\makeatletter
\@ifundefined{HCode}
{\documentclass[screen,CRCHIM,Unicode,biblatex,published]{cedram} 
\addbibresource{crchim20240611.bib}
\newenvironment{noXML}{}{}
\def\tsup#1{$^{{#1}}$}
\def\tsub#1{$_{{#1}}$}
\def\thead{\noalign{\relax}\hline}
\def\tbody{\noalign{\relax}}
\def\endthead{\noalign{\relax}\hline}
\def\tabnote#1{\vskip4pt\parbox{.95\linewidth}{#1}}
\usepackage[figuresright]{rotating}
\RequirePackage{etoolbox}
\def\jobid{crchim20240611}
\def\inlinefig#1{\includegraphics{#1}}
\newenvironment{xxcenter}{\begin{center}}{\end{center}}
\def\xsection#1{}
%\graphicspath{{/tmp/\jobid_figs/web/}}
\graphicspath{{./figures/}}
\usepackage[T1]{fontenc}
\usepackage{comment}
\newcounter{runlevel}
\usepackage{upgreek}
\let\MakeYrStrItalic\relax
\def\Smallskip{\smallskip}
\csdef{Seqnsplit}{\\}
\def\refinput#1{}
\def\back#1{}
\def\0{\phantom{0}}
\def\hyphen{\mbox{-}}
\newenvironment{inftab}{}{}
\def\xmorerows#1#2{#2}
\usepackage{multirow} 
\def\nrow#1{\@tempcnta #1\relax%
\advance\@tempcnta by 1\relax%
\xdef\lenrow{\the\@tempcnta}}
\def\morerows#1#2{\nrow{#1}\multirow{\lenrow}{*}{#2}}
\def\botline{\\\hline}
\def\Lbreak{\newline}
\def\ubreak{\break}
\def\xvarnothing{\emptyset}
\def\mn{\phantom{$-$}}
\def\og{\guillemotleft}
\def\fg{\guillemotright}
\def\bgamma{\boldsymbol{\gamma}}
\def\rmDelta{\Delta}
\makeatletter
\g@addto@macro{\UrlBreaks}{\UrlOrds}
\gappto{\UrlBreaks}{\UrlOrds}
\makeatother
\DOI{10.5802/crchim.331}
\datereceived{2024-07-23}
\dateaccepted{2024-08-29}
\ItHasTeXPublished
}{\documentclass[crchim]{article}
\def\CDRsupplementaryTwotypes#1#2{}
\def\CDRdoi{10.5802/crchim.331} 
\newenvironment{sidewaystable}{\begin{table*}}{\end{table*}}
\def\href#1#2{\url{#1}}
\def\xsection#1{}
\newenvironment{xxcenter}{}{}
\def\Smallskip{}
\let\newline\break
\def\ubreak{}
\let\xvarnothing\emptyset
\makeatletter
}
\makeatother

\usepackage[T1]{fontenc}
\usepackage{upgreek}

\dateposted{2025-02-19}
\begin{document}
%\copyeditor{RB}

\begin{noXML}

\CDRsetmeta{articletype}{clarification}

\title{Fluorinated triazolamers: preferential conformations and
activities on islet amyloid polypeptide (hIAPP) aggregation}

\alttitle{Triazolam\`{e}res fluor\'{e}s : conformations et
activit\'{e}s pr\'{e}f\'{e}rentielles sur l'agr\'{e}gation du
polypeptide amylo\"{i}de des \^{i}lots pancr\'{e}atiques humains
(hIAPP)}

\author{\firstname{Sandrine} \lastname{Ongeri}\CDRorcid{0000-0002-2118-7324}\IsCorresp}
\address{BioCIS UMR 8076 CNRS, Building Henri Moissan, Universit\'{e}
Paris-Saclay, 17 avenue des sciences, 91400 Orsay, France}
\email[S. Ongeri]{Sandrine.ongeri@universite-paris-saclay.fr}

\author{\firstname{Beno\^{i}t} \lastname{Crousse}\CDRorcid{0000-0002-2042-9942}\IsCorresp}
\addressSameAs{1}{BioCIS UMR 8076 CNRS, Building Henri Moissan,
Universit\'{e} Paris-Saclay, 17 avenue des sciences, 91400 Orsay,
France}
\email[B. Crousse]{Benoit.crousse@universite-paris-saclay.fr}

