyl-grafted ethylene bridge and, to
a lesser extent the N-methyl substituents, increase the level of
Zn({${\bmathcal{L}}$}) versus Cu({${\bmathcal{L}}$}) at a given time
(before the thermodynamic equilibrium is reached). The weaker effect of
L$'$ on the arrest of Cu(A${\upbeta}$)-induced Asc consumption may be in
line with the reported \textit{cis}-${\upbeta}$ arrangement (i.e. with
the two phenolato moiety being in \textit{cis}) of ligand L$'$ in
{[}Cu$_{\mathrm{2}}$(HL$'$)$_{\mathrm{2}}${]}$^{2+}$
species~\cite{45} unobserved with other ligands, for which
\textit{trans} arrangements (i.e. all the endogenous atoms from
{${\bmathcal{L}}$} are in the equatorial plane) were reported~\cite{25,
46,47,48}. We can indeed anticipate that such \textit{cis}-${\upbeta}$
arrangement is more appropriate for Zn(II) versus Cu(II) binding in
contrast to the \textit{trans} arrangement which is more appropriate
for Cu(II) versus Zn(II). 


\subsubsection{Ligand $\text{L}'_{\mathrm{Me}}$}\label{sec3.2.2}

Among the series, ligand $\mathrm{L}'_{\mathrm{Me}}$ holds a specific place with
very slow kinetics of Cu extraction from A${\upbeta}$ both in the
absence (reaction (1)) and presence of Zn(II) (reactions (2)--(6),
Scheme~\ref{sch3}). In the absence of Zn(II), this is shown in the experiments
with a ``short'' (i.e. 300~s) incubation time (Figure S5, panel
A). Indeed, since all the Cu(II) is not extracted from A${\upbeta}$
after such a short time, when Asc is added, its consumption is induced
by the remaining Cu(A${\upbeta}$). To complete the reaction of Cu
extraction from A${\upbeta}$, more than 18 h are required (Figure S6).
In the presence of 10 equiv. of Zn(II), this is slightly worse (the level
of Asc. consumption lessening in the presence of 10~equiv. of Zn(II)
after 24 h equals that in the absence of Zn(II) after 18 h, Figure~\ref{fig2},
panel D to be compared with Figure S6). This is due to other reactions
coming at play, namely reactions (2)--(6), as described previously
for the other ligands. The specific properties of the
$\mathrm{L}'_{\mathrm{Me}}$ ligand may be linked to the combination of the
presence of the 1,2-cyclohexyl bridge and the N-methyl group, which
makes possible a \textit{cis}-${\upbeta}$ arrangement of the ligand
(i.e. with the two phenolato moiety being in \textit{cis}), described
in the solid-state for Cu($\mathrm{L}'_{\mathrm{Me}}$)~\cite{49}. However this
geometry is not recovered in the thermodynamically-stable complex
formed in aqueous solution as probed by EPR and to a lesser extent
UV--vis signatures. But it may exist transiently and slow down the
Cu(II) complex formation (reaction (1)), favor and/or accelerate
Zn($\mathrm{L}'_{\mathrm{Me}}$) complex formation versus Cu($\mathrm{L}'_{\mathrm{Me}}$)
complex (reaction (3) versus (2)), and/or disfavor and/or slow down
reaction (6) with respect to other ligands in the series. Because the
difference between the ability to prevent Cu(A${\upbeta}$)-induced ROS
formation without Zn(II) (Figure S6) and 10 equiv. of Zn(II) 
(Figure~\ref{fig2}D) is fairly weak, we can anticipate that reaction (1) has the most
important contribution. 

In addition, the trend
$\mathrm{L}'_{\mathrm{Me}}$~${<}$~L$'$~${<}$~L$_{\mathrm{Me}}$~$\ll$~L in the
ability to stop Cu(A${\upbeta}$)-induced ROS formation in the presence
of Zn(II) is reminiscent of recently published data using a series
of pentadentate ligands ($^{\mathrm{p}}$L) based on an ethylene bridge
(possibly appended with a cyclo-hexyl moiety: $^{\mathrm{p}}$L$'$, with
a N-propyl group: $^{\mathrm{p}}$L$_{\mathrm{pro}}$ and both:
$^{\mathrm{p}}\mathrm{L}'_{\mathrm{pro}}$) (Scheme S1). Once chelated with
Mn(II), the ligand exchange with Cu(II) that corresponds to reaction
(6) here, follows the order: $^{\mathrm{p}}\mathrm{L}'_{\mathrm{pro}} <
^{\mathrm{p}}$L$'$~${<}$~$^{\mathrm{p}}$L$_{\mathrm{pro}} <
^{\mathrm{p}}$L (Scheme S2)~\cite{28}.        

\subsection{Kinetic impact of Zn(II)}\label{sec3.3}

In brief, the study of the series of ligands {${\bmathcal{L}}$} reveals
an impact of Zn(II) on Cu(II) extraction from A${\upbeta}$ that is
apparent during the Asc consumption experiments and which is beyond
only thermodynamic considerations. Indeed, if Cu,Zn(A${\upbeta}$) and
the ligands are incubated long enough, all the ligands can prevent
Cu(A${\upbeta}$)-induced ROS production. However, in the course of Asc.
consumption (no incubation) or when the incubation is short (300~s),
ligands {${\bmathcal{L}}$} can be differentiated by their efficiency to
stop Cu(A${\upbeta}$)-induced ROS production in the presence of various
Zn(II) levels. Their ability to stop Cu(A${\upbeta}$)-induced ROS
production strongly depends on subtle variations in the ligand scaffold
because all the ligands studied here were built on the very same first
coordination sphere. Thermodynamic selectivity is now a parameter
considered in the design of Cu-targeting ligands in the context of
AD~\cite{21, 22}. In the present study, we also show that it is
important to consider the effect of Zn(II) which can modify the rate of
Cu(II) extraction from A${\upbeta}$ by a ligand and can thus prevent
the formation of the corresponding Cu(II) complex resistant to Asc.
reduction and the associated arrest of Cu(A${\upbeta}$)-induced ROS
production. 

\subsection{Perspectives}\label{sec3.4}

Among the tested ligands, the simplest L appears to be the ideal
candidate with respect to its ability to stop Cu(A${\upbeta}$)-induced
ROS formation in the presence of a biologically relevant ratio of the
competing Zn(II) ion. Further studies on this ligand series will aim to
relate the \textit{in vitro} data obtained here with their ability to relieve
the cellular toxicity induced by ROS produced by Cu(A${\upbeta}$) with
and without Zn, as reported for other ligands~\cite{50,51,52,53,54}. Since L is
expected to be partly neutral at pH 7 based on potentiometric data
reported for $^{\mathrm{G}/\mathrm{S}}$L$_{\mathrm{Me}}$~\cite{24, 25},
and thus to fulfill Lipinski's rules, it appears as a good candidate
for blood--brain barrier penetration by passive diffusion and thus for
further \textit{in vivo} studies on AD animal models.\looseness=-1

\section*{Declaration of interests}

The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

\section*{Acknowledgments}

Sonia Mallet-Ladeira is acknowledged for providing
the X-ray crystallographic data of related Cu(II)
complexes.\looseness=-1 



\back{}

\section*{Supplementary data}

Supporting information for this article is available on the journal's
website under \printDOI\ or from the author.  

\CDRsupplementaryTwotypes{supplementary-material}{\cdrattach{crchim-255-suppl.pdf}}

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