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\DOI{10.5802/crchim.362}
\datereceived{2024-09-03}
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\title{One-step introduction of the hexafluoroisobutyl group, synthesis
of (\textit{S})-5,5,5,\tralicstex{5\textquotesingle}{$5'$},\tralicstex{5\textquotesingle}{$5'$},\tralicstex{5\textquotesingle}{$5'$}-hexafluoroleucine, and its
incorporation into peptides and proteins}

\alttitle{Introduction du groupe hexafluoroisobutyle en une \'{e}tape,
synth\`{e}se de la (\textit{S})-5,5,5,\tralicstex{5\textquotesingle}{$5'$},\tralicstex{5\textquotesingle}{$5'$},\tralicstex{5\textquotesingle}{$5'$}-Hexafluoroleucine
et son incorporation dans les peptides et les prot\'{e}ines}

\author{\firstname{Aline} \lastname{Delamare}}
\address{Univ. of Bordeaux, CNRS, Bordeaux, INP, CBMN, UMR 5248, IECB,
F-33607 Pessac, France}

\author{\firstname{Guillaume} \lastname{Naulet}}
\addressSameAs{1}{Univ. of Bordeaux, CNRS, Bordeaux, INP, CBMN, UMR 5248,
IECB, F-33607 Pessac, France}

\author{\firstname{Sandeep} \lastname{Mummadi}}
\addressSameAs{1}{Univ. of Bordeaux, CNRS, Bordeaux, INP, CBMN, UMR 5248,
IECB, F-33607 Pessac, France}

\author{\firstname{Gilles} \lastname{Guichard}\CDRorcid{0000-0002-2584-7502}}
\addressSameAs{1}{Univ. of Bordeaux, CNRS, Bordeaux, INP, CBMN, UMR 5248,
IECB, F-33607 Pessac, France}

\author{\firstname{Guillaume} \lastname{Compain}\CDRorcid{0000-0002-9556-1016}\IsCorresp}
\addressSameAs{1}{Univ. of Bordeaux, CNRS, Bordeaux, INP, CBMN, UMR 5248,
IECB, F-33607 Pessac, France}
\email[G. Compain]{g.compain@iecb.u-bordeaux.fr}

\shortrunauthors

\keywords{\kwd{Fluoroalkylation} 
\kwd{S\textsubscript{N}2\tralicstex{\textquotesingle}{$'$} mechanism} 
\kwd{Hydrofluorination} 
\kwd{Hexafluoroleucine} 
\kwd{Fluorinated peptide} 
\kwd{Fluorinated protein}}

\altkeywords{\kwd{Fluoroalkylation} 
\kwd{M\'{e}canisme S\textsubscript{N}2\tralicstex{\textquotesingle}{$'$}} 
\kwd{Hydrofluoration} 
\kwd{Hexafluoroleucine} 
\kwd{Peptide fluor\'{e}} 
\kwd{Prot\'{e}ine fluor\'{e}e}}


\begin{abstract}
The isobutyl motif is a highly prevalent functional group in medicinal
chemistry, commonly present in numerous drugs and bioactive compounds.
In biological systems, it notably corresponds to the side chain of the
amino acid leucine. The replacement of this
branched chain by the hexafluoroisobutyl group provides a significantly
more hydrophobic moiety while maintaining a close global morphology.
This modification has been overlooked due to the absence of general,
simple, and/or efficient synthetic methods for introducing such a group
into small molecules, amino acids, peptides, and proteins. In this
account, we review our latest contributions to this field, highlighting
the development of a general one-step method to incorporate the
hexafluoroisobutyl group using a liquid fluorinated reagent. This
method involves a tandem mechanism that overcomes the
S\textsubscript{N}2\tralicstex{\textquotesingle}{$'$} mechanism usually observed with
\tralicstex{\textit{α}}{${\rmalpha}$}-(trifluoromethyl)vinyl groups. This methodology was
successfully applied to the synthesis of
(\textit{S})-5,5,5,\tralicstex{5\textquotesingle}{$5'$},\tralicstex{5\textquotesingle}{$5'$},\tralicstex{5\textquotesingle}{$5'$}-hexafluoroleucine (Hfl).
Additionally, we summarize the research conducted on incorporating this
valuable amino acid into peptides and proteins. Overall, this work
opens new perspectives in organofluorine chemistry, medicinal
chemistry, and protein engineering, which are also discussed herein.
\end{abstract}