\keywords{\kwd{Fluorine}\kwd{Triazole}\kwd{Peptidomimetics}\kwd{Foldamers}\kwd{Amyloids}\kwd{Type 2 diabetes}}

\altkeywords{\kwd{Fluor}\kwd{Triazole}\kwd{Peptidomim\'{e}tiques}\kwd{Foldam\`{e}res}\kwd{Amylo\"{i}des}\kwd{Diab\`{e}te de type 2}}

\dedicatory{Dedicated to Dani\`{e}le Bonnet-Delpon on the occasion of
her 75th birthday}

\thanks{CNRS, French Ministry of Education and Research}

\begin{abstract}
This account summarizes our work on the synthesis of fluorinated
peptidomimetics integrating the \textit{N}-CF\tsub{2}R triazole
functions. Emphasis is placed on the development of fluorinated
foldamers possessing preferential conformations. We have shown that
these foldamers are potential modulators of amyloid protein
aggregation.
\end{abstract}

\begin{altabstract}
Ce compte rendu r\'{e}sume nos travaux de synth\`{e}se de
peptidomim\'{e}tiques fluor\'{e}s int\'{e}grant la fonction triazole
\textit{N}-CF\tsub{2}R. L'accent est mis sur le d\'{e}veloppement de
foldam\`{e}res fluor\'{e}s poss\'{e}dant des conformations
pr\'{e}f\'{e}rentielles. Nous avons montr\'{e} que ces foldam\`{e}res
sont des modulateurs de l'agr\'{e}gation de prot\'{e}ines
amylo\"{i}des.
\end{altabstract}

\maketitle

\twocolumngrid

\end{noXML}

\section{Introduction}

Peptides and proteins can form specific folded structures necessary for
their biological functions. This folding is ensured by intermolecular
or intramolecular hydrogen bonds, between the carbonyl groups and the
amine protons of the amide bond, as well as by electrostatic and
hydrophobic interactions of the side chains. Artificial foldamers are
repetitive synthetic structures that adopt folded molecular
configurations, thus mimicking the structural properties of
biomolecules~\cite{1}. With the increasing use of therapeutic
peptides~\cite{2,3a,3b}, interest in peptide-based foldamers capable of
mimicking secondary structures is growing rapidly~\cite{4a,4b,5a,5b}. Indeed,
peptidomimetic foldamers offer solutions to overcome two main issues of
short peptides: their lack of stability against proteolysis and their
low ability to adopt specific conformations, thereby limiting their
\mbox{practical} use in certain therapeutic fields such as the inhibition of
protein--protein interactions.

Fluorinated molecules have generated considerable interest in medicinal
chemistry, with approximately 150 reaching the pharmaceutical market
and accounting for nearly 30\% of all newly approved
drugs~\cite{6,7,8}. Furthermore, incorporating fluorinated amino acids
into peptides and proteins has increased their biological activity and
propensity to form secondary structures due to fluorine's ability to
enhance local hydrophobicity and significantly strengthen hydrogen
bonds~\cite{9,10a,10b}. The addition of fluorinated amino acids can also
improve chemical and enzymatic stability~\cite{8,9} although in some
cases, the effect is less pronounced with direct fluorinated analogues
of natural amino acids~\cite{10b}. Additionally, fluorine labeling is a
significant advantage for studying biological events using
\tsup{19}F NMR spectroscopy, such as the structure and function of
biomolecules, enzymatic mechanisms, ligand--biomolecule interactions,
metabolic pathways, and for \tsup{19}F MRI medical imaging~\cite{11}.
However, to our \mbox{knowledge,} very few examples of fluorinated foldamers
have been reported~\cite{12a,12b,13,15}. Regarding peptidic foldamers,
fluorinated peptoids~\cite{13}, CF\tsub{3}-Aib~\cite{14a},
CF\tsub{3}-${\upbeta}$-peptides~\cite{14b}, and
CF\tsub{3}-pseudoprolines~\cite{14c} adopt helical conformations, while
oligomers of \textit{cis}-2-amino-1-fluorocyclobutane-1-carboxylic
acid~\cite{12a} and 2-fluoro-aryl substituted ${\upalpha}$,${\upbeta}$
2,3-amino acid~\cite{12b} adopt a strand-like secondary structure. As
part of our interest in fluorinated peptidomimetics and foldamer
construction, we are currently exploring the potential of fluorinated
foldamers. This new class of foldamers, noted by a few chemists to date for
their conformational properties~\cite{12a,13,15,14a,14b}, remains
entirely unknown in medicinal chemistry and as potential drug
candidates. Our laboratory has been interested in designing new
fluorinated foldamers based on \textit{N}-difluoromethyl triazole
units. As a proof of concept of their benefit in medicinal chemistry,
the design of potential modulators of protein aggregation in the field
of type 2 diabetes (T2D) was investigated.\looseness=-1

T2D is a significant public health challenge, currently affecting
around 400 million individuals worldwide. This figure is projected to
surpass half a billion by 2045, yet no causal treatment exists for
this condition~\cite{20,21}. T2D belongs to the large family of
diseases called amyloidosis caused by the pathological aggregation of
amyloid proteins~\cite{22,23,24}. The aggregation of human islet
amyloid polypeptide (hIAPP, also known as amylin) plays a role in the
death of pancreatic ${\upbeta}$-cells in T2D patients through a
mechanism that is only partially understood. The hIAPP undergoes a
conformational change that leads to an aggregation pathway, forming
toxic soluble oligomers and fibers. These aggregated species adopt
${\upbeta}$-sheet rich conformations, and amyloid deposits are found in
the pancreas of over 95\% of diabetic patients~\cite{25,26,27}.
Preventing both the presence of soluble transient oligomers and the
later aggregated forms of hIAPP is essential to mitigate cell toxicity
caused by membrane disruption and other less understood phenomena
induced by hIAPP aggregation~\cite{28,29,30}. This approach must be
investigated to develop a viable therapy for T2D.

%{\vspace*{-4pt}}

\section{Synthesis of aminomethyl-1,4-triazolyl-difluoroacetic
acid}\label{sec2}

%{\vspace*{-4pt}}

To start, we designed and prepared an {original} fluorinated
peptidomimetic by {incorporating} {\textit{N}-difluoromethyltriazole
into its structure.} 
The aminomethyl-1,4-triazolyl-difluoroacetic acid named N-difluoromethyl
1,4-Tz amino acid (1,4-Tz-CF\tsub{2}), N-difluoromethyl 1,4-Tz, or
1,4-Tz-CF\tsub{2} amino acid can be considered an analogue of 
Gly-difluoroGly dipeptide, which is unstable \cite{31}.

The pseudotetrapeptide was obtained by copper-catalyzed dipolar
cycloaddition from an azide and an alkyne carrying amino acid residues.
This reaction was carried out in the presence of copper sulfate
pentahydrate, sodium ascorbate, and finally TBTA as a ligand
(Scheme~\ref{sch1}).

\begin{scheme*}
{\vspace*{2pt}}
\includegraphics{sc01}
{\vspace*{5pt}}
\caption{\label{sch1}Synthesis of \textit{N}-CF\tsub{2} triazole.}
\vspace*{-4pt}
\end{scheme*}

We demonstrated that the introduction of one monomer 1,4-Tz-CF\tsub{2}
in short peptides could induce extended structures. NMR, DFT, and X-ray
crystallography showed the interactions between fluorine atoms and
neighboring protons~\cite{31}. The 2D \tsup{19}F-\tsup{1}H NOE NMR
experiments allowed identifying correlations between the fluorine atoms
and the proton of the triazole and the NH of phenylalanine. These
observations were in agreement with the X-ray diffraction analyses. The
structure identified within the crystal lattice highlights the
distances between the two protons (triazole proton/NH phenylalanine)
and a fluorine atom, which are 2.58~\AA{} and 2.29~\AA{} with
${\upvarphi}$ angles of 88\textdegree\ and 107\textdegree,
respectively. This information tends to confirm the proton--fluorine
interactions stabilizing an extended local conformation
(Figure~\ref{fig1}).

\begin{figure*}
\includegraphics{fig01}
\vspace*{-4pt}
\caption{\label{fig1}Interactions between fluorine atoms and
neighboring protons~\cite{31}.}
\end{figure*}

In view of these particular characteristics of this
\textit{N}-CF\tsub{2}-triazole unit, we were interested in
incorporating it into oligomers to investigate their specific
conformations and their potential as modulators of hIAPP amyloid
aggregation. We therefore focused our attention on different and
original classes of fluorinated triazolamers.