\begin{altabstract}
Le motif isobutyle est un groupement tr\`{e}s r\'{e}pandu en chimie
m\'{e}dicinale, que l'on retrouve dans de nombreux m\'{e}dicaments et
compos\'{e}s bioactifs, mais aussi en biologie puisqu'il correspond
\`{a} la cha\^{i}ne lat\'{e}rale de la leucine. Le remplacement de
cette cha\^{i}ne ramifi\'{e}e par le groupement hexafluoroisobutyle
hautement fluor\'{e}e permet d'obtenir un motif mol\'{e}culaire
beaucoup plus hydrophobe, tout en conservant une morphologie globale
proche. Cette modification a \'{e}t\'{e} n\'{e}glig\'{e}e en raison de
l'absence de m\'{e}thodes synth\'{e}tiques g\'{e}n\'{e}rales, simples
et/ou efficaces pour introduire un tel groupe dans les petites
mol\'{e}cules, les acides amin\'{e}s, les peptides et les
prot\'{e}ines. Dans cet article, nous passons en revue nos
derni\`{e}res contributions dans ce domaine, notamment le
d\'{e}veloppement d'une m\'{e}thode g\'{e}n\'{e}rale pour incorporer le
groupe hexafluoroisobutyle en une \'{e}tape \`{a} l'aide d'un
r\'{e}actif fluor\'{e} liquide. Cette m\'{e}thode implique un
m\'{e}canisme en tandem qui permet de d\'{e}passer le m\'{e}canisme de
S\textsubscript{N}2\tralicstex{\textquotesingle}{$'$} g\'{e}n\'{e}ralement observ\'{e} avec les
groupements vinyliques \tralicstex{\textit{α}}{${\rmalpha}$}-trifluorom\'{e}thyl\'{e}s. Cette
m\'{e}thodologie a \'{e}t\'{e} appliqu\'{e}e avec succ\`{e}s \`{a} la
synth\`{e}se de la (\textit{S})-5,5,5,\tralicstex{5\textquotesingle}{$5'$},\tralicstex{5\textquotesingle}{$5'$},\tralicstex{5\textquotesingle}{$5'$}-hexafluoroleucine
(Hfl). De plus, nous r\'{e}sumons les recherches men\'{e}es sur
l'incorporation de cet acide amin\'{e} d'int\'{e}r\^{e}t dans les
peptides et les prot\'{e}ines. Dans l'ensemble, ce travail ouvre de
nouvelles perspectives en chimie organique du fluor, en chimie
m\'{e}dicinale et en ing\'{e}nierie des prot\'{e}ines, qui sont
\'{e}galement discut\'{e}es.
\end{altabstract}

\thanks{{Agence Nationale de la Recherche} (ANR, Grant ANR-20-CE06-0008),
Minist\`{e}re de l'Enseignement Sup\'{e}rieur, de la Recherche et de
l'Innovation (MESRI), R\'{e}gion Nouvelle Aquitaine}

\thanks{\textbf{Note}. This article has been submitted in response to
an invitation of the editorial committee for a special issue on
fluorine chemistry.}

\maketitle

\twocolumngrid

\end{noXML}

\section{Introduction}

Fluorine is an essential element in pharmaceuticals, with approximately
20\% of drugs containing at least one fluorine
atom~\cite{bib1,bib2,bib3,bib4}. In many cases, the introduction of
fluorine into molecules has significantly improved molecular
properties~\cite{bib5,bib6,bib7}. \mbox{Fluorination} affects several
parameters, including the \mbox{hydrophilicity}/lipophilicity balance, the
metabolic stability of drugs, and the binding affinity to a target. Due
to its small size, the replacement of hydrogen by fluorine leads to minimal
steric change while increasing hydrophobicity and polarity. In
addition, organofluorinated compounds have \mbox{significant} 
\mbox{applications} in
chemical biology~\cite{bib8}; for example, the so-called
\textit{fluorous effect} can be used to study and manipulate biological
systems in an orthogonal manner. In addition, polyfluorinated groups
are valuable probes for performing $^{19}$F NMR studies and are used as MRI
contrast agents in biomedical imaging~\cite{bib9,bib10,bib11}.
Polyfluorinated groups, such as CF$_{3}$, are often required to observe
NMR signals. The incorporation of fluorine into amino acids has
therefore proved particularly useful in this
context~\cite{bib12,bib13}. Fluorinated amino acids have attracted much
attention in the drug industry, whether used individually or as
components of bioactive peptides or peptidomimetics~\cite{bib3,bib14}.
Fluorinated analogues of canonical amino acids are of particular interest
because they can be incorporated into natural bioactive peptides to
modulate their biological properties or to study protein conformation,
binding, dynamics, and interaction with membranes using\break $^{19}$F
NMR~\cite{bib8}.