\section{Synthesis of homotriazolamers}\label{sec3}

Initially, we based our attention on the successive coupling of
1,4-Tz-CF\tsub{2} amino acid to prepare homotriazolamers, trimer
\textbf{1} and tetramer \textbf{2}, (Scheme~\ref{sch2}, foldamers
containing only triazole units)~\cite{32}. 
Triazolamers \textbf{1} and \textbf{2} were prepared according to 
Scheme~\ref{sch2} by carrying out sequences of click chemistry and amidation
with propargylamine and ammonia, and finally Boc cleavage under acidic
conditions.

\begin{scheme*}
{\vspace*{5pt}}
\includegraphics{sc02}
{\vspace*{-3pt}}
\caption{\label{sch2}Synthesis of homotriazolamers \textbf{1} and
\textbf{2}.}
\vspace*{4pt}
\end{scheme*}

\section{Synthesis of heterotriazolamers}\label{sec4}

Next, we designed fluorinated heterotriazolamers by intercalating
natural amino acids between the 1,4-Tz-CF\tsub{2} units in order to
compare the conformations of these structures with those of
homotriazolamers \textbf{1} and \textbf{2}. These natural amino acids
were chosen according to the amyloidogenic sequence of hIAPP,
N\tsub{21}N\tsub{22}F\tsub{23}G\tsub{24}A\tsub{25}I\tsub{26}L\tsub{27},
recognized as mainly responsible for its aggregation. The specific
lateral chains of the natural amino acids should allow interaction
with the amyloidogenic sequence of hIAPP, while the 1,4-Tz-CF\tsub{2}
units should act as ${\upbeta}$-sheet breaker elements disturbing
local intermolecular interactions.

Thus, four fluorinated heterotriazolamers \textbf{3}--\textbf{6} were
prepared for our study. Here also, foldamers were prepared first by
click chemistry reactions with amino acids, then by peptide coupling
reactions, and finally by Boc deprotection and acetylation
(Scheme~\ref{sch3})~\cite{33}.

\begin{scheme*}
{\vspace*{-2pt}}
\includegraphics{sc03}
{\vspace*{7pt}}
\caption{\label{sch3}Fluorinated heterotriazolamers
\textbf{3}--\textbf{6}.}
{\vspace*{-3pt}}
\end{scheme*}

In the remainder of our study, the foldamers were subjected to
conformational evaluations by NMR and molecular dynamics.

\section{Conformational analysis NMR}\label{sec5}

The conformational studies of oligomers \textbf{1}--\textbf{6} were
carried out by NMR in CD\tsub{3}OH, a polar solvent avoiding solubility
problems in water. 
Many experiments such as 1D (\tsup{1}H,\tsup{19}F) and 2D
(\tsup{1}H-\tsup{1}H TOCSY, \tsup{1}H-\tsup{1}H ROESY,
\tsup{1}H-\tsup{19}F HOESY, \tsup{1}H-\tsup{13}C HSQC, and
\tsup{1}H-\tsup{13}C HMBC) were recorded at 283~K and 313~K, respectively. 
The main
results obtained from these different experiments are presented below.

In trimer \textbf{1} and tetramer \textbf{2}, the amide NH bonds are
not engaged in stable intra- (or inter-) molecular hydrogen after
the calculation of the temperature coefficients. Furthermore, no
long-distance correlation was observed for trimer \textbf{1} and
tetramer \textbf{2}. Their 2D \tsup{19}F-\tsup{1}H HOESY analyses
confirmed short-distance correlations within each
\textit{N}-difluoromethyl triazole unit, between the two fluorine
atoms, the triazole protons, and the NH of the subsequent unit, already
observed for the monomer~\cite{32}. So the rotation around the
difluoromethyl group is restricted, either due to the steric hindrance
caused by the two fluorine atoms or because of a stabilizing
electrostatic interaction between the fluorine atoms and the
neighboring protons. Furthermore, no long-range \tsup{19}F-\tsup{1}H
hetero-NOE correlations between different units were detected,
supporting an extended conformation~\cite{32}.