Recently, efforts have been directed toward the development of
synthetic routes to access emerging polyfluorinated motifs~\cite{bib15}
that could be highly profitable for the applications mentioned above.
However, the widespread use of specific fluorinated motifs depends on
their synthetic accessibility, which requires efficient synthetic
processes using affordable and safe fluorinated reagents. Our recent
research has focused on the introduction of polyfluorinated analogues of
the isobutyl group, a hydrophobic branched alkyl chain frequently
encountered in medicinal chemistry research programs and present in
many drugs, such as tetrabenazine and bromocriptine. In addition, the
isobutyl moiety is the side chain of leucine, the most abundant
hydrophobic amino acid, making it ubiquitous in proteins. It is
typically found in the hydrophobic core of proteins and plays a key
role in many protein assemblies, such as leucine zippers~\cite{bib16}.
Replacing the isobutyl side chain with a hexafluoroisobutyl group
significantly increases hydrophobicity due to the increased side-chain
volume while maintaining the overall morphology of the amino
acid~\cite{bib17,bib18}. This group also exhibits a significant dipole
moment, probably comparable to that of a water
molecule~\cite{bib19,bib20}.


Several research groups have investigated the effect of replacing
leucine with (\textit{S})-5,5,5,$5'$,$5'$,$5'$-hexafluoroleucine
(Hfl) in peptides or proteins on folding,
self-assembly~\cite{bib21,bib22,bib23,bib24,bib25,bib26,bib27,bib28,bib29,bib30,bib31}, 
and biological
properties~\cite{bib32,bib33,bib34,bib35,bib36,bib37,bib38}. However,
the synthesis of the corresponding amino acid typically requires
multiple synthetic steps to attach the hexafluoroisopropyl synthon to
the ${\upbeta}$-carbon of the amino acid (Figure~\ref{fig1}), which
significantly limits its wider application in various fields such as
drug discovery and chemical biology~\cite{bib27,bib39,bib40,bib41}. To
establish a concise and versatile method for introducing this
fluorinated group, we developed an efficient single-step procedure to
incorporate the entire fluorinated chain using a non-gaseous
fluorinated reagent. This method required overcoming critical chemical
hurdles, such as the unwanted elimination of fluoride ions. We have
successfully synthesized enantiopure Hfl using this process and have
begun to explore its potential in foldamer chemistry and for modulating
biologically relevant peptides and proteins. In addition, we have
recently reported a cell-free approach to incorporate this highly
fluorinated residue into amyloids for high-resolution magic-angle
spinning\break $^{19}$F NMR.

\begin{figure}
\includegraphics{fig01}
\caption{\label{fig1}Key disconnections to build or introduce the
hexafluoroisobutyl group.}
\end{figure}

\begin{scheme}
\includegraphics{sc01}
{\vspace*{6pt}}
\caption{\label{sch1}(A) Elimination of HBr on
2-(bromomethyl)-1,1,1,3,3,3-hexafluoropropane under basic conditions
leads to hexafluoroisobutene. (B) First attempt to introduce the
hexafluoroisobutyl group on a ketoester; hexafluorinated compound
\textbf{1} is formed as a minor product and pentafluorinated compound
\textbf{2} is obtained predominantly.}
{\vspace*{-6pt}}
\end{scheme}

\begin{figure*}
\includegraphics{fig02}
\caption{\label{fig3}Scope of the hexafluoroisobutylation reaction.
$^{\mathrm{a}}$2 equiv of the electrophile was used.
$^{\mathrm{b}}$Estimated yield using NMR.}
\end{figure*}