Concerning heterotriazolamers \textbf{3} and \textbf{4} with
amino acids, long-range \tsup{1}H-\tsup{1}H ROE correlations were
observed, indicating folded conformations. In both \textbf{3} and
\textbf{4}, ROEs were observed between the CH\tsub{3} of Ala-3 and the
Hc of the two triazoles, TZL-2 and TZL-4. In \textbf{4}, correlations
between CH\tsub{3} of Ala-3 and H${\upalpha}$ of Phe-1 and between
H${\upbeta}$ of Phe-1 and Hc of TZL-2 were also visible
(Figure~\ref{fig2}).

\begin{figure*}
\includegraphics{fig02}
\vspace*{-15pt}
\caption{\label{fig2}NMR interactions of foldamers \textbf{3} and
\textbf{4} in CD\tsub{3}OH (\tsup{1}H-\tsup{1}H and
\tsup{1}H-\tsup{19}F NOE).}
\vspace*{-4pt}
\end{figure*}

The two amino acids both at the N- and C-termini of the TZL-2 unit could
become close together thanks to the flexibility of the C${\upalpha}$ of
the TZL unit. Indeed, this side of the
\textit{N}-difluoromethyltriazole scaffold has a higher flexibility
than the side bearing the two fluorine atoms, which induces a
constraining effect thanks to H--F interactions (Figure~\ref{fig3}). In
the case of the NH\tsub{2}-free analogue \textbf{3}, one ROE was
visible between H${\upalpha}$ of Phe-1 and Hc of TZL-2. The direct
rapprochement between Phe 1 and Ala-3 was not observed~\cite{33}.

\begin{figure*}
\includegraphics{fig03}
\caption{\label{fig3}NMR interactions of foldamers \textbf{5} and
\textbf{6}.}
\end{figure*}

Very similar NMR data were obtained for the longer heterotriazolamers
\textbf{5} and \textbf{6}, confirming their folded conformations. In
particular, many long-range \tsup{1}H-\tsup{1}H ROE correlations were
observed (Figure~\ref{fig3}) (between CH${\upbeta}$ of Asn-1 and Hc of
TZL-2, CH${\upbeta}$ of Phe-3 and Hc of TZL-2 and TZL-4 [only TZL-4 for
6], CH\tsub{3} of Ala-3 and Hc of TZL-4 and TZL-6, and CH${\updelta}$
of Leu-7 and Hc of TZL-6). Interestingly, a very long-range
\tsup{1}H-\tsup{1}H ROE correlation was also observed between
CH${\updelta}$ of Leu-7 and Hc of TZL-2 in both \textbf{5} and
\textbf{6}, indicating a close proximity between their N- and
C-termini. In the 2D \tsup{19}F-\tsup{1}H HOESY spectra of
\textbf{3}--\textbf{6}, only the short-range correlations involving in
each 1,4-Tz CF\tsub{2} unit the two fluorine atoms with the proton of
the triazole and the NH of the successive unit were visible. This
result suggests that the two fluorine atoms of each 1,4-Tz CF\tsub{2}
unit maintained the extended constraint at its C-terminal part but were
not involved in the overall folding of the foldamers
(Figure~\ref{fig3})~\cite{33}.

\section{Conformational analysis by molecular dynamics}\label{sec6}

In parallel, molecular dynamic (MD)  simulations of these triazolamers
(\textbf{1}--\textbf{6}) were conducted in explicit methanol solvent to be
as close as possible to NMR conditions. The convergence of our simulations
was evaluated first by visually inspecting the time evolution of RMSD
relative to the initial conformation and that of the radius of gyration
(Figure~\ref{fig4}), and by comparing distributions of the radius of
gyration over time. The distributions of the radius of gyration of
1,4-Tz CF\tsub{2} triazolamers \textbf{1} and \textbf{2} attain high
values, indicating extended conformation (Figure~\ref{fig4})~\cite{32}.

\begin{figure*}
\includegraphics{fig04}
\caption{\label{fig4}Representative structures of the most populated
clusters of 1,4-Tz-CF\tsub{2} homotriazolamers \textbf{1} (left)
and \textbf{2} (right).}
\vspace*{-3pt}
\end{figure*}

On the contrary, MD simulations of compounds \textbf{3} and \textbf{4}
generated conformational ensembles in which the two rather rigid and
extended segments AA-TZL2-AA and AA-TZL4-AA can rotate around the
central AA residue, acting as a hinge and allowing conformational
transitions between hairpin-folded segments and more extended linear
structures (Figure~\ref{fig5})~\cite{33}.

\begin{figure*}
\includegraphics{fig05}
\vspace*{-8pt}
\caption{Compounds \textbf{3} and \textbf{4}.}
\label{fig5}
\end{figure*}

MD trajectory visualizations show that both molecules \textbf{5} and
\textbf{6} are highly flexible. The representative structures of the
most populated clusters of compounds \textbf{5} and \textbf{6} are
displayed in Figure~\ref{fig6}. As~in compounds \textbf{3} and
\textbf{4}, Asn-TZL, Phe-TZL, and Ala-TZL fragments are rather rigid
but can rotate around the two residues Phe and Ala, which act as
bends. Thus, the transient hairpin-like structures or zigzag
conformations angled at these two positions are observed, sometimes
even folding into ladder-like structures similar to short
multistranded ${\upbeta}$-sheets (Figure~\ref{fig6})~\cite{33}.

\begin{figure*}
\includegraphics{fig06}
\caption{\label{fig6}Compounds \textbf{5} and \textbf{6}.}
\end{figure*}

\begin{figure*}
\includegraphics{fig07}
\caption{\label{fig7}Representative curves of ThT fluorescence of one
assay over time showing hIAPP control aggregation (5~${\upmu}$M) in the
absence (red curve) and in the presence of compounds \textbf{1} and
\textbf{4} at compound/hIAPP ratios of 10/1 (purple curves) and 1/1
(green curves). The control curves are represented by orange lines and
the buffer by gray. Solid lines correspond to curves fitted with a
Boltzmann sigmoidal model.} 
{\vspace*{-2pt}}
\end{figure*}

As the activity of these compounds were performed in water, we compared
MD simulations of compounds \textbf{4} and \textbf{6} in explicit water
versus methanol. Both compounds \textbf{4} and \textbf{6} were slightly
more flexible and sampled more diverse conformations than in methanol.
Nevertheless, the transient hairpin-like structures of compound
\textbf{4} were still significantly present in water. Notably,
conformation of compound \textbf{6} appeared slightly more compact but
less structured in water than in methanol.

\section{Activity on hIAPP Fibrilization by ThT-fluorescence experiments} \label{sec7}

The ability of foldamers \textbf{1}--\textbf{6} to interact with hIAPP
fibril formation was first investigated using the in~vitro
Thioflavin T (ThT) fluorescence assay. We only report here triazolamers
that showed some specific effect and activity on amyloid, compared to
the natural peptides FGAIL-NH\tsub{2} and NNFGAIL-NH\tsub{2}. The
aggregation curves of hIAPP in the presence of homotriazolamer
\textbf{1} at \textbf{1}/hIAPP ratio 10/1 (Figure~\ref{fig7}) showed a
shorter $t_{1/2}$ and a higher fluorescence plateau than for the control
experiment, indicating that compound \textbf{1} is capable of
accelerating but also increasing the aggregation of hIAPP~\cite{33}.

\pagebreak
On the contrary, the inhibitory activity of \textbf{4} was dramatically
increased compared to that of the natural peptide parent FGAIL at
\textbf{4}/hIAPP ratio 10/1 (Figure~\ref{fig7}), both on the
fibrilization kinetics ($\rmDelta t_{1/2} = + 112$\% for \textbf{4} vs no
effect for FGAIL-NH\tsub{2}) and on the fluorescence plateau ($\rmDelta
F = 80$\% for \textbf{4} vs $=$\ 36\% for FGAIL-NH\tsub{2}). The
activity was not retained at the lower ratio of 1/1.
%\vspace*{-20pt}

\begin{figure*}
{\vspace*{-2pt}}
\includegraphics{fig08}
{\vspace*{-2pt}}
\caption{\label{fig8}\tsup{1}H NMR experiments.}
{\vspace*{-2pt}}
\end{figure*}
\section{Interaction of homotriazolamer 1 and heterotriazolamer 4 with
hIAPP by NMR}\label{sec8}

The effect of compounds \textbf{1} and \textbf{4} on hIAPP was first
studied using proton NMR experiments (Figure~\ref{fig8}). In
particular, for \textbf{4}, interactions with sequences directly
involved in the hIAPP aggregation process (residues 10--17 and 25--28) were
observed. At 0.5~h, a line broadening was observed in the presence of
\textbf{1} or \textbf{4} (green and blue curves, respectively), which
could be due to their interaction with monomeric hIAPP or to hIAPP
aggregation.