\section{A general method to introduce the hexafluoroisobutyl chain in
a single step}

\subsection{Development of the method}

We started our investigation using
2-(bromomethyl)-1,1,1,3,3,3-hexafluoropropane as an electrophilic
reagent. This reagent is a liquid at room temperature and is easy to
handle. Although this reagent is commercially available, its reactivity
was almost unknown~\cite{bib42,bib43}. In our initial attempts to
alkylate substrates with this reagent, we observed that under 
basic conditions, the reagent
undergoes  a complete ${\upbeta}$-elimination of
HBr within a few minutes, leading to hexafluoroisobutene
(Scheme~\ref{sch1}A). The formation of this unsaturated compound was
confirmed by NMR. Therefore, this unsaturated fluorinated molecule is
rapidly generated in solution and acts as an \mbox{electrophilic} reagent.
However, the fluoroalkylation reaction on ketoesters with the
fluoroalkylating reagent resulted predominantly in a pentafluoroalkene,
with the hexafluorinated derivative being formed as a minor product
(Scheme~\ref{sch1}B)~\cite{bib44}.  To circumvent this problem, we
found that the use of a large excess of tetrabutylammonium fluoride
(TBAF), acting both as a source of nucleophilic fluoride and as a base,
provided the desired compound in good to excellent yields for a wide
range of carbonylated substrates (Figure~\ref{fig3}). The reaction was
suitable with substituted ketoesters, malonates, diketones, Schiff
base esters, and malononitrile. Interestingly, starting from
${\upalpha}$-acetylbutyrolactone, the deacetylated product was
successfully obtained giving direct access to the
${\upalpha}$-fluoroalkylated ester (Scheme~\ref{sch2}). 

\begin{scheme*}
\includegraphics{sc02}
{\vspace*{6pt}}
\caption{\label{sch2}Hexafluoroisobutylation/deacetylation cascade reaction.}
{\vspace*{2pt}}
\end{scheme*}

\begin{table*}
\caption{\label{tab1}Influence of the base on the
hexafluoroisobutylation reaction of a chiral Ni(II) complex
\vspace*{4pt}\tpar{\protect\inlinefig{fx01}}\vspace*{-6pt}}
\tabcolsep=15pt
\begin{tabular}{ccccc}
\thead
Base (equiv) & Time (h) & \multicolumn{3}{c}{Yield}\\ \cline{3-5}
&& \textbf{3} & \textbf{4} & \textbf{5}\\
\endthead
TBAF (10) & \03 & - & 66\% \vspace*{5pt} & - \\
NaOH (4) & \04 & 20\% & \06\% & 55\% \\
LiOH (4) & \04 & 40\% & 15\% & 38\% \\
KOH (4) & \04 & 32\% & \06\% & 44\% \\
\textit{t}-BuOK (4) & \04 & 20\% & 13\% \vspace*{5pt}& 47\% \\
DBU (4) & \03 & - & 84\% & - \\
Et$_{3}$N (4) & 23 & ${>}$95\%\0 & trace & -
\botline
\end{tabular}
\end{table*}

Importantly, hexafluoroisobutene---the key reactive species---is a
gas (bp 14.1~\textdegree C) but can be easily generated in situ from
the liquid brominated precursor with this protocol.

\subsection{Access to (\emph{S})-5,5,5,5$'$,5$'$,5$'$-hexafluoroleucine}

We then decided to apply this methodology to the stereoselective
synthesis of Hfl. We used the robust strategy originally developed by
Belokon et~al., based on the stereoselective homologation of a glycine
Schiff base complex~\cite{bib45,bib46}, and further developed by others,
to access fluorinated amino acids, including
Soloshonok~\cite{bib47,bib48,bib49}, Larionov~\cite{bib50,bib51}, and
Koksch~\cite{bib52}, or other non-canonical amino acids~\cite{bib53}.
We found that the reaction is \mbox{compatible} with the chiral Ni(II) complex
\textbf{3}, which provides the desired alkylated complex \textbf{4} in
66\% yield (Table~\ref{tab1}). In contrast, none of the alkali metal
bases commonly used with this nickel complex (NaOH, LiOH, KOH, \textit{t}-BuOK)
were suitable for the reaction, which predominantly gave the
elimination product \textbf{5}, similar to the results observed with
ketoesters. Remarkably, we showed that the organic base
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) also favors the formation of
the hexafluorinated compound, with a 84\% yield~\cite{bib54}. This
study highlights the crucial role of the nature of the base and its
$\mathrm{p}K_{\mathrm{a}}$ in determining the outcome of the reaction.

\begin{scheme*}
{\vspace*{-2pt}}
\includegraphics{sc03}
{\vspace*{6pt}}
\caption{\label{sch3}Hydrolysis of the fluoroalkylated Ni(II) complex
to synthesize Fmoc- and Boc-protected Hfl and the unprotected form.}
{\vspace*{-2pt}}
\end{scheme*}

The procedure is suitable for large-scale synthesis, and the hydrolysis
of the Ni(II) complex produced free enantiopure Hfl as well as its
\textit{N}-Fmoc and \textit{N}-Boc derivatives (Scheme~\ref{sch3}).
This route provides easy access to this building block for use in a
wide range of applications.

\subsection{Mechanistic aspects}

In situ NMR experiments were performed and allowed the identification
of several intermediates. First, the brominated electrophile underwent
an elimination of HBr under basic conditions, leading to
hexafluoroisobutene after a few minutes (Scheme~\ref{sch4}).
Hexafluoroisobutene, with its electron-poor double bond, is highly
electrophilic and reacts with the deprotonated substrate via an
$\mathrm{S}_{\mathrm{N}}2'$ mechanism, leading to the pentafluoroalkene
intermediate. 

\begin{scheme*}
{\vspace*{-2pt}}
\includegraphics{sc04}
{\vspace*{6pt}}
\caption{\label{sch4}Proposed general mechanism.}
{\vspace*{-2pt}}
\end{scheme*}

Using an excess of fluoride anions (TBAF), we have shown that
hydrofluorination of the newly formed double bond is favored, yielding
the saturated hexafluorinated chain. With alkali metal bases, the
fluoride released in the medium forms inorganic fluoride salts that are
not nucleophilic enough to react with the fluorinated alkene. In
contrast, the organic base DBU generates a DBU${\bullet}$HF salt that
preserves the nucleophilicity of the fluoride ions released in situ and
acts as a fluorinating agent, as demonstrated by our in situ NMR
experiments (Figure~\ref{fig7}A). In this reaction, the DBU${\bullet}$HF
salt thus enabled the successful hydrofluorination of the
pentafluorinated intermediate without the need for an external source
of nucleophilic fluoride ions (Figure~\ref{fig7}B). In a more general
context, it is well known that ${\upalpha}$-(trifluoromethyl)vinyl
groups generally react with nucleophiles via an
$\mathrm{S}_{\mathrm{N}}2'$ mechanism, leading to ${\upbeta}$-fluoride
elimination~\cite{bib55,bib56,bib57}, which is observed in
${\upalpha}$-CF$_{3}$-carbanion chemistry~\cite{bib58}. In our study,
we showed that this reaction can be successfully reversed in the
presence of nucleophilic fluoride ions, restoring the saturated chain
containing two CF$_{3}$ groups.\looseness=-1

\begin{figure*}[p!]
\includegraphics{fig03}
\caption{\label{fig7}(A) In situ NMR experiments showing the
hydrofluorination reaction of the pentafluoroalkene with
DBU${\bullet}$HF. (B) Proposed mechanism of the hexafluoroisobutylation
reaction when DBU is employed.}
\end{figure*}