The interaction of compounds \textbf{1} and \textbf{4} with hIAPP was
then investigated using \tsup{19}F NMR experiments (Figure~\ref{fig9}).
For the achiral homotriazolamer \textbf{1}, which contains three
CF\tsub{2} groups, the \tsup{19}F NMR signals appear as three very
close singlets around 88~ppm. When \textbf{1} was mixed with 
50~${\upmu}$M hIAPP, a change in signal intensities around 88
ppm was observed (green spectrum compared to the orange spectrum of the
compound alone; see Figure~\ref{fig9}). In the case of compound \textbf{4},
mixing it with hIAPP resulted in a roughly 40\% reduction in the
\tsup{19}F signal intensity in the 86--88 ppm region compared to
\textbf{4} without hIAPP (blue spectrum compared to red spectrum; see
Figure~\ref{fig9}).
\vspace*{20pt}
%\pagebreak

\begin{figure*}
\includegraphics{fig09}
\caption{\tsup{19}F NMR spectra of \textbf{1} and \textbf{4}.}
\label{fig9}
\end{figure*}

\pagebreak
The observed differences in signal intensities can be attributed to the
presence of two distinct populations of the compounds in the mixture:
those bound to hIAPP and those not bound to hIAPP in a mixture
containing a 5 molar excess of compounds, specially in the case of 
heterotriazolamer~\textbf{4}.

\section{Effect on hIAPP oligomerization by native and ion mobility mass spectrometry experiments}\label{sec9}

Mass spectrometry (MS) has been used as a valuable technique to
identify oligomeric species formed during the early oligomerization
process of amyloid peptides and proteins. MS experiments revealed
differences between the two foldamers \textbf{1} and \textbf{4}, in
particular regarding the nature, number, and shape of noncovalent
complexes of foldamers with hIAPP monomers/oligomers formed. Foldamer
\textbf{1}, which accelerates fiber formation in ThT
experiments, interacts slightly with the hIAPP monomer while the
noncovalent complexes hIAPP/\textbf{1} and hIAPP oligomers disappear
very quickly~\cite{33}.

On the contrary, foldamer \textbf{4}, which is a retarder of the
fibrilization process in ThT assays, forms numerous noncovalent
complexes almost exclusively with the hIAPP monomer, which are more
compact and stable over time. We 
can therefore hypothesize that
foldamer \textbf{4} interacts more strongly with hIAPP monomers,
sequestering them and favoring a compact conformer that may not be 
prone to aggregation.

\section{Conclusion}\label{sec10}

We demonstrated that fluorinated triazolamers based on
aminomethyl-1,4-triazolyl-difluoroacetic acid (1,4-Tz-CF\tsub{2}) are
readily available synthetically. Experimental and computational
conformational studies showed that homotriazolamers adopt extended or
worm-like chain structures, while heterotriazolamers are structured
more like hairpins. Some of the triazolamers have given rise to very
interesting but opposite activities on hIAPP, with one homotriazolamer
accelerating and one heterotriazolamer inhibiting hIAPP aggregation.

This study highlights the potential of hitherto unknown fluorinated
peptidomimetic foldamers as modulators of amyloid aggregation as well
as for wider implications in medicinal chemistry. Our findings affirm
the value of fluorine ``labeling'' in investigating biological events,
such as ligand--biomolecule interaction structures via \tsup{19}F NMR
spectroscopy. In addition, this work paves the way for utilizing
fluorinated foldamers in \tsup{19}F MRI medical imaging.

The interest of substituting the methylene of the
aminomethyl-1,4-triazolyl-difluoroacetic acid to mimic other
AA-difluoroGly dipeptide to increase the affinity towards other target
proteins will be soon provided.

{\vspace*{30pt}}
\break
\section*{Declaration of interests}

%{\vspace*{-2pt}}

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.

%{\vspace*{-2pt}}

\section*{Acknowledgments}

{\vspace*{-2pt}}

The authors gratefully acknowledge all the students and collaborators
who have contributed to this work. They thank the  CNRS and the French Ministry of
Education and Research for their financial support. 
They also warmly thank the French Fluorine Network (GIS-FLUOR)  for its
unconditional support.
{\vspace*{-2pt}}

\back{}

\printbibliography
\refinput{crchim20240611-reference.tex}

\end{document}