\section{Incorporation of Hfl into peptides and peptidomimetics}

The incorporation of Hfl into peptides has previously been achieved
using solid-phase peptide \mbox{synthesis} (SPPS). The SPPS allows the rapid
preparation of peptides up to 30--40 residues in length, either manually
or using automated peptide synthesizers. However, despite its
similarity to canonical leucine, the integration of Hfl into peptides
required optimization of synthetic procedures. The Boc strategy has
been the most commonly used approach to obtain Hfl-containing
sequences, particularly by the Kumar
group~\cite{bib25,bib31,bib32,bib59,bib60} and the Marsh
group~\cite{bib24,bib28,bib33,bib61}. This method uses the in situ
neutralization/{\textit{N}},{\textit{N}},{\textit{N}},{\textit{N}}-tetramethyl-{\textit{O}}-(1H-benzotriazol-1-yl)uronium 
hexafluorophosphate  protocol described by Schn\"{o}lzer
et~al.~\cite{bib62} and 4-methylbenzhydrylamine resin. Coupling
of Boc-protected Hfl sometimes required a prolonged coupling step
(${>}$2~h)~\cite{bib60}. The Boc strategy is robust but requires HF
treatment at the end of the elongation process to release the peptide
from the resin, which is a limitation for research laboratories that
are not equipped to safely handle anhydrous HF. In some cases, the Boc
strategy has been chosen over the more practical Fmoc strategy because
of the ineffective synthesis of Hfl-containing peptides with the latter
method~\cite{bib28}. MALDI-TOF analyses revealed that the coupling
yields for the two residues following Hfl incorporation were very low.
However, the reason for this remains unclear. Nevertheless, the
incorporation of Hfl into other peptide sequences has been successfully
achieved using the Fmoc strategy on Wang, Rink amide, and
Fmoc-PAL-PEG-PS resins~\cite{bib22,bib30,bib34,bib38} with a reduced
amount of coupling reagents relative to the molar amount of
Fmoc-Hfl-OH. Racemization of the Hfl residue was also described in one
case during peptide synthesis, resulting in two diastereoisomers with
identical masses (L/D ratio: 10/4)~\cite{bib22}. Finally, the Schepartz
group reported ${\upbeta}$-peptides containing ${\upbeta}$-Hfl to
investigate their supramolecular properties in the foldamer
field~\cite{bib63}. The fluorinated oligomers were synthesized using
Fmoc chemistry on Wang resin with PyAOP, HOAt, and DIEA for the
coupling steps. Exploration of alternative coupling agents and
optimization of conditions for deprotection of the amine protecting
group to further improve the synthesis of Hfl-containing peptides and
generalize their potential use are areas where our group is currently
focused. In addition, diversifying the types of hexafluorinated
monomers that can be used in foldamer chemistry is certainly a
promising direction for future research.

\section{Incorporation of Hfl into proteins}

There are several approaches to synthesize proteins containing
non-canonical amino acids, such as those using genetic code expansion
or SPPS combined with native chemical
ligations~\cite{bib64,bib65,bib66}. Some of these strategies have been
used to incorporate fluorinated amino acids~\cite{bib12}. In the case
of leucine analogues, several fluorinated derivatives have been
successfully incorporated into large proteins using \textit{E.~coli}
strains auxotrophic for leucine~\cite{bib67,bib68,bib69,bib70}; for
example, the introduction of (\textit{S})-5,5,5-trifluoroleucine into
several proteins has been achieved using this method. However, the
incorporation of Hfl still remains challenging with this approach and
requires engineered cells overexpressing Leu-tRNA synthetase to
overcome the unfavorable formation of the activated ester between Hfl
and its corresponding tRNA~\cite{bib27}. Importantly, this approach
does not result in complete incorporation of the fluorinated amino acid
due to the presence of the canonical amino acid in the cells, resulting
in a mixture of modified proteins. On the other hand, cell-free protein
expression offers a powerful alternative to in-cell
methods~\cite{bib71,bib72,bib73,bib74} and allows the incorporation of
non-natural amino acids using the translation
machinery~\cite{bib75,bib76,bib77}. This method allows the synthesis of
proteins that are difficult to produce in cell culture, including those
that are toxic or prone to aggregation. In collaboration with the
Loquet group at CBMN and IECB, we showed that the introduction of Hfl
into amyloid fibrils can be successfully achieved~\cite{bib78}. The
three Leu residues of the HET-s (218--289) prion were completely
replaced by Hfl within this 72-residue protein. Interestingly, the
fluorinated HET-s was well assembled and remained pathological. 
Solid-state $^{19}$F NMR reveals five out of six relatively narrow CF$_{3}$
signals (the sixth is certainly overlapped by the others) reflecting
the different local environment of the CF$_{3}$ groups within the
folded protein and a well-ordered conformation of the Hfl side chains
(Figure~\ref{fig8}). This method is thus well suited for the
incorporation of highly fluorinated residues and nicely complements
existing approaches for using this fluorinated amino acid to study
protein structure and function.

\begin{figure}
\includegraphics{fig04}
\caption{\label{fig8}$^{19}$F NMR at 60~kHz magic-angle spinning
solid-state experiments of cell-free synthesized HET-s containing three
Hfl residues.}
\end{figure}

\section{Conclusion and outlook}

In summary, our work has led to several key developments with promising
prospects.
\begin{itemize}
\item We have developed a general method for incorporating the
hexafluoroisobutyl group into carbonylated compounds. This process
allows the introduction of a fluorinated chain in a single step through
a tandem mechanism using a non-gaseous fluorinated electrophile. In
particular, this process overcomes the ${\upbeta}$-fluoride elimination
($\mathrm{S}_{\mathrm{N}}2'$ mechanism) thanks to an in situ
fluorination reaction when using an excess of fluoride sources or DBU
as a strong organic base, allowing the formation of the hexafluorinated
chain. The $\mathrm{S}_{\mathrm{N}}2'$ mechanism can thus be reversed.
Future work could explore the application of tandem
$\mathrm{S}_{\mathrm{N}}2'$/hydrofluorination reactions to other
${\upalpha}$-(trifluoromethyl)vinyl groups. This method now makes it
possible to introduce this fluorinated group and investigate its
potential interest in drug discovery programs. In addition, this
\mbox{fluoroalkylation} reaction was successfully applied to the enantiopure
synthesis of Hfl, which can now be produced on a large scale using a
chiral Ni complex. Furthermore, although fluoride elimination was not
desirable for synthesizing the fluorinated chain, the central acidic
proton of highly fluorinated---though not perfluorinated---groups,
next to the CF$_{3}$, could be used to promote fluoride ion
elimination. This, in turn, could potentially trigger the \mbox{degradation}
of the side chain, which may be of interest in the search for
degradable fluorinated motifs~\cite{bib79}.
\item We have demonstrated the potential of DBU${\bullet}$HF as a
fluorinating agent. The nucleophilicity of the fluoride ions in this
salt has yet to be determined and compared to other fluoride sources,
as well as its stability at high temperatures. This salt could
potentially be ({i}) less acidic than other widely used
nucleophilic fluoride sources such as Et$_{3}$N${\bullet}$3HF and
({ii}) less prone to Hofmann elimination compared to
TBAF~\cite{bib80}. DBU${\bullet}$HF could thus complement the existing
arsenal of fluorinating agents and find a wide range of applications in
fluorine chemistry.
\item The introduction of the Hfl into peptides has been previously
achieved by standard SPPS methods, including Boc and Fmoc strategies.
However, problems such as inefficient coupling steps and residue
racemization have been reported. There is potential for further
improvements in the synthesis of peptides containing this important
fluorinated residue, which would enhance its application in peptide
science and foldamer chemistry. Although there are some studies on
bioactive peptides containing
Hfl~\cite{bib32,bib33,bib34,bib35,bib36,bib37,bib38}, in-depth studies
on the rational design of peptide ligands to accurately incorporate Hfl
and exploit the full potential of this highly fluorinated group to
enhance (bio)molecular recognition properties are still needed.
\item Finally, we have shown that cell-free protein expression is not
only effective but particularly well suited for introducing Hfl into
large proteins---in our study, amyloid fibrils---that can be difficult
to synthesize using other approaches, such as SPPS or using auxotrophic
bacterial strains. This method opens new avenues for studying protein
structure and function with this highly fluorinated residue.
\end{itemize}

\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 affiliation other than their research organizations.

\section*{Funding}

This work has been supported by grants from {Agence Nationale de la
Recherche} (ANR, ANR-20-CE06-0008), {Minist\`{e}re de l'Enseignement
Sup\'{e}rieur, de la Recherche et de l'Innovation} (MESRI), and
{R\'{e}gion Nouvelle Aquitaine}. They are gratefully acknowledged.

\section*{Acknowledgments}

The authors thank the IECB Biophysical and Structural Chemistry Platform (BPCS),
CNRS UAR 3033, INSERM US001, and University of Bordeaux, especially Estelle Morvan
for NMR facilities and Dr. Brice Kauffmann for X-ray diffraction
analyses. They thank Dr. Antoine Loquet and his team for the fruitful
collaboration on cell-free synthesis of Hfl-containing amyloids.

\CDRGrant[ANR]{ANR-20-CE06-0008}

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